Progress in Brain Research Volume 29 Brain Barrier Systems

P R O G R E S S IN B R A I N R E S E A R C H V O L U M E 29

BRAIN BARRIER SYSTEMS

PROGRESS I N BRAIN RESEARCH

ADVlSORY BOARD W. Bargmann

H. T. Chang E. De Robertis J. C. Eccles J. D. French H. Hyden

J. Ariens Kappers S. A. Sarkisov

J. P. Schade F. 0. Schmitt

Kiel Shanghai Buenos Aires Canberra

Los Angeles Goteborg Amsterdam Moscow

Amsterdam Brookline (Mass.)

T. Tokizane

Tokyo

J. Z. Young

London

PROGRESS I N BRAIN RESEARCH V O L U M E 29

BRAIN B A R R I E R SYSTEMS

EDITED

ABEL LAJTHA New York State Research Institute for Neuroc1ienii.str.v and Drur Addiction, and Department of Bioclieniistry, College of’ Physicians arid Surgeons, Colrtiiihiu University, New York

AND

DONALD H. FORD Department of’ Anatoniy, State University of’ New York, Downstate Medical Center, Brooklyn, New York

ELSEVIER P U B L I S H I N G C O M P A N Y A M S T E R D A M / LONDON / N E W Y O R K

1968

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0 1968 BY ELSEVIER PUBLISHING COMPANY, AMSTERDAM

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List of Contributors

H. M. ADAM,Department of Pharmacology, University of Edinburgh Medical School, Teviot Place, Edinburgh (Scotland). CHARLES F. BARLOW,Department of Neurology, Harvard Medical School, Boston, Mass. (U.S.A.). CLAUDE F. BAXTER, Neurochemistry Laboratories, Veterans Administration Hospital, Sepulveda, California (U.S.A.). LOUISBAKAY,Division of Neurosurgery, State University of New York at Buffalo, School of Medicine, 462 Grider Street, Buffalo, New York (U.S.A.). R. BLASDFRG,New York State Research Institute for Neurochemistry and Drug Addiction, Ward’s Island, New York (U.S.A.). MILTONBRICHTMAN,Laboratory of Neuroanatomical Sciences, National Institute of Neurological Diseases and Blindness, Department of Health, Education and Welfare, Bethesda, Maryland (U.S.A.). S. CLAYMAN, McGill University Cancer Research Unit, McIntyre Medical Sciences Building, 3655 Drummond Street, Montreal (Canada). MIROBRZIN,Institute of Pathophysiology University of Ljubljana, Ljubljana (Yugoslavia). R. V. COXON, University Laboratory of Physiology, Oxford (England). CHRISTIAN CRONE,Department of Physiology, University of Copenhagen, Institute of Medicine, Juliane Maries Vej 28, Copenhagen (Denmark). T. Z. CSLKY,Department of Pharmacology, University of Kentucky, College of Medicine, Lexington, Kentucky (U.S.A.). R. W. P. CUTLER, Neurology Service of the Children’s Hospital Medical Center, Peter Bent Brigham Hospital. 300 Longwood Avenue, Boston, Mass. (U.S.A.). D. H. DEUL,N.V. Philips-Duphar, Weesp (The Netherlands). J. DOBBING, Department of Physiology, London Hospital Medical College, London (England). PHILIPDUFFY,Department of Pathology, Division of Neuropathology, College of Physicians and Surgeons, Colombia University, New York, N.Y. (U.S.A.). K . A. C. ELLIOTT, Department of Biochemistry and the Montreal Neurological Institute, McGill University, Montreal (Canada). J. FOIXH-PI,Department of Biochemistry, Harvard Medical School and McLean Hospital, Boston, Massachusetts (U.S.A.). D. H. FORD,Department of Anatomy, State University of New York, Downstate Medical Center,:450 Clarkson Avenue, Brooklyn, New York (t!.S.A.). C. M. FRENCH, Department of Physiology, The London Hospital Medical College, Turner Street, Londonl( England).

VI

LIST O F C O N T R I B U T O R S

STANLEY GINSBURG, Saul R. Korey Department of Neurology, Albert Einstein College of Medicine, Bronx, N.Y. (U.S.A.). LEONARD GRAZIANI, Saul R. Korey Department of Neurology, Albert Einstein College of Medicine, Bronx, N.Y. (U.S.A.). JANNY A. HAISMA, Biochemistry Department, Institute of Psychiatry, State University, Groningen (The Netherlands). P. L. IPATA, lstituto di Chimica Biologica dell’Universit8 di Pisa (Italy). J. JONGKIND, Netherlands Central Institute for Brain Research, IJdijk 28, Amsterdam (The Netherlands). J. ARlENs KAPPERS, Netherlands Central Institute for Brain Research, IJdijk 28, Amsterdam (The Netherlands). ROBERTKATZMAN,Saul R. Korey, Department of Neurology, Albert Einstein College of Medicine, Yeshiva University, Eastchester Road and Morris Park Avenue, Bronx, N.Y. (U.S.A.). IGOR KLATZO,Section on Clinical Neuropathology, National Institute of Neurological Diseases and Blindness, Department of Health, Education and Welfare, Bethesda, Maryland (U.S.A.). HAROLD KoENiC;, Department of Neurology and Psychiatry, Northwestern University Medical School and Neurology Service, VA Research Hospital, 333 East Huron Street, Chicago, 111. (U.S.A.). ABELLAJTHA, New York State Research Institute for Neurochemistry and Drug Addiction, Ward’s Island, New York (U.S.A.). GiuLio LEw, Istituto Superiore di Smith, Viale Regina Elena 299, Rome (Italy). A. LOWENTHAL, Fondation Born-Bung pour la Recherche, Department of Neurochemistry, Berchem-Antwerpen (Belgium). P. MANDEL, Centre de Neurochimie, Centre National de la Recherche Scientifique, Strasbourg (France). H. MCILWAIN, Department of Biochemistry, Institute of Psychiatry, The Maudsley Hospital, Denmark Hill, London, S.E. 5 (England). FREDERICK MINARD,Abbott Laboratories, Scientific Division, North Chicago, Ill. (U.S.A.). K. D. NEAME,Department of Physiology, University of Liverpool, Liverpool 3 (England). G . D. PAPPAS,Department of Anatomy, Albert Einstein College of Medicine, Yeshiva University, Eastchester Road and Morris Park Avenue, Bronx, N.Y. (U.S.A.). H. PAPPIUS, Department of Neurology and Neurosurgery, McGill University and Montreal Neurological Hospital, 3801 University Street, Montreal (Canada). E. PASCOE,Netherlands Central Institute for Brain Research, IJdijk 28, Amsterdam (The Netherlands). G . QUADBECK, Tnstitut fur Pathochemie und allgemeine Neurochemie, Pathologisches Blnstitut der Universitat Heidelberg, Berliner Strasse 5, Heidelberg (Germany). D. P. RALL,Department of Experimental Therapeutics, National Cancer Institute, Department of Health, Education and Welfare, Bethesda, Maryland (U.S.A.).

LIST OF CONTRIBUTORS

VII

B. M. RIGOR,Department of Pharmacology, University of Kentucky College of Medicine, Lexington, Kentucky (U.S.A.). S. ROBERTS, Department of Biological Chemistry and the Brain Research Institute, UCLA Center for the Health Sciences, Los Angeles, California 90024 (U.S.A.). CARLOA. Rossi, lstituto di Chimica Biologica, Dell’Universita di Pisa, Pisa (Italy). J. P. SCHADE, Netherlands Central Institute for Brain Research, IJdijk 28, Amsterdam (The Netherlands). P. G . SCHOLEFIELD, McGill University Cancer Research Unit, Mclntyre Medical Sciences Building, 3655 Drummond Street, Montreal (Canada). 0. STEINWALL, Department of Neurology, University of Goteborg, Goteborg (Sweden). VIRGINIATENNYSON, Department of Pathology, Division of Neuropathology, College of Physicians and Surgeons, Colombia University, New York, N.Y. (U.S.A.). WALLACE W. TOURTELOTTE, Department of Neurology, University Hospital, University of Michigan Medical Center, Ann Arbor, Michigan (U.S.A.). D. B. TOWER,Laboratory of Neurochemistry, National Institute for Neurological Diseases and Blindness, Department of Health, Education and Welfare, Bethesda, Maryland (U.S.A.). J. F. L. VAN BREEMEN, Biochemistry Department, Institute of Psychiatry, State University, Groningen (The Netherlands). C. J. VAN DEN BERG, Netherlands Central Institute for Brain Research, IJdijk 28, Amsterdam (The Netherlands). Department of Physiology, University of Montreal, Montreal, Nico M. VAN GELDER, Quebec (Canada). DIXONM. WOODBURY, Department of Pharmacology, University of Utah College of Medicine, Salt Lake City, Utah (U.S.A.). B. D. WYKE,Neurological Laboratory, Department of Applied Physiology, Royal College of Surgeons of England, Lincoln’s Inn Fields, London-WC 2 (England).

Vlll

Other volumes in this series:

Volume I : Brain Mechanisms Specific and Unspecific Mechanisms of Sensory Motor Integration Edited by G. Moruzzi, A. Fessard and H. H. Jasper Volume 2: Nerve, Brain and Memory Models Edited by Norbert Wiener? and J. P. Schade Volume 3 : The Rhinencephalon and Related Structures Edited by W. Bargmann and J. P. Schade Volume 4: Growth and Maturation of the Brain Edited by D. P. Purpura and J. P. Schade Volume 5 : Lectures on the Diencephalon Edited by W . Bargmann and J. P. Schadk Volume 6: Topics in Basic Neurology Edited by W. Bargmann and J. P. SchadC Volume 7: Slow Electrical Processes in the Brain by N. A. Aladjalova Volume 8: Biogenic Aiuines Edited by Harold E. Himwich and Williamina A. Himwich Volume 9: The Developing Brain Edited by Williamina A. Himwich and Harold E. Himwich Volume 10: The Stritctitre und Function of the Epiphysis Cerebri Edited by J. Ariens Kappers and J. P. Schadt Volume 11 : Organization of the Spinal Cord Edited by J . C. Eccles and J. P. Schade Volume 12: Physiology of Spinal Neurons Edited by J . C. Eccles and J. P. Schade Volume 13 : Mechanisms of Neural Regeneration Edited by M. Singer and J. P. Schadd Volume 14: Degeneration Pafterns in the Nervous Sysfem Edited by M. Singer and J. P. Schade

IX Volume 15: Biology of Neuroglia Edited by E. D. P. De Robertis and R. Carrca Volume I6 : Horizons in Neuropsychopharmacology Edited by Williamina A. Himwich and J. P. Schade Volume 17: Cybernetics of the Nervous System Edited by Norbert Wiener? and J. P. SchadC Volume 18 : Sleep Mechanisms Edited by K. Akert, Ch. Bally and J. P. SchadC Volume 19: Experimental Epilepsy by A. Kreindler Volume 20 : Pharmacology and Physiology of the Reticular Formation Edited by A. V. Valdman Volume 21A : Correlative Neurosciences. Part A : Fundamental Mechanisms Edited by T. Tokizane and J. P. Schade Volume 21 B: Correlative Neurosciences. Part B: Clinical Studies Edited by T. Tokizane and J. P. SchadC Volume 22: Brain reflexes Edited bij E. A. Asratyan Volume 23 : Sensory Mechanisms Edited by Y.Zotterman Volunie 24: Carbon Monoxide Poisoning Edited by H . Bour and I. McA. Ledingham Volume 25: The Cerebelluni Edited by C. A. Fox and R. S. Snider Volume 26: Developmental Neurology Edited by C.G.Bernhard Volume 27: Slructure and Function of the Limbic System Edited by W.Ross Adey and T. Tokizane Volume 28: Anticholinergic Drugs Edited by P. B. Bradley and M. Fink Volume 30: Cerebral Circulation Edited by W.Luyendijk

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Preface

I n recent years, many important developments have occurred in many research disciplines that are directly related to brain permeability. In morphological fields, the fine resolution provided by electron microscopy has resulted in new concepts of membrane structure. Electron microscopy, combined with autoradiography, have contributed significant information concerning dynamic events occurring in and around such membranes. Permeability measurements with drugs have led to new theories of the physical and chemical requirements which determine the penetration of drugs into the central nervous system. Measurements of permeability during development and in later more mature stages of life have resulted in theories that relate brain permeability and function and, as in senescence, brain permeability and pathology. Recent work with normal metabolites in the brain has shown complex active transport mechanisms to be present in the walls of the various cell types making up the nervous system. Cerebral transport phenomena have been shown to have a considerable degree of selectivity and specificity and seem highly significant in controlling the mechanisms of brain metabolism. Inasmuch as these membranes and transport systems sometimes appear to restrict entry of materials into the brain, it is apparent that they represent, in part at least, what has been termed the “Blood-Brain Barrier”. Thus, the brain-barrier system may determine which metabolites can gain access to the brain, may determine the level of these metabolites available to various brain parts and brain cells, and may determine the rate of supply and the rate of elimination. It has also been suggested that specific transport processes may be interfered with in pathological states (i.e. amino acidurias, and phenylketonuria), and therefore that alterations of permeability can be involved in altered mental function. It is obvious from this very brief survey that great advances have been made recently on a number of fronts, in such areas as anatomy, physiology, neurochemistry, and pharmacology. Despite these advances, however, it is not uncommon to attend meetings and learn that the failure of almost any compound to enter the brain is due to the Blood-Brain Barrier, or that the only amino acid capable of penetrating the brain is glutamine. Most textbooks of neuroanatomy treat the concept of “Brain-Barrier” as being too complicated for discussion or provide some very structural rigid concept for restricting entry of most compounds. Although a number of conferences have been planned to consider these important advances in cerebral permeability, barriers or transport, none has taken place in recent years. Thus it was the purpose of the conference held in Amsterdam from September 26 to 30, entitled “Brain Barrier Systems” to gather together leading investigators from America and Europe who have been working on the various anatomico-bio-

XI1

PREFACE

chemical-physiologic aspects of the complex membrane systems in the brain, and attempt to define as concisely as possible our state of knowledge today about these “barriers”. It is most fortunate that so many of the outstanding contributors to this field could come and participate at the conference. Many more investigators with important contributions are missing from the volume, because many of the authors had previous commitments and so were, to our regret, unable to participate. Whiletheir absence is certainly felt, it was fortunate that it was possible to have contributions on most of the important aspects of the problem. The conference, therefore, could show the relationship and interdependence of the disciplines concerned with barrier phenomena, and it was highly successful in clarifying the nature of the investigations still required to permit us to fully understand how these “membranes” serve to maintain, or influence, normal brain function. In planning this conference, we were delighted to obtain the cooperation of the Netherlands Central Institute for Brain Research, which has taken care of all the local arrangements. We were indeed fortunate to be able to gather in the stimulating atmosphere provided by this castle (De Hooge Vuursche), which by its nature led us to many fruitful hours of discussion after the close of the formal meetings. Unfortunately, it was not feasible to hide microphones along the paths in the beautiful garden and in the woods surrounding the conference to record all the free discussions that went on till late in the evening hours. We are also indebted to the Office of Naval Research for their interest, both intellectual and financial, in the support of this conference. Additional financial support was provided by the drug houses of E. R. Squibb and Sons, Organon, Abbott Laboratories, and the Warner Lambert Research Laboratories. Without the assistance of the Brain Institute, the Office of Naval Research, and the research directors of the above drug companies, all the best intentions and hope for having a conference dealing with the importance of the various Brain-Barriers in the neurobiologic system would have long ago foundered. Thus, it is proper that we should express our thanks for their interest. Finally, thanks are due to all the participants for their enthusiastic participation, for the excellent contributions, and for the stimulating discussions, all of which made the conference such a success and makes the present book an excellent summary of the problems. DONALD H. FORD AEELLAJTHA

Contents

List of Contributors Preface . . . . . .

................................ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V XI

STKUCTUKAL CONSIDERAIIONS The composition of nervous membranes J.Folch-Pi.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 The intracerebrdi movement of proteins injected into blood and cerebrospinal fluid of mice Milton W. Brightinan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Electron microscopic cytochemistry and rnicrogasometric analysis of cholinesterase in the nervous system Virginia M. Tennyson, Miro Brzin and Philip Duffy . . . . . . . . . . . . . . . . . 41 The fine structuie of the choroid plexus: Adult and developmental stages Virginia M. Tennyson and George D. Pappas . . . . . . . . . . . . . . . . . . . . 63 Transport and effects of cationic dyes and tetrazolium salts in the central nervous system Harold Koenig . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 The evidence that ganglioside, a metal, and chemical energy are involved in the serotonin household of synaptic vesicles from brain D. H . Deul, Janny A. Haisma and J. F. L. van Breemen . . . . . . . . . . . . . . . . 125

CEREHKOSPINAL FLUID Cerebrospinal fluid transport R.V.Coxon. . . . . . . . . . . . . . . . . . . . . The choroid plexus as a glucose barrier T. Z. Csaky and B. M. Rigor. . . . . . . . . . . . . . Transport through the ependymal linings David P. Rail . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . .

135

..............

147

..............

159

SUBSTRATES FOR BRAIN METABOLISM Mechanisms of metabolite transport in various tissues P. G . Scholelield and S. Clayman . . . . . . . . . . . . . . . . . . . . . . . . . . 173 A comparison of the transport system for amino acids in brain, intestine, kidney and tumour K. D. Neame. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Transport as control mechanism of cerebral metabolite levels Abel Lajtha . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Regional differences in cerebral amino acid transport Giulio Levi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .219

CONTENTS

XIV

Influence of elevated circulating levels of amino acidson cerebral concentrations and utilization of amino acids Sidney Roberts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Specificity of cerebral amino acid transport: A kinetic analysis Ronald G . Blasberg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 A possible enzyme barrier for y-aminobutyric acid in the central nervous system Nico M. van Gelder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

ION MOVEMENTS Ion movements in isolated preparations from the mammalian brain 273 Henry Mcllwain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cation exchange in blood, brain and CSF Robert Katzman, Leonard Graziani and Stanley Ginsburg . . . . . . . . . . . . . . .283 Distribution of nonelectrolytes in the brain as affected by alterations in cerebrospinal fluid secret ion Dixon M. Woodbury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297

FACTORS INFLUENCING BARRIER FUNCTION Changes in barrier effect in pathological states Louis Bakay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 Clinical importance of alterations in barrier G . Quadbeck. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Drug influence on the barrier G.Quadbeck. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 Transport inhibition phenomena in unilateral chemical injury of blood-brain barrier Oskar Steinwall. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357 Changes in blood-brain permeability during pharmacologically induced convulsions Robert W. P. Cutler, Antonio V. Lorenzo and Charles F. Barlow . . . . . . . . . . . . 367 The effect of hypothermia on electric impedance and penetration of substances from the CSF into the periventricular brain tissue Igor Klatzo, Choh-Luh Li, Don M. Long, Anthony F. Rak, Miroslaw J. Mossakowsky, Levon 0. Parker and Louis E. Rasmussen . . . . . . . . . . . . . . . . . . . . . . 385 Changes in brain accumulation of amino acids and adenine associated with changes in the physiologic state Donald H. Ford . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401 The development of the blood-brain barrier John Dobbing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417 Intrinsic amino acid levels and the blood-brain barrier Claude F. Baxter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429

.

SPACESIN

THE

CENTRAL NERVOUS SYSTEM

Introduction to session on brain spaces K. A. C. Elliott. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451 Spaces in brain tissue in vitro and in vivo Hanna M. Pappius . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455 Delineation of fluid compartmentation in cerebral tissues Donald B. Tower . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465 Thiocyanate in the brain and the size of the extracellular space C. M. French . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .485

xv

CONTENTS

Some spaces and barriers in postmortem multiple sclerosis Wallace W. Tourtellotte and Julius A . Parker . . . . . . . . . . . . . . . . . . . Inhibition of sheep brain 5-nucleotidase by nucleoside triphosphates P. L . lpata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions A . Lajtha and D . Ford . . . . . . . . . . . . . . . . . . . . . . . . . . . .

..

493

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527

.. Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

535 539

547

Structural Considerations

The Composition of Nervous Membranes J. F O L C H - P I McLean Hospital, Belniont, and Department of Biochemistry, Harvard Medical School, Boston, Mass. ( U S A . )

The literature on the chemistry of membranes is much too vast to allow a meaningful review within the space and time available for this presentation. Therefore, instead of attempting such an impossible task, the subject matter of this presentation will be limited to the discussion of four groups of compounds which occur mainly, if not exclusively, in the nervous system and all of which are clearly identifiable as membrane constituents. These compounds are gangliosides, proteolipids, polyphosphoinositides and neurokeratin. Gangliosides are neuronal components; proteolipids, polyphosphoinositides and neurokeratin are myelin components. Since even our information on these compounds - and specially the sum total of our uncertainties about them - is much too vast for adequate presentation in the time available, the following comments will bear mainly on those aspects of their chemistry that are specially pertinent to their function as membrane constituents. GANCLIOSIDES

In 1941, Klenk isolated from brain a new aniino acid to which he gave the name of iieuruminic acid. In 1942, he described a group of new brain glycolipids that were

characterized by the presence of neuraminic acid and which were otherwise constituted by a lipid moiety, presumably a ceramide, and a carbohydrate moiety presumably consisting of one or more monosaccharides. He named them gangliosides because their distribution in the tissue suggested that they were components of ganglion cells. Subsequent work by Klenk and other workers established that neuraminic acid was present in gangliosides usually as an N-acetyl derivative, and that it was identical with siulic acid which had been isolated by Blix from submaxillary mucin a few years previously (Klenk, 1936); that the carbohydrate moiety of gangliosides contained hexosamine(s) in addition to neutral sugars; and, finally, especially after the introduction of thin layer chromatography, that gangliosides comprised a large number of closely related chemical compounds. The chemistry of gangliosides has been the object of recent authoritative reviews, to which the reader is referred for a detailed discussion of the subject (Svennerholm, 1964; Ledeen, 1966). In summary, gangliosides are complex glycolipids consisting, Referenrrs p. 13-14

2

J. F O L C H - P I

according to Kuhn and Wiegant (1963), of a lipid moiety in the form of a ceramide, and of a carbohydrate moiety in the form of a tetrasaccharide, as follows: galactose (1 -3)N-acetylgalactosamine( 1-4)gaIactose( I -4)glucose( 1- 1)ceramide

To this backbone are attached, I , 2 or 3 sialic acid residues constituting respectively mono-, di- or trisialogangliosides. Variations in the carbohydrate moiety have been reported and at present upward to 12 different gangliosides have been recognized. In addition, the ceramide moiety, although consisting mainly of sphingosine and stearic acid, contains also higher and lower homologs of sphingosine, and a host of different fatty acids, thus multiplying several times the number of individual gangliosides that occur in nature. In parallel with this chemical work, it was observed that, although gangliosides were extracted from brain tissue with conventional lipid solvents, they were easily soluble in water as undialyzable solutes. In aqueous solutions they appeared to be monodisperse (Folch et al., 1951), with an apparent molecular weight which was first computed, from ultracentifuge data, as being 250 000 and which by other methods of measurement employing different parameters has been given values ranging from 180 000 to 400 000. This observation led to the assumption that a physically homogeneous high molecular weight compound was being dealt with, and to it was given the name of strandin (Folch et al., 1951). With advances in the chemistry of gangliosides it became apparent that strandin was a polymeric form of gangliosides, and even a critical micellar concentration of 0.015 % was suggested (Howard and Burton, 1964). Since it had been observed that preparations of gangliosides contained small amounts of polypeptides (Folch et a / . , 1951; Folch and Lees, 1959) or proteins, it has been also suggested that these presumed protein contaminants might play a part in determining the remarkable homogeneity of the micellar solutions of strandin (Rosenberg and Chargaff, 1956). That gangliosides are, at least in part, membrane components appears to be a reasonable assumption on the basis of their distribution in the nervous system, of their rate of accumulation during brain development (Folch, 1955), of histochemical evidence (Diezel, 1959, and of their distribution among subcellular fractions of brain tissue (Wolfe, 1961 ; Wherrett and Mcllwain, 1962; Seminario et al., 1964; Burton et al., 1964; Eichberg et al., 1964; Spence et a/., 1964). In addition, a consideration of some of the properties of gangliosides clearly points to them as being exceptionally well designed as membrane constituents: the presence of the carboxyl group of sialic acid which permits binding with organic and inorganic cations, the presence of the lipophilic groups of the ceramide and of the hydrophilic groups of the carbohydrate moiety which permit interaction with many different substances including proteins, lipids, and many small molecule substances, and the ability to form micelles of fairly uniform size. Indeed, it is not surprising that gangliosides have been implicated by many workers in different membrane functions : cation transport (Mcllwain, 1962), acetylcholine release at the presynaptic membrane, synaptic inhibition, receptor function for serotonin (Burton et al., 1964), for tetanus toxin (Van Heyningen, 1963), which parallel the well established function of sialic acid as a viral receptor in red

COMPOSITION O F NERVOUS MEMBRANES

3

blood cells, just to mention a few highlights in a considerable literature dealing with possible functions of gangliosides. We will close this brief survey by discussing the interaction of gangliosides with sodium, potassium, calcium and magnesium, and an apparent effect of the presence or absence of polypeptide on the behavior of the resulting complexes. As expected, all four cations combine with gangliosides, presumably by simple electrostatic bonds. These combinations are reversible, and each cation can displace the others from combination with the ganglioside, the divalent cations being more effective than the monovalent. However, the calcium-ganglioside complex is much less polar than either the free ganglioside or the complexes of ganglioside with the other cations (Quarles and Folch-Pi, 1965). Thus, when these salts of gangliosides are dissolved in the biphasic system chloroform : methanol water 8 : 4 : 3, v/v/v, free ganglioside, and its sodium, potassium or magnesium salts remain in the upper (polar) phase. On the other hand, the calcium complex will remain in the upper phase at low and at high concentrations of calcium ions, but at intermediate concentrations of these ions, it will partition into the lower, least polar phase. This effect of calcium appears to require the presence of small amounts of other lipids, sulfatides being especially effective in this action. In addition, the presence of polypeptide will tend to produce an accumulation of calcium gangliosidate at the interphase. All these interactions illustrate the dramatic changes that may occur in the physical properties of gangliosides, hence on their possible behavior as membrane constituents, and they also point to a possible crucial influence of the presence of small amounts of polypeptides on ganglioside properties. The effect of polypeptides does not appear to be a general protein property. Since the concentrations of calcium that effect the change in polarity of the ganglioside fall in part within the physiological range of concentrations of calcium, it is clear that the observations on the model employed may have implications for the behavior of gangliosides in vivo.

+

Proteolipids, polyphosphoinositides and neurokeratin These three groups of substances are closely related biochemically and anatomically. As will be detailed below, these are myelin constituents and, since myelin itself is formed by the infolding of the plasma membrane of the satellite glial cells around the axons, it is obvious that myelin components are membrane components by definition. In addition, polyphosphoinositides are constituents of both proteolipids and neurokeratin and proteolipids and neurokeratin appear to be very closely related. Since polyphosphoinositides are components of both proteolipid and neurokeratin, it might be pertinent to review highly their history. In 1941 Folch and Woolley (1942) reported the occurrence of inositol as a constituent of brain lipids. Subsequent work resulted in the isolation of an inositol-rich lipid fraction (Folch, 1949) which appeared to have as constituents, inositol diphosphate, glycerol and fatty acids in integral molar ratios and to which the name diphosphoinositide was given (DPI). Later work, using chromatographic techniques, showed, that besides diphosphoinositide, there was a triphosphoinositide (Dittmer and Dawson, 1961 ; Brockerhoff and Ballou, 1961), References p. 13-14

4

J. F O L C H - P I

and that, in fact, the latter might well be the most abundant of the two, DPI possibly being derived by partial dephosphorylation of TPI. Proteolipids - The name proteolipid was introduced in 1951 by Folch and Lees to designate substances consisting of a protein moiety and a lipid moiety and characterized by a complete insolubility in water and solubility in some organic solvents, especially in chloroform : methanol mixtures. The name is intended to emphasize that proteolipids are lipoproteins which behave like lipids. The original observation that led to the discovery of proteolipids was that chloroform : methanol extracts of brain, presumably freed of nonlipid material by water washing, contained protein material (Folch and Lees, 195I). This protein material remained in chloroform through successive water washings, i.e., it was not only soluble in chloroform but insoluble in water. The protein material could be obtained by simply taking to dryness the extract, and extracting the residue with chloroform : methanol. Apparently, in the course of drying the protein underwent some rearrangement that resulted in the loss of its original solubility in chloroform : methanol. As a consequence, the protein remained as an insoluble residue. It contained 14 % N, 1.75 % S and, after acid hydrolysis, 91 % of its nitrogen could be recovered as free amino acids. Its amino acid composition revealed a preponderance of monoamino-mono-carboxylic acids, a high concentration of methionine and cysteine (or cystine) and a relatively small concentration of acidic and of basic amino acids. The material was resistant to the action of trypsin, pepsin, papain and erepsin. Later, it was found to be hydrolyzable by pronase. Distribution of proteolipids. - Although especially abundant in nervous tissue, proteolipids are also found in a wide variety of animal and vegetable tissues. Bovine tissues contain the following amounts of proteolipid protein (mg/g tissue weight) : heart, 3.5; kidney 2.0; liver, 1.6; lung, 0.95; uterus, 0.6; biceps, 0.4. In spinach chloroplasts they represent 2-4 % of dry weight (Zill and Harmon, 1961). These values are only indicative because the yields obtained may have been incomplete. In the nervous system, proteolipids are found at highest concentration in white matter (20-25 mg/g wet tissue) and at about 1/5 this concentration in gray matter. They are present in peripheral nerve at only 1/20 to 1/80 the concentration in white matter (Folch et al., 1958), which may well indicate a qualitative difference between peripheral and central myelin. They are absent from fetal brain and their appearance and progressive accumulation is concurrent with myelination (Folch, 1955). In a study of 28 different anatomical areas of the human nervous system, Amaducci (1962) has observed marked and consistent differences from one anatomical area to another. He has shown that the highest concentration of proteolipids occurs in central white matter, with 1/5 to 1/10 as much in gray matter, and only 1/20 to 1/80 as much in peripheral nerve. Within this general pattern the concentration of proteolipids decreases progressively from cerebral white matter to spinal cord white matter, with cerebellar white matter showing an intermediate value. In spinal cord itself, the concentration of proteolipids appears to decrease in the anterolateral columns

COMPOSITION OF NERVOUS MEMBRANES

5

from rostra1 to caudal levels, while no such gradient is found in the posterior columns. The anterior and posterior spinal roots contain proteolipids at substantially lower concentrations than are found in spinal cord white matter, but this amount is still several times that present in peripheral nerves. The concentration of proteolipids found in gray matter from various areas shows no clear pattern. On the basis of all these observations it had been assumed that, in the central nervous system, proteolipids were in part myelin components. Later work on isolated myelin has established that this is the case and that proteolipids are the main protein found in myelin (Autilio, 1966). In gray matter and in non-neural tissues, especially in heart, proteolipids have been traced to mitochondria at least in part. Ptirifcation of ,i.hite matter proteolipids - Hitherto proteolipids have been obtained from tissue only by extraction with chloroform : methanol. From the extracts thus obtained proteolipids have been prepared in various states of relative purity by the “fluff” method, by emulsion-centrifugation, by dialysis, or by chromatography. The “fluff” method (Folch and Lees, 1951), the one originally used for the preparation of proteolipids, is based on the tendency of proteolipids to concentrate at interfaces. The chloroform : methanol extract is allowed to equilibrate with at least five-fold its volume of water; a biphasic system consisting of a chloroformic phase and an overlying water-methanol phase is eventually obtained. The proteolipids are in part concentrated at the interface as a fluff and, in part, in the chloroformic phase. By further handling, proteolipids A and B are obtained from the fluff, and proteolipid C from the chloroformic phase. These preparations contain from 20 to 70 % protein, and are, otherwise, purely operational concentrates of proteolipids. The emulsion-centrifugation procedure (Folch e t a ] . , 1959) is based on the difference of density between free lipids and proteolipids. A washed chloroform-methanol extract of white matter is taken to dryness in vacuum. The resulting residue is emulsified in 30-fold its weight of water, and the emulsion is centrifuged at 4600 g for 1 h. The supernatant is decanted, the residue suspended in the same amount of water as before and the new suspension centrifuged as before. The whole cycle is repeated twice more. The third and fourth supernatants are water clear or only slightly opalescent. The residue from the fourth centrifugation is again suspended in the same volume of water as before, and the new suspension centrifuged at 200 g for 10 min. The supernatant is decanted. The residue is soluble in chloroform containing small amounts of methanol and water. It is a crude proteolipid preparation that contains approximately 30-40 % protein, 40 % phosphatides, and 12-15 % each of cerebrosides (including sulfatides) and cholesterol. This crude preparation can be purified further by extracting in succession, twice with 70-fold its weight of ethyl ether, and twice with 80-fold its weight of ethanol. The final residue represents a total preparation of white matter proteolipids with the composition given in Table I. Preparatiori ofproteolipids by dialysis - Since proteolipids are high molecular weight compounds, they can be separated from free lipids by dialysis in organic solvents. Murakami et a/. (1962) used dialysis in the purification of brain heart proteolipids Rrfcrmrrs p. 13-14

TABLE I A V E R A G E C O M P O S I T I O N O F B R A I N W H I T E MATTER P R O T E O L I P I D F R A C T I O N S P R E P A R E D BY D I F F E R E N T M E T H O D S

(Unless otherwise noted, all components are expressed at % of the respective fraction)

Procedure and Fraction

Fluff method Proteol. A Proteol. B Proteol. C Emulsion-Centrifugation Crude Concentrated Dialysis Chromatography I I1 111 Water-soluble proteolipid protein

Yield, fresh tissue

Proreolipid protein

Phosphatides

Cerebrosides

Cholesterol

E 1 em/'% ar 280 mp

(wig)

( 7);

( %)

I %I

( %)

20 10 10

12-20

5-15

3540

30

50 20

55-65

25-30

traces traces traces

a 5 0 25-30 20

35-45 55-65 70

40 25 20-25

5-1 3

7 1 0.2

6 - 8 9 -11 10.5- 13.5

4

95 95 95

2- 5 2 2

-

-

13-14 13-14 13-14

99-100

1

-

1622

4 7

3- I 2- 5

-

COMPOSITION O F NERVOUS MEMBRANES

3.0

I II 'I

2.5

g

t

I

2.0

01

+ U t

7 5 2 5 4 5 J: 7 0 3 0 6

80 20 3

8515l\!

2

I

'

60409

7

/I

I I I

1.5

v,

z

LL]

n

a

1.0

0 k-

CL

0.5

0

5

10

15

20

25

30

TUBE NUMBER (each 5 m l )

Fig. 1 . Chromatography of proteolipids obtained by the emulsion-centrifugation method on a silicic acid column. A 10 mm inner diameter column packed with 4 g silicic acid was used. It was loaded 1 2 8 0 mp = 8.1. The ratios on the upper line express the prowith 69 mg proteolipids Elcm portions of chloroform, methanol and water of the eluting mixture. - Optical density at 280 mp; _ _ _ _ amount of P.

and Thompson et al. (1963) have applied it to the purification of myelin proteins. In the case of brain white matter, the proteolipid and free lipid mixture obtained from a washed lipid extract, or partially purified proteolipid preparations are dissolved in chloroform-methanol 2 : I and the solution placed in a cellophane dialysis tubing previously washed with water and with chloroform-methanol, and dialyzed against the solvent mixture. The system is shaken gently, the diffusate is changed daily and the dialysis allowed to proceed until the diffusate is free of solutes. Usually 7 days suffice. The composition of such preparations is given in Table I.

Chromatography of proteolipids - Matsumoto et al. ( 1 964) have chromatographed the concentrated proteolipid preparations prepared by the emulsion centrifugation procedure, on silicic acid columns. The details of such a chromatographic run is given in Fig. 1. It shows that the first two peaks obtained are free lipids, with little or no protein, and that they are followed by three peaks consisting mainly of protein. It is noteworthy that the last protein peak can only be eluted by chloroform-methanol 1 : 1 containing HCI. This last fraction shows solubility properties different from those of the starting preparation in the sense that in the biphasic system chloroform-methanolwater 8 : 4 : 3 (v/v/v), the original proteolipid is found quantitatively in the chloroformic phase, whereas the proteolipid recovered from the last chromatographic Rrferencrs p. 13-14

8

J. F O L C H - P I

fraction has a definite partition between the two phases, the methanolic-water phase containing about 1/5 as much proteolipid as the chloroformic phase. Properties and composition of proteolipids - All proteolipid preparations described above are soluble in chloroform or in mixtures ofchloroform with methanol and water. They are completely insoluble in water and in aqueous solutions and in the biphasic system chloroform-methanol-water 8 : 4 : 3 (v/v/v), they will concentrate quantitatively in the chloroform phase. All the proteolipid preparations have been found to be resistant to the action of pepsin, trypsin, papain and erepsin. This resistance is not due to the presence of lipids, because it is found in the water-soluble proteolipid protein ( v i ) which is free of lipids, and in the insoluble denatured proteolipid protein described below. The only enzyme that attacks proteolipids is pronase, although the extent of this susceptibility has not been determined exactly. The chemical composition of the various proteolipids is given in Table I. Composition given for proteolipids A, B and C is merely indicative because both yield and composition vary widely according to the exact conditions followed in preparation. The other methods of preparation yield more consistent products. An important fact illustrated by this table is that the amount of lipids in proteolipids may vary from 60 % (in crude emulsion-centrifugation proteolipid) to less than 5 % in the three chromatographic fractions without any change in general solubility properties. Proteolipid protein - It has been isolated as an insoluble material by drying from solutions in biphasic systems (Brockerhoff and Ballou, 1961) or by exposure toalkaline pH’s at certain ionic strengths (Webster and Folch, 1961). At pH 8 or 9, proteolipids can split, with liberation of free lipids and, of protein, as an insoluble material, provided the medium contains ions at sufficient concentration. At pH 8.8 between ionic strengths 0.001 and 1 .O the proportion of proteolipid split is proportional to the logarithm of the ionic concentrations; this fact suggests that the mechanism of splitting is by ionic competition. These insoluble proteolipid proteins still contain small amounts of lipids. The lipid content can be reduced by extraction with hot chloroform : methanol; the lipid most firmly bound to the protein appears to be a polyphosphoinositide mainly triphosphoinositide. It can be removed only with chloroform : methanol acidified with HC1 to 0.04 N concentration (Pritchard and Folch-Pi, 1963). The amino acid composition of the different proteolipids has been estimated repeatedly by different methods and in different laboratories with wholly concordant results. These are that the amino acid patterns of the different white matter proteolipid preparations are identical or so similar as to be indistinguishable from each other. Table I1 gives the amino acid composition of preparations obtained by emulsion centrifugation of the chromatographic parallel fractions obtained from them, and the water soluble proteolipid (v.i.). For more meaningful comparison, serine, the concentration of which varies with the amount of phosphatidyl serine present, has been oomputed uniformly at 6 % of total amino acids on a molar basis; methionine and

T A B L E I1

P

A M I N O A C I D C O M P O S I T I O N O F B R A I N W H I T E MATTER P R O T E O L I P I D P R E P A R E D B Y DIFFERElvT M E T H O D S

(Results expressed as Amino Acids

Leucine Isoleucine Valine Glycine Threonine Serine Proline Aspartic Acid Glutamic Acid Histidine Arginine Lysine Tyrosine Phenyl Alanine Alanine Methionine Half cystine

Washed total Lipid extract

11.2 7.6 9.5 8.3 11.5 (1KO)* 2.2 2.9 2.4 2.7 2.2 7.4 3.0 8.0 9.0 (6.0)**

Crude Proteolipid

o’,

of total a-amino acid N recovered from acid hydrolysates) Concentrated Proteolipid

I

Chrontatographic Fractions II

III

Water Soluble Proteolipid Protein

11.1 5.9 7.3 9.7 8.4 (7.3)* 2.7 3.7 4.5 2.1 1.7 6.9 4.8 8.3 11.0

11.4 4.9 6.7 10.7 8.3 (7.4)* 3.1 4.0 5.8 1.9 2.0 3.8 4.9 8.3 12.0

11.8 5.3 6.7 9.6 9.0 (5.5)* 3.0 4.3 4.3 1.7 2.7 3.9 4.4 8.6 13.3

11.1 4.9 6.5 10.5 9.0 (5.2)* 2.3 3.9 3.8 1.8 3.1 4.3 4.9 8.2 13.5

11.4 4.8 6.4 10.8 8.8 (5.0)* 2.4 3.9 4.2 2.0 3.2 4.4 4.9 8.2 13.2

11.1 4.9 6.9 10.3 8.5 (8.5)* 2.8 4.2 6.0 1.8 2.6 4.3 4.6 7.9 12.5

(6.0)**

(6.0)**

(6.0)**

(6.0)**

(6.0)* *

(6.0)**

* Serine computed uniformly at 6 % of total Amino Acids. ** Methionine and half cystine computed at 6 % of total Amino Acids.

c)

0

=! 0

2: 0

-I

Z

m

w < 0

10

J. F O L C H - P I

half-cystine have also been computed jointly at 6 %, a concentration consistently found when these amino acids are estimated independently. Tryptophan, which is destroyed by acid hydrolysis, is not included. From optical density values at 280 mp, it can be computed to amount to 3 % or higher. It can be seen that the amino acid composition is indistinguishable from preparation to preparation and very similar to the composition of total chloroform-methanol soluble protein. The same holds true for proteolipid A, B and C, and proteolipids separated by dialysis. However, in the latter case, 10 to 15 % of the protein is found in the diffusate. This dialyzable fraction is protein and not a simple mixture of amino acids. The amino acid composition of the dialyzable material is different from that of the undialyzable proteolipid in that the former contains relatively more glycine, alanine and perhaps threonine. This may represent a real difference or a differential loss of amino acids in the course of hydrolysis because of the presence of a large concentration of lipid in the diffusate. The amino acid pattern of proteolipids has the following features: (a) A relative scarcity of acidic and of basic amino acids; aspartic and glutamic amount jointly to less than 10 % and arginine, lysine and histidine amount jointly also to less than 10 %; (b) A wealth of methionine and half-cystine, as is to be expected from the high concentration of sulfur in proteolipid protein (1.75 %); (c) A relative abundance of the so-called non-polar amino acids, i.e., amino acids that when combined in a peptide chain, offer only non-polar groups to the medium; leucine, isoleucine, valine, glycine, proline, phenylalanine and alanine amount to 57-58 % of total amino acids. If tryptophan is added, over 60 % of amino acids are non-polar ; (d) The relatively high concentration of tryptophan as indicated by the high optical density at 280 mp. On the basis of the least abundant residue, proteolipid protein is computed to comprise 125 amino acid residues of an average size of 100, which gives a minimal molecular weight of 12,500 for the protein moiety of proteolipids (Folch-Pi, 1959). Water-solubleproteolipidprotein - If proteolipids are dialyzed in chloroform : methanol containing HCI to 0.04 N concentration, and then the composition of the outer phase is slowly changed to pure water by gradually decreasing the organic solvent content of the successive outer phases, the retentate is found to consist of protein essentially free of lipids (Tenenbaum and Folch, in press). This preparation has an amino acid pattern indistinguishable from that of the starting proteolipids. It is soluble in acidified aqueous solutions and in chloroform. Apparently, it is the result of a conformational change of the original proteolipid protein (Zand, 1966). The dramatic change in solubility properties is concomitant with the removal of triphosphoinositide. Organization of the proteolipid molecule - Although no complete model of the proteolipid molecule can yet be formulated, it is clear that the lipids in proteolipids exist in different types of binding. Triphosphoinositide is almost certainly bound by an

COMPOSITION O F NERVOUS MEMBRANES

11

electrostatic bond. Other lipids, mainly phosphatidylserine, are bound by ionic linkages which can be dissociated by ionic competition. Finally, other lipids must be bound by more labile types of association. Of these three types of bonds, the first two most likely occur in vivo, while the third type most likely represents in vitro associations. The peculiar solubility properties of proteolipids, which remain unchanged even when the lipid content is reduced to 5 % or less, must be explained in terms of the protein moiety. Since the proteolipid molecule must present a non-polar surface, a tertiary structure must be postulated which would bring to the surface the non-polar groups of the amino acids, while retaining their polar groups in the core of the molecule. A possible structure would involve the stabilization of a particular conformation by triphosphoinositide which, being a polyanion, could combine with the cationic charges of the protein, thus orienting them towards the core of the helical structure and leaving an outer surface occupied mainly by non-polar groups. The release of triphosphoinositide concomitantly with the transformation of proteolipid protein to a water-soluble form would be in favor of this explanation. Neurokeratin - The name of neurokeratin was given by Ewald and Kuhne (1874-77) almost a century ago, to the gastric juice-resistant, pancreatic juice-resistant, fraction of brain proteins. The material was obtained by defatting brain tissue by exhaustive extraction with ethanol and ethyl ether, and submitting the defatted residue to the action of gastric juice and of pancreatic juice, in succession. The final product was an insoluble protein material, rich in S (1.7 %) and free of P. On the basis of its distribution in the nervous system, of the increase in its concentration in temporal relationship to myelination, and of some histochemical evidence, it was concluded that neurokeratin was a myelin constituent, and the name was adopted by histologists to designate the protein framework of the myelin sheath. LeBaron and Folch (1956) were able to prepare neurokeratin by a procedure milder than that used by earlier workers. The trypsin and pepsin resistant material obtained from white matter and designated Trypsin resistant protein residue (TRPR) was resistant to the action of proteolytic enzyme, was characterized by general insolubility, and contained 1.7 % S. In brief, it was very similar to classic neurokeratin except for the important difference that it contained about I .7 % P, almost all of which corresponded to polyphosphoinositide, presumably combined in it by an electrostatic linkage. They also showed that the classical procedure for preparation of neurokeratin resulted in the complete destruction of the constituent polyphosphoinositide, thus yielding a P-free product. The amount of polyphosphoinositide (PPI) present in TRPR accounts for the bulk of the PPI of brain tissue. In the original description of proteolipids (Klenk, 1941), it became obvious that there were many similarities between neurokeratin and proteolipid protein: general insolubility, high sulfur content, relationship to the myelin sheath, similar amino acid composition. The suggestion was made that neurokeratin might, in fact, be a product of breakdown of proteolipids. This suggestion was given further credence by References p.

13-14

J. F O L C H - P I

12

the finding that neurokeratin in its “native” state contained polyphosphoinositide in electrostatic combination just as is the case with proteolipid. This suggestion has been both reinforced and complicated by the recent work on isolated myelin. As already mentioned, it has been found that isolated myelin is completely or almost completely soluble in chloroform : methanol. Operationally this means that neurokeratin and TRPR, which are prepared from the chloroform : methanol insoluble fraction of white matter cannot be prepared from isolated myelin, since it yields no chloroform : methanol insoluble fraction. This forces the conclusion that if, indeed, neurokeratin is a myelin constituent, it exists in it in a form that is soluble in chloroform : methanol after isolation of myelin. This would place neurokeratin in the same category as proteolipids. On the other hand, these observations raise the question of the mechanism by which neurokeratin would become insoluble in chloroform : methanol, and why the same thing would not apply to the proteolipids. There is as yet no answer to these questions. Polyphosphoinositides (PPI) - Many facts pertaining to the discussion of PPI have already been mentioned and the following will only complement them and attempt a brief synthesis of our present knowledge on these interesting compounds. PPI are found mainly in combination in TRPR, which accounts for 80 to 90 per cent of white matter PPT, the balance being found mainly in proteolipids. They are clearly myelin TABLE 111 C O M P A R I S O N O F LEVELS O F P O L Y P H O S P H O I N O S I T I D E S I N D E V E L O P I N G R A T B R A I N A N D O F T R Y P S I N RESISTANT PROTEIN RESIDUE

(TRPR) IN

Rat brain

Age

TPI

DPI

Mouse brain TRPR Total

l(g P/g brain 2 days 4 days 7 days 10 days 16 days 17 days 19 days 34 days 35 days 40 days Adult

8.5 9.2 12.2

-

2.7 3.3

-

11.9 15.5

19.4 44.4

5.7 8.7

25.1 53.1

-

-

11.7

54.5

-

42.8

-

D E V E L O P I N G MOUSE B R A I N

-

TRPR

fresh wt. 0.063 0.115 0.19 0.38 0.40 0.67 0.67 -

TRPR-phosphorus p g P/g brain 5.0 9.4 15.2 30.4

-

32.0 53.6 53.6 -

Results for rat courteously supplied by Doctors J. Eichberg and G. Hauser. Results from mouse obtained or computed from Folch, 1955. Rats were decapitated, the head dropped in liquid nitrogen, and the brain removed without thawing. Mice were anesthesized with ether, the brain removed surgically and placed in a weighing bottle in dry ice. The time elapsed between removal of brain from the living body and its freezing was the time required for the actual freezing of the tissue once placed in contact with the chilled glass wall. The good fitting of values for PPI-P for rat with the values for mice should be regarded as fortuitous. The important analogy is the slope of the increase, which is essentially the same in both species.

COMPOSITION O F N E R V O U S MEMBRANES

13

constituents; they are found in myelin at much larger concentration than in other subcellular fractions of white matter (Eichberg and Dawson, 1965); they are found only in very small amounts, if at all, in non-neural tissues. They appear at the time of myelination and they increase in concentration with the gradual accumulation of myelin. Table 111 gives results on this point, courteously supplied by Doctors J. Eichberg and G . Hauser. They show that from 7 days, before myelination, to 34 days, the concentration of PPI-P increased 5-fold ; for comparison, the concentration of TRPR in the mouse at similar ages is given, both as amount of TRPR and as P (Folch, 1955). There is a remarkable analogy between the total amount of PPI-P and of TRPR-P, a fact that, although not unexpected, bears out strongly the myelinic nature of PPI. Numerous observations attest that PPI exhibits a high rate of P turnover, a fact in sharp contrast with the generally low level of metabolic activity of other myelin components. As yet, no evidence has been forthcoming relating this high metabolic activity of PPI-P to neural function. The marked neural character of PPI, their high metabolic activity, suggests that they must play some crucial role in nerve tissue. What this role is can only be established by further work. ACKNOWLEDGEMENT

The original work described in this discussion was supported by Grants NB-00130 and NB-02840 of the National Institute of Neurology and Blindness, National Institutes of Health. REFERENCES AMADUCCI, L. (1962) The distribution of proteolipids in the human nervous system. J. Neurochem., 9, 153-160. AUTILIO, L. (1966) Fractionation of myelin proteins. Fed. Proc., 25, 764. BLIX,G . (1936) The carbohydrate groups of the submaxillary rnucin. Z. Physiol. Chem., 240.43-54. BROCKERHOFF, H . A N D BALLOU,C. E. (1961) The structure of the phosphoinositide complex of beef brain. 1.Biol. Cherri., 236, 1907-1911. BURTON,R. M., HOWARD,R . E., BAER,S. AND BALFOUR, Y. M. (1964) Gangliosides and acetylcholine of the central nervous system. Biochirn. Biophys. Acta, 84, 441441. DIEZEL, P. M. (1955) Bestimrnung der Neuraminsaure im histologischen Schnittpraparat. Narurwiss., 42, 487-488. DITTMER, J. AND DAWSON, R. M . C. (1961) The isolation of a new lipid triphosphoinositide, and rnonophosphoinositide from ox brain. Biochem. J., 81, 535-540. EICHBERG, J. AND DAWSON, R. M. c. (1965) Polyphosphoinositides in myelin. Biochent. J., 96, 644650. EICHBERG, J., WHITTAKER, V. P. A N D DAWSON, R. M.C . (1964) Distribution of lipids in subcellular particles of guinea-pig brain. Biocheni. J., 92, 91-100. EWALD,A, AND KUHNE,W. (1874-1877) Verharrdl. Naturhist.-Meif.,1, 457. FOLCH,J., (1949); Brain diphosphoinositide, a new phosphatide having inositol rnetadiphosphate as a constituent. J. Biol. Cheni., 177, 505-519. FOLCH,J., ARSOVE, S. AND MEATH, J. A. (1951) Isolation of brain strandin, a new type of large molecule tissue component. J . Biol. Chert?.,191, 819-831. FOLCH,5. AND LEES,M. (1959) Studies on the brain ganglioside strandin in normal brain and in Tay-Sachs’ disease. Arner. J. Dis. Child., 97, 730-738. FOLCH,J. (1955) Composition of the brain in relation to maturation. Biochernisrry of rhe Developing Nervous System, H. Waelsch, Editor, Academic Press, New York, p. 121.

14

J. F O L C H - P I

A N D WOOLLEY, D. W. (1942) Inositol, a constituent of a brain phosphatide. J . Biol. Chem., 142,963-964. FOLCH,J. AND LEES,M. (1951) Proteolipids, a new type of tissue lipoproteins - their isolation from brain. J. Biol. Chem., 191,807-817.

FOLCH,J.

FOLCH,J., LEES,M. AND CARR,S. (1958) Studies of the chemical composition of the nervous system. Exp. Cell Res., Suppl., 5, 58-71. FOLCH,J., WEBSTER, G . R. AND LEES, M. (1959) The preparation of proteolipids. Fed. Proc., 18, 228. FoLcH-Pi, J., (1959); etudes Rkentes sur la chimie du cerveau et leur rapport avec la structure de la gaine myklinique. Exp. Ann. Biochim. Med., 21,81-95. HOWARD, R. E. AND BURTON,R. M. (1964) Studies on the ganglioside micelle. Biochini. Biophys. Acia, 84,435-440. KLENK, E. (1941) NeuraminsBure, das Spaltprodukt eines neuen Gehirnlipoids. Z. Physiol. Chem., 268,50-58. KLENK,E. (1942) Uber die Ganglioside, eine neue Gruppe von Zuckerhaltigcn Gehirnlipoiden. Z. Physiol. Chem., 273, 76-86. KUHN,R.AND WiEcAND-r, H. (1963) Constitution of ganglio-N-tetraose and the ganglioside GI. Chem. Ber., 96,866-880. LEBARON,F. N. AND FoLcii-PI, J. (1956) The isolation from brain tissue of a trypsin-resistant protein fraction containing combined insolitol, and its relation to neurokeratin. J . Neurocliem., 1, 101-108. LEDEEN, R. (1966) The chemistry of gangliosides: A review. J . Amer. O i l Chemists' SOC.,43,57-66. LOWDEN, J. A. AND WOLFE,L. S. (1964) Studies on Brain Gangliosides 111. Evidence for the location of gangliosides specifically in neurones. Canad. J . Biocheni., 42, 1587-1594. MATSUMOTO, M., MATSUMOTO, R. AND FOLCH-PI,J. (1964) The chromatographic fractionation of brain white matter proteolipids. J . Neurochem., 11, 829-838. MCILWAIN, H. (1962) New factors connecting metabolic and electrical events in cerebral tissue. In Ulirasirticture and Metabolism of the Nervous System, Research Publication Association Research Nervous Mental Disease, XL. Williams and Wilkins Co., Baltimore (page 43). MURAKAMI, M., SEKINE, H. AND F U N A H A S H I , s. (1962) Proteolipid from beef heart muSCk-'. Application of organic dialysis to preparation of proteolipid. J. Biochem., 51, 431435. PRITCHARD, E. G . AND FOLCH-PI, J. (1963) Tightly bound proteolipid phospholipid in bovine brain white matter. Biochim. Biopliys. Acia, 70,481483. QUARLES, R. A N D FoLcH-PI, J. (1965) Some effects of physiological cations on the behaviour of gangliosides in a chloroform-methanol-water biphasic system. J . Neurochem., 12,543-553. ROSENBERG, A. AND CHARGAFF, E. (1956) Nitrogenous constituents of an ox brain mucolipid. Biochim. Biophys. Acia. 21, 588-589. SEMiNARio, L. M., HREN, N. A N D GOMEZ,G. J. (1964) Lipid distribution in subcellular fractions of the rat brain. J. Neurochem., 11, 197-209. SPENCE, M. W. AND WOLFE,L. S. (1964) The isolation of a ganglioside-rich membrane fraction from new-born rat brain. Sixth Intern. Congr. Biochem., New York, Abstr., V-,5118, 418. SVENNERHOLM, L. (1964) The gangliosides. J. Lipid Res., 5, 145-155. TENENBAUM, D., AND FOLCH,J., (1966); The prepraration and characterization of water-soluble proteolipid protein from bovine brain white matter. Biochim. Biophys. Acta, 115,141-147. THOMPSON, E.B., KIES, M. W. AND ALVORD,JR. (1963) Isolation of a n encephalitogenic phospholipid-protein complex by dialysis of myelin in organic solvents. Biochem. Biophys. Res. Comm., 13, 198-204. VAN HEYNINGEN, W. E. (1963) The fixation of tetanus toxin, strychnine, serotonin and other substance by gangliosidc. J. Cen. Microbiol., 31,375-387. WEBSTER, G. R. AND FOLCH,J. (1961) Some studies on the properties of proteolipids. Biocliini. Biophys. Acia, 49, 399-401. WHERREIT, J. R. AND M C ~ L W A IH. N , (1962) Gangliosides, phospholipids, protein and ribonucleic acid in subfractions of cerebral microsomal material. Biochem. J., 84,232-337. WOLFE,L. S . (1961) The distribution of gangliosides in subcellular fractions of guinea-pig cerebral cortex. Biocheni. J., 79, 348-355. WOOLLEY, D. W. A N D GOMMI, B. W. (1964) Serotonin Receptors: V, Selective destruction by neuraminidase plus EDTA and reactivation with tissue lipids. Narure, 202, 1074-1075. ZAND,R. (1966) Physical chemical studies on the solution properties of bovine brain white matter proteolipids. Fed. Proc., 25, 736. Z u , L. P. AND HARMON,E. A. (1961) Chloroplast proteolipid. Biochim. Biophys. Acta., 53, 579-58 I.

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DISCUSSION Monday afternoon KATZMAN: I t has been many years since Dr. Folch-Pi characterized and identified the inositol phosphotides and proteolipids and identified the polymer form of inositol phosphate, and it is very exciting to me to see that all these materials are still in the forefront of research. It is most interesting, as Dr. Folch-Pi reported, that the triphosphoinositides, which are so actively turning over, are major constituents of the proteolipids. I would like to ask Dr. Folch-Pi specifically whether it can be demonstrated that in the isolated purified myelin the triphosphoinositides turn over so rapidly. In other membranes where there is a rapid turnover of phosphoinositides, it seems that only a small fraction of phosphoinositide is turning over, and the bulk is not so active. FOLCH-PI:1 don’t think that the triphosphoinositides are especially prominent. The monophosphoinositides, as I am sure you are aware, have a different function from the other inositides and they have a different distribution. Perhaps they are connected with synaptic membranes. In relation to the polyphosphoinositides in myelin, I am not aware that the precise kinetics of a single component’s turnover has been measured. KATZMAN: Has it been shown that the phosphate that is turning over in the purified myelin is specifically the phosphate of the triphosphoinositides? FOLCH-PI: Dawson and others have actually shown the incorporation of P-32 and they have isolated various cell fractions.

MANDEL:There is a turnover of triphosphoinositol in the myelin sheath, but this is quite low compared to the turnover of phosphoinositides in other parts. What is peculiar is that the highest turnover of phosphoinositides is of cardiolipin in the myelin sheith. FOLCH-PI:There is some indication of turnover of cardiolipins, which are the polyanions in mitochondria, but the bulk of the polyphosphoinositides is definitely in the myelin. In isolated proteolipids, LeBaron and Hauser showed years ago that phosphoinositides had a very high turnover of phosphate there. LAJTHA: Can make you any statement on the composition of the proteolipid fractions of the various particulate fractions, and on differences between gray matter and white matter. What I am really driving at is whether you can make any statement about the differences in the composition of the various membranes, whether membranes of glia versus neuronal membranes or particulate membranes. CSAKY:I note that you have isolated the protein from the proteolipids. Could you describe the properties of this fraction, particularly whether you think that this is a typical structural protein? Does it have a high molecular weight? Does it consist of long, thread-like particles? Does it have very high viscosity? Does it respond in its physicochemical properties very readily to ions, and things like that? FOLCH-PI:Some of those measurements have actually been made, although not all of them. However, they are more indicative than exact. Taking the question of molecular size, for example, by the technique of the least abundant component, we get a molecular size of the order of about 12,00015,000. By other physicochemical measurements, for example, sedimentation, diffusion or light scattering, we get somewhat wider scatter with a minimum value of about 20,000 or 30,000. All of these measurements are, of course, done in organic media, since these compounds are insoluble in water. Therefore I would consider every value that we obtained rather comparative only. By Sephadex L-20, molecular size is more between 30,000 and 100,000. Most likely a low polymer formation occurs. We didn’t measure viscosity, again, for the reason that we would have to do that in chloroform and therefore it would have doubtful meaning. But there are people who have done that, for example, Dr. Zahn in Ann Arbor, and Dr. Onkley. We have some information about the tertiary structure, using nuclear magnetic measurements, and in chloroform we get a highly helical structure of about 85 per cent. When the proteins are passed into the water phase, an unfolding of the helixes occurs and they become random coiled.

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J. F O L C H - P I

CSAKY:But would you be willing to say that it could be considered a s a structural protein of the myelin sheath? FOLCH-PI:I think there is very little doubt about that, although I would not make a statement on all membranes. In heart the bulk of the proteolipids are found in mitochondria, and I would think that they must be the components of the structural membranes. The amino acid turnover is very low in these components so that they show a chemical stability, and although not identical, the amino acid composition is fairly similar to Green’s so-called protein isolated from mitochondria. LAJTHA: You don’t think that the lack of turnover is only apparent and is d u e to lack of permeability, that is, the precursor amino acid that you add from the outside doesn’t penetrate to the inside of the myelin sheath, and therefore it is not incorporated. FOLCH-PI:Now we are talking about the mechanism of the stability or how it is actually obtained. It is possible that it is only an apparent one, as you say, but this is very real as far as the living body is concerned. TOWER:Perhaps we are making a mistake when we are talking about fhe structural protein. Wc certainly are dealing with tissues, and the brain in particular, that have many, many membranes with different functions. Our laboratory has some preliminary results which indicate that in membranerich fractions, subfractionated from cerebral microsomal fractions, there is a protein fraction (not necessarily a single protein, although it comes out on a column in a single peak) which exhibits an amino acid composition of about 30 per cent glutamyl plus aspartyl residues. This, theoretically, at least, provides a set of very high negative charges on these molecules (not provided by phosphates in this case but by carboxyl groups), and since this represents a major portion of the proteins of the endoplasmic reticulum, which, in turn, many of us consider may have an important role in transport, it poses some very interesting possibilities. COXON:Dr. Folch-Pi referred to some interactions between ions and the lipids that he is studying. Can he make any statements about selective affinities as between calcium and magnesium and potassium and sodium? FOLCH-PI:There is certainly a difference between the divalent ions and the monovalent ions. The divalent ions have about 500 times higher affinity; something of that order. This is, of course, just a comparative figure. This may be specific for gangliosides, and the carboxyl group of the sialic acid may have an important role. Calcium there certainly displaces sodium, etc. A very interesting point here is that the calcium salt of gangliosides is mainly non-polar, while the magnesium salt is very polar and the polarity changes very much according to the calcium concentration of the medium with which the ganglioside is in contact, and where such shifts happen is around the physiological levels of thc ions. If calcium is somehow sequestered and the ganglioside is facing a relatively low calcium concentration or there would be high magnesium there, there would be a part of the membrane which would be rather lipophobic, but as soon as more calcium came, and I don’t want to speculate how this would occur, that part of the membrane would become much more lipophilic. Therefore, one could postulate this as part of the mechanism of the actual movement of macromolecules in the membrane. This is attractive because it seems to be reversible and doesn’t particularly require energy, and the concentrations required are within the physiological range. The size, from molecular weight measurements, is about 70 Angstroms of these compounds, which well fits within the usual structural arrangement as we picture the membranes. DOBEING: I wonder if you could make any statement about the turnover of these particles that we are discussing, especially since they are often buried beneath several layers, of, for example, proteolipids which, in themselves, do not turn over very rapidly? Is it possible to account for all the turnover as being the turnover of only the exterior part of the sheaths? FOLCH-PI:Now we are in the field of pure speculation. Amaducci did a very careful study several years ago, trying to correlate proteolipids with myelin structures. He not only confirmed what was already known that peripheral nerve contains very little proteolipids, but from a number of structures, such as the optic nerve and the corpus callosum, the brachial plexus and the sciatic nerve, he could

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correlate very well with the sum of the circumferences of the fibers. If you take into account that myelin is usually about half the thickness of the fiber that it surrounds, the myelin is usually related to the surface of the fiber. The smaller the fiber, the greater the sum total of the circumference is, of course, per unit area of the brain. And then he had a very good correlation all the way from the corpus callosurn, which has about 27 parts of proteolipids per loo0 of wet weight basis, down to the brachial plexus which has less then 1 per cent. That could then mean that in the myelin spiral proteolipids would not be distributed uniformly but the distribution somehow would be correlated with the circumference. This could be done by two ways, by having either more or less in the outer turn or the inner turn of the spiral. If you now then equate proteolipids and triphosphoinositides, then you can say that the triphosphoinositides are not equally distributed, and if you postulate that they are richer in the outer turn of the spiral, then you would have the triphosphoinositides much more available than other forms. DOBEING: I f one follows the rate of development of myelin by following the accumulation of the various lipid components, their deposition occurs at different times. On Dr. Folch's hypothesis, it should follow that there would be a different timing for the deposition of the phosphoinositides and o f other components, such as, for example, cholesterol. Could this not be studied in small well-defined areas? FOLCH-PI:I don't think all data were really good enough to put weight on such correlations. In general fashion we have tried such measurements but the data are not really good enough to make too many statements definite. I t did follow the general increase in niyelination and so it showed general correlation. Dawson has done quite a number of such measurements but didn't go into very great detail.

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The Intracerebral Movement of Proteins Injected into Blood and Cerebrospinal Fluid of Mice M I L T O N W. B R I G H T M A N Laboratory of’ Neuroaiiatoiirical Sciences, National Itistilute of Neurological Diseases and Blindness, National Itistitrites of Health, Public Health Service, U. S. Department of Health, Education, and Welfare, Bethesda, Maryland (U.S.A.)

INTRODUCTION

The entry of radioactively labelled protein from blood into cerebrospinal fluid (CSF) and into cerebral tissue is much slower and less in amount (Fishman, 1953; Bering, 1955), than that of tritiated water and certain ions (Sweet and Locksley, 1953; Bering, 1955; Bakay, 1956). The barrier that impedes the entry of labelled protein into the CSF has been interpreted as being responsible also for the exclusion of intravascularly injected acid dyes which, because of their negative electric charge, bind to serum proteins (Tschirgi, 1952). The anatomical site of this barrier to protein movement has been ascribed by light microscopists to the vessel wall (e.g., Broman, 1949) or its surrounding sheath (Tschirgi, 1952). Morphological details of these structures are best resolved by electronmicroscopy, the technique recently used to follow the intracerebral course taken by intravenously administered peroxidase. This protein does not apparently cross the endothelium of cerebral vessels (Reese and Karnovsky, 1967). It has long been known, however, that there are certain regions of the brain where the vessels do not retain acid dyes (Goldmann, 1913), presumably bound to protein, or colloidal particles (Dempsey and Wislocki, 1955; Pappas and Tennyson, 1962; Klatzo et al., 1962). One of these regions is the choroid plexus. The impression gained from these published observations is that, in general, large colloidal particles including some proteins leave the choroidal vessels and accumulate primarily within the perivascular connective tissue stroma (Wislocki and Leduc, 1952; Dempsey and Wislocki, 1955; Brown, 1961 ; Klatzo, et al., 1962; Pappas and Tennyson, 1962). In contrast, certain dyes such as trypan blue (King, 1938; Wislocki and Leduc, 1952), despite its protein-binding tendency, and proflavine hydrochloride (Rodriguez, 1955) are able to go beyond the stroma into the surrounding epithelial cells. The first portion of the present account is an electron microscopic description of the passage of a foreign protein (horseradish peroxidase) from the blood into the stroma and the epithelium of the choroid plexus. When labelled proteins are in.jected into the cerebral ventricles (Bowsher, 1957) Rrfircnir, p. 36-37

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M. W. B R I G H T M A N

or cisterna magna (Lee and Olszewski, 1960) rather than into the blood, there does not appear to be a barrier to their entry into the surrounding brain parenchyma. The second portion of this paper is concerned with this direction of transfer and offers a brief, preliminary description of the movement of peroxidase from ventricular CSF not only toward the vessels of the choroid plexus but also toward the vessels of the cerebral parenchyma. Some observations on the passage of the protein ferritin in this direction are also included. Karnovsky's cytocliemical method ( I 965) of visualizing peroxidase activity is a sensitive one because activity of a few molecules of this enzyme yields a very electron dense reaction product. Six mg of horseradish peroxidase were injected, under Avertin anesthesia, into the femoral vein of mice weighing about 20 g. Fifteen to thirty minutes later the entire periventricular neuropil and choroid plexus were fixed by perfusion through the cerebral ventricles of 3 % glutaraldehyde in phosphate buffer at pH 7.4 (Brightman, 1965a). After immersion for 4 to 16 h in this fixative at about lo" C, frozen sections (about 120 p thick) werecut and incubated at about 25" C for 15 min in a substrate containing 3-3' diaminobenzidineand hydrogen peroxide(Karnovsky, 1965). The sections were then washed in buffer, immersed in phosphate-buffered I 7;osmium tetroxide for 1 to 2 h and prepared for electronmicroscopy. A few blocks, about I p thick, were not frozen, but were otherwise processed in the same way. Henceforth, the terms enzyme (or protein) and reaction product will be used interchangeably. I . M O V E M E N T O F P E R O X I D A S E F R O M C H O R O I D A L B L O O D TO E P I T H E L I U M

A . Endothelial passage

The choroidal vessels, unlike the parenchymal vessels of the brain, have a fenestrated endothelium. The fenestrae are not pores. Although the available techniques led earlier investigators to conclude that the fenestrae were perforations (Maxwell and Pease, 1956) or interruptions (Wislocki and Ladman, 1958), they are now known to be spanned by a thin diaphragm (Pappas and Tennyson, 1962) formed by the apposed outer leaflets of the endothelial unit membrane (Luft, 1966; Wolff, 1966). In such vessels occurring in other organs as well (e.g., Farquhar, 1961), the only continuous part of the endothelial cells are these fused outer leaflets. In tissue fixed 15 min after the intravenous administration of peroxidase, the very dense, amorphous reaction product usually appeared as a coating on the luminal surface of the thicker, non-fenestrated portions as well as the diaphragms of the endothelium (Figs. 2, 3 and 4). Occasionally, and presumably as the result of improper fixation, the dense substance seemed to take the place of the diaphragm and extended from the lumen into the basement membrane of the vessel. In undamaged capillaries, however, the enzyme was restricted to the lumen and appeared to be adsorbed to the entire luminal surface, including its tiny invaginations or pinocytotic pits. In two instances similar pits on the contraluminal surface were coated and it is presumed that these had traversed the endothelium as cytoplasmic vesicles that had originated from the luminal plasmalemma (Fig. 3). Many of these pinocytotic pits and vesicles within the endothelial cytoplasm were completely filled with reaction product.

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Fig. I . Part of a villus from the choroid plexus consists of three epithelial cells with a capillary lumen at the lower right and ventricle at the upper left. The dense, black material is rp (reaction product) of peroxidase that had been injected intravascularly. x 13 000.

Vesicles containing some reaction product were more numerous in the endothelium of choroidal vessels than in that of parenchymal ones. The role of the junctions between these endothelial cells in the passage of protein across the vessels is as yet equivocal. In most cases, peroxidase penetrated the junction for only a short distance (Fig. 2). In several instances, however, reaction product Rcfcwnres p. 36-37

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M. W. B R I G H T M A N

Fig. 2. A fenestrated, choroidal capillary contains pinocytotic vesicles that are eithec rimmed or filled with rp. The fenestrae are indicated by arrows. One junction (JI), between endothelial cells, is cut transversely and contains no rp. Two junctions (Jz and J3), are only partially filled with rp whereas a fourth (54) is more completely filled. The stromal space (S), lined by basement membrane (BM) lies between the vessel and base of an epithelial cell (right margin). x 39 000.

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Fig. 3. In Figs. 3 and 4, the vessel lumen is at the bottom of the figure. A fenestrated, choroidal capillary contains vesicles rimmed by rp at both luminal and contraluminal surfaces. A portion of an epithelial cell is at the top of the figure. x 54 000.

Fig. 4. The junction betwecn these endothelial cells of a choroid plexus capillary is filled with rp. x 73000.

appeared to occupy most or all of the junction (Fig. 4). It is possible that some of the peroxidase within the junction did not enter exclusively from the lumen but was deposited also by vesicular transport. Thus, pinocytotic vesicles arising at the luminal surface may have transported their content to the walls of the junction (Fig. 4) or to the contraluminal surface (Fig. 3). In the latter case, peroxidase may have subsequently diffused from the basement membrane back into the junction.

B. Epithelial entry After crossing the endothelium of the choroid plexus the protein entered the perivascular connective tissue space. This stromal space is bounded by two basement membranes, one apposed to endothelium, the other to epithelium (Fig. 2). The enzyme R r f i r i v w s p. 36-37

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M. W. B R I G H T M A N

Fig. 5. The stromal space, running through the middle of the micrograph, is full of rp that lines the tortuous, communicating spaces between epithelial cell invaginations. Peroxidase has been pinocytosed by numerous vesicles, one of which (arrow, center - left) is continuous with the infolded plasmalemma of an epithelial cell. x 33 000.

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25

penetrated the endothelial basement membrane, some became affixed to collagen fibrils within the space, and appreciable amounts moved across the stromal space to penetrate the epithelial basement membrane. From this site, the enzyme was incorporated within numerous pits and vesicles arising from the highly folded basal and lateral plasmalemmas of the epithelial cells (Figs. I and 5). Many such vesicles migrated to the interior of the cells (Figs. 1 and 6), some moving very close to the free, ventricular surface (Figs. I , 6 and 7). This surface is much more plicated than the lateral or basal surfaces (Figs. 1, 6, 7 and 8), yet the folds are too pleomorphic to constitute a striated or brush border (Maxwell and Pease, 1956). Though the peroxidaseladen vesicles had moved very close to the bases of these free evaginations, none seemed to make contact with their plasmalemma which, moreover, did not appear to have become coated with peroxidase. Occasionally, a flat, broad, inverted, V-shaped cistern intervened between the ventricular surface and the vesicles. Neither these cisterns (Figs. 8, 9 and 10) nor those of the granular endoplasmic reticulum received protein from the vesicles, but vacuoles and multivesicular bodies did. So dense and complete was the filling of the matrix within the multivesicular bodies that the enclosed vesicles appeared to be negatively stained (Figs. 1 and 6). As in the case of fenitin (Brightman, 1965a), these vesicles remained free of peroxidase. C. In tercellular movement

Protein moved between adjacent epithelial cells concurrently with its entry into these cells. The infoldings of the basal and lateral portions oftheircell membranes form many deep, narrow channels (Maxwell and Pease, 1956) that communicate with the large stromal space (Figs. I and 5). Peroxidase left this large space to enter the intercellular channels. These were usually lined with reaction product or sometimes completely filled by it as far as a conical stricture of the interspace (Figs. I and 8). At the stricture, occurring near the ventricular surface, the outer leaflets of adjacent cell membranes presumably approximated each other closely enough to exclude peroxidase. Apical to these appositions the interspace often became distended for a short distance, then it narrowed and was less indented than the space basal to the stricture. Thus a luminal, narrow neck opened into the ventricle but usually contained no protein (Fig. 8). It was evident that peroxidase had moved between adjacent epithelial cells only as far as the strictures and not beyond them into the ventricular CSF. 11. M O V E M E N T O F P E R O X I D A S E A N D F E R R l T l N F R O M V E N T R I C L E I N T O

CHOROIDAL EPITHELIUM A N D CEREBRAL PARENCHYMA

A . Clioroidal entry

Preliminary experiments have demonstrated that 30 min after the injection into the cerebral ventricles of 0.5 mg of peroxidase, a small amount was pinocytosed by the epithelial cells of the choroid plexus. Pinocytotic activity was carried out by that part of the ventricular plasmalemma between the bases of the surface evaginations and Rcfcrmws p . 36-37

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M. W. B R I G H T M A N

Fig. 6. The apical region of an epithelial cell contains many vesicles and at least two multivesicular bodies (B) laden with rp. The ventricle appears at the top. x 34 000.

not by the portion covering them (Fig. 10). The number of vesicles that had engulfed peroxidase was small and they were confined to the apical regions of the cells. Similarly, in one experiment where 35 mg of ferritin had been administered intraventricularly at the same time that 5.0 mg of peroxidase was injected intravenously, the number of ferritin-containing vesicles in the apical cytoplasm was small in contrast to the numerous peroxidase-laden vesicles that had arisen from the folded basal and

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27

Fig. 7. The apical cytoplasm of an epithelial cell contains three peroxidase-laden vesicles, one of which lies very close to this surface. Pleomorphic evaginations of the cell surface o x u p y the ventricle into which ferritin (appesring as micelles about 60 A wide) had been injected. x I17 000.

lateral surfaces (Fig. 7). Neither intraventricular peroxidase nor ferritin passed between adjacent epithelial cells of the choroid plexus in discernible amounts. B. Epetdymal passage

Both proteins, however, passed readily between neighbouring ependymal cells to enter the interstices of the underlying cerebral neuropil. Either protein flowed along the I50 to 200 A-wide clefts between glial and neuronal processes to enter synaptic clefts, as did ferritin in the rat brain (Brightman, 1965b). This continuity ofthe synRc.lcnvico p. 36-37

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M. W. B R I G H T M A N

Fig. 8. The interspace between two epithelial cells is lined with rp as far as the conical stricture near thc ventricular lumen appearing at the top. x 73 000.

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29

Fig. 9. A flattened, agranular cistern occurs commonly near the luminal surface of an epithelial cell. 137 000. Fig. 10. Three pinocytotic invaginations ( * ) are formed by the luminal plasmalemma between the bases of evaginations. A somewhat flattened vesicle contains peroxidase that had been injected into the ventricle (at top). x 128 000. \A

aptic cleft with the rest of the interspaces has recently been confirmed with the use of saccharated iron oxide (Pappas and Purpura, 1966). Peroxidase passed between perivascular glial end-feet ((,f Brightman, 1965b) to spread into the basement membrane surrounding the endothelium of parenchymal capillaries (Fig. 1 1). Either protein was then able t o enter the endothelial pits that communicate with this membrane (Fig. 12). These flask-like invaginations in the contraluminal plasmalemma of endothelial cells thus received either peroxidase or ferritin that had moved extraReferences p. 36-37

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M.

W.

BRIGHTMAN

cellularly from the ventricular CSF. Ferritin also occurred in vesicles within the endothelial cytoplasm but, lacking serial sections, it is unkown whether the vesicle interiors retained continuity with the basement membrane in another plane. None of these vesicles containing protein has yet been observed in contact with the luminal surface of the endothelium; evidence of vesicular transport from outer to luminal surface has, therefore, not yet been obtained. Peroxidase which left the perivascular basement membrane to enter the junctions between endothelial cells could move only a short distancerbefore being stopped. Presumably, the tight junction that blocks the intercellular movement of peroxidase from the blood side of the endothelium (Reese and Karnovsky, 1967) also excludes protein that spread from the parenchymal side. It appears at the present time, that the junctions between endothelial cells of parenchymal capillaries are closed to peroxidase whereas those of choroid plexus vessels are open to it. C. Pinocytosis by presynaptic terminals

Throughout the subependymal neuropil, peroxidase or ferritin spread between glial and neuronal processes and were pinocytosed by these processes. For example, large coated pits in the membranes of dendrites were filled with reaction product. Of special interest, however, was the incorporation of either protein by presynaptic terminals (Figs. 13A and B). Peroxidase or ferritin that had entered the synaptic cleft and the remainder of the periterminal interspace was pinocytosed by the bouton plasmalemma. Small invaginations of the plasmalemma contained peroxidase or one or more molecules of ferritin. Though such pits have not yet been observed i n the membrane fronting the synaptic cleft, it is likely that they form here too (Westrum, 1965). Indeed, in one instance, a peroxidase-laden-vesicle was found very close to a synaptic cleft. Within boutons, protein-containing vesicles had separated from the plasmalemma and had become interspersed among the synaptic vesicles. These pinocytotic vesicles were of the same size as synaptic vesicles and were thus considerably smaller than those occurring, for example, in ependymal and choroidal epithelium. These small pinocytotic vesicles further resembled synaptic vesicles in being non-coated. DISCUSSION

The anatomical barriers to the movement ofperoxidase from blood to ventricular CSF consist of: (a) the structures (which are probably tight junctions) between the apices of the choroidal epithelial cells and, perhaps, (b) their ventricular surface. This surface has been regarded as the barrier to the movement of fluorescent proflavine dyes out of the choroid plexus, though the only part of the epithelial cells enclosing demonstrable dye was the nuclear membrane rather than any portion of the cell membrane itself (Rodriguez, 1955). The conclusion would have been more acceptable if, for example, the basal and lateral plasmalemma had been stained, whereas the apical portion had not. Although no peroxidase-laden vesicles were observed in contact with the ventricular plasmalemma, it is possible that some of the

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Fig. I I . Peroxidase, injected intraventricularly, has entered the perivascular basement membrane ( B M ) from the interspace (arrow) between glial end-feet. x 93 000. Fig. 12. Peroxidase has spread from the perivascular membrane into several pits within the contraluminal plasmalemma of a parenchymal capillary. x 1 I 1 000.

vesicles fused with this membrane and released peroxidase that was immediately washed away by CSF. Such a vesicular transport of a small fraction of intravascularly injected protein could account for the slow entry o f a small amount of labelled albumin into the ventricles observed by others (Fishman, 1953 and Bering, 1955). The ventricular plasmalemma of the choroid epithelial cells is capable, nonetheR&rciu

L’S

p . 36-37

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Fig. 13A. Peroxidase has permeated the interspaces, including synaptic clefts (SC) and has been pinocytosed by vesicles (V) interspersed among synaptic vesicles within presynaptic terminals. x 54000. Fig. 13B. Fertitin has left the cerebral ventricle to enter the neuropil interspaces from which it is pinocytosed by neural processes such as this bouton. The plasmalemmal invaginations (arrows) each contain a molecule of ferritin as does the vesicle (V) that has pinched off from the membrane. x 91 000.

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less, of some pinocytotic uptake of peroxidase. However, a substance that is incorporated into a cell by pinocytosis does not necessarily move across the cell by vesicular transport. Vesicles that had imbibed peroxidase from the ventricle remained in the apical cytoplasm. In contrast, the vesicles that had engulfed protein from the perivascular space had also moved across the cell toward the ventricle. Thus, if vesicular transport does occur, it appears to proceed from the blood-side toward the ventricle and not in the opposite direction. It appears unlikely, therefore, that vesicular transport could account for the nondiffusional movement of nonelectrolytes from a ventricular bath to choroidal blood as suggested by Welch and Sadler (1966). Yet, this possibility cannot be discounted at present because of the imprudence, as emphasized by Bering (l955), of extrapolating between results obtained with different classes of substances. The endothelium of the choroid plexus does not act as a barrier to the movement of intravascularly administered peroxidase but, instead, may allow peroxidase to cross by means of two mechanisms. Perhaps the more important of these may be an intercellular migration along the junctions between endothelial cells. Thorium dioxide, injected into the blood, also enters these junctions in the choroid plexus of the rabbit (Pappas and Tennyson, 1962). The second mechanism is that of vesicular transport, though this route has not here been demonstrated unequivocally. For example, figure 3 may be interpreted in two ways. It may represent the transcellular movement of peroxidase-containing vesicles from the luminal to the contraluminal surface or it may as likely illustrate the uptake by the contraluminal plasmalemma of peroxidase that had permeated the basement membrane after intercellular passage, rather than after vesicular transport. If vesicular transport does occur, it probably takes place across portions of the endothelium that are at least 3001( thick (Brightman, 1965b). More attenuated parts of the endothelium do not contain pinocytotic vesicles (Pappas and Tennyson, 1962). Glial sheets thinner than about 300A do not give rise to focal invaginations and it has been suggested that cell processes in general must attain this minimal thickness before their cell membranes are capable of pinocytotic activity (Brightman, I965b). Throughout the brain, some neurons such as those in the periglomerular regions of the olfactory bulb (Fig. 14) are nearly surrounded by uniformly thin glial wrappings (Reese and Brightman, 1965). Similar thin glial wrappings form a multi-layered, cup-like enclosure around certain synapses in the thalamus of the cat, as we have seen in Dr. Pappas’s presentation (Pappas et a/., Fig. 6, 1966) and in the dorsal motor nucleus of the vagus nerve (Fig. 15) in the rat (Richardson, 1967). This glial-neural configuration implies that the only way in which a large molecule can penetrate the enclosed pericellular and synaptic clefts from the glial channels would be by travelling between the attenuated glial sheets, not across them. Therefore, in the immediate vicinity of such perisomatic and synaptic clefts, the only cell processes that could contribute large molecules, by means of transcytoplasmic vesicular transport, would be neural, not glial. Although the intact fenestral diaphragms are impervious to peroxidase, they may be sites where smaller molecules readily cross the endothelium. Regardless of the R(:lcrcn(xv p . 36-37

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M. W. B R I G H T M A N

Fig. 14. Two small neurones are partly surrounded by several wrappings of very thin glial sheets (identified by their content of glycogen granules). Periglomerular region of rat olfactory bulb. Os01fixation. (Reese and Brightman, 1965). ,” 15 000. Fig. IS. A stack of thin glial sheets cups that portion of a presynaptic terminal opposite its synaptic cleft (SC). Dorsal motor nucleus of a rat. KMnOA fixation. (Courtesy of K. C. Richardson). i 30 000

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mechanism, the choroidal endothelium allows the passage of peroxidase whereas the parenchymal capillaries do not. Disregarding the risk of extrapolating between results obtained with peroxidase and those with radio-iodinated albumin, one may infer that what little albumin does leave the blood to enter the ventricular CSF (Bering, 1959, does not move across parenchymal, subependymal vessels (Reese and Karnovsky, 1967) but rather across the vessels of the choroid plexus. This inference, however, is at variance with Bering's findings (1955) that such entry into the ventricular CSF occurs after removal of the choroid plexus from an isolated ventricle. The distribution within the cerebral parenchyma of ferritin or peroxidase that had been injected into the cerebral ventricles confirms the observation that the ependyma does not stand as a barrier to many substances including proteins (Lee and Olszewski, 1960; Klatzo et a/., 1962). The results suggest further that the extracellular clefts, about 150-200 A wide, are hydrated channels permitting the movement of hydrophilic substances such as protein. The channels, nevertheless, are variable in content and width (Brightman, 1965b). Those between perivascular end-feet are too narrow to allow passage of ferritin and were, consequently, interpreted as being closed or fused junctions (Brightman, 1965b). The present results, however, support the observation that these particular appositions are narrow but patent (Reese and Karnovsky, 1967) as they are in more primitive species (Kuffler and Nicholls, 1966)and permit the extracellular movement of peroxidase into the perivascular basement membrane. At the ventricular surface, the contents of the intercellular channels are in ultimate communication with the CSF. But our observations tell us nothing about whether the compositions of the fluids are equivalent. The pinocytosis of protein by glial and neuronal processes from their extracellular clefts may signify, conversely, that substances are added to the cleft fluid. The composition of the ambient parenchymal fluid may be thus modulated from cell to cell and could ultimately be quite unlike that of the ventricular fluid. The ability of cerebral nerve terminals to pinocytose ferritin and peroxidase is of particular interest. These proteins are not only incorporated by large (about 80 to 100 p wide), coated vesicles (Brightman, 1965b), characteristic of many different cell types in various species, but are also taken up by presynaptic terminals in vesicles indistinguishable from synaptic vesicles. These pinocytotic vesicles are derived from the bouton plasmalemma; they are smaller than the usual pinocytotic ones, are noncoated, and become interspersed among synaptic vesicles inside the bouton. However, the identification of these pinocytotic vesicles as synaptic has yet to be established. SUMMARY

Following its intravascular injection in mice, the protein, horseradish peroxidase, crosses the fenestrated endothelium of the choroid plexus to enter the stromal space. From there, the protein is pinocytosed by choroid plexus epithelial cells within which peroxidase-containing vesicles migrate as far as the ventricular surface. Concurrently, the enzyme moves between these epithelial cells until stopped by a conical Rrtc~rcwi('s p. 36 37

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M. W. B R I G H T M A N

stricture of the interspace near the ventricular lumen. Unlike parenchymal vessels, the capillaries of the choroid plexus allow the passage of protein that is then taken up by the choroidal epithelium. When peroxidase or ferritin is injected into the cerebral ventricles, relatively little is pinocytosed by the epithelial cells of the choroid plexus and none passes between them. However, either protein readily moves between ependymal cells to penetrate the extracellular clefts of the neuropil. From these channels the proteins are pinocytosed by glial and neuronal processes. In presynaptic endings the plasmalemma forms pits and vesicles incorporating the proteins. These vesicles are morphologically identical with synaptic vesicles among which they become interspersed.

R E F E R E NCES BAKAY, L. (1956) The Blood Brain Barrier. G . C . Thomas, Springfield, Ill. (p. 40-76). BERING,E. A. JR. (1955) Studies on the role of the choroid plexus in tracer exchange between blood and cerebrospinal fluid. J . Nertrosurg., 12, 385-392. BOWSHER, D. (1957) Pathways of absorption of protein from the cerebrospinal fluid: an autoradiographic study in the cat. Anat. Rec., 128, 23-40. BRIGHTMAN, M. W. (1965a) The distribution within the brain of ferritin injected into cerebrospinal fluid compartments. 1. Ependymal distribution. J . Cell Biol., 26, 99-123. -(1965b)The distribution within the brain of ferritin injected into cerebrospinal fluid compartments. II. Parenchymal distribution. Anrer. J. Anat., 117, 193-220. BROMAN, T. ( 1949) The permeability of the cerebrospinal vessels in normal and pathological cortrlitiotrs. Einar Munksgaard, Copenhagen (p. 7-19). BROWN,P. (1961) Albumin, connective tissue and the blood-brain barrier. Bull. Johrrs H o p k i m Hosp., 108, 200-207. DEMPSEY, E. W. AND WISLOCKI, G. B. (1955) An electron microscopic study of the blood-brain barrier in the rat, employing silver nitrate as a vital stain. J . Biophys. Biochcm. Cyrol., 1, 245-256. FARQUHAR, M. G . (1961) Fine structure and function in capillaries of the anterior pituitary gland. Altgiology, 12. 270- 292. FISHMAN, R. A. (1953) Exchange of albumin between plasma and cerebrospinal fluid. Amer. J. Physiol., 175, 96-98. GOLDMANN, E. E. (1913) Experimentelle Untersuchungen iiber die Function der Plex. chorioid. und der Hirnhaute. Verh. cleutsch. Ges. Chirurgie, 42, 107-1 13. KARNOVSKY, M. (1965) Vesicular transport of exogenous peroxidase across capillary endothelium into the T-system of muscle. J . Cell. Biol., 27, 49A. KING,L. (1938) The hematoencephalic barrier. Arch. Neurol. Psychiat., 41, 51-72. KLATZO, I., MIQUEL, J. AND OTENASEK, R. (1962) The application of fluorescein labeled serum proteins (FLSP) to the study of vascular permeability in the brain. Acta Neuropathol., 2, 144-160. KUFFLER, S . W. AND NIcHoLLS, J. G . (1966) The physiology of neuroglial cells. Ergeb. Physiol. Biol. Chem. E x p ~ lPharnrakol., . 57, 1-90. LEE,J. c. AND OLszEwsKI, J. (1960) Penetration of radioactive bovine albumin from cerebrospinal fluid into brain tissue. J . Neurol.. 10, 814-822. LUFT.J. (1966) The ultrastructural basis of capillary permeability. In: The /irflamt~ra/oryProcess, B. W . Zweifach, (Ed.:, 1, Ch. 3, 121-159. MAXWELL, D. S. AND PEASE,D. C. (1956) The electron microscopy of the choroid plexus. J . Biophys. Biochem. Cytol., 2, 467-474. PAPPAS,G. D. AND TENNYSON, V. M. (1962) An electron microscopic study of the passage of colloidal particles from the blood vessels of the ciliary process and choroid plexus of the rabbit. J . Cell Biol., 15, 227-240. PAPPAS,G . D. AND PURPURA, D. P. (1966) Distribution of colloidal particles in extracellular space and synaptic cleft substance of mammalian cerebral cortex. Nature, 210, 1391-2.

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PAPPAS, G. B.. COHEN,E. B. A N D PURPURA, D. P. (1966) Fine structure of synaptic and non-synaptic neuronal relations in the thalamus of thecat. The Thalamus. D. P. Purpura and M. D . Yahr (Eds.). Columbia Univ. Press, New York (p. 47-75). R ~ E S FT.. S. A N D B R I G H T M A N , M. w. (1965) Electron microscopic studies on the rat olfactory bulb. Anat. Rec., 151, 492. REESE,T. S. AND KARNOVSKY, M. J. (1967) Fine structural localization of a blood-brain barrier for exogenous peroxidasc. J. CeN Biol. (In Press). RICHARDSON, K. C. (1967) Personal communication. RODRIGUEZ, L. A. (1955) Experiments on the histologic locus of the hemato-encephalic barrier. J . Conrp. Neitrol., 102, 27-39. SWEET, W. H.A N D LOCKSLEY, H. B. (1953) Formation, flow, and reabsorption of cerebrospinal fluid in man. Prac. SOC.Exptl. Biol. Men., 84, 397405. TSCHIRGI, R. D. (1952)Blood-brain barrier, The Biology of Metrtal Health andDisease, Paul B. Hoeber Inc. (p. 34-46). WLLCH,K. A N D SADLER, K. (1966) Permeability of the choroid plexus of the rabbit to several solutes. Arne,.. J. P ~ J J s ~210, o ~ . ,652-660. WESTRUM, L. E. (1965) On the origin of synaptic vesicles in cerebral cortex. J. Physiol., 179 (Proceedings) 4-6. WISLOCKI, G. B. A N D LADMAN, A. J. (1958) The fine structure of the mammalian choroid plexus. Ciba Fobrrnrlatiotr S.vniposiunr on the Cerebrospinal fliiid, Wolstenholme and O'Connor (Eds.). London: Churchill (p. 55-79). WISLOCKI, G. B. A N D LEDUE,E. H. (1952) Vital staining of the hematoencephalic barrier by silver nitrate and trypan blue, and cytologicat comparisons of the neurohypophysis, pineal body, area postrema, intercolumnar tubercle and supraoptic crest. J. Comp. Neurol., 96, 371414. WOLFF,J . (1966) Elektronenmikroskopische Untersuchungen iiber die Vesikulation in dem Kapillarendothel. Z. Zel&wsch., 73, 143-164. DISCUSSION

R. KATZMAN: I would like to ask Dr. Brightman if he can estimate the rate at which the materials move through these pinocytotic vesicles. Dr. Rall's evidence that there is no pinocytosis for inulin would be true only if the time of movement in a pinocytotic vesicle was slower than diffusion. And I wonder if there is some estimate in terms of how many seconds or minutes it takes your marker to move through, e.g., 30 / I . M. BRIGHTMAN: I cannot answer this with certainty. All I can say is that within the shortest time interval (15 minutes), peroxidase had left the choroidal blood, crossed the stromal space and was pinocytosed by the choroidal epithelium. The pinocytotic vesicles, within that interval, moved across these cells (about 15 1) long) to the opposite, ventricular surface. As for the intercellirlar movement from the ventricle across the ependyma, even so large a molecule as ferritin can penetrate the interspaces for at least 30 / t in about 15 minutes, probably less. T. 2. CSAKY: Could you give any indication as to what makes the vesicles move, what is the driving force ? From your pictures it appears that there is a constant ratio gradient. But d o you have any better evidence? Is it a coniplex diffusion? Does it always move away from the place where you put your marker? M. BRIGHTMAN: That is correct. T. 2. CSAKY:But what is the driving force that makes it move?

M . BRIGHTMAN: I can only guess. Cytoplasmic streaming? A pumping activity on the part of the epithelial cells? Although the vesicular traffic appears to be from the blood toward the CSF side of the epithelium. the vesicles ending up near the CSF surface have mixed origins. The lateral (as well as the basal) plasmalemma gives rise to vesicles which need only move a very short distance before reaching the ventricular surface. Moreover, we do not know which proportion of laterally vs basally derived vesicles coalesce, en route, with multivesicular bodies, and so never reach the free surface. But what the forces are that favor this basal to luniinal movement remains unknown.

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D. M. WOODBURY: Did you try the movement of particles across capillaries in other tissue, e.g., muscle capillaries as compared with those in the brain? M. BRIGHTMAN: This is a very good question, anticipating the work of Karnovsky, who found that peroxidase molecules can pass between adjacent endothelial cells in heart muscle capillaries. When Reese and Karnovsky then examined cerebral capillaries, they made the important observation that in this type of blood vessel, the spaces between the endothelial cells were closed to peroxidase. The cerebral endothelium stands as a barrier to the movement of this protein. In the choroid plexus, peroxidase is able to cross the endothelium, in as yet an undetermined way, but the epithelium acts as the barrier to passage into the ventricular fluid.

D. M.WOODBURY: So there is a distinct difference then? M. B R I G H T M A N : Very much so; so that pinocytosis, I daresay, could be rather unimportant, or simply auxillary. D. M. WOODBURY: But what about the smaller molecules? M. BRIGHTMAN: 1 would very much like to find them, but as far as visualizing them, we are limited. D. M . WOODBURY: So you cannot really make any conclusions about these smaller molecules? M. BRIGHTMAN: NO.

K. A. C. ELLIOTT: 1 wondered if Dr. Brightman could straighten me out a little bit: Sometimes you spoke about these particles as though they were just large, inert particles; sometimes you indicated that they were proteins. Now if I am right, they were both proteins and I wonder how this would affect reactions within the tissue. Also, what was the effective size? They could be hydrated and then they would have an effective size that would not be reflected by the molecular weight. And if great big proteins can get in, what about substances like the globulins of the tissue? M . BRIGHTMAN: Both tracers were proteins, apoferritin having a molecular weight of about 480,000 and peroxidase about 40,000.Some sort of binding between protein and cell membrane is a necessary prelude to pinocytosis, as Brandt has so convincingly shown. But the visualization of a protein coat (i.e. peroxidase reaction product) on the cell surface is difficult to interpret. A recent study has shown that many cross-linkages are formed between the dialdehyde, glutaraldehyde, and two different proteins. Thus, the “binding” of one protein (tracer) to another protein (cell membrane) may be purely an event of fixation rather than a linkage that had occurred during life. However, this objection is overcome by the facts that pinocytosis does take place and can be performed only by the responsive membrane of a living cell. Pinocytosis cannot be carried out after fixation. K. A. C. ELLIOTT:What about globulin? Wouldn’t that go in in the same way? M. BRIGHTMAN: 1 think it would.

D. M . WOODBURY: I am glad to hear that the electronmicroscopists d o admit occasionally that there (ire some artefacts! R. V. COXON:Could I first say by way of an addition to what Dr. Brightman has said, that Dr. Simpson-Morgan in my laboratory did study the passage of ferritin out of the cardiac capillaries about a year ago. He found that the process seemed to be determined by the nature of the perfusion fluid. He found that if he kept it perfused with blood, there was practically no escape of ferritin from the capillaries at all. If on the other hand, blood was replaced with saline, then there was a very free penetration of the ferritin into tissue spaces. I would like to, if 1 may, put a perhaps rather wild question to Dr. Brightman: Does he think that his work, showing the uptake of these rather large particles by cells of the nervous system, may give any substance to the claim that has been made that you can transfer learning from one animal to another?

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M. BRIGHTMAN: I have no comment on that question. J. DOBEING:May 1 comment on Dr. Brightman's remarks. First, if he accepts that the BloodBrain Barrier is in the endothelial wall (or rather in the space between it and the parenchyma, such as exists in the heart muscle), we can all go home. But there are other differences than this intracellular gap betwcen cardiac capillaries and brain capillaries. After all, the cardiac capillary is not predominantly ensheathed by glial endfeet. In his other remark towards the end of his paper where he said that the Blood-Brain Barrier was damaged (by which he meant the endothelium) and this material came rushing in; he presuniably damaged more than the endothelium here too.

M. BRIGHTMAN: I would like to say that even though there is a marked difference in the so-called nonBlood-Brain-Barrier areas, within the area postrema and the choroid plexus, there are perivascular connective tissue spaces in both. Olsjewski, for example, found that radioactive proteins got out of the capillaries into the large connective tissue spaces of the area postrema but usually went no further; very little entered the cerebral parenchyma. Some barrier, presumably glial appeared to exclude the protein from the surrounding brain. Now, I emphasize again that I an1 describing barriers to the movement of colloids. When injected into the blood, peroxidase cannot cross the cerebral endothelium to even reach the capillary basement membrane let alone the glia as Reese and Karnovsky have shown. However, as I've illustrated, peroxidase injected into the cerebrospinal fluid compartments has no trouble in reaching the cerebral endothelium. The protein readily moves between the perivascular sheath of glial cells to reach the capillary wall. The glial cells, therefore, do not act as a barrier to the extracellular passage of proteins froni cerebral ventricle to capillary and I see no reason why they would in the opposite direction. The role of the glia and endothelium in the niovenient of amino acids and electrolytes may be, on the other hand, a very different matter. B. D. WYKE:I would like to suggest, contrary to Dr. Dobbing's remark, that we may be even further froni going home than before we arrived. Following the very stimulating dialogue this morning between Dr. Tower and Dr. Csaky on the interesting subject of glucose transport, I would like to suggest for the record that a distinction should be drawn between the mechanisms, whatever they are, of blood and CSF-exchange, and blood and brain exchange. I think it was very noticeable that Drs. Ford and Lajtha, in titling this symposium, p l t the word "systems" in the plural and not in the singular. I feel very strongly that there are at least two systems for glucose exchange (and perhaps more), and that their behaviour is probably very different (Wyke, 1965, in Generd Airuesthesiu, Evans and Gray (Eds.), p. 157, Butterworth, London). If I may, for the benefit of the non-clinical members of the group, illustrate this difference from a human study, 1 would like to remind you of the syndrome of relative cerebral hypoglyceniia that we first described nearly 10 years ago (Wyke, 1959, E/i,c/r.oeticep/i.din. N~wop/i.v.sio/.,/ I : 602). This is a rare condition in which young adults develop the symptoms and the E.E.G. changes of hypoglycemia, although with blood and CSF glucose concentrations that are entirely within normal ranges statistically, both in the fasting and non-fasting states. The only way you can keep these patients symptom free (and the EEG normal) is by maintaining their blood glucose concentrations at figures between 130 and 200 mgm per 100 mL, so that they are in fact in a hyperglycemic state, and some of them are secreting glucose in the urine continuously. However, this does not elevate their CSF-glucose concentrations by more than 10% at the very most, and very often less than that. So here in these patients, you can drive glucose through the Blood-Brain Barrier, whatever that may be, and restore the cerebral metabolic activity to normal by raising the arterial blood glucose concentration, but you cuii not drive it into the CSF to any significant degree. Thus, it seems to me that in this abnormal condition one has a difference in the behavior of the glucose exchange mechanisms between the blood and the brain on the one hand, and between the blood and CSF on the other. Perhaps somebody will have a comment on that? G. PAPPAS: I think this is a rather important aspect. If I might summarize in general: ifyou introduced marker particles into the CSF fluid, the uptake by the choroideal plexus epithelium is via pinocytotic vesicles or vacuoles at the base of the villi. If you introduce marker particles, on the other hand, into the basal portion of these cells, the uptake is much different. They progress via the extracellular space for a while and then later may be pinched up in the vacuoles and vesicles, and we have not clearly shown their cntcring into the cerebrospinal h i d . So that even on a small scale, what you say does seem to hold up rather well, that therc is a difference between the two barriers.

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T. Z. CSAKY: May I point out again that indeed we have to distinguish between the transport of sugar from the capillary into the brain and the sugar exchange between the blood and CSF. When I talked about the choroid plexus I referred to the latter, while the blood-to-brain transport, I believe, is mediated through the glia cells. The two systems are different morphologically and most likely also functionally. One is an epithelial transport mechanism, the other is the glial transport. While we have some limited idea about the epithelium we are in the dark about the transport in the glia.

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Electron Microscopic Cytochemistry and Microgasometric Analysis of Cholinesterase in the Nervous System V I R G I N I A M. T E N N Y S O N , M l R O BRZIN*

AND

PHILIP DUFFY

Departnierit of Pathology, Division of Neuropatliology, College of Physicians and Surgeoris of Columbia University, New York ( US.A.) , arid *Iristitrite of Pathopliy.siology, University of Ljubljana, Ljubljana ( Yugoslavia).

INTRODUCTION

Techniques from different disciplines have been combined in the present investigation in order to provide more meaningful information than would be obtained otherwise. Electron microscopic-cytochemistry and microgasometric analysis have been used to study acetylcholinesterase in the sympathetic and dorsal root ganglia of the frog, using tissue blocks or isolated cells. In addition, some observations on the development of cholinesterase in the nervous system of the embryonic rabbit and human are presented. Cholinesterase activity of isolated sympathetic and dorsal root neurons has been studied by use of the Cartesian diver (Giacobini, 1957, 1959), but the magnetic diver (Brzin et al., 1964; Brzin and Zeuthen, 1964; Brzin et a/., 1965, and Pavlin, 1965) used in the present studies, permits a more sensitive method for quantitative determination of the activity of the enzyme. Isolated neurons have previously been examined with the electron microscope (Roots and Johnston, 1964, 1965; Johnston and Roots, 1965), but no microchemical studies were done on those cells. These investigators (Roots and Johnston, 1965) suggested that in isolated neurons the absence of a physical boundary would undoubtedly alter rates of penetration of substrates into the cell, and recommended that metabolic studies of these cells be monitored by the electron microscope. In previous studies from this laboratory (Brzin et a/., 1966a, 1966b, Tennyson e t a / . , 1966a), isolated neurons, which had been examined with the electron microscope following microgasometric analysis, showed a relationship between chemically measured activity and the morphological condition of the sample. Neurons having intact plasmalemmas and sheath cells showed lower activity values than those in which these structures were ruptured or absent. Ultracytochemistry was then applied to individual neurons, which had been analyzed microgasometrically (Brzin et al., in press). I t was shown that the neural plasmalemma is the ultimate permeability barrier to the substrates acetylcholine and acetylthiocholine. Satellite sheath cytoplasm Rcfhicalanalysis according to the method of Lineweaver and Burk of the initial inflsx 0; a-aminoisobutyric acid (AIB), alone and in the presence of an amino acid inhibitor (L-alanine and L-leucine), is presented in Fig. 1. The line drawn between the (Va) of individual points for control AIB influx is linear and extrapolates to a V,, 2.5 ,umole/ml intracellular water/minute; the transport constant Ka calculated from the slope of the line and Va is 1.3 mM. In the presence of an amino acid inhibitor (Lalanine or L-leucine), the individual points also fit along a straight line and extrapolate

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to a V,,, similar to the control, i.e., V, + b z V, (Table I). This indicates that the inhibition of AlB flux by these amino acids is a competitive inhibition. The transport constant in the presence of inhibitor ( K , + b) was determined and the inhibitor constant Ki calculated as previously discussed; the value of Ki for L-alanine and L-leucine inhibition of AIB influx was I .9 mM and 8.9 mM respectively. The inhibitor constants Ki for other amino acids were determined similarly and are given in Table 2. In each case the inhibition appeared to be a competitive inhibition (Table I). The transport constants for the inhibitors themselves ( K b ) were determined by a similar graphical analysis as previously described and are also presented in Table 2. I t can be seen that four amino acid inhibitors (glycine, L-alanine, malanine, cycloleucine) all within the small neutral group of amino acids have similar Ki and Kb values. The values of Ki and K b for the other four amino acids (L-methionine, Lproline, L-histidine, and L-leucine) in contrast are quite dissimilar. In othe words the ratio Kj/Kb is close to unity for the first four amino acids but not for the latter four amino acids. This would indicate from the previous discussion that the first four amino acids are predominatly transported by the same carrier system(s) mediating the transport of AIB over the concentration range studied. Since AlB is classified as a small neutral amino acid and the first four amino acids (glycine, L and D-alanine, and cycloleucine) are also recognized to be within the small neutral classification, their transport into the cell would be expected to be mediated by the same carrier system(s) (Blasberg and Lajtha, 1966). The fact that the K i / K b ratio is somewhat greater than unity for all of these amino acids could be due to experimental error; however, it may suggest some as yet unresolved differences in the transport of neutral amino acids with short side chains. Since the KI/Kb ratio for the latter four amino acids (L-methionine, L-proline, Lhistidine, and L-leucine) is at least 6, it is apparent that these amino acids are not predominantly transported by the same carrier system(s) mediating AIB influx. This group of amino acids does interact with AIB competitively for transport, however, which indicates that these amino acids have some affinity to the carrier(s) mediating AIB influx or that AIB is transported into the cell to some extent by the carrier system(s) primarily responsible for the transport of the amino acid inhibitor, or possibly that both conditions apply. A number of models for substrate-inhibitor interaction or carrier-inhibitor interaction could be constructed to explain the observed inhibition in terms of a multi-carrier transport reaction. Whatever the model, it is apparent that AIB flux is representative of small neutral amino acid transport. Although AIB influx is inhibited competitively by neutral amino acids with long side chains (methionine, leucine, and possibly histidine) as well, the transport of AIB and the latter amino acids are predominantly mediated by different carrier systems over the coiicentration range studied. The specificity of L-phenylalanine, L-arginine, and L-aspartate transport is summarized in Table 3. These amino acids are representatives of thelarge neutra1,large basic, and acidic amino acid transport classes, respectively (Basberg and Lajtha, 1966). The transport constants K a and V, were determined similarly to AIB as were the inhibitor constants Ki; in each case the inhibition appeared to be a competitive inhibition R i p k r m w s p. 256

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(Table I). The specificity of phenylalanine transport is essentially the mirror image of that for AIB with respect to the neutral amino acid inhibitors. The KJKb ratio for the first three inhibitors (glycine and L- and D-alanine) is significantly greater than that for the latter four inhibitors (cycloleucine, histidine, methionine, and leucine). The Ki/Kb ratio indicates that the small neutral amino acids (glycine and L- and D-alanine) are not predominantly transported by the same carrier system(s) mediating phenylalanine. Those amino acids with long side chains (cycloleucine, methionine, leucine, and possibly histidine), however, would be expected to be transported by the same carrier system(s) mediating phenylalanine influx; this is indicated by the Kt/Kb ratio for these amino acid inhibitors. Cycloleucine has been included in both the small neutral and large neutral amino acid transport classes because the Kr/Kh ratio was close to unity for the inhibition of both AIB flux and phenylalanine flux. This condition would exist if cycloleucine were transported to a similar extent into the cell by the carrier systems predominantly mediating AIB and phenylalanine at the concentration studied. The specificity of arginine transport demonstrates that only lysine of the amino acid inhibitors studied has a Ki/Kb ratio close to unity. This would be expected if the large basic amino acids were primarily transported by a carrier system which is relatively specific for amino acids with a long cationic side chain. Neutral amino acids inhibited arginine influx to a limited extent, but the Ki/Kb ratio indicates that neutral amino acids are predominanlty transported by another carrier system(s). In previous studies it has been shown that large basic amino acids (lysine and arginine) have little or no effect on neutral amino acid influx (Blasberg and Lajtha, 1966). This would suggest that large basic amino acids have little or no affinity to the carrier systems mediating neutral amino acid transport. The inhibition of aspartate by other acidic amino acids (L- and D-glutamate) demonstrates that the C4 and Cg acidic amino acids are transported by the same carrier system(s). Although glycine inhibits aspartate transport to some extent, the Ki/Kb ratio clearly indicates that glycine is primarily transported by a carrier system other than that which predominantly mediates aspartate flux. Other amino acids with neutral or long basic side chains have been shown to have essentially no effect on acidic amino acid influx; similarly, acidic amino acids have been shown to have little or no effect on neutral or basic amino acid influx (Blasberg and Lajtha, 1966). This suggests that C4 and C5 acidic amino acids are primarily transported by a carrier system that is relatively specific for amino acids with an anionic side chain. CONCLUSIONS

( I ) The mediated passage of amino acids into brain cells can be described phenomenologically by Michaelis-Menten kinetics; the kinetic constants for the transport reaction are formally equivalent to Michaelis-Menten constants K, and Vmax. (2) Transport constants for amino acid influx in brain slices must be determined from initial uptake experiments, which essentially measure the initial unidirectional flux; transport constants for influx cannot be determined from steady state uptake

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experiments, since amino acid exodus from brain cells is a mediated process and demonstrates saturation kinetics. (3) Amino acids within the same transport class have similar maximum transport rates ( V d . (4) Amino acid inhibition of amino acid transport into brain cells appears to be a competitive inhibition. ( 5 ) A comparison of the Ki/Kt, ratio for the transport inhibition of four amino acids (u-aminoisobutyric acid, L-phenylalanine, L-arginine, and L-aspartate), as representatives of different amino acid transport classes (small neutral, large neutral, large basic, and acidic amino acids, respectively), demonstrates that at least four transport systems mediate amino acid passage across brain cell membranes and indicates that an amino acid may enter the cell by more than one transport system. (a) The small neutral amino acids are predominantly transported by the same carrier system(s) mediating AIB influx; large neutral amino acids inhibit AIB flux competitively but are not primarily transported by the same carrier system(s) at the concentration studied . (b) Large neutral amino acids are predominantly transported by the same carrier system(s) mediating phenylalanine influx; small neutral amino acids inhibit phenylalanine flux competitively but are not primarily transported by the same carrier system(s) at the concentration studied. (c) Cycloleucine appears to be transported by two carrier systems (AIB’s carrier system and phenylalanine’s carrier system) to a similar extent at the concentration studied. (d) Lysine and arginine are transported by the same carrier system(s), which is relatively specific for amino acids with long cationic side chains. (e) Aspartate and glutamate are transported by the same carrier system(s), which is relatively specific for c 4 - C ~amino acids with an anionic side chain. I n summary, amino acid transport in brain slices involves a number of carrier systems, which can be partially characterized by the particular group of amino acids it predominantly mediates across the cell membrane. These transport systems apparently do not possess absolute specificity, since a number of amino acids appear to have some capacity for transport by carrier systems other than the one which primarily mediates their passage into the cell. A kinetic analysis of amino acid transport and amino acid inhibition of transport provides one method to evaluate the number and characteristics of the carrier systems in greater detail. ACKNOWLEDGEMENTS

The experimental work was done at the New York State Research Institute for Neurochemistry and Drug Addiction. I want to thank Dr. A. Lajthafor his hospitality and his advice, Dr. S. R. Cohen for his criticisms of the manuscript, and Mr. A. Mazeika and Z. Ronay for expert technical assistance. The investigation was supported in part by Grant No. NB04360 form the U.S. Public Health Service (to Dr. A. Lajtha.) RrJerenres p.

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REFERENCES P. G. (1962a) Amino acid transport in brain cortex slices. 1. ABADOM, P. N. A N D SCHOLEFIELD, Relation bEtween energy production and the glucose-dependent transport of glycine. Canad. J. Biochem., 40, 1575-1 590.

-, (1962b) Amino acid transport in brain cortex slices. 11. Competition between amino acids. Canud. J. Biochem., 40, 1591-1602.

-, (1962~)Amino acid transport in brain cortex slices. 111. Utilization of energy for transport. Canud. J. Biochern., 40, 1603-1618. BLASBERG, R. AND LAJTHA,A. (1965) Substrate specificity of steady state amino acid transport in mouse brain slices. Arch. Biochem. Biophys., 112, 361-377. -, (1966) Heterogeneity of mediated transport systems of amino acid uptake in brain. Brain Res., 1, 86-104.

CHRISTENSEN, H. N. (1962) Biological Transport. W. A. Benjamin, New York (p. 54-65). DIXON, M. A N D WEBB,E. (1964) Enzymes. Academic Press, New York (p. 87-90). ELLIOTT, K. A. c. A N D VAN GELDER, N. M.(1958) Occlusion and metabolism of y-aminobutyric acid by brain. J. Neurocheni., 3, 28-5 I . GUROFF,G., KING,W. AND UDENFRIEND, S. (1961) The uptake of tyrosine by rat brain in vitro. J . Biol. Chem., 236, 1773-1777.

GUROFF, G. AND UDENFRIEND, S. (1962) Studies on aromatic amino acid uptake by rat brain in vivo. J. Biol. Chem., 237, 803-806.

HEINZ,E. (1954) Kinetic studies on the “influx” of glycine I-CYinto the Ehrlich mouse ascites carcinoma cell. J. Biol. Chern., 211, 781. LAtiIRI, s. A ND LAJTHA,A. (1964) Cerebral amino acid transport in vitro. I. Some requirements and properties of uptake. J. Neurochem., 11, 77-86. LAJTHA, A. (1961) Exchange rates of amino acids between plasma and brain in different parts of the brain. Regional Neurochemistry. S. S. Kety and J. Elkes (Eds.). Pergamon Press, Oxford (p. 19-24). -, (1962) The brain barrier system. Neurochemistry. K. A. C. Elliott, I. H. Page, and J. H. Quastel (Eds.). Charles C. Thomas, Springfield (p. 399-430). LAJTHA,A. AND MELA,P. (1961) The brain barrier system. 1. The exchange of free amino acids between plasma and brain. J. Neurochem., 7, 210-217. LAJTHA, A. AND TOTH,J. (1961) The brain barrier system..II. Uptake and transport of amino acids by the brain. J. Neurochem., 8, 216-225. LAJTHA,A., BLASBERG, R. AND LEVI,G. (1966) Control of cerebral amino acid concentrations. Significance of Changes in Plasma Amino Acid Patterns, Rutgers University Press, New Brunswick, New Jersey, In press. LEVI,G., BLASBERG, R. AND LAJTHA,A. (1966) Substrate specificity of cerebral amino acid exit in vitro. Arch. Biochem. Biophys., 114, 339-351.

LINEWEAVER, H. AND BURK,D. (1934) The determination of enzyme dissociation constants. J. Amer. Chern. SOC.,56, 658.

NEAME, K. D. (1962) Uptake of L-histidine, L-proline, L-tyrosine, and L-ornithine by brain, intestinal mucosa, testis, kidney, spleen, liver heart muscle, skeletal muscle, and erythrocytes of the rat in vitro. J. Physiol., 162, 1-12. -, (1964) Effect of amino acids on uptake of L-histidine by rat brain slices. J. Neurochem., 11,67-76. OXENDER, D. L. AND CHRISTENSEN, H. N. (1963) Distinct mediating systems for the transport of neutral amino acids by the Ehrlich cell. J. Biol. Chem., 238, 3686-3699. STERN,J. A., EGGLESTON, L. V., HEMS,R. AND KREBS,H. A. (1949) Accumulation of glutamic acid in isolated brain tissue. Biochem. J . , 44, 410-418. TALLAN, H. H. (1962) A survey of the amino acids and related compounds in the nervuus tissue. Amino Acid Pools. J. T. Holden (Ed.). Elsevier, Amsterdam (p. 471-485). TSUKADA, Y.,NAGATA,Y., HIRANO,S. AND MATSUTANI, T. (1963) Active transport of amino acids into cerebral cortex slices. J. Neurochem., 10, 241-256. T. (1961) The concept of carrier transport and its corollaries in WILBRANDT, W. AND ROSENBERG, pharmacology. Pharmacol. Rev., 13, 109-183.

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DISCUSSION P. G. SCHOLEHELD: We faced the choice of measuring steady state levels or initial velocities for the determination of Km-values several years ago. I think there are many factors that have to be considered, and the reason that we rejected initial velocities was very simple; it was that initially a tissue, whether it is brain slice, or pancreas, or ascites cells, always contains amino acids inside the cell, and these can always exchange with the amino acid that is added to the medium. So when initial velocities are measured there is always a danger. When we measure steady state values all the amino acids inside the cell have been swept out or have exchanged as much as they possibly can and this would be a relatively sniall amount compared with the amount that one puts in the medium. We tried to substantiate this directly by preincubating cells or tissues to get rid of endogenous amino acids, and then we found somewhat different Km values. So that this, I think, is a note of warning, if 1may.

R. BLASBERG: To answer your point: our initial velocities were calculated from 3-minute intracellular uptake values, and for most of the amino acids, intracellular uptake was approximately linear between 2 and 5 minutes. Undoubtedly there is reflux and physical diffusion of amino acid between the intracellular and extracellular phases during this period, but we believe this to be relatively sniall. Secondly, our tissue is pre-incubated for 30 minutes in amino acid-free medium, during which time the endogenous amino acids will tend to equilibrate with the medium. Whether exchange processes significantly alter the calculated unidirectional flux, I cannot really answer, except to say that the Lineweaver-Burk plots are linear below 2 mM for most amino acids. This concentration is considerably higher than the endogenous tissue level of most amino acids. In any event, Dr. Scholefield’s point is a good one and must be kept in mind in any kinetic study of transport processes. A. LAJTHA:May I ask you, Dr. Scholefield, if you have any evidence of moles of ATP used per mole amino acid transported in your system at least approximately? No, we have never really calculated this. It is rather difficult to assay ATPP. G. SCHOLEFIELD: utilization in ascites., We did some years ago attempt to study 32Pturnover, to get after this but it is difficult because the 3?P turnover is determined mainly by the rate at which 32Penters the ascites cells or the brain slice. I f brain slices or ascites cells are incubated with 3zP it will take about a n hour to get a steady state label in ATP, whereas, assuming a P/O ratio of three and a normal rate of oxygen uptake, all the ATP should be labelled within, I think, 45 to 60 seconds. Study of 3eP turnover is therefore of no use in efforts to answer this question from studies in which an intact cell preparation is used. Perhaps I could make one point to Dr. Mandel on this general topic. In studies of brain slices we tried to elevate the level of ATP in the slice, and we found adenine of little use since its presence could lead to perhaps 5 or 10% increase. Addition of adenosine led to an increase of the total ATP level of the slice so our in vitro experiments would lend some support to your in vivo experiments.

K. D. NEAME:I would just like to comment on the relationship between the review slides I gave on the various transport systems and Dr. Blasberg’s presentation. The systems that I gave were based, as I indicated, on a multitudinous amount of work by many people, but essentially it rested on the interpretations of net uptake, that is, uptake expressed as influx plus the effect of efflux over a certain time, or, as Dr. Lajtha indicated, the steady state situation where there is a balance. Whereas Dr. Blasberg’s presentation was on influx only, and some of Dr. Scholefield’s comments were on intracellular amino acids coming out of the tissue, Dr. Blasberg’s comments seem to correlate very well with the steady state situation as regards the different transport systems. They seem to agree very well indeed. D. B. TOWER: But is it not true that if you pre-incubate brain slices for 30 minutes under optimal conditions you reach a steady state? You are simply putting in a tracer to see what is going on. Is that not correct? A. LAJTHA: No, it is not tracer amounts that were added in Dr. Blasberg’s experiment at 30 minutes. We wanted to measure uptake rather than exchange. It is interesting that endogenous amino acid levels in brain slices d o not disappear if you incubate them for 30 minutes. You get an ATP level that is about half of the in vivo levels, but the amino acid levels seem to be about the same in the incubated

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slice as they were in the living brain. This high endogenous pool was the reason for our using 2 mM amino acids in the incubation medium. The level reached in the slice with 2 mM medium concentrations is way above the endogenous levels, even after 2 minutes. Therefore 1 would not think that the exchange with the small amount of endogenous amino acid would be very significant. When there is a lower concentration in the medium - perhaps below 0.1 mM - then exchange with endogenous amino acids may contribute to initial uptake rates. Our measurements therefore, were mostly of net uptake. At present we are measuring exchange which shows somewhat different behavior. T. Z. CSAKY:I would like to ask Dr. Scholefield: what is the other source of energy if ATP is rejected? If there are other supplies of energy, how does digitalis act in inhibiting the active transport? P. G. SCHOLEFIELD: I think the key word in what I said is direct. I have been trying to find out whether ATP may not be the direct material involved. If, for example, phosphoprotein is involved, the ATP may cause a conformational change, and the conformational change of the phosphoprotein may be what is directly involved in the transport.

T. Z. CSAKY:And where does the ouabain enter into this whole picture?

P. G. SCHOLEFIELD: In the phosphorlation-of the phosphoprotein. P. MANDEL: But this is quite rapid. The phosphorylation of phosphoprotein is a very rapid phenomenon. P. G. SCHOLEFIELD: While we are discussing amino acids and cerebral systems I think the phenomena of the excitatory amino acids should be mentioned. There are extremely rapid interactions between amino acids and cerebral systems, whereby amino acids (including some which are not metabolized at all) can cause cell-firing within seconds or milliseconds of their application to the cerebral cortex in vitro; corresponding phenomena occur in vivo. 111 vitro this was shown to depend on a relationship between glutamic acid and sodium ions. The metabolic relationship between glutamic acid and cerebral systems in vivo and in vitro is already complex. The mechanism for this immediate provoking of cell-firing appears to be that glutamic acid increases permeability to sodium, and this also occurs within milliseconds. The acid allows the entry of sodium, and this is through the initiation of part of the normal mechanism that causes cell firing. The membrane potential is lowered through entry of positive ions, and this can cause cell-firing in vivo. In vitro there occurs a diminution in membrane potential, and this sets in motion processes which normally include the entry of sodium. The system is normally restored by sodium extrusion: the sodium pump comes into operation. This accounts for the loss of creatine phosphate that takes place, and as Dr. Elliott mentioned some time ago, the entry of potassium and the entry of water afterwards are a secondary phenomenon to the action of the sodium pump, which is removing the sodium and bringing in potassium at the same time.

A. LAJTHA:I just wanted to say that, from what we know about the reactions in which ATP participates, and about ATP turnover, one would expect that the energy rich compounds always equilibrate fairly rapidly with each other. So probably the ATP levels fluctuate quite rapidly and in parallel fashion with an intermediate that, in turn, is, as Dr. Scholefield said, more directly utilized.

T. Z. CSAKY:I think it is very important, physiologically, that the ATP we talk about is not identical to the chemists’ interpretation. ATP is a system, an equilibrium between the adenosine compounds and creatine phosphate, and it is fairly rapidly turned over. So, physiologically, when we talk about ATP, what we talk about is really higher energy phosphates.

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A Possible Enzyme Barrier for r-Aminobutyric Acid in the

Central Nervous System N l C O M. V A N G E L D E R Departirient of Physiolog??. Fuatlty of’ Medicitre Uiriversity of’ Moiiireal, Montreal, @(e. (Canada)

In 1958 van Gelder and Elliot demonstrated that high concentrations of y-aminobutyric acid (GABA) in blood did not result in an elevation of brain GABA levels. Their work indicated that, as is the case for many compounds, a barrier exists which restricts the passage of GABA from blood to nervous tissue. The nature of the barrier is unknown, but recent histochemical studies on the distribution of GABA-a-ketoglutarate transaminase in the mammalian central nervous system (van Gelder, 1965a, b) suggested that the barrier may be in part a reflection of high GABA-transaminase activity in the walls of cerebral blood vessels and in the cells lining the cerebrospinal fluid spaces (see below). In order to explore this suggestion further, a study was made of the distribution of GABA-transaminase in kidney and liver. GABA appears to pass freely from blood to kidney, since as much as fifty per cent of the total GABA injected can be recovered from urine (van Gelder and Elliott, 1958). Similarly, the concentration of GABA in liver after injection parallels that of the blood. In view of these findings, an enzyme barrier similar to the one observed in the central nervous system should not exist in these organs. The present results indeed suggest that no such barrier prevents GABA from being filtered by the kidney from blood since very little GABA-transaminaseappears to be present i n either the blood vessels of the kidney or the capsule surrounding this organ. In liver also, GABA-transaminase does not appear to be concentrated particularly around blood vessels, and it may even be present in below normal concentrations whenever the vessels are surrounded by hepatic connective tissue. METHODS

The histochemical procedures for the localization of GABA-transaminase have been described previously (van Gelder, I965a). Frozen sections of brain are incubated in a 0.5 % agar-saline medium (pH 7.4) having the following composition: Nitro BT, 2.0 mg/ml; NAD, 2 mg/ml; (1-ketoglutarate, 5 mg/ml; GABA, 5 mg/ml. Incubation is carried out at 40” for 10 min i n a moist chamber. The method is based on the fact that, in the course of the metabolism of GABA to succinic acid, NADH is formed, which in turn reduces a tetrazolium salt (Nitro BT) RcWenic’s p. 268

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to its formazan form. Formazan is insoluble and will precipitate out at sites in the tissue where the combined presence of GABA-u-ketoglutarate transaminase and succinic semialdehyde dehydrogenase have caused the conversion of GABA to succinic acid. For further details of the procedure as well as determination of the specificity of the reaction for GABA metabolism, see van Gelder, 1965a and 1966. RESULTS

When sections of mammalian brain are incubated with a transaminase medium (see Methods), strong formazan precipitation occurs at sites where GABA-transaminase and succinic semialdehyde dehydrogenase are present in combination (van Gelder, 1965a). Although the presence of both enzymes is a prerequisite for the reaction, recent studies (van Gelder, 1966) suggested that the transamination step is ratelimiting with respect to the histochemical demonstration of GABA metabolism. The ependymal cell layer which lines the cerebrospinal fluid spaces is among the sites in the central nervous system which exhibit strong GABA-transaminase activity. This is demonstrated, for example, in Fig. I , which shows the central canal in the

Fig. 1. GABA-u-ketoglutarate transarninase in central canal of mouse. A heavy zone of formazan precipitation, indicative of strong transaminase activity, is present in ependyrnal cells which line the cerebrospinal fluid space. Activity is directed towards the lumen of the canal. These results apply to all cerebrospinal fluid spaces and are also obtained with brain tissue of monkey or rabbit. Section (10 p ) through cervical region ( x 400 magnification).

cervical region. The heavy layer of formazan in the ependymal cells which is directed towards the lumen of the canal indicates that GABA can be rapidly metabolized in this region. Similar results are obtained in other areas of the cerebrospinal fluid

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Fig. 2. GABA-transaminase activity in mouse cerebral blood vessels. 2a = Control: section incubated in a medium lacking GABA. 2b = Experimental: consecutive section incubated in the same medium after GABA had been added. Enzyme activity, visualized by formazan precipitation, surrounds all blood vessels and is also present in the pia-arachnoid. a = artery; bl = blood vessels; pa = pia-arachnoid. Section (10 p ) through mouse brain stem ( x 400 magnification).

spaces, where the choroid plexus is also found to show a pronounced histochemical reaction. With the possible exception of very large blood vessels, a layer of GABAtransaminase activity appears to surround cerebral blood vessels as well. Fig. 2 shows two consecutive brain sections, one of which was incubated with a control medium lacking GABA while the other was incubated in the same medium after Rcfiwnces p. 268

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GABA had been added. As illustrated here, the production of formazan is dependent on the presence of GABA (van Gelder, 1965a), but once GABA metabolism has been initiated by the addition of GABA to the medium, the resulting tetrazolium reduction is heavily concentrated around cerebral blood vessels of various sizes, as well as the pia-arachnoid (pa). In sections incubated in this manner, most capillaries appear to be outlined by a formazan precipitate. Results such as these suggest that diffusion of GABA from the cerebrospinal fluid spaces or from the blood must occur through a zone of strong GABA-transaminase activity. It would appear that these zones act as a barrier against the penetration of exogenous GABA into the brain parenchyma. In order to investigate the distribution of GABA-transaminase in kidney and liver, which do not appear to possess a barrier for GABA, frozen sections of these organs were incubated with the same medium as that used for the brain sections. Figs. 3a and

Fig. 3. GABA transaminase activity in mouse kidney. A relatively large artery (a) is shown, partially surrounded by proximal tubules (p). Control section (3a) exhibits a certain degree of non-specific tetrazolium reduction, but enhancement of staining in proximal tubules of experimental section (3b) indicates the presence of GABA-transaminase. Little increase in formazan production has occurred in artery of experimental section, as compared to that of control section. Section (10 p); ( x 400 magnification).

4a illustrate that, unlike brain tissue, kidney sections will reduce tetrazolium salts to some extent in the absence of GABA. Such reduction, therefore, cannot be ascribed to the presence of GABA-transaminase. However, after incubation of sections in the medium to which GABA had been added, a strong enhancement of formazan production was observed in certain areas. This enhancement was especially noted in the proximal tubules (p), which are therefore believed to possess considerable GABA-

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Fig. 4. Glomerulus (9) in mouse kidney surrounded by proximal tubules (p). The glomerular parenchyma is stained about equally in control (4a) and experimental (4b) section, indicating low GABA-transaminase activity at this location. Compare to surrounding proximal tubules.

Fig. 5 . Section through mouse kidney showing cortex (c) and outer medulla (m). Only proximal tubules in the outer medulla exhibit appreciable GABA-transaminase activity. Portions of the tubules in the cortex or inner medulla as well as kidney blood vessels (bl) are devoid of enzyme activity. Connective tissue capsule (ca) surrounding the kidney is also not stained. Section (10 p ) ; ( x 100 magnification). Rrfercnces p . 268

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transaminase activity. In contrast, little enhancement in formazan production was seen in blood vessels. This is shown in Fig. 3b and Fig. 4b, which respectively represent a relatively large artery (a) and the glomerular tuft (g) surrounded by proximal tubules (p). In both photographs, no striking difference in formazan precipitation is seen in the blood vessels of the experimental sections as compared with those of the control sections. It is especially noteworthy that little GABA-transaminase activity appears to be present in the capillary tuft of the glomerulus, since it is at this site that GABA must pass into the kidney tubules. Fig. 5 represents a kidney section incubated in the transaminase medium. It shows that neither the connective tissue capsule surrounding the kidney (ca), nor the kidney cortex (c) are stained for GABA-transaminase activity. Only the outer medulla (m), which is composed primarily of the distal portion of the proximal tubules and the descending and ascending loops of Henle, shows appreciable enzyme activity. Close examination of this area revealed that the loop of Henle exhibits no tetrazolium reduction, beyond that found in control sections. The inner medulla is also not stained by GABA-transaminase activity, nor are the walls of the blood vessels (bl). Finally, to determine the specificity of the histochemical reaction for GABAtransaminase in kidney, hydrazinopropionic acid (1 5 mg/kg) was injected into a mouse and its kidney was sectioned two hours later. Such sections, when incubated in the transaminase medium, were identical in appearance to normal sections which had been incubated in a control medium lacking GABA. Hydrazinopropionic acid, which was recently synthesized in this laboratory, is a very strong and probably quite specific in vivo and in vitro inhibitor of GABA-transaminase (to be published). This compound is a close structural analog of GABA. The results obtained with liver sections are to a large extent similar to those obtained with kidney sections. In this organ too, a certain degree of tetrazolium reduction occurs which is not dependent on GABA-transaminase activity (Fig. 6a). The formazan precipitate which is formed in these control sections has a pin-point appearance (Fig. 6b), which is not found in the kidney. The addition of GABA to the medium will result in a considerable enhancement of formazan formation as shown in Fig. 6c. The exact localization of this tetrazolium reduction, due to GABA-transaminase activity, is difficult to determine because of the background of non-specific formazan precipitation. However, Figs. 7a and 7b clearly show that transaminase activity in the connective tissue cells (ct) surrounding hepatic blood vessels is low as compared with hepatic parenchyma, while the bile ducts (bi) appear to have a high activity. The low transaminase activity in hepatic connective tissue is in sharp contrast to the high enzyme activity which surrounds cerebral blood vessels. DISCUSSION

Both Dobbing (1961) and Lajtha (1962), in reviewing studies on the blood-brain barrier, concluded that in addition to anatomical barriers, metabolic processes had to play an important role in preventing a variety of different compounds from penetrating into the central nervous system. Similarly, Barrnett and co-workers in a long

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Fig. 6. GABA-transaminase in mouse liver. As in the kidney, non-specific tetrazolium reduction occurs in control section (6a) which has a characteristic pin-point appearance (6b; x 400). After addition of GABA to the medium (6c) sections exhibit enhanced formazan production. Connective tissue surrounding blood vessels (bl) has low transaminase activity (see Fig. 7). Section (10 p ) ; ( x 73 magnification).

series of histochemical studies (e.g., Rostgaard and Barrnett, 1964; Marchesi and Barrnett, 1965) suggested that enzymes located at membrane surfaces aided in the transport of substances across such membranes. This suggestion presumably implies that, in the course of being transported, compounds are altered in some manner (Rostgaard and Barrnett, 1964). Hrfcwnci.s p. 268

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Fig. 7. Two hepatic veins in an experimental section. Connective tissue (ct) surrounding veins is noticeably lower in GABA-transaminase activity than liver parenchyma. High enzyme activity is seen in bile ducts (bi). Non-specific tetrazolium reduction (Fig. 6b) partially obscures localization of GABA-transaminase in parenchyma. Section (10 p ) : ( x 400 magnification).

The present histochemical data is in agreement with the above observations, since they suggest that in the process of diffusion across cerebral blood vessels or across the cerebrospinal fluid spaces, GABA can be rapidly converted to succinic acid. Little information is available regarding rates of diffusion of amino acids at these sites, but the results of Kuttner, Sims and Gordon (1961) indicate that this process may be slow. Using a-aminoisobutyric acid, which is metabolically inert, these

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authors reported that equilibration of the concentrations between plasma and brain required up to 10 h. This slow diffusion process would presumably allow ample opportunity for the destruction of GABA in passage from blood or cerebrospinal fluid to brain (see also Levin, Garcia Argiz and Nogueira, 1966). The situation i n kidney appears somewhat different, since very little destruction of G ABA seems possible during passage through the glomeruli (Figs. 3 and 4). The rate of removal of GABA from the blood by the kidney would therefore depend simply on the glomerular filtration rate, which varies from approximately 6.5 ml/min/kg in rat to about 120 ml/min/1.73 m2 in man. The results suggest that once GABA has entered the proximal tubule, a certain percentage will be destroyed by GABAtransaminase present in this part of the tubule. The amount destroyed should depend on both the concentration of GABA in the filtrate and the flow rate through the tubule. Once past the descending loop of Henle, the subsequent redistribution of water in the more distal portion of the kidney tubule would tend to concentrate the remaining G ABA. This would account for the high concentrations of GABA which are found in kidney and urine after intraperitoneal or intravenous injections of GABA into animals (van Gelder and Elliott, 1958). The distribution of GABA-transaminase in liver indicates that here, too, no particular barrier exists for the penetration of GABA into this organ. On the contrary, the concentration of the enzyme in connective tissue cells surrounding the blood vessels appears to be below normal when compared to the liver parenchyma or the bile ducts. While the histochemical data as well as pharmacological studies (van Gelder, 1966) tend to support the concept of a metabolic barrier for GABA, it must be pointed out that biochemical evidence so far islacking. Attempts to demonstrate entrance of GABA into the central nervous system of mice (van Gelder, 1966) and rats (Fisher, Hagen and Colvin, 1965) by in viva inhibition of GABA-transaminase with aminooxyacetic acid have been unsuccessful even after injection of as much as 1500 mg/kg of GABA (unpublished data). Possibly, indirect biochemical evidence may have been provided by several studies in recent years which have shown that injections of radioactive amino acids result in labeling of brain constituents without a concomitant net increase in their brain levels (e.g., Bed, Lajtha and Waelsch, 1961 ; Gaitonde, 1965). Contrary to prediction, such data indicated that the specific activities of expected metabolites from these amino acids were often higher in brain than the specific activity of the original amino acid injected, This led to the suggestion that several independent metabolic compartments for the same amino acid exist in brain. If the concept of a metabolic barrier is extended to other amino acids, the simplest explanation of this discrepancy would indeed be that such amino acids are metabolized while passing from blood or cerebrospinal fluid to nervous parenchyma. Bed, Lajtha and Waelsch (1961) have in fact suggested that the compartment for amidation of administered glutamate is located in the cerebral surface membranes or the ependymal lining. More recently, Levin, Garcia Argiz and Nogueira (1966) reported that ventriculo-cisternal perfusion of labeled GABA (or glutamate) in cats resulted in the appearance of radioactivity in the periventricular tissue (3-4 mm deep). At the same time no net increase in the concentration of this amino acid could be demonstrated R~~/r.rcncrs p.1268

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at these sites. These findings are also in accordance with the histochemical demonstration of GABA-transaminase in the ependymal lining of the cerebrospinal fluid spaces. In conclusion, the available data, when taken together, seem to support the suggestion that various enzymes, localized in cells bordering blood and cereborspinal fluid spaces, form a barrier towards the entrance of exogenous amino acids into the central nervous system. It is clear, however, that more direct biochemical evidence must be forthcoming before such an important concept can be accepted. ACKNOWLEDGEMENTS

This work was supported by a grant from the National Multiple Sclerosis Society. I wish to thank Miss Ann Larratt for her valuable assistance. REFERENCES BERL,S., LAJTHA,A. AND WAELSCH, H.(1961) Amino acid and protein metabolism - VI. Cerebral compartments of glutamic acid metabolism. J. Neurochem., 1, 186-197. J. (1961) The blood-brain barrier. Physiol. Rev., 41, 130-188. DOBBING, FISHER, M. A., HAGEN,D. Q. AND COLVIN,R. B. (1966) Aminooxyacetic Acid: Interactions with Gamma-Aminobutyric Acid and the Blood-Brain Barrier. Sci., 153, 1668-1 670. GAITONDE, M. K. (1965) Rate of Utilization of Glucose and 'Compartmentation' of a-Oxoglutarate and Glutamate in Rat Brain. Biochem. J., 95, 803-810. KUTTNER,R., SIMS,J. A. AND GORDON,M. W. (1961) The uptake of a metabolically inert amino acid by brain and other organs. J. Neurochem., 6, 311-317. LAJTHA,A. (1962) Neurochemistry. Illinois. Charles C. Thomas. LEVIN,E., GARCIAARGIZ,C. A. AND NOGUEIRA, G. J. (1966) Ventriculocisternal perfusion of amino acids in cat brain - 11. Incorporation of glutamic acid, glutamine and GABA into the brain parenchyma. J. Neurochem., 13, 979-988. MARCHESI, V. T. AND BARRNETT, R . J. (1964) The localization of nucleosidephosphatase activity in different types of small blood vessels. J. Ultrastruc. Res., 10, 103-1 15. ROSTGAARD, J. AND BARRNETT, R. J. (1964) Fine structure localization of nucleoside phosphatases in relation to smooth muscle cells and unmyelinated nerves in the small intestine of the rat. J. Ultrastruc. Res., 11, 193-207. VAN GELDER, N. M. (1965a) The histochemical demonstration of y-aminobutyric acid metabolism by reduction of a tetrazolium salt. J. Neurochem., 12, 231-237. -, (1965b) A comparison of y-aminobutyric acid metabolism in rabbit and mouse nervous tissue. J. Neurochem., 12, 239-244. -, (1966) The effect of aminooxyacetic acid on the metabolism of y-aminobutyric acid in brain. Biochem. Pharmacol., 15, 533-539. VAN GELDER, N. M. AND ELLIOTT,K. A. C. (1958) Disposition of y-aminobutyric acid administered to mammals. J. Neurochem., 3, 139-143.

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DISCUSSION Did you use a-ketoglutarate for the transamination reaction? H. KOENIG: N. M. VAN GELDER: The transamination reaction requires GABA plus a-ketoglutarate. One then gets succinic semi-aldehyde and glutamate.

H. KOENIG:Glutamate dehydrogenase has exactly the same histochemical distribution as your enzyme (Koenig and Barron, 1962, Acta Neurol. Scan& Suppl., p. 72). This enzyme system, strategically located at the Blood-Brain Barrier, might serve the function of protecting the brain from ammonia, because glutamic dehydrogenase brings about the reductive amination of a-ketoglutarate to form glutamate. (Koenig, 1964, in "Morphological and Biochemical Concepts of Neural Activity", Cohen, M. M. and Snider, R. S. (Eds.), p. 39, Hoeber, New York). N. M. VAN GELDER: 1 would like to point out that there are many enzymes localized in the ependymal cells: e.g., succinic dehydrogenase. H. KOENIG: Glutamate-dehydrogenase occurs not only in the perivascular glial cells but also in the neuropil. Did you look at grey matter for this enzyme?

N. M. VAN GELDER: I did not look for glutamate-dehydrogenase.

P. MANDEL:The first problem is, I think, to demonstrate that there are enzymes which recognize glucose in the intestine; then you can be sure that glucose is going across the intestinal epithelium. You also need enzymes which are able to destroy the substance or complex because it is not the albumin that is going through.

N. M . VAN GELDER:I am not saying that albumin can. The point is how to demonstrate the glucose enzymes as such. If you look at the intestine, it appears that glucose goes through, but it is phosphorylated in the process of penetrating, and dephosphorylated before it enters the blood. P. MANDEL:But anyway, it isgoing through! N. M. VAN GELDER: Yes, but I am suggesting that if glucose arrives in the blood as, e.g., succinic acid, it is just as good a substrate.

P. MANDEL: The second remark is that if you purify GABA-transaminase 100-fold, you also have glutamate-transaminase activity because you cannot separate one from the other. Even with several methods of electrophoresis you always have the two enzymatic activities together. VAN GELDER: But what you are measuring here is not glutamate-transaminase. I have tried these same brain sections with glutamate, and one does not get the tetrazolium reduction with glutamate as substrate, at least not under the experimental conditions employed.

N. M.

P. MANDEL:Perhaps it is because it is altering the cell. Anyway, even if the enzyme is purified, the two activities remain together. A. LAJTHA:For the removal of GABA in the reaction sequence you discuss, you would need an equivalent amount of ketoglutarate to transaminate with. I wonder if there is enough ketoglutarate present in the brain, or whether enough can be generated, especially in experiments in which you administered significant amounts of GABA. As you know, ketoglutarate levels in the brain are rather low; and although enough enzyme may be present at the barrier site you propose, one of the substrates may not be there in sufficient amount to allow the reaction to go to completion.

N. M. VAN GELDER: I agree with you that there has to be a direct biochemical proof before one can really accept this type of blood-brain barrier. I found it attractive, and it struck me just from the distribution of GABA transaminase that an enzymic barrier may exist.

D. B. TOWER:I have two questions. One is in regard to the other tissues where, if memory serves

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me correctly, Eugene Roberts found a GABA-transaminase in large amounts. How do your histochemical qualitative observations coincide with the quantities which he has found for enzyme-activity in the liver and kidney and elsewhere? And the second question relates to the question of the entry of GABA into the CSF, since this is one place where we measured it. We did not use radioactive GABA. GABA was given to human subjects, in doses of several millimols per kg and measured in the blood and in the CSF by Robins’ method which has some disadvantages. 1 think, however, since it was confirmed by paper chromatoaraphy it is fair to say that we were actually measuring GABA. In normal subjects it is quite true that you cannot demonstrate any GABA entering into the central nervous system, but in certain patients with epilepsy you can show this. My question is: if this barrier that you have proposed is the mechanism which normally keeps GABA out of the central nervous system following a systemic administration, what would you anticipate has happened in these seizure patients to modify the barrier? I don’t expect a specific answer to this question, but I wonder if you have thought about this problem. It is a puzzling problem because it relates to a very important fundamental question, namely the modification of the barrier systems. We are going into that tomorrow, but here is one example which is already presented to us, where in certain seizure patients (not all) there is apparently a modification of whatever the mechanism is that normally excludes GABA. N. M. VAN GELDER: Offhand I can suggest at least two possible explanations. First of all: a lower substrate level, assuming for the moment that there is enough, and I am not saying that there is, but assuming that there is enough u-ketoglutarate in normal brain tissue, then in the seizure areas there may just not be enough a-ketoglutarate available to allow for transamination. A second possibility may be that in many of the seizure patients one finds abnormal circulation which may completely alter the permeability of the blood vessels, or even the transaminase distribution in these blood vessels; but I don’t know anything about this at the moment. With respect to the second question, the chemical results in liver and kidney appear to correlate well with the biochemical data of Roberts. C. F. BAXTER: We have some experimental evidence on the movement of GABA out of the cerebral ventricles which would tend to corroborate Dr. van Gelder’s findings. In collaboration with Dr. M. Rubinstein we have found that there is also a high concentration of GABA metabolizing enzyme around the ventricular walls in the rat. The technique used was virtually identical to that described by Dr. van Gelder. In other experiments we have injected stereotaxically 1 [14C]GABA into the lateral ventricle of rats. Five to 45 minutes after the injection, the rats were frozen in liquid nitrogen and radioautographs prepared of frozen brain sections. These autographs showed that the movement of radioactivity from the ventricle to the more distal parts of the brain was surprisingly slow. At the same time 1 [“TIGABA injected intraventricularly, is rapidly metabolized (Baxter, 1963). It is quite possible, therefore, that deep penetration of radioactivity from 1 [14C]GABA into cerebral tissues is hindered by the rapid metabolism of the carboxyl group of this amino acid in areas adjacent to the ventricle. This concept agrees with Dr. van Gelder’s observations. In the mouse it is more difficult to show slow penetration from the ventricle into brain tissues except into the cerebellum. However, it is apparent that there is an effective barrier which prevents the movement of GABA out of the ventricle into the rest of the body. This barrier is compromised if the vascular bed of the brain tissue is damaged during the injection procedure. This finding is illustrated by the figure.

We injected mice intraventricularly with 2 [WIGABA using the Haley technique (1957). After various time periods each mouse was quick frozen, embedded in methyl cellulose and radioautographs made of longitudinal microtome sections. These techniques were developed in Sweden (UW berg, 1954). The top third of the figure shows a typical section. The middle third shows a radioautograph which grossly indicates the distribution of 2 [I4C]GABA injected 25 minutes earlier. It can be seen that radioactivity is almost exclusively confined to the central nervous system and parts of the spinal cord. A little activity in the urine (bladder) can also be detected. There is very little radioactivity in liver and kidney. This result should be compared with the bottom radioautograph in the figure. In this experiment the intracerebral injection of the isotope was accompanied by rupture of a small cerebral blood vessel, observable by the fact that a little blood was on the injection needle when it was withdrawn.

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Under these conditions, a substantial amount of radioactivity in liver and kidney was detectable within 5 minutes after intracerebral injection. These incidental findings clearly illustrate some of the difficulties encountered when attempting to determine the "normal" rates at which substances move in vivo within the brain and from the brain into other body tissues. REFERENCES BAXTER,C. F. (1963) Cerebral Metabolism of Some Amino Acids in v i v a Fed. Proc., 22 : 301. HALEY,T. J., AND MCCORMICK, W. C. (1957) Pharmacological Effects Produced by lntracerebral Injection of Drugs in the Conscious Mouse. British J. Pharinacol. Chetiiother., 12 : 12-1 5. U L L B ~ RS. G ,(1959) Autoradiographic Studies on the Distribution of Labeled Drugs in the Body. Progress in Nitclear Energy - Series 6,22,29-35. N. M. VAN GELDER:I find Dr. Baxter's observation very interesting. One would almost suspect that if the barrier works in one way, structures forming the barrier may just as well work in the other way in the reverse direction. This would support the fact that even when you load up GABA-levels in the brain to fantastic amounts, you don't find anything in the blood. As a matter of fact, you don't find much GABA in the CSF either. So it looks as if the barrier would indeed work both ways. All 1 am saying is that the localization of GABA transaminase seems to be compatible with an enzymic barrier but I am not suggesting that it is the only barrier. G. PAPPAS:I just wanted to ask Dr. French if he has any comments to make about the action of rhodanase, or anybody else, about rhondanase and thiocyanate?

C . M. FRENCH: It is an enzyme which breaks or converts thiocyanide, and it seems to have an equilibrium position such as the cyanide. It is present in very small concentrations, probably 0.1 mequiv./l, whereas the thiocyanate will be at something like I 0 0 rnequiv./l. This was the point I was making when asked what causes the definite ion movement; that very small quantities of thiocyanate d o get converted to cyanide. The quantity is so small that it does not affect the analysis of how much thiocyanate there is present. On the other hand, it may have a marked effect on the function of the cells. Kogan has suggested that this is the cause of the reduced arterial venous oxygen difference. He suggested that cyanide is blocking oxygen uptake. If thiocyanate i s converted to cyanide, it is an indication of course, that the thiocyanate can become intracellular, and it would support the concept that some cells are resistant to cyanide. It occurs mainly in vitro in red cells of rats. I have been able to show a conversion of thiocyanate into cyanide in vitro, but I have not been able to show this in vivo.

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Ion Movements

Ion Movements in Isolated Preparations from the Mammalian Brain HENRY Mcl LWAIN Department of Bioclieriiistry, Institute of Psychiatry (British Postgraduate Medical Federation. University oj’lotidon), Maudsley Hospital, Denmark Hill, London, S. E.5 (England)

INTRODUCTION

This contribution is concerned mainly with the barriers or restrictions to diffusion which operate at the cellular level in the brain. At cellular level is manifested that manipulation of barriers which is the most characteristic property of any neural system. In about a millisecond, restrictions normally imposed on the movements of Na and K are greatly altered : diminished to a fraction of their previous value and then reasserted, these changes occurring in the defined sequence which produces the nerve action potential. Demonstration and understanding of such events need co-ordinated observations by chemical and electrical means, and these are greatly furthered by separating the neural system concerned from the organism of which it forms part, and examining it in vitro. This practice has long been established in relation to amphibian peripheral nerve and brilliantly applied to the giant axons of crustacea and squids. Application to the central nervous system may have been delayed by exaggerated opinions of the damage caused in preparing the tissue samples, and for this reason the following appraisal is given (Mcllwain, 1956; Mcllwain and Joanny, 1963). A P P R A I S A L OF ISOLATED TISSUE P R E P A R A T I O N S FROM THE B R A I N

To obtain portions of the brain for biochemical work, a usual practice is to prepare sheets about 30-150 mg in weight and 0.35 mm in thickness. This allows access at their outer surfaces to materials normally exchanged with the blood stream at the cerebral capillaries. Note that this implies immediately that one category of barrier phenomena important in vivo is eliminated in using isolated tissues suspended in aqueous media; those operating between the blood capillaries and the extracellular fluids of the brain. Such simplification is a major reason for using in vitro techniques. In particular, opportunity is given in vitro for direct observation of events between extracellular and intracellular phases, which in vivo may need indirect computation. In appraising such systems it is valuable to know their area of cell surface (i) in reRrfirenrrs p. 280

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TABLE I C O M P U T E D AREAS IN CEREBRAL C O R T I C A L SPECIMENS

Data

(i) Area of external surfaces mmz (ii) Cut area, mrn2 (iii) Neuronal area (partial), mm2 (iv) Cut area, as approx. % of neuronal area (v) Capillary area, mmL

Valites, per 100-nig sample 600 300 82,000

0.1 1,000

Notes

Cut 0.35 mrn thick

-

Rabbit cervical vagus, 60,000 mm2/100mg Giant axon, 0.5

-

Most data are approximate only, and for rat, guinea pig or rabbit: for further details, including the parts referred to in line (iii) see text.

lation to the external area of tissue sample, and (ii) in relation to the area of capillaries which normally traverse the tissue sample. Data summarized in Table I involve the following considerations. (i) A tissue slice of 100 mg, prepared 0.35 mm in thickness from the surface of the brain, has an external area of about 600 mm2, of which 300 mm2 has been formed by cutting. In the cerebral cortex of the rabbit, SchadC and Baxter (1960) calculated the volume and surface area of neuronal components from microscopical measurements. The cell body, apical and basal dendrites with branches (but not including the axons leaving the cortex) gave total areas of 850 mm2/mm3 of tissue. At a specific gravity of about 1.04 (Thudichum, 1884), this corresponds to 82 600 mm2/100 mg. The measured portions of the neuronal surface thus presented an area over 270 times the total cut area. The elements which had been measured totalled only about 1 1 % of the volume of the cortex, and it is thus likely the newly-cut surface represents no more than 0.1 % of the cell-surfaces of the tissue. The cell bodies, it may be noted, formed only 4 % of the volume of neurones. Data consistent with this, but indicating the variation occurring in different cortical areas and in different species, are given by Scholl (1956), Tower (1954) and Heller and Elliott (1954). In a preponderantly non-myelinated mammalian nerve, the rabbit cervical vagus, Keynes and Ritchie’s (1965) data indicate that as used in experiments involved with ion movement, the specimens contained non-myelinated fibers, at a density of a similar order, namely one which totalled 60 000 mm2/mg. Comparison with giant axons as investigated by Hodgkin and Keynes (1955, 1956) indicates that axons 0.2-0.6 mm in diameter and 4 to 10 cm long have been used. Taking 0.5 mm diameter and 50 mm length as typical sizes, the area of the two ends of a cylinder of this magnitude is found to be 0.5 % of the area of the curved surface. (ii) The external area of tissue samples, prepared from the mammalian brain by sectioning at 0.35 mm intervals, approaches that of the blood capillaries which they carry. Estimates of capillary area can be made from the data of Cragie (1938); they are approximate only. In the rat brain the average capillary diameter was 2.9 p, and the average length of capillaries in the cerebral cortex was 1 100 mm/mm3. This gives

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the value of c 10 mm2/mm3 for capillary area per unit volume, which is included in Table I. For variation in different parts of the brain and other circumstances see Cragie (1938) and Horstmann (1960). Thus the external surfaces which must form the route of supply to tissue sections of the type described, are quite similar in area to that of the capillary walls which form the route of supply in vivo. This is understandable, for diffusion factors must condition the biological structure and are also involved in the calculations which lead to choice of the dimensions of the tissue samples. E L E C T R I C A L E VI D E N C E

In most neural systems the observations which required the supposition of rapidly adjustable ion-barriers, were first made electrically. Electrical observations with isolated cerebral tissues may therefore be described first, though they are of relatively recent development and come largely from our own laboratory. The phenomena observable depend greatly on the part of the brain which is sampled and maintained in vitro. The piriform lobe is the most versatile of these so far examined; its position in the rat or guinea pig brain is described by Allison (1953), Cajal (191 I , 1955) and Valverde, 1965). These data indicate that a sample taken superficially from the surface of the lobe, as a sheet 0.3 mm or more in thickness, can be expected to contain the fibres ofthe lateral olfactory tract, and also much ofthe neurons on which these fibres terminate. For in vitro experiments, we(Yamamoto and McIlwain 1966; Mcllwain 1966) have cut sheets 0.35 mm thick, for these can still be adequately oxygenated, and sufficiently large to include most of the surface of a piriform lobe of the guinea pig: that is, the portion medial to the rhinal fissure. These are about 7 x 14 mm, and 35 mg in weight. They have been immersed in bicarbonate-buffered balanced salt mixtures, with glucose and equilibrated with 0 2 - 5 %COz, in the slice chamber of Gibson and McIlwain (1965). After 20-30 min preincubation, incubating fluid has been withdrawn so that the tissue rested on a nylon grid, now at the surface of the liquid and with its upper surface in the moist gas phase. On this upper surface, formerly part of the ventral surface of the brain, the lateral olfactory tract could be seen and two stimulating electrodes were placed at its anterior end. Effects of applying to these electrodes brief, isolated pulses of rectangular time-voltage relationships were then examined by placing recording electrodes on the tissue. With extracellular recording electrodes on the lateral olfactory tract itself at distances of I to 10 mm from the stimulating electrodes, the first brief response to effective stimuli was biphasic and travelled along the tract at 12 m/sec (Yamamoto and Mcllwain, 1966). This was concluded to represent conduction along the myelinated fibres of the tract itself. A response of different characteristics, however, preponderated when the recording electrodes were moved away from the tract and placed on the surface of the pre-piriform cortex. This was a negative wave of much longer duration (10-20 msec), which was of maximum amplitude about 1.5 mm from the tract. The conducted response was lost at distances more than about 0.7 mm from the tract. Response to paired and repetitive stimuli, and also the effects of some inhibitory agents, suggested that the negative wave represented a post-synaptic potential. Ri~erc~ PJnp. ~ 280

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In several characteristics the responses now observed in vitro resembled those seen in the piriform cortex in vivo on stimulation of the lateral olfactory tract (MacLean et a/., 1957). The magnitude of respiratory responses suggested that stimulation at the lateral olfactory tract affected an appreciable proportion of tissue beyond the tract itself (McIlwain, 1966). Electrical activities such as those described normally imply appreciable resting potentials at the excitable cell membrane, detectable by intracellular electrodes. Ample evidence for this has been obtained in samples isolated from the mammalian brain. Observations made with micropipette electrodes showed (Li and McIlwain, 1957; Hillman and McIlwain, 1961; Gibson and Mcllwain, 1965) resting membrane potentials of about -60 mV in mammalian neocortical samples, relative to extracellular fluids. Moreover, the ionic basis for these potentials was adumbrated by observing their requirement for sodium and diminution on increasing the extracelM a r potassium concentration. Spike potentials were observed in vitro in such tissues, which involved a transitorily positive potential at the peak of the spike. It therefore appears likely that the sequence of changes in Na and K permeability, established in other neural systems, operates in the cerebral samples in vitro. Because of the opportunity which such systems afford for obtaining fundamental data, they have been explored by chemical methods now to be described. T A B L E I1 ION C O N C E N T R A T I O N A N D D I S T R I B U T I O N I N I N C U B A T E D C ER EBR A L N EOC OR TEX

Values based on: Constituent

Potassium: [Klr, mM Ratio [Kl{/[Klp Sodium: [Nali, m M Ratio [Na]i/[Na]p Chloride: [Clli, mM Ratio [Cl]i/[Cl]c

( a ) Passive distribution of CI-

( b ) lnirlin deterniitiation

136 22 41 0.3 1 14.5 0.108

131 21 53 0.35 33 0.25

Data from Gibson and Mcllwain (1965), for guinea pig cerebral cortex after incubation in media which contained (mM): NaCI, 124; KCI, 5; KHzPO.1, 1.24; MgSOJ, 1.3; CaCIz, 2.6; NaHCOz, 26 and glucose, 10 in equilibrium with 5 % COz in 0 2 . Suffix i, intracellular; e, extracellular. Values ( a ) based o n observed membrane potential, at which passive distribution of CI- gives Cle/ CIi of 9.3. Values (b) assume that all the extracellular space and none of the intracellular space is accessible to inulin. CHEMICAL ANALYSIS A N D ISOTOPIC MEASUREMENTS

Direct chemical analysis of samples of cerebral tissues which have been maintained satisfactorily in vitro, shows that among the major diffusible ions the tissues are enriched in K and are poorer in Na and CI than the fluids in which they are incubated.

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This gives an immediate demonstration of some of the most important barriers in cerebral tissues and also qualitative basis for some of the electrical phenomena just described. Quantitative statements need knowledge also of the amount of extracellular space in the tissue. In tissues as complex structurally as are those of the brain, calculation of the extracellular space involves simplifying assumptions, a matter discussed more fully in other sessions of this meeting. Ion gradients and resting potential Two different assumptions are used in alternative calculations of the data of Table I1 and these give consistent accounts of the distribution of Na and K ; that of C1 is less satisfactory. Among these ions the effects of K must preponderate in normal, unstimulated cerebral tissues, for the resting membrane potential is immediately affected by change in Ki and to a smaller extent and more slowly by change in Na or CI. However, the equilibrium potential corresponding to a K gradient of 21 or 22 (Table 11) is -83mV, markedly more negative than the observed-60mV. Presumably, therefore, the Na gradient is normally partly effective. With the relationship (i) derived from constant-field equations : (i)

v=-

RT

F

In

+ b “la, + c [CIle [Kli + b “ l a i f c [Clli

[Kle

(see Hodgkin and Katz, 1949) and for the present ignoring the C1 gradient, the K and Na values of Table I can be used to calculate a value for b. This factor gives the permeability of the tissue to Na as a ratio of its permeability to K+, and the value obtained is 0.06. Thus the resting tissue may be concluded to present a greater barrier to Na than to K . Values obtained for the squid axon, normal or perfused but also unstimulated were of 0.01-0.08 (Baker et al., 1962; Hodgkin, 1965), but on excitation the value can rise momentarily to 20, corresponding to the positive overshoot at the peak of the action potential. Following addition of KCI, the time-course of the diminution of membrane potentials in cerebral tissue was measured sufficiently accurately to merit comparison with calculated values (Gibson and McIlwain, 1965). Calculation was based on a diffusion equation and on equation (i). Quite close agreement was found when b was taken as 0.06, c as 0.5 and allowance made for diffusion of KCI in the available extracellular space. Net movements on stimulation Alterations in many metabolic properties of isolated cerebral tissues occur on electrical stimulation in vitro (see McIlwain, 1966; McIlwain and Rodnight, 1962). Included are increase in tissue Na and decrease in K. These changes which are in the direction of the concentration gradient for each ion show some simple relationships to characteristics of the applied electrical pulses (McIlwain and Joanny, 1963). The stimuli, ofcharacteristics similar to those which in vivo produce motor or other responRcyerences p. 280

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T A B L E 111 ION MOVEMENTS I N I N C U B A T E D C E R E B R A L N E O C O R T I C A L S A M P L E S

Movement

Tissue not stimulated 42K influx and efflux 24Nainflux and efflux Tissue maximally stimulated Net loss of K 42Kefflux Net entry of Na “Na influx

Rate

-~

( i ) Observed, pequiv./glh

( i i ) Computed pequiv.lcmz/h

330-400 175-21 5

45 21

240480 600-150 460 1050-1 I80

44 82 56 136

~-

Tissues were preincubated underconditionssimilar to thoseofTable I1 and reached stable composition before electrical stimuli were applied or isotopically-labelled salts were added; data from Cummins and McIlwain (1961), Keesey and Wallgren (1965) and Keesey et a / . (1965). A range of values in column (i) implies different conditions of measurement, and mean values have been used in calculating the data of column (ii). Column (ii) in addition uses the value of 82000 mm2 of neuronal surface1 100 mg of tissue, derived as explained in the text.

ses from the cerebral cortex, may be applied to electrodes which surround the tissue samples while they are immersed in media such as that quoted in Table 11. With stimuli of rectangular time-voltage relationship, the Na and K movements increased with duration of pulse between 0.03 and 0.4 msec; threshold corresponded to potential gradients of about l V/mm. Applied at frequencies between 2 and 30/sec, the stimuli resulted in initial changes of Na and K which were approximately equal and of between 5 and 6 mpequiv./g tissue/pulse (Table 111). A similar rate of K movement per pulse was subsequently observed (Keynes and Ritchie, 1965) for a mammalian non-myelinated peripheral nerve : the rabbit vagus. Movements of Na and K shown isotopically The applicability of equation (i) to data from isolated cerebral tissues implies that the tissues are permeable to the ions concerned, but does not indicate the magnitude of such permeability. Measurements using salts of 24Na or 42K show that an appreciable proportion of the tissue content of Na and K can undergo exchange each minute (Table 111). Entry of 24Na into the cellular compartments of incubated cerebral tissues was found readily separable from its movements into the extracellular phase (Keesey and Wallgren, 1965; Keesey et al., 1965). The greater part of the intracellular Na exchanged rapidly, at some 250 pequiv./g tissue/h; only in small proportion exchanged less freely. The electrical stimulation which altered the tissues’ net content of Na also altered greatly its rate of movement. The rate of turnover increased 4-6

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279

fold and became about 1100 pequiv./g tissue/h, implying an exchange of about one third of the intracellular Na each minute. I t will be noted that stimulation caused an immediate increase in Na efflux, even though the net movement of the ion was inwards; emphasizing that stimulation did indeed increase the permeability of the tissue to the ion, rather than prompting a unidirectional transfer. Comparable phenomena were found also in relation to potassium ions (Table 111; Cummings and McIlwain, 1961). In neocortical samples from the guinea pig, the normal flux of K+ was greater than that of Na+ and was increased by electrical stimulation. Again, stimulation increased both influx and exflux of 42K although the net result was loss of K. Applied pulses caused increased efflux of K from tissues maintained under a variety of conditions, including some conditions under which maximal gradients in K were not maintained. Thus in absence of added glucose, when stimuli did not increase tissue respiration or glycolysis, they nevertheless diminished tissue K. This gives support to the view that increase in ion permeability is a primary result of stimulation rather than its resulting from the major metabolic changes which usually accompany it. COMMENT

Electrical and ionic events of tissues from the mammalian brain are thus similar in several respects to events in simpler neural systems. Much can be ascribed to permeability barriers which show specificity to simple ions, and which can be caused to undergo equally specific modification by chemical and electrical means. The chemical nature of the cell membrane at the points at which it is penetrated by Na and K requires specification in cerebral as in other excitable tissues. In the more thoroughly-investigated systems, characterization of the groups interacting with Na and K still does not go far: the probable involvement of multiplycharged sites at some of which Ca may play a regulating role (Hodgkin, 1965). Certain actions of Ca in peripheral neural systems are reproduced in the brain and an examination of Ca in relation to excitation of cerebral tissues showed that it conditioned tissue Na and K content (Lolley, 1963; Lolley and McIlwain, 1964). Polybasic and polyacidic peptides and lipids also interact in conditioning tissue excitability and ion content (McIlwain et a/., 1961). A proposal relating ion passage to membrane potential postulated pores lined by molecules which carried acidic groupings on chains a few carbon atoms in length (Mcllwain, 1963). Others have related the sequence of Na and K movements to ion displacement at acidic groupings at the outer and inner cell surfaces (Blank, 1965); further chemical characterization may come from the susceptibility of cerebral ion movements to protoveratrine, to local anaesthetics and to tetrodotoxin (Wollenberger, 1955; McIlwain and Joanny, 1963; Hillman el al., 1963 and McIlwain, 1967). The manner in which the Na and K gradients normal to the brain have beenestablished, is deliberately not approached in the present account. The energy-consuming, pump mechanisms merit discussion at a meeting comparable to the present one for the utilization of energy-rich compounds which is necessary to active Na and K Rrfirences p . 280

280

H. M C I L W A I N

movements is giving valuable insight into the processes involved. I t is to be noted, however, that in the maintenance of many differential concentrations in neural systems the role of energy-assisted movements may preponderate over that played by barrier phenomena. REFERENCES ALLISON, A. C. (1953) Biol. Rev. (Cambridge), 28, 195. BAKER, P. F., HODGKIN, A. L. AND SHAW,T. I. (1962) J. Physiol., 164, 355. BLANK,M. (1965) J. Coll. Sci., 20, 933. CAJAL,S. R. Y (1911, 1955) Studies on the Cerebral Cortex, translated Kraft, L. M. Lloyd-Luke, London. E. H. (1938) Proc. Ass. Res. Nerv. Mental Dis., 18, 3. CRAGIE, CUMMINS, J. T. AND MCILWAIN, H. (1961) Biochem. J., 79, 330. GIBSON,I. M. A N D MCILWAIN, H. (1965) J . Physiol., 176, 261. HELLER, I. H. AND ELLIOTT,K. A. C. (1954) Canad. J . Biochem. Physiol., 32, 584. HILLMAN, H. H., CAMPBELL, W. J. AND MCILWAIN,H. (1963) J. Neurocheni., 10, 325. HILLMAN, H. H. AND MCILWAIN, H. (1961) J. Physiol., 157, 263. HODGKIN, A. L. (1965) The conduction of the nervous impulse. University Press, Liverpool. HODGKIN, A. L. AND KATZ,B. (1949) J . Physiol., 108, 37. HODGKIN, A. L. AND KEYNES, R. D. (1955) J . Physiol., 128,28. - (1956) J. Physiol., 131, 592. HORSTMANN, E. (1960) in: Structure and function of the cerebral cortex. Tower and Schadk (Eds.). Elsevier, Amsterdam (p. 59). KEESEY, 5. C. AND WALLGREN, H. (1965) Biochem. J., 95, 301. KEESEY, J. C., WALLGREN, H. AND MCTLWAIN, H. (1965) Biocheni. J., 95, 289. KEYNES, R. D. AND RITCHIE, J. M. (1965) J . Physiol., 179, 333. LI, C. L. A N D MCILWAIN, H. (1957) J. Physiol., 139, 178. LOLLEY, R. N. (1963) J. Neurochem., 10, 665. LOLLEY, R. N. AND MCILWAIN, H. (1964) Biochem. J., 93, 12P. MCILWAIN, H. (1956) Physiol. Rev., 36, 355. - (1966) J. Physiol., 185, 65P. - (1966) Biochemistry and the central nervous system. 3rd ed. Churchill, London. - (1967), J. Physiol., 190, 39 P . MCILWAIN, H. AND RODNIGHT,R. (I962) Practical Neurochemistry, Churchill, London. MCILWAIN, H. AND JOANNY, P. (1963) J. Neurochem., 10, 313. R. J. AND CUMMINS, J. T. (1961) Biochem. J., 81, 79. MCILWAIN, H., WOODMAN, MACLEAN, P. D., ROSNER, B. S. AND ROBINSON, F. (1957) Amer. J. Physiol., 189, 395. SCHADE,J. P. AND BAXTER, C. F. (1960) Inhibition of the Nervous System and y-Aniinohutyric Acid, E. Roberts et a/. (Eds.). Pergamon, New York. SHOLL,D. A. (1956) Progress in Neurobiology, 1, 324. J. A. Kappers, Editor. Elsevier, Amsterdam. THUDICHUM, J. L. W. (1884) A Treatise in the chemical constitution o j t h e Brain. Bailliere, Tindall & Cox, London. TOWER,D. B. (1954) J. Comp. Neurol., 101, 19. VALVERDE, F. (1965) Studies 011 the Piriform Lobe. Harvard University Press, Mass. YAMAMOTO, C. AND MCILWAIN, H. (1966) Nature, 120, 1055. WOLLENBERGER, A. (1955) Biochem. J . 61 68.

I O N M O V E M E N T IN M A M M A L I A N B R A I N

28 1

DISCUSSION D. B. TOWER:I think all of us owe Dr. Mcllwain a debt of gratitude. What you saw today represents many years of work, and much thought in devising these very ingenious experiments. For those of us who work with incubated slices it is gratifying to see how close one really comes in vitro to the situation in vivo. I t is of tremendous value to be able to do a number of observations in v i m which one could not do otherwise with confidence. Obviously the best preparation is the living brain in situ, but this presents many technical problems in studying the questions which we are considering hcre. I f you can work at them in isolation, and vary one parameter at a time as Dr. Mcllwain has indicated, you can really gain a great deal of understanding. With that little complement I would like to ask one question now, and that is about the chloride: We have done some calculations similar to yours about the potentials that one would expect, based on passive distribution of chloride in comparison to caiculations with the Hodgkin-Huxley type of equation. There are some peculiar abnormalities with chloride as you suggested in one slide, especially when one deals with a medium in which you alter some of the ionic constituents. There seems to be a disparity between the resting potentials which one would expect for a distribution of chloride on a passive electrochemical basis. 1 wonder if you have thought about this enough to give us some ideas, because frankly, I am puzzled as to how to explain some of the apparent behavior that chloride shows in these in vitro preparations. H. MCILWAIN: We hope to make a more detailed study of chloride, but we have not yet done so. The problems posed by chloride are being examined in several systems. Those who are studying muscle, for example, have shown somewhat similar features in potassium and chloride distribution. It is still debated whether there is an active distribution of chloride or a passive one. Because of tte cellular complexity of cerebral tissues, I hesitate to draw a conclusion from our data. K. A. C. ELLIOTT: I really wanted to say more or less the same thing as Dr. Tower. I am very impressed with this work. and the stage in this work which Dr. Mcllwain has reached. I feed that over the years the electrophysiologists have tended to dismiss Dr. Mcllwain’s approach, perhaps because it was done by a biochemist, or perhaps because it was done on a tissue slice. I think that the sort of work he is doing now is going to bring us to the highly desirable stage of marrying electrophysiology to biochemistry. We will reach the stage where the writing of the recording pen will not just be. telling us about an electrical phenomenon which means nothing; it will be an index of a chemical event, which is what we want. Dr. Mcllwain has already led to this with a lot of hisearlier studies. R. KATZMAN: One of the problems that has arisen in understanding the electrolytes in the central nervous system has been the very large amounts of sodium in brain, as compared to other tissues. I t has been postulated that this has been extracellular or in the glia, but your evidence would indicate that this is intraneuronal. If the sodium is this high within the neurons, this would indicate that the action potential ought to have a very small overshoot. Have you in fact measured the action potential, and does it have a small overshoot? H. MC~LWAIN: The overshoots were small in our preparations. Our preparations contain a higher concentration of sodium ions than in vivo, even though we have looked very carefully a t the medium constituents and so forth, in order to analyze this. It is fairly certain t o be due in part to sectioning of the tissue, but it is often overlooked that the brain in vivo has some 20-25% of extracellular space. R. KATZMAN: In other words; you feel that this may not necessarily reflect what is going on in vivo in terms of the sodium concentration. May this be in part due to the incubation situation then? H. M C ~ L W A IINthink : we do get a high sodium content, and this is due not so much to the particular fluid that we useirr vitro, but is contributed by the obvious fact that the tissue has been sectioned, and we actually gain sodium beyond that found in normal preparations in vivo.

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283

Cation Exchange in Blood, Brain and CSF ROBERT KATZMAN, L E O N A R D G R A Z I A N I

AND

STANLEY GINSBURG

Saul R . Korey Departnierit of Neiirology, Albert Einstein College of Medicine, Bronx, N . Y. (U.S.A.)

In the presence of marked changes in serum and body electrolytes the constancy of brain and CSF cations, K, Ca, and Mg, has been repeatedly confirmed (Bradbury et al., 1963; Katzman, 1966; Kemeny et al., 1961; Leiderman and Katzman, 1953; MacIntyre and Davidson, 1958; Oppelt et al., 1963a,b; Schain, 1964; Wallach et al., 1964). In the case of these cations, one factor in maintenance of the brain level may be the slowness of exchange across the blood-brain barrier. Thus, injected 42K, which exchanges with most body tissues within minutes, was shown by Katzman and Leiderman (1953) to exchange very slowly with brain K. Twenty-four to 36 h were required for half of the brain K to exchange, the rate of 42K influx being about 3 mequiv./kg/h in a brain containing 100 mequiv./kg of total K (Table I). Adrenalectomy which alters body and muscle electrolytes and causes a rise in serum electrolytes of over 50% did not alter the influx rate or total K. However, it is well established that CSF K is maintained independently of serum K. The value for CSF K of 3 mM is remarkably constant among species of mammals and some invertebrates extending back to sharks (Rall and Cserr, unpublished observations), even though the serum concentrations of the varying species are quite different. Such constancy of CSF K would certainly have survival value, if this represented the extracellular fluid K, for then cell membranes would have a rather constant environment biasing their membrane potentials. In searching for the mechanisms underlying this constancy of CSF K, early experiments were carried out in which the specific activity of the CSF was compared to that of plasma after intravenous and intracisternal injections (Table 11). It is apparent that equilibrium between CSF and plasma is reached much more rapidly than between brain and plasma. Moreover, much of the intracisternal injection is carried rapidly from the CSF, appearing both in plasma and in adjacent brain, particularly the medulla. With the development of ventriculocisternal steady state perfusions the movement of isotopes between plasma, CSF, and brain can be more accurately analyzed and the effects upon such movement of various factors such as serum concentration, inhibitors, etc. studied. Although ventriculocisternal perfusions have been utilized for many years particularly by Leusen (1950) and by Bhattacharya and Feldberg (l958), the application of this method to the measurement of ion flux has been developed largely by Pappenheimer and his associates (1961, 1962), by Heisey (1962), and by Davson ReJiwncrs pp. 293-294

284

R. K A T Z M A N et

al.

and Pollay (1963). Pappenheimer el al. (1961) introduced the use of inulin dilution as a measure of the rate of formation of CSF occurring during the ventriculocisternal perfusion. During a steady state ventriculocisternal perfusion at the inflow rate, Vt, ml/min, assuming that inulin does not diffuse across ependyma, glia, or arachnoid, then Vf, the newly formed CSF can be measured by inulin dilution

v,

=

vs

(Ct - CO)

ml/min

/.(

L O

(Heisey et al., 1962) where Ct is inflow concentration of inulin, and Cois outflow concentration of inulin. The rates of formation of CSF measured by this method varies from 0.0096 in the rabbit (Davson and Pollay, 1963) to 0.37 in man (Rubin et al., 1966). Recently, TABLE I* EFFECT O F ADRENALECTOMY O N BRAIN

K

AND

K - F L U XI N

Kbr

No.

Normal Adrenalectomy

* From Leiderman and Katzman,

Influx

mequiv./L

mequiv./kg we, weigh, nieqctiv./kg/h

5.48 f 0.1 7.60 f 0.2

100.9 f 0.9 98.7 f 1.2

Kpl

30 18

RATS

2.89 2.97

1953.

Ames et al. (1965a) have demonstrated that the rate of CSF produced is related to the pCO2 of the blood. We have, indeed, found in a series of cats perfused during nembutal anesthesia without artificial respirations, that the Vf of 0.022 was almost 20% greater than the Vf of 0.016 observed in anesthetized animals of the same size in whom artificial respiration had been carried out and in whom hyperventilation had probably occurred (Graziani, in preparation). The second use of inulin data has been the determination of inulin clearance as a TABLE 11* SPECIFIC ACTIVITY O F

CSF

A N D BRAIN AFTER I N T R A C I S T E R N A L A N D I N T R A V E N O U S I N J E C T I O N SO F

Intravenous Intracisternal

2h 10 min 60 rnin 100 rnin

42K I N

RATS

S A CSFISA plasma

SA brainlSA plasma

0.63 30,000 85

0.085

* From Katzman and Leiderman, 1953.

S A medullalSA C S F

0.03 0.97

C A T I O N E X C H A N G E IN B L O O D , B R A I N A N D

CSF

285

measure of perfusate absorbed or diverted into the subarachnoid space, Va. During a ventriculocisternal perfusion, some perfusate may not be recovered through the outflow catheter but instead may be diverted into the subarachnoid spaces and later absorbed at the arachnoid villi. Va must be known in order to carry out further calculations; it may be determined easily, since it is identical to inulin clearance. Assuming, inulin is removed from cerebrospinal fluid spaces as part of bulk absorption of CSF at Va ml/min, and steady state perfusion at inflow Vt ml/min, outflow Vo ml/min then

v,

vr Cr - v o co =

ml/min

CO (Heisey et al., 1962) With the measurement of Va, it now becomes possible to study ion flux by adding isotope either to the ventricular perfusate or giving the isotope intravenously. In the succeeding paragraphs, the equations used in the study of this ion flux are explicitly presented. Although several of the equations may appear to be trivial, their inclusion has become necessary in order to show step by step how coefficients derived from isotopic data can be used to measure ion flux per se. This is important in view of the criticisms of isotopic data recently expressed by authors such as Nims (1966).

Eflux coeficient With the addition of an isotope to the simulated spinal fluid, the efflux of this isotope into brain and plasma can be expressed as a clearance coefficient as shown by Cserr (1964, 1965) and Katzman et a/. (1965). Thus, for an isotope C* the efflux equation becomes k e f f l u x C* = (kvp kVBr) C* = Vi Ci* - Vo Co* - Va Co* (1) where kcfPlux is the ml of perfusate cleared of C*/min; is the average concentration of C* in the ventricle (taken arbitrarily as either the simple arithmetic average of Ct* and Co* or as the exponential mean); Vi is the perfusion rate in ml/min; V, is the cisternal effluent i n ml/min, and Va is the measure of the perfusate absorbed elsewhere in the system as determined by the inulin clearance. This efflux coefficient can be separated into components kvp and kvBr representing the movement into plasma and brain, respectively.

+

c*

Transport coeficient, plasma to ventricle

Provided the efflux coefficient has already been determined, the movement of an isotope from plasma to ventricle can be measured as a clearance coefficient of the plasma. During steady state ventriculocisternal perfusion with non-radioactive perfusate, if C* is perfused intravenously and assuming that brain uptake of C* from plasma is negligible then the following material balance holds R+rences

p p . 293-294

R. K A T Z M A N et al.

286

+

+

k p v C*P = Vo C*o Va C*o k e f t l u x C* (2) where k p v is the transport coefficient from plasma to ventricle, or clearance of c* from plasma in ml/min.

Isotopic j u x e s

The model we are dealing with is a 3 compartment system in which a constant flow is maintained through the middle compartment, the CSF.

In this system, we ignore the exchange that must occur between plasma and brain. At steady state the system is described by 6 unidirectional isotope fluxes, J * , J*r J*PV J*BV = J * o J*VP J*VB -(3) where J * t = Vr C*r, J*o = ( V o Va) C*o,the sum of J * V P J*VB = kefrlux c*, J*PV = kruriux C*P. An approximation which permits a separate calculation of JVP and JVB is obtained by determining the ratio of counts remaining in the brain and the counts lost to the plasma during a recovery experiment following isotopic perfusion. To determine JEW*, we use the fact that the brain is not appreciably labeled during the influx measurements. If our perfusate is initially free of both isotope and parent species, then the non-radioactive isotope which appears in the effluent can be treated as if it were a tracer coming from the brain. Here,

+

J*BV = J*PV

+

+

+

)1

+

+

(S.A.) effluent

S.A. effluent

(S.A.) plasma

S.A. plasma

(1 -

(4)

where S.A. is the specific activity in counts/min/moles, (C*/C). All of the isotopic fluxes are now calculated. Flux of parent species The unidirectional fluxes between compartments I and 2 ofparent species in moles/min may be determined simply by utilizing the general expression that J12 = J * i 2 / ( S . A . ) i

(5)

where J is the flux in moles/min, and S.A. is the specific activity in counts/min/mole. We may then rewrite equation (3) Jr JPV J B ~ V= J o JVP JVB (6) Among the simplifying assumptions in this analysis are the following: I . During the time course of the experiments inulin does not diffuse appreciably into the brain or blood vessels.

+ +

+ +

CATION EXCHANGE IN BLOOD, B R A I N A N D

CSF

287

2. That the rate of exchange of isotope between plasma and brain is negligible in the time course of the experiment. This is probably a reasonable assumption for K and Mg, less so for Ca, and not valid for Na. 3. The interfaces between CSF and brain and CSF and plasma are each treated as homogeneous entities even though it is evident they include ependymal surfaces, pia-glial surfaces, etc. We have applied this analysis to the study of the exchange of K (Katzman et a/., 1965), Mg (Ginsburg and Katzman, in preparation), and Ca (Graziani et a/., 1967) in the anesthetized cat. Similar studies for K have been carried out by Cserr (1965) and Bradbury and Davson (1965) with comparable results. In determining the fluxes, the clearance of isotope from the perfused fluid, kcflux, must be measured first, since this value is needed for the later calculations. The values obtained for clearance of 42K, ZRMg, and 45Ca during ventriculocisternal perfusion T A B L E 111 C A T I O N F L U X FROM P E R F U S A T E T O P L A S M A A N D B R A I N

No. of animals

K" Ca" MgC

7 9 4

Rate of perJision. Vi, nil/niin

0.19 0.19 0.09

coejicient

kernux

0.096

0.025 0.032

90-riiin Perfusion Counts % Counts recovered 0;

I0

in brain

21.9 4.8 8.0

kr1.

k\.Hr*

*

plasma'

15.3 8.6 15.3

0.041 0.016 0.021

0.055 0.009 0.011

* Estimated as difference between total counts perfused and counts recovered in cisternal effluent, brain, and accounted for by Va. * * kvl%ris underestimated here, since re-entry of isotope into perfusate from adjacent brain tissue has not been taken into account. a Katzman et a/., 1965. Graziani et a/., 1965. Ginsburg and Katzman, 1966. in the cat are shown in Table 111. It is evident that the clearance of 42K is much greater than that of 4SCaand 28Mg, which are similar to each other. The value for the clearance of 42K, 0.096 ml/min, is 4 to 5 times the usual rate of formation of spinal fluid in the ventriculocisternal system of the cat. At the end of a perfusion experiment a portion of the isotope cleared can be found in the brain, the rest having entered the circulation. The distribution of the cleared isotope between brain and plasma is estimated by carrying out 90-min perfusions. The animal is then sacrificed, and the total percentage of perfused material recovered in the effluent and accounted for by Va and the per cent recovered in the brain are determined and then the amount entering the plasma is estimated by difference. Here again, a different pattern for K emerges where a greater proportion enters the brain, than for Ca and Mg, where the greatest proportion is lost to the plasma (Table 111). The pattern of movement of the isotope into brain tissue is shown in Fig. 1 for 42K. Rrfercnccs pp. 293-294

288

R. K A T Z M A N et al.

Fig. I . Distribution of isotope in cat brain following 90-min ventriculocisternal perfusion with perfusate containing 42K. The numbers represent the per cent of total counts in entire perfusate received from each region of the brain.

As was expected, the highest concentration is immediately adjacent to the ventricles,

but it should be also noted that there is very considerable recovery of the material from the base of the brain stem. It would appear that the isotope can move equally well across the ependymal surface into the brain stem and across the subarachnoid, pia-glial surface. This is a useful situation ir. which to compare the movement across these two surfaces, since the brain stem is more or less cylindrical, and the surface areas of the ventricle and of the base are at least of the same order of magnitude. With the determination of the efflux coefficient, the rate of the unidirectional flux can be calculated. However, this in itself gives no information about the mechanism of transport. Information can be obtained about mechanisms by determining the unidirectional fluxes or efflux coefficients while parameters such as cation concentration are varied. One can also use such measurements to determine the effect of inhibitory agents such as ouabain. The effects of variations in K, Mg, and Ca concentrations of the perfusate upon clearance of the respective isotopes have been previously reported (Katzman et a / . , 1965; Ginsburg et al., in prep.; Graziani et al., 1965) and are summarized in Table 1V. Ineach instance, theclearancecoefficient is independent of the mean CSFconcentration of cation. Hence, the flux of the cation from the CSF is directly proportional to this concentration. This is consistent with but does not establish the existence of a simple diffusion process. Once the efflux coefficient has been determined, the influx coefficient, kpv, can be obtained by utilizing equation (2). From this value and the serum concentration, the influx JPVcan be calculated. Values of Jpv obtained for K, Ca, and Mg are shown in Table V. By determining the specific activity of the effluent and assuming little labeling of the brain during the time course of the perfusions, the influx JBV can also

C A T I O N E X C H A N G E I N BLOOD, B R A I N A N D

CSF

289

TABLE IV

K, Ca,

I N D E P E N D E N C E O F C L E A R A N C E C O E F F I C I E N T S , kernuS, A N D C O N C E N T R A T I O N S O F AND

0- I

3- 4 9-10

0.91 0.95 0.89

Mg

IN PERFUSATE

0 -0.08 1.2-1.7 1.8-2.5

0.026 0.021 0.024

0 4.04 0.08-1.2 2.1 -2.5

0.049 0.038 0.047

be calculated. Again, the flux of K is much greater than that of Ca and Mg. The flux of K from brain to ventricle is greater than from plasma to ventricle, however, the reverse is true for Ca and Mg. The effect of alterations in plasma concentrations of the cations upon kpv and upon the flux from plasma to ventricle is very different from that of the effect of alterations of CSF concentrations upon keeflux and flux from ventricle to plasma and brain. In studies of Mg exchange, our initial data indicate that the flux from plasma to ventricle is independent of plasma cation concentration; under the circumstances, kpV is inversely proportional to plasma concentration. I n studies of Ca exchange, the influx coefficient has been found to be a hyperbolic function of the plasma concentration (Graziani et al., 1966). Flux from plasma to TABLE V U N I D I R E C T I O N A L F L U X OF C A T I O N I N P E R F U S E D C A T

JPV pM/niin J I N pM/rnin Jv I’ J v 15

0.121 0.178 0.039 _K 0.057 K

0.026 0.013 0.021 Mg 0.01I M g

0.026 0.015 0.016 0.009 Ca

The units of the coefficients are nil/min. K, etc. average concentration of K in ventricle in pM/ml.

ventricle could be resolved into a major component that was concentration independent and presumably represented movement via a carrier-mediated or active transport and another component which was proportional to serum concentration and represented a concentration-dependent, presumably diffusional, component. The equation found was: 0.0037 Cap1 JVP = k v p Cap1 = 0.0156 with 0.0156 pMlmin representing the carrier-mediated; 0.0037 (ml/min) Cap1 (,uM/ml) representing the diffusional transport. We have not carried out similar experiments with K, but the data of Ames et al. (1965b) on K content of the choroid plexus fluid as a reflection of seru I K concen-

+

Rtfi.ri*nc.cspp. 293-294

-

R. K A T Z M A N et al.

290

tration and the data of Cserr as reported in her thesis (1964) both indicate that the movement of K from plasma to ventricle is similar to Ca; largely a concentration independent flux with the addition of a small diffusional component. Thus, the evidence from these studies indicates an asymmetrical type of transport system in which the movement out of CSF acts as if it were diffusional, whereas the movement into CSF appears to be largely carrier-mediated. The interpretation of these findings in the case of K is made more difficult because of the known effect of K on the electrical gradient. It has been shown by Held et a/. (1964) that alterations of K concentration within the CSF alters the electrical potential between the ventricle and venous system. If this is so, the efflux of K should not depend solely upon the internal concentration of K, as it does, but should also be modified by the changing electrical gradient. The asymmetry of the exchange process cannot be understood in terms of a single membrane between blood and ventricle. It may be interpreted to indicate at least two membranes in series with different functional capacities. Thus, the efflux from CSF is across a membrane which acts as a diffusional barrier and is not the site of the electrical potential difference. However, the movement from plasma into ventricle is via some specialized nondiffusional process such as carrier mediation or active transport. The effects of inhibitors upon the system are in the process of being studied. When added in usual concentrations to the CSF some of the ordinary inhibitors such as dinitrophenol produce little effect upon ion transport, but perhaps this may be due to their inability to penetrate into essential regions. So far no attempts have been made to correlate simultaneously the effects of inhibitors upon ion transport and upon other functional parameters such as oxygen utilization by the ependymal or other cells. Most striking effects are produced by the introduction of ouabain in concentrations of 10-4 to lo-’ M. Here, there is a marked alteration in K exchange. The K concentration in the cisternal effluent becomes elevated. This elevation in effluent K is attributable both to a decrease in efflux of K from the CSF and an increase in movement of K from brain into CSF. Here, ouabain may exercise an effect upon cellular permeability as well as upon the active transport process. Acetazolamide reduces formation of spinal fluid by approximately one-half, but it has little effect upon the influx of K from plasma to ventricle. The effect of ouabain upon the movement of Ca and Mg is currently under study. In carrying out these calculations of isotope exchange the question may arise as to whether the values obtained describe transport of cation as such. This can be readily checked in the ventriculocisternal perfusion experiments, since the coefficients derived from isotopic studies can be applied to the prediction of the total concentration of cation, when the ventriculocisternal system is perfused with perfusates containing varying initial concentrations. In this computation one applies equation (7) substituting the flux coefficients as appropriate; Jt

+

JPV

4-J B ~ V= Jo f JVP

+

JVBr

C A T I O N E X C H A N G E IN B L O O D , B R A I N A N D

co =

CSF

29 1

Vi Ci iJ r v 1- J J ~~ vkeffit1.u Cip

Vi

+ Vf +

(7) krffiux/z

The values to be used in the numerical solution of this equation are those in Table V. In Fig. 2, the relationship of C, to Ci has been plotted for K, Mg, and Ca using this equation. In each instance, the actual measured values are also shown. It can be seen that there is an excellent fit of the equations to the measured values of Co in each instance. In addition, in the case of ouabain a good fit is also obtained. This would suggest that although ouabain alters both the influx K and the clearance coefficient, that the me-hanism in each case is not basically altered; that is, that efflux is still via diffusion, although at a different rate, and influx via a concentration independent system. The exchange of cations occurred throughout the entire ventricular subarachnoid space. In experiments in which the exchange coefficients had been determined in perfusions between lateral ventricle and aqueduct, lateral ventricle and fourth ventricle, and lateral ventricle and cisteriia magna, the value of the coefficients increases proportionately as the area of circuits exposed to the perfusate increases. This is shown in Table VI. Subarachnoid perfusions in which the area being perfused is difficult to estimate but which has been carried out between the parasagittal space and the cisterna TABLE VI C O M P A R I S O N OF E X C H A N G E C O E F F I C I E N T S I N R E G I O N A L P E R F U S I O N S

Ventriculocisternal Lateral 3rd 4th ventricle (kaolin prepdrdtion) Lateral 3rd ventricle (aqueduct) Ccrebrdl subarachnoid Cisternal-lumbar

0.092

0.026

0.011

0.041

0.022 0.002

0.008 0.0034 0.034 0.012

0.33 0.29

0.06

magna yield clearance coefficients that are several-fold greater than that of the ventriculocisternal perfusicn. A more reproducible system is that of cisternal lumbar sac perfusion in which appreciable exchange is shown along the subarachnoid space enclosing the spinal cord. Again, this is several-fold larger than that seen in the ventriculocisternal region, but the circuit’s area is considerably greater. In the cases of studies of Ca and Mg exchange, the flux between the subarachnoid ventricular space and brain is less than that between CSF and blood. In the case of K, however, there seems to be a very vigorous exchange between brain and CSF, and a slower exchange between brain and blood. This has raised the question as to whether the K i n the extracellular space of the brain may be in equilibrium with CSF K. If such an equilibrium existed, it would certainly be of considerable importance for R r J ; ~ r c n w spp.

293-294

al.

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Kout

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Fig. 2. Relationship of cation concentration in cisternal effluentto cation concentration in ventricular perfusate. The calculated steady state curves derived from equation (7) utilized the numerical data in Table V.

C A T I O N E X C H A N G E I N BLOOD, B R A I N A N D

CSF

293

CSF K is maintained at the level of 3 mM in all mammalian and many vertebrate species, whereas the blood K may be 50% or more above this. In addition, the blood level is quite variable within different animals of the same species as well as in the same animal from time to time. The CSF level varies only between 2.6 and 3.2 mM. A low extracellular K maintained at a constant level could provide a bias for the resting membrane potential which would lead to a large K equilibrium potential E K and which would, therefore, provide a reasonable membrane potential, even if the membranes were somewhat leaky. It should be noted, however, that the hypothesis that an equilibrium does exist between brain extracellular K and CSF K, while consistent with all known facts, has not been directly determined at the present time. SUMMARY

It has been our purpose to set down in a formal way the simple equations describing isotopic flux during ventriculocisternal perfusion experiments and to relate these equations to the measurement of flux of the parent cation. The effect of variation in cation concentration upon cation flux has been measured. The present evidence suggests that K, Ca, and Mg are cleared from the ventriculocisternal system by a concentration dependent system consistent with simple diffusion. In contrast, the flux from plasma to ventricle is largely concentration independent, presumably involving a carrier-mediated transport system. This asymmetry indicates that the blood-CSF barrier is not a single membrane system. Instead, there may be membranes with different properties which are in series. Alternatively, influx and efflux of cations may occur at separate sites. ACKNOWLEDGEMENT

This work was supported by National Institute of Health grants NB-03356, NB01450, and 5T1 MH-6418, U.S. Public Health Service. REFERENCES

AMES,A., 111, HIGHASHI, K. AND NESBETT, F. B. (1965a) Effect of pC02 acetazolamide and ouabain on volume and composition of choroid plexus fluid. J . Physiol., 181, 516-524. - (1965b) Relation of potassium concentration in choroid plexus fluid to that in plasma. J. Physiol., 181, 506-515. BHATTACHARYA, B. K. AND FELDBERG, W. (1958) Perfusion of Cerebral Ventricles: Effects of drugs on outflow from the cisterna and aqueduct. Brit. J. Pharniacol., 13, 156-162. BRADBURY, M. W. B. AND DAVSON, H. (1965) The transport of potassium between blood, cerebrospinal fluid and brain. J. Physiol., 181, 151-174. BRADBURY, M. W. B., STUBBS, J., HUGHES, I. E. AND PARKER, P. (1963) The distribution of potassium, sodium, chloride and urea between lumbar cerebrospinal fluid and blood serum in human subjects. Clin. Sci., 25, 97-105. CSERR,H. F. (I 964) Excharige of Substances Between Ventricular Fluid, Plasma and Brain with Special Reference to Potassiuni. (Doctoral Thesis), Boston, Harvard University. -( 1965) Potassium exchange between cerebrospinal fluid, plasma and brain. Amer. J. Physiol., 209, 1219-1226. DAVSON, H. AND POLLAY, M. (1963) Influence of various drugs on the transport of and PAH across the cerebrospinal fluid-blood barrier. J . Physiol., 167, 239-246,

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al.

GINSBURG, S. AND KATZMAN, R. (1966) Mg exchange between blood, brain and CSF in cat. In preparation. GRAZIANI, L., ESCRIVA, A. AND KATZMAN, R. (1965) Exchange of calcium between blood brain, and cerebrospinal fluid. Anier. J. Physiol., 208, 1058-1064. GRAZIANI, L. J., KAPLAN, R. K., ESCRIVA, A. A N D KATZMAN, R. (1967) Calcium flux into CSF from blood, brain, and spinal cord. Amer. J. Physiol. (in press). HEisEY, S. R., HELD,D. AND PAPPENHEIMER, J. R. (1962) Bulk flow and diffusion in the cerebrospinal fluid system of the goat. Amer. J. Physiol., 203, 775-78 I . HELD,D., FENCL,V. A N D PAPPENHEIMER, J. R. (1964) Electrical potential of cerebrospinal fluid. J. Neitrophysiol., 27, 942-959. KATZMAN, R. (1966) Effect of electrolyte disturbance on the central nervous system. Ann. Rev. Me& 17, 197-212. KATZMAN, R.,GRAZIANI, L., KAPLAN, R. AND ESCRIVA, A. (1965) Exchange of cerebrospinal fluid potassium with blood and brain. Arch. Neurol., 13, 513-524. KATZMAN, R. AND LEIDERMAN, P. H. (1953) Brain potassium exchange in normal adult and immature rats. Amer. J. Physiol., 175, 263-270. KEMENY, A., BOLDIZSAR, H. AND PETHES, G . (1961) The distribution of cations in plasma and cerebrospinal fluid following infusion of solutions of salts of sodium, potassium, magnesium, and calcium. J. Neurocliem., 7, 218-227. LEIDERMAN, P. H. AND KATZMAN, R. (1953) Effect of adrenalectomy, desoxycorticosterone and cortisone on brain potassium exchange. Amer. J. Physiol., 175, 271-275. LEUSEN, 1. (1950) The influence of calcium, potassium, and magnesium ions in cerebrospinal fluid on vasomotor system. J. Physiol., 110, 313-329. MACINTYRE, 1. AND DAVIDSSON, D. (1958) The production of secondary potassium depletion, sodium retention, nephrocalcinosis and hypercalcaemia by magnesium deficiency. Biochem. J. 70,456-462 NIMS,L. F. (1966) Biologic barriers and material transfer. In: Head Injury, W. F. Caveness and A. E. Walker (Eds.), London, Lippencott. OPPELT, W. W., MACINTYRE, 1. AND RALL,D. P. (1963a) Magnesium exchange between blood and cerebrospinal fluid. Anier. J. Physiol., 205, 959-962. OPPELT, W. W., OWENS, E. D. A N D RALL,D. P. (1963b) Calcium exchange between blood and cerebrospinal fluid. Lqe Sci., 2, 599-605. PAPPENHEIMER, 5. R., HEISEY,S. R. AND JORDAN, E. F. (1961) Active transport of diodrast and phenolsulfonphthalein from cerebrospinal fluid to blood. Amer. J. Physiol., 200, 1-10, PAPPENHEIMER, J. R., HEISEY, S. R., JORDAN, E. F. AND DOWNER, J. (1962) Perfusion of the cerebral ventricular system in unanest hetized goats. Amer. J. Physiol., 203, 763-774. RUBIN, R. C., HENDERSON, E. S., OMMAYA, A. K., WALKER, M. D. AND RALL,D. P. (1966) The production of cerebrospinal fluid in man and its modification by acetazolamide. J. Neurosurg., 25, 430436. SCHAIN, R. J. (1964) Cerebrospinal fluid and serum cation levels. Arch. Neurol., 11, 330-333. WALLACH, S., BELLAVIA, J. V., SCHORR, J. AND REIZENSTEIN, D. L. (1964) Tissue distribution of electrolytes Ca47 and Mg** in acute hypercalcemia. Amer. J. Physiol., 207, 553-560.

CATION EXCHANGE IN BLOOD, B R A IN A N D

CSF

295

DISCUSSION

D. B. TOWER:I think it is only fair since I rose to congratulate Dr. MCILWAIN that I d o the same for Dr. Katzman. You have been in this particular field for a long time, and I think we owe quite a debt to you, and also to some of the later workers like Held, Cserr, and Pappenheimer who have contributed considerably to the techniques, but perhaps have not been interested in the nervous system. There is one point I would like to comment on, that Dr. Katzman passed over rather quickly, and that is the effect of ouabain. For many years people have been using ouabain in a sort of in vitro system. Not so very long ago we had a conference at the N.T.H., and the main question was: what does it do in vivo? Nobody seemed t o know. Shortly after that there came a publication from Dr. Katzman and I think almost simultaneously one from Dr. Cserr at Harvard, in which they gave an answer to our question. As you saw today, it does have an effect which is - at least qualitatively quite similar to what occurs in vitro. Also, if you give too much for too long a period of time, seizures occur, which is also quite interesting. L. BAKAY: I just want to add one thing: I think this is a most ingenious way to use perfusions for the calculations that were obviously impossible to obtain by injecting tracers into a system with a semi-stagnant cerebrospinal fluid. R. KATZMAN: I would like to point out that even though this kind of analysis leads to equations that do fit the data quite well for points which were not originally included in arriving at the coefficients, that in fact it is not really a pure system because of the existence of heterogeneous interphases. Within the brain we do not have compartmental mixing, but there is mixing in CSF and blood which is much better. L. BAKAY:The main disadvantage of injecting tracers directly into the cerebrospinal fluid system is their unequal distribution in that compartment. There is superficially a much higher activity than in the deeper structures, and the isotopes do not mix very well within a reasonable time along the entire nervous system. unless you use barbotage. By barbotage it can be mixed. However, you don't know quite how much artefact you have induced, because by barbotage you will necessarily reduce the pressure to almost zero when you withdraw the fluid, and involuntarily increase it when you inject it back. You made a remark that with the subarachnoid-cisternal perfusion method it is difficult t o assess the subarachnoid surface with which this fluid is in contact. This is quite true. I think in an animal like the rabbit or rat the effective subarachnoid surface in a short experiment could be considered just the plain surface of the brain, without any consideration for its gyral configuration. From data I have, the circulating cerebrospinal fluid did not seem to go into the sulci. The circulating fluid remained in contact with the top of the gyri so t o speak, at the surface of the brain-and did not go into the spacewithin the sulci very much. I also have very much admired Dr. Katzman's work and would like to ask him whether H. MCILWAIN: he has one particular application of it; that is: to understand the time course of spreading depression. As you know, the phenomena of spreading depression has been attributed to ion movements. One suggestion with respect to it is that there may be a diffusion of potassium ions extracellularly in the brain,*and you may have some information about the extent to which any potassium that is liberated extracellularly from a local source, is removed or diluted. I am wondering if the data fit in with your calculations. R. KATZMAN: T have not made that calculation. Brindley, et a/. (1960, Neurology, 23 : 246) have shown by a local perfusion of the cortical surface that when spreading depression occurs in the area being perfused, the potassium flux from the brain markedly increases. H. MCILWAIN:It would be valuable to try to do it, I think, because there are two candidates as possible intermediaries which are very different: that is glutamate ions and potassium salts.

H. KOENIG: T would like to add my plaudits, to both Dr. Mcllwain and Dr. Katzman. I think it is remarkable that despite the gradient from the ventricular ependymal surface to the depths of the

296

R. K A T Z M A N et

al.

brain, there is such an excellent penetration of radioactivity at 1 mm intervals? Was this your glutaraldehyde fixed preparation or were these frozen materials?

R. KATZMAN: They were frozen materials. H. KOEMG:Have you checked this with your gluteraldehyde-perfused material which you claimed retains and preserves quite precisely the ions as well as other macromolecular constituents? R. KATZMAN: The gluteraldehyde perfusion retained rather well the electrolytes in the brain - that is, within 5 % of the actual barrier. We were not taking any chances.

H. KOENIG:This opens the door to analysis of experimentally induced metabolic lesions which are known to affect neural excitability and function in general. I should like to say a word about the in vitro experiments. I had the privilege a few days ago of seeing an in virro preparation of the pyriform cortex, a system which permits the analysis of synaptic transmission in Professor Mcllwain’s laboratory. The effects of altering and manipulating the chemical environment can be investigated in such a system. There are some structural and biochemical abnormalities in brain slices maintained in vitro. For example, there are quite remarkable changes in the metabolism of purine and pyrimidine nucleotides in brain slices (Ramuz et al., 1964, J . Neurochem. 11 : 827). Despite these changes, neurons in tissue slices seem to behave properly, e.g., they have a proper membrane potential and so on. H. MCILWAIN: Some workers have been doing even more drastic things to neural systems: current studies with giant axons involve the cytoplasm from the interior of the neuronal membrane.

D. B. TOWER:I just want to make a little correction for the record. Dr. Elliott was the first to show a swelling in brain slices in 1946. J. P. SCHADE:In ouabain-treated preparations one finds a drop of the average membrane potential. This may, among other things, be explained by the decreased ATP-content of these brains. Do you have any idea about the membrane potential in your preparations? R. KATZMAN: I have no measurements of membrane potentials. We have not measured these during perfusion, but we have looked a t some behavioral changes. These occur rather rapidly during perfusion.

B. D. WYKE:I am sorry to persist, but there is just one question which worries me. It is really sort of supplementary to Professor Mcllwain’s question about the transcerebral potential. As I understood it from the slide that you just showed, this potential does not oscillate with changes in the potassium concentration in the perfusate. Yet one knows (Tschergi, R. D. and J. L. Taylor, 1958, Amer. J . Physiol., 195 :7; Wyke, B. D., 1963, Brain Function and Metabolic Disorders, London, Butterworths) that if one manipulates the hydrogen ion concentration either in the serum or in the CSF, this potential does change. Do you have any comment about this in relation to what you said about the independence of the transcerebral potential from potassium ion changes? Do you, for instance, get changes in the hydrogen ion concentration of the perfusate which might counteract the possible variations in transcerebral potential due to the potassium change?

R. KATZMAN: The potentials as shown by Dr. Held (Held et a/., 1964, J. Neurophysiol., 27 : 942) change with shifts in potassium very clearly. The point I was making was that with our flux-equations we can account for the changes in terms of concentrations without the electrical factor. There is nowhere that we need to put in a term for the electrical changes that are known to occur. This is very disturbing. The data we have, and the data that Dr. Cserr has, and the data from Drs. Bradbury and Davson are very similar. What seems t o be very reproducible is that the potassium flux looks as if it were simple diffusion from the inside. From the outside of the system it looks like it is a carrier system. This is probably due to the fact that there are membranes in series rather than a single membrane, and it is probably a very complicated system,

297

Distribution of Nonelectrolytes and Electrolytes in the Brain as Affected by Alterations in Cerebrospinal Fluid Secretion D l X O N M. W O O D B U R Y * Department of Pharrriacology, Uiiiversity of Utah College of Medicine, Salt Lake City, Utah ( U.S.A.)

One of the intriguing problems in the field of “blood-brain barrier” physiology is the relation between the cerebrospinal fluid (CSF) and the extracellular fluid of the brain. As has been described by Davson (1956, 1963, 1965) and others (Reed and Woodbury, 1963; Woodbury, 1965b), the CSF, because of its active secretion by the choroid plexus, its bulk flow through the ventricles and subarachnoid cavities, and its exit through the arachnoid villi, acts as a “sink” for substances entering the brain interstitial space from the brain capillaries across the “blood-brain barrier”. Because the transfer rate of most substances across the capillary-brain interface is slower than their rate of egress from the CSF by bulk flow, their concentration in the interstitial fluid of the brain is lower than plasma, but higher than in CSF, provided that the substances do not enter the CSF across the choroid plexus or, if they do, provided that they enter at a rate slower than that across the brain capillaries. Thus, indicator substances normally used to measure extracellular fluid volume cannot be used to measure brain interstitial volume because of this fact. An additional factor that maintains a lower concentration of substances in brain and CSF than in plasma is their active transport out of the CSF across the choroid plexus. Such substances as monovalent inorganic anions, probably potassium, and organic anions and cations are transported out of the CSF in this manner. It is the purpose of this paper to summarize the effects of bulk flow of CSF and of active transport across the choroid plexus on the distribution of nonelectrolytes and electrolytes in the brain. In Fig. I , taken from the work of Ferguson and Woodbury (1967), is shown schematically a summary of the various factors involved in transfer of substances into and out of the brain and CSF as discussed above. In young animals the flow of CSF through the arachnoid villi is sluggish but progressively increases as the choroid plexus matures. Its maturation occurs at about 3-8 days after birth. There is a large extracellular space in the brain of young animals with little or no barrier to entry of substances from the plasma, as will be shown below. Increasing CSF flow, barrier formation between blood and brain, and decreasing extracellular space all contribute to decreased concentration of substances in the brain and a lowering of the brain/

* Recipient of a Public Health Service Research Career Award (No. 5-K6-NB-13,838) from the National Institute of Neurological Diseases and Blindness, National Institutes of Health. Ri!Termcrs p p . 312-313

D. M. W O O D B U R Y

298

PLASMA

J

RACH NO ID ViLLI

CHOROI PLEXU

1

PLASMA

r

Fig. 1 . Schematic drawing of the various factors that regulate the exchange of substances between plasma, brain, and cerebrospinal fluid in young as compared with adult rats. The filled arrows indicate the movement of solutes and the open arrows the flow of cerebrospinal fluid. The width of the arrows is proportional to the rate of flow of the solutes across the various boundaries. The thicker boundary line at the brain-plasma interface of the adults as compared to the young is an indication of the greater development of the “blood-brain barrier” in the older group. See text for further discussion. (From Ferguson and Woodbury, 1967.)

plasma ratio for substances found in the interstitial and cellular spaces. However, the brain/CSF ratio continues to estimate the extracellular space as long as no barrier forms between brain and CSF. This aspect will be discussed later. Transport out of the CSF across the choroid plexus also helps to lower the level of substances in the CSF and also in the brain. The concentration of a substance in the CSF and brain is thus determined by the difference between its rate of entrance into brain from plasma and its rate of exit from the CSF, either by bulk flow through the arachnoid villi or by transport across the choroid plexus, or by both processes. Substances such as Na and CI that are transported across the plexus are in approximately the same concentration in CSF as in plasma and hence are little affected by bulk flow. These relationships in developing rats are shown clearly by the data presented in Fig. 2 (Ferguson and Woodbury, 1967). The ordinates are the ratios of CSF/plasma (left side) and brain (cerebral cortex)/plasma (right side) in per cent of [Wlinulin for different aged animals, and the abscissa is time in hours. The uptake data were obtained simultaneously for CSF and brain in animals that varied in age from -4 days (1.2 g) (4 days before birth, or 17 days after conception) to 26 days (60 g) and adults (250 g). It is evident from Fig. 2 that inulin concentrations in the CSF and brain are considerably higher in the young than in the adult animals and that they progressively decrease with age. It is also evident that the brain spaces decrease puri pussu with the CSF spaces as the animals mature. A steady-state distribution is reached in about 24 h in most cases. Since even in the youngest animals the CSF/ plasma ratio is about 0.9, the level in the CSF is not the same as in the plasma. Hence, the inulin space of the brain measured in terms of the plasma still underestimates the extracellular space but is very close to it in the 4-day-prenatal rats. However, as shown in Fig. 3 (Ferguson and Woodbury, 1967), the inulin space calculated from the

CSF

A N D NONELECTROLYTES A N D ELECTROLYTES I N B R A I N

299

ratio of brain to CSF inulin concentrations probably does measure fairly accurately the extracellular space of animals 16 days of age or younger. The ordinate is the brain/ fluid ratio of [“Tlinulin and [14C]sucrose activity and the abscissa is age of the rats i n days. In fetuses tested 4 days before birth (-4 days old), the brain/plasma and brain/CSF ratios for inulin approach one another, an indication that at this age the two ratios measure nearly the same space. If no CSF flow is present, then the two ratios would be equal. This condition is present in fetuses tested 6 days before birth (15 days post conception), a time also when the choroid plexus first begins to

0 4 4

Fig. 2. The simultaneous uptake of [Wlinulin into cerebral cortex (called brain in subsequent figures) and cerebrospinal fluid (CSF) of rats during maturation as a function of the time after injection of the inulin. The ordinate on the left is the ratio of CSF to plasma water [14C]inulin concentrations 100 (space in per cent); on the right, the ratio of brain to plasma water [14C]inulinconcentrations 100. The abscissa in both graphs is time in hours. All the animals were bilaterally nephrectomized for 24 h. See text for discussion. (From Ferguson and Woodbury, 1967.) 1

form as described by Dr. Virginia M. Tennyson in this symposium. Although both ratios decrease with increasing age, the ratios of brain/plasma and brain/CSF continuously diverge, probably as a result of increasing flow of CSF and of barrier formation to inulin transfer between plasma and brain. These two mechanisms would decrease the brain/plasma ratio presumably without affecting the brain/CSF ratio since there is a rapid rate of diffusion between brain and CSF. Between 16 and 26 days of age the brain/CSF ratio begins to increase and continues to rise to the adult values. The increase at this time probably is due to decreased permeability of the ependymal lining of brain to inulin so that brain to CSF exchange is slowed. Thereafter, inulin does not measure the interstitial volume of the brain. Since the volume of the brain interstitial space in adult rats is about 13-14 % as measured by the technique of ventricular-cisternal perfusion with inulin (Woodward et al., 1967), the curve of the brain/CSF ratio of inulin has been extrapolated to this value (indicated by an asterisk in Fig. 3) and the combined solid and dotted line represents the true extraRsfi.rcnc.rs pp, 312-313

300

D. M. W O O D B U R Y

cellular space of the brain as animals mature. The space decreases from about 48 ”/, in 4-day prenatal rats to 13 to 14 % in adults. The curve for sucrose is at all times higher than that for inulin but parallel to it, a fact that indicates that this substance probably penetrates cellular structures in the brain not penetrated by inulin. Since secretion of CSF and transport across the cells of the choroid plexus profoundly affect the levels of substances in the CSF and brain, and since both processes are active and require energy, it is possible to change the CSF and brain concentrations of these substances entering the CNS across the brain capillaries by inhibition or stimulation of the choroid plexus transport processes. Alterations of this transport system influence the levels either by altering bulk flow rate or by altering movements of the transported ions. If CSF secretion rate is changed then the concentrations of all substances entering the CSF from the brain are also changed. However, if only a single transport system is affected, then the distribution only of the substances transported by that system is changed unless that substance is a necessary part of the CSF secretory system.

g

60-

x

5

::

50-

. c . 2

40-

30-

20-

10-

Fig. 3. Relation between the brain to fluid (CSF or plasma water) ratio x 100 (space in per cent) of [14C]inulin and [14C]sucrose and the age in days after or before birth of rats. See text for discussion. (From Ferguson and Woodbury, 1967.)

Before considering the effects of substances that alter CSF secretion or choroid plexus transport processes, it seems worthwhile to discuss these transport systems of the choroid plexus and their relation to the distribution of ions in the CSF and brain. In Figure 4 is depicted a schematic representation of the choroid plexus cells of the rabbit and the distribution of ions in these cells, in the CSF, and in the interstitial space surrounding the choroid plexus capillaries. Below the diagram is a summary of the observed CSF/plasma ion-distribution ratios for rabbits and dog, the two species for which potential measurements between these two fluids have been measured, and the ratios that would be expected if the ions were passively distributed according to a 5 mV potential (CSF positive) for the dog and a 14 m V potential for the rabbit. The potential measurements in the dog are by Held et al. (1964) and

+

+

CSF

A N D N O N E L E C T R O L Y T E S A N D E L E C T R O L Y T E S IN B R A I N

301

those in the rabbit are by Welch and Sadler (1965). The CSF/plasma ratios are taken mainly from the works of Davson (1956; 1963), Held et al. (l964), and Schain (1964) but are in the same range as for other species including the rat. It is seen that most of the ions listed with the possible exception of Ca++and CI- are not distributed passively. Na t- and Mg++appear to be transported inward from plasma to CSF and K+ appears to be transported outward from CSF to plasma. Of the anions, only CIappears to be inwardly directed and most results suggest that it is passively distributed. However, data to be presented later, and other data (Rall, 1964), suggest that C1is transported actively from plasma to CSF. It appears, therefore, that Na+ and C1are the main ions involved in secretion of CSF. The anions, bicarbonate (HCO3-), iodide (I-), thiocyanate (SCN-) and, as will be shown later, perchlorate ( C D - ) , are transported out of the CSF. In the upper part of the diagram these transport systems are shown in relation to the membranes of the ependymal cells illustrated

CSF

CHOROID PLEXUS

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I .48

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0.72

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0.84

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Fig. 4. The upper diagram is a schematic representation of the transport systems in, andthevoltage gradients across, the choroid plexus cells. The lower table is a summary of the observed CSF/plasma concentration ratios of the indicated ions in the rabbit and in the dog as compared with the calculated ratios for passive distribution according to the observed potential difference between blood and CSF determined for these two species. The voltage values for the rabbit were measured by Welch (1965); for the dog, by Held ef a/. (1964). See text for discussion. Reji,rences pp. 312-313

302

D. M. W O O D B U R Y

schematically in this figure. In the lower of the three ependymal cells the potential gradients across the cells as determined by Welch and Sadler (1965) are depicted. The ependymal cell membrane on the CSF side has a potential of -64 mV (inside of cell negative) and the one on the interstitial fluid side has a potential of -50 mV. Thus a potential difference of +I4 mV, CSF side positive, exists across the cells. In the ependymal cell shown in the middle of the diagram, the cation transport system is shown with the size of the letters indicating the probable concentration of the substances in the different compartments. K+ and Ca++ are higher on the interstitial fluid side and Na+ and Mg++on the CSF side. In the cell, K+ and Mg++are probably high and Na+ is low, as is the case with most other mammalian cells, although no experimental data are available to support this postulation. In muscle and other cells Mg+ is concentrated (30 : 1) but not as much as would be expected from the musclecell membrane potential of -90 mV and its double positive charge (1000 : I ) ; consequently, Mg++,like Na+, must be pumped out of cells (see Woodbury, 1965a). This is probably accomplished by the same carrier system that transports Na+.However, the transport of Na+ inward and of K+ outward, which process is assumed to be coupled, must take place in the membrane on the CSF side of the cell. Thus the pump of the choroid plexus, assumed to be a coupled Na+ and Mgki for K+ pump, is in the membrane of the CSF side. The movement across the membrane of the interstitial side is probably passive for all three cations since it is along their eleotrical or chemical gradients. The mechanism of transport of Ca++is not known but appears to be passive. In the ependymal cell illustrated at the top of the diagram, the anion transport system is depicted. Chloride ion appears to distribute passively across the membrane on the interstitial side of the cell. It then is probably actively transported into the CSF across the membrane on the CSF side. The outward transport of the anions I-, C104-, SCN- and probably HC03- is thought to be coupled to inward chloride movement. The location of the anion pump with respect to this membrane is necessitated by the observations of Welch (1962a, 1962b) that the choroid plexus can concentrate I- and SCN- ions in vitro. Since this is the case and if only a single pump is present, it must be in the CSF side of the membrane in order to concentrate I- and SCNin cells and still maintain a concentration difference between CSF and plasma. However, a pump in both membranes could also accomplish the same result but there is no evidence for this one way or the other. The consequences of inhibiting the various pumps of the choroid plexus for the concentration of various substances in the CSF and brain can now be considered. In Figs. 5-9 are shown the effects of inhibition of the anion transport system. In Figure 5 , modified from the figures of Reed et al. (1965), are shown the effects of a large load of iodide on the distribution of [l3l]Iiodide ion and [14C]inulin in CSF and brain. Iodide saturates the I- pump and thereby decreases the exit of the I- from the CSF by this route. The major route of I- exit, therefore, is via the arachnoid villi by bulk flow, provided that this flow is not also inhibited by large loads of iodide ion. That bulk flow appears to be little affected is demonstrated by the relatively minor change in CSF [“Clinulin space (which is a measure of CSF bu1kflow)with increasing loads of iodide. The brain space of inulin also increases slightly but in-

CSF

A N D NONELECTROLYTES AND ELECTROLYTES I N BRAIN

303

significantly. The effect on [1311]iodidespace, however, is striking. Increasing doses of iodide progressively elevate the CSF ['3ll]iodide space and concomitantly the brain [131l]iodide space is also increased. Thus the I- space of CSF increases from 2 to 43 % and that of brain from 2 to nearly 12 %. It is clearly evident that saturating the carrier for transport of I- out of the CSF raises not only the CSF but also the brain levels of this anion. The concentrations in these two fluids thus more closely approach each other and also the concentration in the plasma. Since the exchange rates between CSF and interstitial space of brain of this anion are rapid, the ratio between the two (labeled (brain/CSF) 1311 in Fig. 5) is a measure of the volume of distribution of the I- in the brain, especially in the carrier-iodide loaded animals in which the concentrations are more nearly equal in the two fluids. The calculated brain/CSF ratio is 1.04 in controls and levels out at about 0.26 in the carrier-iodide loaded animals. This volume of distribution is equal to that of CI- and suggests that I- distributes like C1- in the brain when 1- transport is blocked. If the final distribution volume is taken as 26 %, then the calculated concentration of I- ion in brain at any one iodide load is its actual concentration in the whole brain divided by the volume of water in which it is distributed, namely, 26 % in this case. Thus the brain concentrations of I- for the four 100-

nosoSO -

7

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4J-

30

-

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'\, __---------_ _ _ _ _ _--------a ___-

.

a

=.A-

V

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I

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*' 0

B cR sPil H 1 O l

BRAIN-Ci4-

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I

10 0

50

L L q I N CARRILR l O O l D E I N J L C T F O

Fig. 5. Effects of iodide loading on the 4-h distribution of intraperitoneally-injected[13Ll]iodideand [L4C]inulinin brain and CSF of rats bilaterally nephrectomized for 4 h. The ordinate is the space (brain/plasrna, CSF/plasma, brain/CSF) in per cent and the abscissa is mequiv/kg body weight of carrier iodide injected into theanimals.The figure is drawn from the data of Reed et al. (1965).See text for discussion.

different iodide loads used in this experiment (Fig. 5 ) are

I .9

5.8 =

0.26

1.3,

8.6 = 22.3,

~

0.26

11.7 = 33,

~

0.26

and

_ _ = 45,

0.26

as compared to the corresponding CSF levels of 2, 22, 33 and 43 and plasma levels of 100. Thus in the controls the concentration of I- in brain water is higher than that of CSF but less than that of plasma. However, in the iodide-loaded animals the concentrations in brain and CSF are equal but less than in plasma. These data indicate Ki,J./Privrcspp. 312-313

D. M. W O O D B U R Y

304

that I- enters the CSF from the brain capillaries via the interstitial fluid of the brain and not through the choroid plexus. They also indicate that the barrier to entrance is between the capillaries of the brain and the interstitial fluid of the brain and that there is free exchange of iodide between the brain and CSF, as already mentioned. The effects of intraperitoneal administration of other inhibitors of transport processes on [13ll]iodide and [14C]inulindistribution in brain and CSF are shown in Fig. 6 (Reed et al., 1965). The inhibitors used and the rationale for their use were:

C1‘-INULIN

4 HOUR SPACE

1 0 0 1 0 ~HOUR ~ ~ ~S P4A C E

I. : S i p n ~ l l r o n l l y d i l l e r m l 110.

c

L0”llOl “ O I U e ,

ll

Fig. 6. Effects of various transport inhibitors on the 4-h distribution of intraperitoneally administered [13LI]iodideand[14C]inulin in brain and CSF of rats bilaterally nephrectomized for 5 h. The ordinate is space of brain or CSF in per cent. The Inhibitors used together with their doses are indicated along the abscissa. The data used to construct this figure were obtained from the paper of Reed eta/. (1965). See text for discussion.

perchlorate ion, a competitive inhibitor of I- transport in the thyroid ; acetazolamide, a carbonic anhydrase inhibitor that has been shown to inhibit CSF secretion; and oirabain, an inhibitor of the Na+-K+ active transport system of cells and also of the I- uptake by the thyroid gland. Of the three inhibitors used, only perchlorate ion had a profound effect on the distribution of I-. It markedly increased the concentration of [13ll]iodide in both CSF and brain. The [“Clinulin space was little affected. After intraperitoneal injection, acetazolamide and ouabain, unlike perchlorate, had no significant effect on the distribution of [Wlinulin and [131I]iodide; but, on intracisternal injection, they markedly increased the concentrations of [14C] inulin and [13ll]iodide in CSF and brain (Reed et al., 1965). The changes induced by ouabain and acetazolamide on distribution of intracisternal injected [“Clinulin were greater than with I-, an observation that indicates these agents affect I- distribution by inhibition of CSF secretion with its resultant decreased bulk flow, not only through the CSF but also through the brain substance, a process previously suggested by Reed and Woodbury (1963). An inhibition of bulk flow through brain tissue would be expected to decrease levels in the brain relative to the CSF to the same extent for both I- and inulin even though both diffuse across membranes at different rates. This is precisely what happened after acetazolamide and ouabain. For example, the 4-h

CSF

A N D NONELECTROLYTES A N D ELECTROLYTES I N B R A I N

305

CSF/brain ratios for [l"l]iodide and [14C]inulin, respectively, after intracisternal administration in controls were 2.5 and 3. I , whereas after acetazolamide they were both 11.2. One of the questions concerning anion transport across the choroid plexus is when it begins developmentally. To answer this, the ability of the choroid plexus to transport I- was tested in 8-day-old rats. In addition, the comparative inhibitory effect of a perchlorate ion load on this system in young as compared to adult animals was also

-

01 0 1 2

I

I

8 TINE

21

I N Houris

Fig. 7. Effects of perchlorate treatment on the [l311]iodide and [*4C]inulin spaces in per cent (ordinbrain in 8-day-old and adult rats as a function of time in hours (abscissa) after intraperitoneal injection of the tracers. The adult rats were bilaterally nephrectomized for 5 hand the 8-day-old rats for 24 h. The duration of nephrectomy, however, only slightly affects the volume of distribution of these substances. See text for discussion.

ate) of

measured. The results are shown in Fig. 7. The brain [13lI]iodide and [14C]inulin spaces are the ordinate and the abscissa is time in hours after injection of [13lI]iodide or [14C]inulin. The uptake experiments were done on 8-day-old control and perchlorate-treated rats and the results were compared with the 4-h brain I- and inulin spaces of adult control or perchlorate-treated rats. The brain uptake of I- is very rapid in 8-day-old rats and reaches a peak of about 5 % in 40 min, it falls off very gradually thereafter. Perchlorate (5 mequiv./kg) elevated the brain space of 8-day-old rats to about 14 "/, at 4 h, compared to the control value of 4.5 % measured at the same time. Thus the brain space was increased 3.1 times by this inhibitor of 1- transport. The levels of I- in the CSF were not measured in this experiment. The uptake of [14C]inulin by the brain of the 8-day-old rats was much slower than that of ['3lI]iodide, but in the controls it reached a larger volume of distribution. In this experiment perchlorate did not affect the volume of distribution or the rate of entrance of inulin in the brain of 8-day-old rats. Since the control I- space is less than the inulin space and the Ispace is increased by perchlorate ion to a value greater than the inulin space, it is apparent that the 1- pump is actively working in these young animals. However, that it is less active than in the adult is evident from the effect of this anion on the Rcfc~rrnccspp, 312-313

306

D. M. W O O D B U R Y

[13ll]iodide space of the adult animals. The 4-h [13lI]iodide space was 1.8 % in control animals, a value less than the corresponding value of 8-day-old animals, but it was increased to 7.2 % by perchlorate in a dose of 3.4 mequiv./kg. This represents an increase of 7.2/1.8 or 4 times the control value as compared to 3.1 times for the young rats in which a larger (5 mequiv./kg) dose was used. Thus the inhibitor exerted a greater effect in the adults than in the young animals. This suggests that the active transport is less active in the 8-day-old animals. The inulin space of the adults was slightly increased by perchlorate but the effect was not significant. In the thyroid gland, anions besides 1- are transported by the anion pump and concentrated in the gland. The next question, then, is whether these same anions are also transported by the choroid plexus system and, if so, whether they inhibit transport across the plexus. In Fig. 8 is shown the effect of perchlorate ion (5 mequiv./kg) on6

0 1 2

4

6

8 12 T I M E I N HOURS

24

Fig. 8. The effect of a perchlorate ion load on the uptake of radioactive perchlorate ion (36CIO~-) by brain and CSF of adult rats. The ordinate is [36Cl]perchloratespace of brain and CSF in per cent and theabscissa is time in hours after intraperitoneal injection of the 36CIOc in rats bilaterally nephrectomized for 24 h. See text for explanation.

the uptake of radioactive perchlorate (36ClO4) by brain and CSF of nephrectomized adult rats. The ordinate is [36Cl]perchlorate space in per cent and the abscissa is time in hours after injection. It is clear that, as in the case for an iodide load, stable perchlorate markedly increased the volume of distribution of 36C104 in both CSF and brain. Inasmuch as the plasma levels remained constant, these increases are due to a marked rise in the concentration of 36C104 in CSF and brain tissue. Since the CSF/ plasma ratio of the control was only 0.1 at equilibrium and since there was no appreciable change in the [Wlinulin space on perchlorate treatment, it is evident that this anion, like I-, is transported out of the CSF and that its transport can be self blocked. Again, an increase in CSF concentration increased the level in the brain such that it approached that of the plasma. Since brain-CSF exchange is rapid for this ion, as it is for I-, the brain/CSFratio of 3sC104 in the perchlorate-treated group measures quite accurately its volume of distribution in the brain. This value is 17.5/54

CSF

A N D NONELECTROLYTES A N D ELECTROLYTES IN BRAIN

307

x,

100 = 32.5 a value close to the chloride space. These data indicate that C104as well as I- distributes in the brain as does CI- ion. The effects on 36CIO~ transport in the choroid plexus of some of the monovalent anions that inhibit iodide transport across the choroid plexus and in the thyroid are shown in Fig. 9 (Chow and Woodbury, 1967). The ordinate is 36C104 space in % of wet weight of brain and skeletal muscle. CSF was not measured, but, as just dei

CONT.

CCHEBrAL CORTEX--SKELtTAL

‘I01

NoSCN

MUSCLE-

Fig. 9. Effects of various monovalent anions (5 mequiv/kg) on the [3RCI]-perchloratespaces of brain and skeletal muscle one hour after intraperitoneal injection of the tracer in adult rats. The anion inhibitors were given 2 hours before sacrifice of the animals. The figure in each bar is the number of animals used for that group. See text for discussion. (From Chow and Woodbury, 1967.)

monstrated, inhibition of anion transport in the choroid plexus by monovalent anions is always accompanied by an increase in both CSF and brain concentrations; hence an effect of a monovalent anion to increase the brain space generally indicates blockade of this transport system. It is evident that iodide, perchlorate, and thiocyanate (in doses of 5 mequiv./kg) all block the anion transport system in the choroid plexus. The order of effectiveness is perchlorate > iodide > thiocyanate. In this experiment the radioactive perchlorate space of muscle was little affected by these monovalent anions. Another question that arises is the effect of a perchlorate-ion load on the distribution of substances other than I - and C104- in brain and CSF. In the schematic diagram of the choroid plexus system shown in Fig. 4 it is indicated that CI- is probably secreted into the CSF coupled to outward transport of I-, C104-, etc. If this is the case, inhibition of outward transport of I- or C I O c with perchlorate ion should inhibit inward CI- transport and decrease the CSF level of CI-. Also, so4=concentration is lower i n the CSF than in the plasma (Van Harreveld, 1966), and the possibility exists that this doubly charged anion might also be transported across the choroid plexus into blood. However, its distribution, like that of inulin, could equally be accounted for by its slow penetration across the capillary-brain interface, coupled with its carriage out of the CSF by bulk flow through the arachnoid villi. If movement of Sod- out of the CSF involves the anion transport system of the choroid plexus, Rc+riwii>\ p p . 312-313

D. M. W O O D B U R Y

308

then a large dose of perchlorate should compete with Sod= for the carrier, as it does for I-, and block outward so4=transport. The result would then be an increase in SO4= levels in the CSF and brain. If only bulk flow is involved in SO4= exit from the CSF, it should behave like inulin when CSF flow is altered. Therefore, the effects of 5 mequiv/kgdoses ofperchlorate ion on the distribution of 36C104, 36C1, 35S04 and ['4C]inulin in CSF and brain were determined in nephrectomized rats. The results of these studies are indicated in Fig. 10. The spaces are shown on the ordinate and the various treatments and isotopes used are presented along the abscissa. The entire uptake curves were determined in both control and perchlorate-treated groups, but only the equilibrium values determined at the times indicated below each isotope listed are depicted in the figure. As demonstrated previously in Fig. 8, a perchlorate load increased the volume of distribution of 36C104 in both CSF and brain. The

Cl%i

24 D W I

c1J6 LI

hOYI,

SJ504 1 how,

Clq

- lwull~ n ~ W I

Fig. 10. Effect of perchlorate treatment on the distribution of 36CI04,36CI,35S04,and [14C]inulinin brain and CSF of adult rats at the times after injection indicated along the abscissa. The ordinate is the space of brain or CSF in per cent. The animals were bilaterally nephrectomized for 24 h. See text for discussion.

increase in the CSF is greater than that in brain. In contrast to its effect on CSF 36c104, perchlorate treatment decreased the concentration of W I in the CSF; but, like 36C104, it increased the 36CI space of the brain. (The stable CI (35C1)space in CSF was also decreased and the 35Cl space of brain increased.) Thus, in this case a decrease in CSF concentration was accompanied by an increase in brain space. Although the changes were not significant, perchlorate slightly and consistently at all time periods increased the 35S04 space of the CSF and also of the brain. It had, however, no significant effect on (14C)inulin spaces in brain or CSF. Since ["Tlinulin spaces were not affected by perchlorate treatment and S5S04 levels were affected slightly, these observations suggest that the low CSF as compared to plasma may to a small extent be accounted for by active transport via the choroid plexus anion system. Further work is obviously necessary to prove this point. The decrease in 36Cl space and the increase in 36C104 space in the CSF induced by perchlorate treatment are in

CSF

AND NONELECTROLYTES AND ELECTROLYTES IN B R A I N

309

harmony with the concept that the two ions are coupled in their transport, as already discussed. As was the case for I-, the C104- space of 9-day-old rats is increased by perchlorate loading but not to the same extent as in the adult (Barham, G. B. and Woodbury, D. M., unpublished observations). This effect is illustrated in Fig. 1 I . The ordinate is

CONTROLS

I}

\Clq-

I

C1j604 S P A C E

)Ci4-INULIN

SPACE!

I N U L I N SPACE

I,/PERCHLORATE I

I

rsr PLASMA

BRAIN PLASMA

9 - DAYS- OLD

ADULTS

Fig. 1 1 . Effect of perchlorate ion (5 mequiv/kg) on the brain/plasma, CSF/plasma and brain/CSF [B8C104]-and[14C]inulinratios (spaces) in 9-day-old as compared to adult rats. The asterisks indicate that the value for the spaces of the perchlorate-treated group marked is significantly different ( P < 0.05) from the corresponding space of the controls. Values plotted from the unpublished observations of G . B. Barham and D. M. Woodbury. See text for discussion.

the 36C104 or [14C]inulin space in per cent. The 9-day-old animals are shown on the left and the adults on the right. Perchlorate spaces are represented by the total height of the bar and inulin spaces by the filled-in areas. The controls are represented by the bar on the left and the treated animals by the bar on the right. In the young rats, perchlorate treatment increased the CSF 36ClO.4 space, which is already high in these animals, from 55 to 88 % (1.6 times) and increased the brain space from 20 to 43% (2.15 times). The [“Wlinulin spaces were also increased by perchlorate: in the CSF from 17 to 26% (1.53 times) and in the brain from 6 to 8.5% (1.42 times). It is evident that blockade of WIO4 transfer by perchlorate ion appears also to inhibit the secretion of CSF as measured by the CSF [“Clinulin space. The anion transport system thus appears to be involved in CSF secretion. In adults, as already described, perchlorate increased the CSF space 5.4 times as compared to I .6 times in the young rats, and increased the brain space 2.5 times as compared to 2.15 times in the young. The inulin space is apparently not altered by perchlorate loads in the adult probably because it is so small that the kind of change observed in the young could not be detected in the adult. It is apparent that anion transport of C104- seems to be less developed in the young, as was shown to be the case for I- in Fig. 7. The brain/CSF ratios give the correct C104- and inulin distribution in brain. In this case, the difference between the 36ClO4 space and [14C]inulin space is a measure of brain intracellular 36C104. In the 9-day-old rats perchlorate increased markedly the brain/CSF 36C104 space and deRcJi.rences pp. 312-313

D. M. W O O D B U R Y

310

creased the [“C]inulin (interstitial) space. Thus intracellular 36C104 levels were increased. In adults the brain/CSF ratio is decreased by perchlorate for the reasons already discussed. However, intracellular 36C104cannot be determined in adult rats from the difference between 36C104and inulin spaces as it can in young rats, because the apparent [14C]inulin space (brain/CSF ratio) is larger than the 36C104space. This situation is generally the case when the blood-brain barrier is developed, as already discussed (see Fig. 3). Although not shown here, the CSF/plasma and brain/plasma CI- ratios were markedly decreased by a perchlorate load in young rats. Hence the reciprocal relation between CI- and C 1 0 ~transfer across the choroid plexus in adults and reflected also in the brain levels is clearly present in the young rats and further supports this hypothesis, especially since perchlorate treatment had little effect on Naf and K+ concentrations in the CSF. Another anion that blocks iodide accumulation in the thyroid and is concentrated by choroid plexus cells (Welch, 1962b) is thiocyanate. Pollay (1966) has shown that large doses of this ion produce the same effects on CSF and brain SCN- and Ispaces as do iodide and perchlorate loads on [13ll]iodide and 36C104 spaces. Thus SCN- is also actively transported out of the CSFacross thechoroid plexus, as indicated in Fig. 4. The data of Pollay are summarized in Fig. 3 (p. 277). We have further evidence to indicate the intimate relations that exist between changes in CSF secretion as a result of transport of substances across the choroid plexus and the concentration of substances in the brain, but the data herein presented adequately illustrate these relationships. One point, however, that needs amplification is the effect of inhibition of CSF flow on the levels of nonelectrolytes and electrolytes in CSF and brain. One facet is shown in Fig. 12. The effects of ouabain and acetazolaamide on CSF and brain inulin spaces in developing rats were studied in order to

‘I

BRAINKSF

20

CSFlPLlSYA

Fig. 12. Effects of acetazolamide and ouabain in the indicated doses on the [%]inulin spaces (brain/ plasma, CSF/plasma, brain/CSF) in rats of different ages after birth. Note that the dose of ouabain used (2 mg/kg ) is considerably lower than the dose necessary to produce an effect in adults (25mg/kg), as seen in Fig. 6. Doses higher than this produced convulsions and death in the young animals, but 25 mg/kg did not produce convulsions or deaths in adults. Data obtained from unpublished observations of R. K. Ferguson and D. M . Woodbury. See text for discussion.

CSF

A N D NONELECTROLYTES A N D ELECTROLYTES I N B R A I N

311

ascertain their influence in a case where the brain barrier is not developed and changes can be easily detected. The ordinate is the [14C]inulin space in per cent, and the abscissa is age of the animals in days after birth. The CSF/plasma and brain/plasma ratios of the controls decrease progressively from 3 to 16 days of age. Ouabain, which inhibits N a t and K+ transport in both the CSF and the brain, increased the CSF [14C]inulin space markedly at 16 days of age but only slightly at 3 days of age. It had very little effect on the [14C]inulin space of brain as measured by the brain/plasma ratio. In this case the change in CSF concentration was not reflected in changes in brain concentration, probably because swelling of the brain cells, caused by the increased cell Na+ and the decreased cell K+ concentrations induced by the direct effect of ouabain on nerve cells, results in a decrease in extracellular fluid volume of brain in the tight box that encloses the brain. That a decrease in extracellular fluid volume occurs with ouabain is evident from the data for the brain/CSF ratios shown in the top graph of Fig. 12. As discussed earlier the brain/CSF ratio in per cent is a measure of the interstitial space of the brain in animals 16 days of age or younger. Thus, ouabain decreased the brain/CSF space in 16-day-old rats and this was accompanied by a marked increase in water, Na+ and CI- and a decrease in K+ concentrations of the brain, an indication that cerebral swelling had occurred. Inhibition of CSF secretion by acetazolamide (which appears to block CI- transport) increased markedly the CSF [14C]inulin spaces at 9 and particularly at 16 days but had little effect at 3 days. The brain [“Tlinulin space was also increased at all three time periods but the effect was greater at 16 days. However, the brain/CSF ratio was decreased by acetazolamide at 9 and 16 days but not at 3 days. Thus this agent, like ouabain, decreased the extracellular space of the brain, again possibly by causing swelling of nerve cells. These age-dependent effects of metabolic inhibitors of transport on inulin distribution provide further evidence that the transport systems are less well developed in young rats. Acetazolamide presumably acts by inhibiting carbonic anhydrase in the choroid plexus and the brain, and it is well known that this enzyme is low in activity in the brain of young animals (Millichap et al., 1957; Karler and Woodbury, 1960). The relative lack of effect of acetazolamide in such animals is readily explained by the paucity in this enzyme. The lower effectiveness of ouabain, which inhibits Na+-K+ transport in adults, in 3-day-old rats is also probably due to the fact that the cation transport portion of the CSF secretory system is immature at this age. Our studies of CSF electrolytes with age have demonstrated that this system is not completely mature until after 3 days of age. Observations on Naf, K+ and CIconcentrations in CSF and brain at different ages and the effects of ouabain and acetazolamide thereon have clearly shown that the same close relationship between CSF and brain fluids exists for cation and anion transport. Subsequent papers will report these data. SUMMARY

The studies reported in this paper demonstrate that there is an intimate relationship between the CSF and the extracellular fluid of the brain. This relationship was deR(fcrenrcs ppr31.2-313

312

D. M. W O O D B U R Y

monstrated by use of substances that block the formation of CSF, by inhibition of either the cation or the anion transport systems in the choroid plexus, both of which appear to be involved in CSF secretion. Anion transport out of the CSF across the choroid plexus was shown to be present for iodide, perchlorate and thiocyanate ions and possibly for sulfate. This outward transport is thought to be coupled to inward chloride transport, since inhibition of outward anion transport is always accompanied by inhibition of inward chloride transport. Inhibition of anion transport across the choroid plexus resulted in an increase in the CSF/plasma ratio (space) of concentrations of the various anions tested. The increase in CSF/plasma ratio was accompanied by an increase in the brain/plasma ratio of the same anions. Thus a change in the CSF concentration of a substance is accompanied by a corresponding change in the brain concentration. This same relationship between CSF and brain also holds when cation transport in the choroid plexus is blocked. Further evidence for this concept was obtained from studies in developing rats. The formation of CSF in rats begins about 6 days before birth and by the ninth day after birth it has matured, as measured by the ability of the choroid plexus to transport anions and cations. During this period of increasing flow of CSF and increasing active transport of cations and anions across the plexus, the direction of change of nonelectrolytes (such as inulin), anions (such as iodide, perchlorate, thiocyanate, and chloride), and cations (such as sodium and potassium) in the CSF is the same as in the brain. Thus these data in maturing animals again demonstrate the intimate relation between CSF and brain extracellular fluid. ACKNOWLEDGEMENT

Original investigations reported herein were supported by a United States Public Health Service Research Program Grant (No. 5-POI -NB-04553) from the National Institute of Neurological Diseases and Blindness, National Institutes of Health. REFERENCES CHOW,S. Y. AND WOODBURY, D. M. (1967) Distribution of C136-perchlorateion in brain and muscle of rats and guinea pigs. Amer. J . Physiol., submitted. DAVSON, H. (1963) The cerebrospinal fluid. Ergeb. Physiol. Biol.Chem. Exptl. Pharmakol., 52, 20-73. -(1965) The extracellular space of the brain. Biology of Neurogliu. E. D. P. De Robertis and R. Carrea (Eds.). Progr. Brain Res., 12, Amsterdam, London and New York. Elsevier Publishing Co. (pp. 124-1 34). R. K. A N D WOODBURY, D . M. (1967) Penetration of C14-inulinand C14-sucroseinto brain FERGUSON, and cerebrospinal fluid of developing rats. With comments on measurement of brain extracellular space with age. Arch. Neurol., submitted. HELD,D., FENCL, V. AND PAPPENHEIMER, J. R. (1964) Electrical Potential of cerebrospinal fluid. J . Neurophysiol., 21, 942-959. KARLER, R. AND WOODBURY, D. M. (1960) Influence of aging on intracellular distribution of carbonic anhydrase. Fed. Proc., 19, 133. MILLICHAP, J. G . (1957) Development of seizure patterns in newborn animals. Significance of brain carbonic anhvdrase. Proc. SOC.Exptl. Biol. Merl., 96, 125-129. M . (1966) Cerebrospinal fluid transport and the thiocyanate space of the brain. Amer. J . POLLAY, Physiol., 210, 275-279,

CSF

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313

RALL.D. P. (1964) The structure and function of the cerebrospinal fluid. The Cellular Furictioris of Memhratie Tramport. J. F. Hoffman, Editor. Englewood Cliffs, New Jersey. Prentice-Hall Inc. (pp. 269-282). REED,D. J. AND WOODBURY, D. M. (1963) Kinetics of movement of iodide, sucrose, inulin and radioiodinated serum albumin in central nervous system and cerebrospinal fluid of rat. J. Physiol., 169, 816-850. REED,D. J., WOODBURY, D. M., JACOBS,L. A N D SQUIRES, R. (1965) Factors affecting distribution of iodide in brain and cerebrospinal fluid. Anier. J. Physiol., 209, 757-764. SCHAIN, R. J. (1964) Cerebrospinal fluid and serum cation levels. Arch. Neurol., 11, 330-333. VAN HARREVELD, A., AHMED,N. A N D TANNER, D. J. (1966) Sulfate concentrations in cerebrospinal fluid and serum of rabbits and cats. Anier. J . Physiol., 210, 777-780. WELCH,K. (1962a) Active transport of iodide by choroid plexus of the rabbit in vitro. Ar7ier. J . Phvsiol., 202, 157-760. -( 1962b) Concentration of thiocyanate by the choroid plexus of the rabbit in vitro. Proc. SOC. Exprl. Biol. Med., 109, 953-954. WELCH,K. A N D SADLER, K. (1965) Electrical potentials of choroid plexus of the rabbit. J . Neurosurg., 22, 344-351. WOODBURY, D. M. (1965a) Physiology of body fluids. Chapter 45 in Physiology and Biophysics. 19th ed., T. C. Ruch and H. D. Patton (Ed.). Philadelphia, W. B. Saunders Co. (pp. 871-898). -( l965b) Blood-cerebrospinal fluid-brain fluid relations. Chapter 47 in Physiology arid Biophysics. 19th ed., T. C. Ruch and H. D. Patton (Eds.). Philadelphia. W. E. Saunders Co. (pp. 942-950). WOODWARD, D. L., REED,D. J. A N D WOODBURY, D. M. (1967) The extracellular space of rat cerebral cortex. Atner. J. Physiol., 212, 367-370.

DISCUSSION T. Z. CSAKY:What were the animals you used in these studies? D. M. WOODBURY: They were all rats. T. Z. CSAKY:Two mg/kg of ouabain in a rat is, if you calculate the concentrations, about 7 mols. Is not that just too little in a rat to have any effect? D. M. WOODBURY: Too little? I think most people would be amazed at how large the dose was. T. Z. CSAKY: We used two doses: one was 26 mg in one instance, in the second it was 2 mg. D. M. WOODBURY: These are very large doses of ouabain. T. Z. CSAKY:Not for a rat. That is right. Rats are very resistant to ouabain, and large doses have to be given D. M. WOODBURY: to get any effect in the adult (at least 25 mg/kg). However, infant rats are very sensitive to it, and if a dose of 2 mg/kg is greatly exceeded most of the animals die. Even at 2 mg/kg some of the animals have seizures. It is apparent, therefore, that ouabain penetrates into the brain of young rats more readily than into the brain of adults. In the rat, the brain is more sensitive than the heart to the effect of ouabain and if it can enter the brain more readily, as it does in the infant, then it attains a higher concentration and produces a greater effect. The electrolyte changes induced in the brain by ouabain in young rats are also much more marked. Sodium is increased and potassium is lost, and seizures occur. None of these effects on the brain are produced by ouabain in adult rats. The seizures induced by ouabain can be prevented by diphenylhydantoin (Dilantin).

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D. B. TOWER:Dr. Woodbury introduced quite a long time ago the importance of the developmental study. I think that is evident here, because some of the questions that we have about these systems cannot be easily answered unless you can sort out the different cells and membranes as they develop. The question I had was about the chloride, particularly in regard to the effect on the brain chloride as opposed to the CSF. I think that it is quite clear from your data, but if you looked earlier than 9 days, which was the earliest you showed us, did you see a difference in the effect of perchlorate on the apparent brain cellular transport system? The reason I ask this question is to know what cells might be involved in chloride transport in the brain, because as you know very well from Brizzee and Jacob's work, there is a difference in the time of development and the proliferation of neurons versus glial cells. A second very quick question: In your ouabain experiments in the adult it looked as if one could conclude from the brain/CSF ratio that you had a considerable decrease in the inulin-accessible space, and hence presumably some swelling. Is this your interpretation? D. M. WOODBURY: Yes, that is what I assumed. Since there is a marked change in the brain concentration of sodium and potassium outside the inulin space, 1 would assume that the swelling caused a decrease in the extracellular space of the brain. In the young animals, even at 9 days, glial cells are not prominent, and I suspect that most of the changes are neuronal in these young animals. We do get some opposite changes in the adult with perchlorate. I did not have time to go into this, but they suggest that possibly glial cells have a different response, although it is hard to sort this out. R. V. COXON:Could I ask Dr. Woodbury a rather simple technical question? How much CSF can you get out of a 17-day rat, in pl? L. BAKAY: This question occurred to me too. D. M. WOODBURY: We gan get about 10-25 ,d from a 17-day-old rat and about 5-15 111from newborn to 9-day-old rats. In fetuses we get about 2-5 pi. R. V. COXON:From where? From the ventricle?

D. M. WOODBURY: From the cisterna. We use capillary tubes similar to those that are used in pulling microelectrodes and make micropipettes out of them. The CSF is remove from the cisterna of anesthetized animals. The fluid is sucked up by capillary action. CSF is actually easier to obtain than is blood. D. H. FORD:The iodide space which you measured, does this include the iodide in the vascular space and that which would perhaps diffuse out? The reason I ask this is that we have done some studies quite a long time ago, where we did radio-autography after injection of sodium iodide, and I would say that over 90% of the activity in our brains was in the vessels.

D. M. WOODBURY: Blood iodide cannot account for the iodide space we measured since we drain all the blood out of the body from the aorta and residual blood left in the brain cannot account for the amount of iodidL in the brain. We have measured the blood volume under these circumstances and find that it is much smaller than the observed iodide space. R. CUTLER: Some of the older anatomists and neurosurgeons have claimed that young animal and human offspring have no arachnoid granulations. I wondered if you have any data bearing on the bulk clearance of the spinal fluid at this age, or whether the increase in CSF/plasma ratio for inulin and the brain/plasma ratio for inulin has resulted in an increased entry rate in this age group.

D. M. WOODBURY: I don't know about the arachnoid granulations. We have not actually measured the clearance in these circumstances. It is certainly a possibility that the slow flow might be due to lack of development of the arachnoid granulations, but it is more likely, and we have evidence for this, that it is due to the fact that the CSF-secretory process has not yet developed. Maybe some of the histologists or anatomists here know about the development of the arachnoid granulations during this period of maturation.

Factors Influencing Barrier Function

Changes in Barrier Effect in Pathological States LOUIS BAKAY Divisiori of Neitroatrgery, Sture Utiiversitj, of New York a1 BuJyirlo, School of Mediciiie, 462 Gride Street, Bitflulo, N . Y . ( U.S.A.)

Diseases of the central nervous system that are severe enough to alter its structural organization result in localized or generalized increase in the permeability of the blood-brain barrier. From an historical point of view, the principle of the bloodbrain barrier was established by Goldman (1913) who based his theory on two postulates. His “first experiment” revealed that the central nervous system does not stain after the administration of a vital dye into the blood stream. The “second experiment” showed that diffuse coloration of the nervous system occurred when the same dye was injected into the cerebrospinal fluid. Although it was not put in the form of a third postulate, it was accepted from the beginning that lack of staining in the “first experiment” did not include those parts of the brain that were not nervous in structure (choroid plexus, meninges, etc.) or those that were affected by a pathological process. In patho-physiological research, the blood-brain barrier still denotes a hypothetical structure or mechanism with specific vulnerability and almost general rate-limiting importance (Edstrom, 1961). Although a great deal of research, utilizing the latest anatomical and chemical methods, has been carried out over the past years to elucidate the nature of the normal blood-brain barrier, a similar sophistication in aim and methodology was less noticeable in studies of the pathological state. This is perhaps understandable when one considers the enormous impact of any change in permeability of the blood-brain barrier in clinical medicine. The increased permeability or absence of the barrier in various lesions became an accepted fact; the main emphasis was placed on the exploitation of this situation for diagnostic or therapeutic purposes without giving too much consideration to the basic factors that are responsible for its occurrence. From a diagnostic point of view, changes in the blood-brain barrier permeability are used for localization of tumors and other lesions by radioactive brain scanning. The same principle allows for the treatment of cerebral infections by antibiotics and of neoplasms by chemotherapeutic agents ; the substances applied for diagnostic or therapeutic reasons have ready access to the lesion while, on the other hand, they are more or less excluded from the normal brain. A complete review of all pathological conditions affecting the transfer of substances from blood to brain would be prohibitive in size. Consequently, I selected a few specific examples of pathological conditions to illustrate some of the basic problems. Kernicterus and other types of bilirubin pigmentation of the brain were chosen, not R c f i r m w r pp. 336-339

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only because of their clinical importance and intriguing patho-physiology, but also because they were historically the first type of lesion where the blood-brain barrier as the cause of the disorder was suspected. The various types of cerebral edema represent a pathological condition where increased barrier and membrane permeability are determining factors. Brain injuries are characterized by derangement of the barrier; this group is followed by a review of a much morecomplex subject, namely, the state of the barrier in tumors, Our knowledge of the latter, although still incomplete, was increased lately by a great flow of information derived from radioactive brain scanning. B I L I R U B I N A N D T H E B L O O D - B R A I N BARRIER

(Kernicterus)

Perhaps the first application of the blood-brain barrier theory to a clinical problem was its implication in the development of kernicterus. It was recognized early that bilirubin staining of certain portions of the brain, particularly that of the basal ganglia, can frequently be seen in icteric newborn while, on the other hand, adults, even with severe and long-lasting jaundice, do not show any bilirubin deposits in the central nervous system except for that of the choroid plexuses and cerebrospinal fluid. It was originally thought that bile pigments deposit in the newborn brain because of the physiologically undeveloped and more permeable blood-brain barrier which, when fully developed and "leakproof", keeps them out of the mature brain. This hypothesis was further strengthened by the discovery that areas of tissue damage in the adult brain (infarcts, hemorrhages, contusions, tumors, etc.) do stain with bilirubin in hyperbilirubinemia. It seemed logical to assume that kernicterus represents a clinical variant of vital dye experiments, trypan blue being simply substituted by a biological pigment, bilirubin. However, as time passed by, investigators became increasingly aware that kernicterus of the newborn is caused by a combination of circumstances; the relative importance of the individual factors is not yet known. Space does not permit a complete coverage of the vast literature on this subject. A comprehensive review on kernicterus up to 1959 was edited by Sass-Kortsik (1961). The results of most investigations indicate that staining of brain tissue by bilirubin in infants represents more than a simple change in permeability of the blood-brain barrier, although an increase in permeability might be a predisposing factor. According to Hugh-Jones et a / . (1960), kernicterus is most frequently observed in erythroblastosis fetalis; it occurs only rarely in nonerythroblastotic premature infants in association with severe hyperbilirubinemia. Nevertheless, it is known that kernicterus and increased bilirubin in the CSF may occur in conditions other than hemolytic disease of the newborn in rough proportion to the degree of hyperbilirubinemia (Stempfel, 1955). The variability in the occurrence of kernicterus is considerable; well-developed and undamaged infants might not develop it even in the presence of very high serum bilirubin levels (Zuelzer and Brown, 1961). At the same time, Harris et al. (1958) observed kernicterus in conjunction with relatively low levels of bilirubin in premature

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infants. It is generally agreed upon that the presence of indirect bilirubin in erythroblastosis fetalis is important, although its concentration in the plasma by itself is not unequivocally associated with the development of kernicterus. Local tissue injury is assumed to be present in addition to prematurity, increased barrier permeability, and hyperbilirubinemia to account for the staining of the central nervous system. The development of kernicterus might be promoted by the specific metabolic vulnerability of certain areas of the newborn brain, by anoxic damage, or by the direct toxic effect of bilirubin. Bilirubin is a toxic substance (Day, 1956; Waters and Bowen, 1955; Ernster et a / . , 1957), and Ernster et a / . (1957) showed experimentally that it is capable of inhibiting oxidative phosphorylation in neural tissue. The localization of ATP depletion and impaired respiration within the hyperpigmented brain suggested to Schenker et al. (I 966) that impaired phosphorylation may be an important feature of kernicterus. It is important to remember that most of the serum bilirubin is albumin-bound. Unbound and potentially neurotoxic bilirubin, available for transport into the brain, does not usually amount to more than a fraction of total bilirubin. Although the relative proportion of protein-bound bilirubin in plasma is variable, Odell (1959) as well as Blanc and Johnson (1959) believe that unconjugated, and consequently, more toxic bilirubin is the cause of brain damage because it leads to direct necrosis of the nerve cells. Diamond and Schmid (1966) presented conclusive evidence that only unbound bilirubin is able to cross the blood-brain barrier and that the pigment level of the nervous system and bilirubin neurotoxicity are related to the unbound, rather than to the total, pigment concentration in the plasma. These investigators used 14C-labeled bilirubin which lent itself to more accurate determination in the brain tissue than analysis based on color changes. Diamond and Schmid (I 966) could not reach definite conclusions concerning the regional deposition of bilirubin in kernicterus. They thought that this might be related to the binding and retention of the pigment by already damaged cells, but they also considered the possibility that “selective areas of the brain are a priori more vulnerable to bilirubin and once damaged, retain the pigment more avidly.” Some of their data could be considered in favor of anoxia or respiratory acidosis being responsible for bilirubin deposition because acidosis enhanced the accumulation of bilirubin in the brain, but this conclusion remains only tentative. The clinico-pathological syndrome of kernicterus observed in rats with congenital deficiency of glucuronyl transferase (Gunn’s, 1938, strain of rats) is similar to human kernicterus (Blanc and Johnson, 1959). These rats were used for the experimental study of kernicterus without any difinite answer as to its etiology, although it is clear from the experiments of Menken et al. (1966) that hyperbilirubinemia is necessary, but not alone sufficient, for the uptake of [“C]bilirubin by the brain. The brain tissue of kernicteric animals contained significantly more isotope than tissue from healthy jaundiced animals. Other experimental models yielded equivocal results. Nuclear jaundice and pigmentation of the nerve cells closely resembling that of human kernicterus were produced in healthy newborn kittens by repeated intravenous K~J.jrrcvtr05 pp.

336-339

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injections of a bilirubin-albumin solution (Rozdilsky and Olszewski, 1961). However, these investigators were unable to obtain the same effect in newborn puppies and rabbits. In these two species, additional damage to the nervous system was necessary in order to produce kernicterus. Recently, Rozdilsky (1966) came to the tentative conclusion that under these experimental conditions, albumin counteracts the toxic action of bilirubin on the brain. While some of the experiments indicated the toxicity of bilirubin as the primary noxious factor in kernicterus, Polani (1954) concluded from his experiments in rats that the neurological damage may be associated principally with hepatic damage. The combination of anoxic damage and hyperbilirubinemia has also been considered to be the cause of kernicterus. As Dobbing (1961) pointed out in his critical review on the blood-brain barrier, anoxic damage is a frequent condition in premature infants, and the regional distribution of damage in cerebral anoxia and in kernicterus is somewhat similar. Lucey et al. (1 964):observed that the commonly observed “physiological jaundice” in newborn rhesus monkeys does not lead to kernicterus; neither does the administration of indirect-reacting bilirubin. However, kernicterus can be produced in newborn monkeys by rendering them hyperbilirubinemic after a period of temporary asphyxia. Chen et al. (1966) described electron microscopic changes in kernicterus of newborn rabbits associated with asphyxia as well as hyperbilirubinemia. The ultrastructural alterations suggested that the access of bilirubin to the intracellular compartments of the brain is dependent upon the effect of acute anoxia. They assume that anoxia provokes an increase in the permeability of the cerebral capillaries and simultaneously increases the permeability of the cell membranes. The importance of cerebral anoxia in the development of kernicterus has not been accepted without reservations. For instance, Malamud (1963) studied the distribution and degree of CNS damage in newborns in various types of hypoxemia and concluded that the findings in anoxic damage caused by perinatal trauma or convulsions differ from those of kernicterus. “The lesions observed in convulsive disorders could best be defined as a hypoxaemic encephalopathy and those in kernicterus as possibly a bilirubin encephalopathy. Their occasional coexistence does not detract from their specificity” (Malamud, 1963). In contradistinction to the kernicterus of infants, bilirubin in jaundiced adults might simply mark abnormal brain tissue. The relationship between the duration and severity of hyperbilirubinemia and the transfer of bilirubin into the CNS or CSF is not clear. Although bilirubin is frequently found in the CSF in various types of hepatitis, leptospirosis, cirrhosis, and biliary obstruction, Galambos and Rosenberg (1959) could not establish a definite correlation between the duration of jaundice and CSF bilirubin concentration nor between the bilirubin and protein content of the spinal fluid. Table I, compiled from our own patients with known plasma bilirubin values and cerebral lesions studied at autopsy, demonstrates a certain degree of correlation between the presence or absence of staining of the lesion and the plasma bilirubin level, particularly that of indirect-reacting bilirubin. However, the number of these cases is not sufficient to draw definite conclusions, particularly concerning the relative

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TABLE I B I L I R U B I N S T A I N I N G O F CEREBRAL LESIONS

__

Cerebral Lesion

Caiise

Total

I.

Hepatitis

2 weeks

61.2 mg

2. Ca, liver 3. Cirrhosis 4. Ca, liver

3 weeks 2 months 1 week

23.3 mg 22.0 mg 13.0 mg

5. Ca, liver 6. Hodgkin’s granuloma, liver 7. Colelithiasis

6 weeks

20.4 mg

12 days Transitory, severe for 8 weeks but resolved 5 weeks before death

9.2 rng 14.0 mg

Staining

Indirect 36.9 mg Infarct (approximately 1 month) 14.1 mg Metastasis 13.2 mg Hemangiorna 4.9 mg Old infarct Recent infarct (few days) 7.5 mg Metastasis ? Contusion 2.0 mg Old infarct Recent infarct (approximately 3 weeks)

-1-

+

+ -

+

-

importance of protein-bound or diffusible bilirubin. There is no valid theoretical reason against the assumption that protein-bound bile pigment deposits in most of these gross brain lesions without difficulty. Summary

One can state that at the present time, the exact mechanism of bilirubin transfer and deposition in the brain of the hyperbilirubinemic newborn is not known. It seems to be certain that the process involves more than increased barrier permeability. However, the relative importance of pre-existing cellular damage (most likely anoxic) versus secondary tissue damage due to bilirubin toxicity in kernicteruscannot be assessed, yet. CEREBRAL EDEMA

Edema of the brain is produced by the increase of its water content. In addition, there is a variable increase of solutes ranging from electrolytes to large protein molecules. It is, therefore, obvious that this condition is of considerable interest from the point of view of the blood-brain barrier because the excess fluid and particulate matter originates in the blood plasma and could hardly pass into the nervous tissue without a change in membrane permeability. This is particularly true in those types of brain edema where the edema fluid is proteinaceous; the presence of serum albumin in the fluid is prima facie evidence of increased barrier permeability since, under normal circumstances, the exchange of large protein molecules between plasma and brain tissue is minimal. In addition to the blood-brdin barrier aspect, recent electron microscopic investigations of edematous brains led to important discoveries concerning the extracellular Refirmwr pp. 336-339

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space, a compartment that must always be considered when attempting to measure barrier permeability. Under normal conditions, the space between the cells of the central nervous system is narrow, although the narrowness of these clefts does not necessarily disqualify them as important pathways of fluid and small molecules (Nichols and Kuffler, 1964). It seems that in the gray matter, edema is almost exclusively intracellular. In the white matter, excess fluid might accumulate, either between the cells, thereby greatly enlarging the extracellular compartment, or in the cells. Studies on cerebral edema clearly indicate that osmotic changes in plasma and brain fluids, both extra- and intracellular, result in rapid changes in fluid and solute transport between the various tissue compartments. The effect of these fluxes on barrier permeability should not be underestimated. Recent experiments by Klatzo, Wisniewsky, and Smith (1965) indicate that such reversible changes in plasma osmolality as that caused by intravascular infusion of 30 % glucose result in a highly abnormal permeability of the cerebral capillaries for proteins. The “leakiness’ of the blood vessels is of short duration and disappears as soon as the plasma osmolality approaches normal values. Edema of the brain is not a single entity. Some of the various types of brain swellings differ not only in the localization and chemical composition of the excess fluid but also in the permeability of the blood-brain barrier. The chemical and structural characteristics of various brain edemas were recently described by Bakay and Lee (1965); the brief presentation which follows will be limited to the observations on barrier permeability.

Cold-induced edema The edematous white matter shows vital coloration after intravascular trypan blue administration in an experimental model where the swelling of a hemisphere is produced by freezing of the cortical surface. Similar staining was seen after the application of Evan’s blue (Clasen et al., 1962) and fluorescein-labeled serum proteins (Klatzo et al., 1962). The vital staining of the swollen white matter is subject to the time which has elapsed from the injection of the dye (Bakay and Haque, 1964). The dye extravasates at the marginal zone of the injured cortex and infiltrates the edematous white matter gradually, reaching complete distribution only 24 h after its administration. The same spatial and temporal distribution was found by using RlSA as a tracer (Bakay and Haque, 1964), this corroborates previous theories that many vital dyes, including trypan blue, are protein-bound in plasma, and their recovery in the abnormal brain tissue corresponds to that of plasma proteins. A similar, gradual diffusion from the point of entrance (the marginal area of the cortical lesion with its leaky blood vessels) into the area rendered edematous was observed for eleztrolytes, particularly for sodium and chloride. However, since the relative distribution of these cations and anions inside and outside the cell membrane is not well-known, under these circumstances, the conclusions concerning the permeability of the blood-brain barrier are best based on the entrance into the brain of proteins for which the barrier is normally impermeable. In summary, the bloodbrain barrier was found to be grossly altered and increasingly permeable for large

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32 I

particles in cortical freezing at the level of the border zone of the lesion. From there, the extravasated components of plasma penetrate the edematous white matter subsequently; however, the permeability of the blood-brain barrier within the region of the edema, itself, is not perceptibly altered. Edema surrounding cerebral neoplasms and other space-taking lesions has long been known to show coloration with vital dyes and to reveal an increased exchange of other substances including proteins with plasma. The edema of this type as well as the changes in barrier permeability are probably identical with that seen after trauma; the propagation of serum exudate into the surrounding brain tissue is facilitated by the leakiness of the neoplastic vessels.

Other traumatic edemas Vital staining was observed in brains rendered edematous by exposure and manipulation (Prados et a/., 1945), by implantation of dry psyllium seed (Sperl et a/., 1957; Samojarski and Moody, 1957), and by inflating a balloon placed in the extradural space (Ishii et a/., 1959). The dyes applied included trypan blue, acridine dyes, Evan’s blue, and di-iodo-fluorescein. In some of these experiments, tissue proteins and electrolytes were studied. Significant increase of albumin was found in the swollen tissue surrounding implanted psyllium (Hauser et a/., 1963). The greatest concentration was found nearest to the psyllium mass; it decreased progressively as the distance ofthe tissue from the lesion increased. Cutler et al. (1964) studied the movement of 1251labeled serum albumin in brain tissue rendered edematous by balloon compression. They found a close relationship between the penetration of albumin and Evan’s blue, indicating that much of the dye is protein-bound. Just as in cold-induced edema, the passage of albumin from blood into brain tissue occurred at the marginal zone of trauma. From this point of entry, the edematous tissue, which was strictly localized in the white matter, was gradually penetrated by serum albumin, indicating a break in the blood-brain barrier at the level of the lesion but not in the edematous white substance.

Injlammatory edema

In inflammatory edema, which is produced by intracerebral injection of bacterial endotoxins, purified protein derivative of tuberculin, and such additional substances as graphite, the permeability of the blood-brain barrier was increased for Geigy blue (Gonatas e t a / . , 1963). Here, again, the edema is located in the white matter and consists of a protein-rich exudate. There is a considerable freedom in exchange of RISA between plasma and edematous tissue (Katzman et al., 1964). Triethy It in - induced edema Triethyltin-induced edema is of considerable interest regarding the permeability of the blood-brain barrier because it seems to be very different from other types of brain Refiwnccs pp. 336-339

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swelling. Edematous changes are limited to the white matter. The excess fluid, at least in its initial stage, is a non-proteinaceous plasma ultrafiltrate situated in intramyelinic vacuoles which are produced by a split in the myelin sheath (Aleu et al., 1963; Lee and Bakay, 1965). There is no pathological alteration in the ultrastructure of the capillaries, glia cells, or in the size of the extracellular space. It is, therefore, perhaps not surprising that the permeability of the blood-brain barrier for large molecules is not affected. The edematous white matter does not stain with trypan blue and other vital dyes, and large molecules such as albumin do not penetrate the edematous tissue from the blood stream (Magee et al., 1957; Kalsbeck and Cumings, 1962: Katzman et al., 1963; Bakay, 1965). This represents a striking contrast with traumatic and inflammatory edemas. The lack of abnormal morphological changes of the vasculature as well as that of the glia cells is undoubtedly responsible for the normal state of the barrier; unfortunately, this normal state of all structures believed to be instrumental in barrier function does not allow us to draw significant conclusions from triethyltin edema as to the morphological basis of the blood-brain barrier. Edema associated nith cerebral anoxia

Edema associated with cerebral anoxia reflects only one aspect of a complex metabolic disturbance. The exact nature of the changes in tissue structure and metabolism is not adequately known; it is particularly difficult to separate the disturbances pertaining to membrane permeability and to the blood-brain barrier in general from those of anoxic damage to the cells, if such a distinction is, indeed, possible. The complexity of varicus factors involved can be illustrated by the fact that there is very early structural damage in the mitochondria (Bakay and Lee, 1967) that are quite resistant in other types of edema, while on the other hand, the fluid accumulation in anoxia is very moderate. Clearly, edema in anoxic brain damage is only one of many alterations. Studies were directed to investigate the permeability of the capillaries and of the blood-brain barrier, in general, under anoxic conditions because a change in permeability was considered to be essential for the development of edema. The findings obtained with the use of vital dyes were contradictory. Broman’s (1949) experiments on cats showed that complete occlusion of the cerebral circulation caused no disturbance in vascular permeability for trypan blue. Grontoft (1954) came to a similar conclusion, stating that in adults, anoxic injury to the blood-brain barrier cannot be demonstrated with trypan blue, although in infants, barrier damage is closely related to the degree of asphyxia. Becker and Quadbeck (1952) used a more easily permeable vital dye, Astroviolett FF. They were able to demonstrate barrier damage in anoxia produced in a high-altitude chamber and in experimental sub-acute carbon monoxide poisoning at a stage when there was no microscopic evidence of tissue damage. Hodges et al. (1958) used fluorescein to study the relationship between hypoxia and blood-brain barrier permeability with special regard to the relative value of various perfusion techniques in vascular surgery. They found that the appearance of fluorescein in the central nervous system was a sensitive indicator of faulty perfusion technique or inadequate pump-oxygenators with cerebral hypoxia as a result.

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It is, of course, conceivable that permeability changes exist for electrolytes and small molecules, even in the absence of gross changes to vital dyes, because most of the vital dyes used are essentially protein-bound. Factors other than hypoxia, alone, may be responsible for the transfer of substances from blood to brain. Arterial increase in C02 concentration, when severe enough, might be a more important factor. Temporary and easily reversible increase in the permeability of the blood-brain barrier for trypan blue was observed in rabbits by Clemedson, Hartelius, and Holmberg (1957) on inhalation of a gas mixture containing 10 % COZor more. In their opinion, hypercapnia should not be disregarded as a cause of cerebral damage, particularly in asphyxia where the component of anoxia “sometimes seems to have been over-emphasized.” Brierley (I 952) noticed an increase in ~erebral,3~P uptake after inhalation of a C02 and 0 2 mixture, but only when the mixture contained 20 % C02. His tentative conclusion was that the increase in the SZP content of the brain was either due to vasodilatation or to increased capillary permeability, or to both. Goldberg, Barlow, and Roth (1961) showed that exposure to 25 % COZ increased the cerebral concentration of phenobarbital, salicylic acid, acetazolamide, and urea in cats. This increase was not related to changes in blood flow and could not be brought up by inhalation of 5 % C02. Exposure to a high C02 content in the inhaled gas mixture with subsequent severe hypercapnia seems to increase the permeability of the blood-brain barrier, even without additional hypoxia. Goldberg, Barlow, and Roth (1963) studied the effect of 25 % COZ on the cerebral uptake of [35S] sulfate and [14C] urea. They found that although the steady-state sulfate space had not changed, the entry of 35s was greatly enhanced within a few minutes after injection. They also observed regional differences in the cerebral deposition of SSS, including an increased concentration in the central core of the white matter, which suggested irregular changes in vascular permeability. Bakay and Bendixen ( 1 963) separated, experimentally, the various factors involved in asphyxia, namely, anoxia, CO2 retention, and increased venous pressure. They found that real brain swelling occurred only when anoxia was associated with hypercapnia. However, the blood-brain barrier was quite resistant under these circumstances. Although there was an increase in the exchange of electrolytes between plasma and brain tissue, vital staining with trypan blue and significant uptake of albumin by the brain from the blood stream occurred only on extreme hypercapnic hypoxia with arterial 0 2 saturation below 20 % to 25 % and an arterial pH below 6.75. Severe anoxic-ischemic lesions and edema develop following the production of carotid ligation and respiratory hypoxia (Plum et al., 1963). Under such circumstances, there is vital staining and marked swelling of the affected portion of the brain. It is obvious, however, that this experimental model represents infarction and total tissue destruction rather than simple anoxic effect.

Summary Although the amount and distribution of the excess fluid varies in different types of brain edema, they all reveal an increase in tissue water as well as sodium and chloride. Rrjerenrrs pp. 336-339

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However, the exchange of large mole-ules (vital dyes, albumin, etc.) between plasma and edema fluid is limited to those cerebral edemas where structural damage to the small blood vessels can be demonstrated. Furthermore, the point of entry into the nervous system for these large particles is the wall of the injured vessel. B R A I N I N J U R I E S A N D THE B L O O D - B R A I N B A R R I E R

Fresh brain wounds stain immediately with vital dyes injected into the blood stream. They also concentrate various radioactive tracers exclusively or to a much greater extent that the surrounding normal brain tissue. Bakay (1960) found that cerebral lesions exchange sodium with plasma rapidly. This results in an early concentration of z4Na in the injured tissue (Fig. I). However, the ratio of 24Na content between injured

Fig. 1. Transverse section of cat brain (A) and corresponding radioautographs 23 min (B), 70 min (C), and 315 rnin (D)after intravenous injection of NaZ4.(From Bakay, 1960).

and normal brain tissue diminishes during the subsequent few hours because a gradual sodium exchange between normal nervous tissue and blood increases. On the other hand, the 24Na concentration of the injured part remainsconstant and then diminishes due to the fact that the radioactive sodium of the lesion remains in free exchange with that of the plasma, and consequently, decreases in linear proportion to the declining plasma 24Naconcentration. Similar observations were made with 23P (Bakay, 1955). A significantly increased uptake of 32P by the lesion when the tracer was given two hours before death still existed six weeks after trauma. In mild traumas, the increased exchange of substances might be limited to small particles such as the increased uptake of S2Pobserved during temporary concussion by Cassen and Neff (1960). However, injuries associated with visible structural alterations

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of brain tissue usually result in the exchange of serum proteins between plasma and brain. The extent of increased barrier permeability around a lesion, as well as its duration, varies according to the physico-chemical characteristics of the substance used for measurement. Lesions produced by focused ultrasound, as described by Bakay et al. (l956), were used to measure the extent of barrier damage with different indicators. Ultrasonic lesions are very suitable for this purpose because they are spherical and easily reproducible. Furthermore, they are surrounded by normal tissue and are not connected to the surface by needle tract. The results shown in Fig. 2 are based on

32

P

7-7

[I3'I]albumin

trypan blue

Fig. 2. Relative size of increased uptake by spherical ultrasonic lesion in brain tissue of 3zP, trypan blue, and ['3'l]albumin.

measurements of freeze-dried, 25 ,u sections and corresponding contact autoradiograms of cat brains thirty minutes after intravascular administrations of the tracers. Calculations of the volume of tissue with increased permeability are even more revealing. They indicate that the total area of increased uptake of RISA and trypan respectively, of that of 32P. blue is 36 % and 30 The duration of increased permeability also varies according to particle size and other unknown factors. The injured blood vessels seal their walls faster for large molecules than for small particles. As a result, increased uptake of small molecules in the lesion can still be observed at a time when the penetration of vital dyes and protein molecules from the lumen of the vessels is already arrested. Tschirgi (1950) emphasized the importance of protein complexes in the permeability of the blood-brain barrier to dyes. Bakay and Haque (1964) demonstrated the striking similarity between trypan blue and 131I-labeled serum albumin in their exchange between plasma and injured brain tissue. This strongly suggests that the great bulk of trypan blue is protein-bound and that this vital dye can be considered, for all practical purposes, a protein (albumin) tracer. In experiments involving the use of radioactive isotopes, the tracer content of the blood has to be taken into consideration since experimentally-induced cerebral

x,

Rifiwtrces pp. 336-33Y

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lesions often interfere with the blood flow, usually in the direction of stagnation. However, the measurements of Broman et al. (1961) indicated that the false increase in tissue radioactivity caused by pooled blood is less significant than the possible error produced by the rinsing of cerebral vessels. The return to normal of increased blood-brain barrier permeability is gradual, associated with the healing of brain wounds. Normal permeability is established much earlier for large particles than for small molecular substances. Abnormal barrier permeability for serum albumin, an “all-or-none’’ phenomenon , since normally, albumin does not penetrate from the vascular network into the brain substance, was first illustrated by autoradiography in brain injuries by Rozdilsky and Olszewski (1957). Lee and Olszewski (1959) studied the permeability of the blood vessels for RISA at various stages of the healing of brain wounds. The area showing albumin uptake decreased gradually with the passage of time from the injury. The barrier was no more permeable for albumin after three weeks. Abnormal permeability for small molecules, such as electrolytes, exists for a considerably longer time. Similar observations were also made in man in the form of brain scanning of patients with various types of head injuries. It is not surprising that abnormal uptake of various radioactive compounds can be seen in areas of cerebral contusion immediately after the injury (Fig. 3). The process of healing eventually results in a restoration of normal permeability; old scars cannot be visualized by scanning. By using relatively large and metabolically inert compounds, the period of increased permeability can be assessed. Van Vliet et al. (l965), for instance, studied post-surgical brain scans with

Fig. 3. Brain scan in head injury, 4 h after i.v. injection o f 750 pC of [203Hg]~hlormerodrin.Increased concentration of the labeled substance can be seen in the contused frontal lobe.

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polyvinylpyrrolidone (PVP) labeled with l 3 l I to determine the effects of surgical trauma on the vascular permeability of the brain. PVP is an inert substance of high molecular weight which is normally excluded from brain tissue. There was an increased uptake in the crmiotomy site in all patients with cortical incision or resection, or after pressure on the cortex by retraction. The intensity of localized radioactivity diminished as the post-operative period increased. Eventually, the scans became normal ; no positive scan was seen after 79 days following craniotomy. The increasing use of brain scanning in man with different radioactive indicators will contribute, in the future, to our understanding of the spatial and temporal aspects of blood-brain barrier permeability. From a basic point of view, explanations of the blood-brain barrier phenomena can be arranged in two main groups. One theory lays emphasis on the permeability of the capillary wall while the other identifies the selective barrier function with the specific function of the nervous tissue proper. When applied to pathological conditions, this second theory would imply that metabolic, rather than vascular, changes within the altered area of the brain are responsible for the increased concentration of various substances. Although investigative work on the barrier permeability of injured brain tissue involved the use of not only metabolically active but also, inert substances, the relative distribution of the various tracers in the intracellular and extracellular compartments at various time intervals after their transfer into the brain cannot be evaluated from the majority of the published results. The relative importance of the different vascular and cellular membranes remains, therefore, obscure. Hess (1955) thought that a PAS-negative ground substance is responsible for the “blood-brain barrier effect” because this substance, presumably consisting of mucopolysaccharides, disappears in brain wounds to be gradually re-elaborated after a while However, this theory cannot be confirmed by electron microscopy which fails to reveal the presence of an intercellular ground substance. A modified hypothesis (Millen and Hess, 1958) was then put forward that the ground substance immediately surrounding the blood vessels may play an important role in the maintenance of the blood-brain barrier, but this assumption also fails to hold up under electron-microscopic scrutiny. Bakay et a/. (1959) measured the uptake of 32P by normal and injured brain tissue by applying the tracer solution directly to the cerebral tissue by supra-cortical or cisternal application excluding, thereby, vascular channels. During the process of absorption of 32P by the brain tissue, there was no increase in concentration of the isotope within ultrasonically-produced lesions when compared with the surrounding normal brain (Fig. 4). This represented a marked contrast to other experiments that demonstrated high uptake of 32P by the same type of lesions after intravascular administration. The conclusion of Bakay et a/. (1959) was that there is no selective concentration of 32P in the lesion once the isotope has been made equally accessible to injured, as well as normal, tissue. The route by which 32Parrives at the lesion site would be irrelevant if the increased uptake by the lesion werecaused by cellular metabolism alone. Similar observations were made with 24Na; intra- and subcortical lesions did not take up more sodium than normal brain tissue after the direct application of an isotonic 24Na solution over the pia-covered surface. This represents a striking R&rciiczs p p . 336-339

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Fig. 4. Upper: Unstained; dry-frozen section of both upper parietal lobes of cat. Arrow points to vitally stained lesion in the lower layers of the cortex, produced by focused ultrasound 4 h before death. Middle: Radioautograph of same section 200 min after application of isotonic solution containing 32P over thepia-covered cortex. Exposure time: 3 days. Lower: Radioautograph of the same section. Exposure time: 4 days. (From Bakay et a/., 1959).

contrast with the fifteen-to-one ratio between injured and normal brain 24Naconcentration within the first hour after intravascular injection of the isotope (Bakay, 1960). Summary

Structural injury to the brain tissue results in an immediate increase of transfer from blood into traumatized brain of substances that normally are transported slowly or not at all. The abnormal exchange comes to an end at a certain stage of the healing process, earlier for macromolecules than for substances of small particle size. There is some evidence that an increase in vascular permeability characterizes the initial phase of altered barrier permeability; the subsequent migration, retention, and participation of the tracer substances within the nervous tissue is the result of a combination of factors that, at the present time, defy detailed analysis. BRAIN TUMORS

Most brain tumors accumulate various substances, such as vital dyes, various radioactive isotopes, etc., from the blood stream. The exchange of these substances between plasma and tumor tissue is relatively free, or at least much faster than their exchange between plasma and normal brain tissue. The concentration of many radioactive compounds is much greater in the tumor than in the surrounding brain tissue, and thus, a tumor/brain concentration ratio exists which can be measured and exploited for diagnostic purposes. This principle is the basis of radioactive brain scanning for the diagnosis and localization of intracranial neoplasms. In order to determine the possible mechanism responsible for the uptake of sub-

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stances by normal and neoplastic brain tissue, one has to consider differences in vascular permeability, size of extracellular space, and metabolism. Vascular permeability Brain tumors, particularly glioblastomas and metastatic tumors, contain abnormal blood vessels. Some of these vessels take the shape of vascular malformations including tortuous, lacunar, and aneurysmal dilatations, glomeruloids, and arteriovenous shunts. Nystrom (I 960) believes that purely mechanical factors are responsible for the irregularities of the vascular lumen: “Dilatation of the wall in shunts, glomeruloids, and lacunar or aneurysmal vessels was considered to be the result of locallyincreased blood pressure bearing upon a defective, and consequently, weak portion of the vessel wall, the elevated intravascular blood pressure being due to narrowing of lumen distal to the defective portion” (Nystrom, 1960). Such abnormalities could easily be responsible for a change in vascular permeability in terms of increased filtration. In addition, there seems to be a profound metabolic (anaplastic) change in the cellular components of the neoplastic vasculature, at least in malignant brain tumors. This is manifested by endothelial proliferation in the wall ofarterioles, venules, and capillaries. Hagerstrand (I 961) considers the vascular proliferation seen in cerebral metastases as a sign of a specific reaction of the blood vessels of the brain because similar changes cannot be seen either in the primary tumors or in their seedings to other organs but the brain. The possibility of increased permeability across the neoplastic capillary wall by heightened active transport, in contrast with increased diffusion through a structurally deficient vessel, must also be seriously considered. Wright (1963) followed the development of vascularization in artificially-induced ependymoma of mice. Initially, the newly formed arteries were thin-walled and had a poorly defined muscular layer. With the passing of time, the cellular arrangement became more anaplastic; this occurred at the same time as other, profound changes in the vasculature, namely, the development of arterio-venous communications and thin-walled sinusoids that emptied into large, tortuous veins. On electron microscopic examination, the blood vessels of glioblastomas reveal signs of increased metabolic processes in the endothelial cells (accumulation of mitochondria and vesicles) as well as vacuolization and marked variation in breadth of the basement membrane (Nystrom, 1960). Nystrom (1960) also pointed out that the vascular changes were much less pronounced in gliomas of a lesser degree of malignancy. The ultrastructure of the blood vessels of astrocytomas and oligodendrogliomas did not differ essentially from that of normal vessels. This, of course, might be one of the reasons why radioactive brain scan is sometimes “negative” in these gliomas. However, the electron microscopic morphology of the capillaries involved in tumor formation has not been adequately studied, and further investigations are needed. According to Torack (1961), capillary changes vary depending on the involvement of the tissue in the pathological process. In the tumor, itself, the endothelium is hyperplastic, and the perivascular zone is enlarged. In the peripheral zone of the neoplasm, the basement membrane is thickened, R i ~ r r c v i1.5 i pp. 336-339

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fenestrated, and filled with cell processes and dense bodies of lipid material. Torack (1961) related these changes to the increased vascular permeability for electrolytes and colloids, and to the phagocytic activity of the pericapillary cells. Although a parallel between embryonic and neoplastic blood vessels, connoting increased permeability of their walls, is frequently drawn, Tani and Ishii (1963) were unable to arrive at a definite conclusion. As far as their ultrastructure is concerned, embryonic capillaries have a poorly developed basement membrane which might explain their leakiness. However, tumor vessels have many additional features, such as the increased metabolic activity of their endothelial cells and perivascular astrocytes, or the close attachment of tumor cells to the basement membrane, as well as the enlarged extracellular space in the vicinity of the blood vessels; any of these structural peculiarities or a combination of them all could be responsible for the increased exchange of material between plasma and tumor tissue.

Blood content of tumor tissue There is considerable variation in the volume of blood per unit of tumor tissue as well as in the speed of its circulation (Ganshirt and Tonnis, 1956) compared to that of normal brain which averages about 0.5 % in the white matter and 2.5 % in the gray matter. Although the blood content of a mature astrocytoma might not be greater than that of normal brain tissue, most tumors contain more blood vessels per unit of tissue than normal brain. Although we do not have enough data, it is obvious that in a vascular tumor, blood volume is a factor that has to be taken into consideration even without assuming that the vascular permeability is increased. In many tumors, the blood vessels are not only numerous but also distended. Consequently, tracers, including radioactive isotopes, can accumulate in a tumor at a time when their concentration in blood is high. Although the distinction between that part of the tracer which is still contained to the vascular lumen and that which has egressed into the surrounding tissue is probably quite arbitrary after a short interval following its injection into the blood stream, the amount pooled in the blood is important in vascular tumors with a slow transcirculation time, such as some meningiomas. Long et al. (1963) emphasized that those substances which produce a high tumor-to-brain concentration ratio are protein-bound in plasma; this statement implies that a significant portion of these tracers remains in the blood stream. Nevertheless, it seems to me that the importance of radioactive blood contained by the tumor as the main source of some positive brain scans has been over-emphasized in the past. Experimental tumor, such as implanted fibrosarGomas (Matthews and Molinaro, I963), revealed a residual blood volume of only 4.6 % which was less than double that of the normal brain and could hardly be held responsible for the accumulation in the tumor of 1311-labeled albumin, although this large molecular tracer remains in the blood for a considerable length of time. Pinocytosis At this point, it is worth reviewing the possible mechanism of the uptake by tumor of

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substances with large molecular weight. Some of these compounds are very valuable in radioactive brain scanning and in the study of altered barrier permeability. It is also known that they are transferred into the neoplastic tissue from the blood stream by other means than simple diffusion. In contrast, they remain almost entirely within the vascular lumen in normal brain tissue. It is not difficult to explain their presence in necrotic brain or tumor tissue: there, the continuity of small blood vessels is broken, and blood or plasma floods the surrounding tissue, carrying with it almost undiluted amounts of radioactive proteins. However, this mechanism does not apply to neoplasms with reasonably intact vasculature. Few attempts were made to determine the transcapillary exchange of large particles, particularly albumin, in brain tumors ; the conclusions are still somewhat tentative. The exact localization of RISA in brain tumors at the cellular level by light microscopy and microscopic radioautography is all but impossible. Tator et al. ( I 965) were unable to distinguish between vascular and cellular factors in the deposition of radioactive serum albumin in various brain tumors. The incorporation of RISA in cells by pinocytosis is strongly suspected because this mechanism of transfer was observed under other pathological conditions (Klatzo and Miquel, 1960). Incorporation of large molecules into cells of the vascular wall is occasionally seen by electron microscopy in normal brain; the number of pinocytotic vesicles formed by endothelial and glial cells increases greatly in traumatized, and presumably, neoplastic nervous tissue. Raimondi (1964) attempted to localize RISA in human brain tumors at the electron microscopic level. In his opinion, serum albumin is transferred into the capillary endothelium and beyond by pinocytosis. This conclusion is supported by the observation that the number and size of pinocytotic vesicles is increased in tumor tissue, an observation which is also shared by Bakay and Lee (1967). Although pinocytosis is obviously operational in the bulk transport of large molecules, its relative importance in the uptake of these compounds by tumor tissue awaits further clarification. Since pinocytosis is a relatively slow, gradual process, it fails to explain completely the increased barrier permeability that is noticeable immediately after the injection of the tracers.

Extracellular space

It has been suggested by several investigators that the main difference in the uptake of various substances between tumor and normal brain tissue is due to the respective size of their extracellular spaces. This reasoning closely follows the argument that the blood-brain barrier effect in normal brain is not caused by a hindrance to passage through the capillary wall, but rather, at the level of the cell membranes of the central nervous system. The relative impermeability of the normal “barrier” would, then, be based on a functionally inadequate extracellular compartment. An extreme view, no longer accepted, denied the existence of any measurable room between the cells; this theory, however, is contrary to both morphological and physiological evidence. Nevertheless, a comparison of the intercellular space of normal and neoplastic Hifcwnc cs pp. 336-339

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brain tissue should be undertaken to clarify this issue. Unfortunately, there are no sufficient data for this purpose. The exact size of the extracellular compartment is a matter of conjecture even under normal conditions. This subject has been reviewed recently by Bakay and Lee (1965). The present estimate of the extracellular space ranges from 5 % to 20 % of volume; most investigators favor the 15 range. However, there is a difference between the gray and white matter in this respect, and regional differences may exist as well. Although a systematic survey on the size of the extracellular space in brain tumors has never been undertaken, electron microscopic observations by Raimondi el al. (1 962) suggest that an anatomically large extracellular compartment is not likely to be the reason for the increased uptake of different substances by a neoplasm. The intercellular clefts in astrocytomas and glioblastomas are probably not significantly wider than in normal brain tissue, except for those spaces that surround degenerating cells. In meningiomas, which almost invariably concentrate radioactive tracers in high concentration, the cells are positioned closely to one another. Whether the “functional” extracellular space, a compartment that might include some intracellular elements and reflects the distribution of primarily extracellular test substances rather than a visible space, is larger in tumors than in normal brain remains a moot question. Some investigators approached this problem by comparing the uptake by tumor of primarily intracellular and extracellular tracers, respectively. However, such a separation is arbitrary because we are not in a position, at the present time, to determine the relative distribution of these substances in the various compartments of the central nervous system. Analogies taken from their behavior in other organs do not necessarily apply to the brain. Matthews and Molinaro (1963), in their studies on transplanted fibrosarcomas, concluded that “for extracellular substances, tumour concentration depends on extracellular tumour spaces.” However, the weakness of their argument is revealed by the statement that the mean extracellular space of the tumor was 75.5 %; after correction for the Donnan effect and the rdtio of plasma to whole blood concentration, it was 43.6 %. This, although feasible in a few selected tumors, seems to be overly large for members of the glioma group. On the other hand, they allowed only 3.9 for the extracellular space of the normal brain; this estimate is too low because after considering that the residual blood content was 2.4 %, it would indicate a virtual absence of space between the cells. The “extracellular” substances used by these authors included 82Br, BzGa, 95Zr, 1Wb, l311, Ag, 95Nb, and 1311 serum albumin. Although some of these ions probably do not enter the cells, the purely extracellular nature of others, to name 82Br, alone, cannot be accepted. Their conclusion remains, therefore, less categoric: “. . . there also appears to be a ‘barrier’ of some kind to uptake of intracellular substances, and so lack of extracellular space will not explain all the results. Thus, there appear to be two possibilities, either (a) the blood-brain barrier is partly due to lack of extracellular space and partly to some difference in cell permeability or uptake in brain cells compared with cells in other organs, or (b) the blood-brain barrier is due to a physical barrier which is impermeable to extracellular substances but slowly permeable to intracellular substances” (Matthews and Molinaro, 1963).

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Cellular metabolism Although brain tumors do not compare favorably with normal brain tissue in many aspects of their metabolism, such as oxygenation and glucose consumption, metabolism might be responsible for their accumulation of a variety of metabolically active substances. Reasonable as this statement might be, it has to be pointed out that no substance has been found which would achieve a concentration in the brain that would exceed that of the initial blood level or those of such metabolically active tissues as liver or kidney. In fact, as Long et al. (1963) pointed out, the seemingly selective concentration of tracers in brain tumors is due to the low normal background rather than to the abnormally high accumulation in the tumor. There is no conclusive evidence that the concentration in tumors of metabolically active agents is significantly greater than that of inert substances of similar physicochemical properties. Although Selverstone and Moulton (1957) have suggested that the relatively high phospholipid fraction of tumor tissue is instrumental in its 32P uptake, the search for compounds that would selectively concentrate in brain tumors because of their participation in specific metabolic processes has not been successful. The latest investigations of Tator et a/. (1966), for instance, have indicated that the uptake of fatty acids by tumor tissue exceeds that of brain and some other normal organs. The fatty acids are partly oxidized as a metabolic fuel and partly utilized in lipid synthesis by gliomatous tissue. Despite these promising characteristics, no truly significant difference was found between the tumor uptake of [1311]oleic acid and 11:jllIserum albumin except during the initial hour of transfer. Experimental studies furnished important data but failed, so far, to supply us with definite proof concerning the role of metabolism. Mundinger (1965) used in vitro models of tumor cell colonies and investigated the uptake by the cells of various compounds used in radioactive brain scanning before and after the blockage of several metabolic processes by specific inhibitors. His conclusion was that [13lI]albumin was taken up by the cells through pinocytosis; 74As and 1311 were thought to be predominantly extracellular in location, and W u - , 206Bi-, and [2°3Hg]chlormerodrin were mostly intracellular. Some tentative conclusions were reached as to the relative role played by active transport and by diffusion in the uptake of the “extracellular” ions. Interesting as these results are, it should be kept in mind that they were obtained in tissue culture under conditions that could yield only very speculative conclusions when applied to conditions in vivo. Matthews and Molinaro (1963) attempted to correlate the uptake of different radioactive substances by tumor tissue and correlate it with their biological characteristics. The experiments were performed in subcutaneously transplanted fibrosarcomas of rats. Their reasoning in doing this was that any tumor, including brain tumors, has its own vascular structure and metabolism which is comparatively independent from where it is growing. This point, however, can be argued because gliomas, the most frequent type of cerebral neoplasms, possess structural and biological peculiarities which make them, even in their most malignant and anaplastic form, inseparably Refirrirws pp. 336-339

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part of the nervous system. Indeed, one of their characteristic traits is that they do not metastatize into any other organs. Matthews and Molinaro (1963) considered 206Bi-, 43K-, SdRb-, 65Zn-, ”Mnand 2vJHgchlormerodrin] as entirely or predominantly intracellular tracers. However, chlormerodrin is almost completely protein-bound in plasma; we do not know how much of this protein complex is broken down and metabolized in neoplastic brain tissue within the time range involved in brain scanning. Matthews and Molinaro ( I 963) concluded that “for intracellular substances, since tumour concentration is approximately constant, the ratio depends on brain concentration and hence, on blood concentration, and substances which are rapidly cleared from the blood give the best ratio”. However, they point out that no selective concentration of any substance by the tumor relative to other organs was found; the accumulation of the isotopes, even the predominantly intracellular ones, did not depend on some special property of tumor metabolism. Summary

Both structural and metabolic peculiarities could be responsible for the accumulation of substances in brain tumors from the blood stream. The relative importance of the different potential factors involved in this process remains uncertain. The theory that the blood-brain barrier is merely a reflection of cerebral metabolism is rather difficult to reconcile with isotope distribution studies (Long et a]., 1963). The data

Fig. 5. Lateral brain scan, 4 h after i.v. injection of 750 pC of [203Hg]chlormerodrinreveals the presence of a parieto-occipital tumor (glioblastoma).

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Fig. 6. Lateral brain scan, 4 h after i.v. administration of 750 pC of [?03Hg]chlormerodrin.Positive concentration of the isotope in a large area within the right hemisphere; this corresponds to a recent infarct of the brain supplied by the right middle cerebral artery.

obtained, so far, can be readily explained only by the theory that a barrier exists based on selective vascular or cellular membrane permeability, and that this selectivity is reduced or lost in tumor tissue. This assumption is based on the following considerations (Bakay, 1956): 1. Substances which accumulate in brain tumors have different physico-chemical characteristics, such as molecular weight, electric charge, solubility, degree of dissociation, etc., without a common denominator. 2. Inert or metabolically unimportant substances concentrate in the lesion as well, or almost as well, as metabolically active compounds. 3. The concentration of many tracers in tumors is of a similar order of magnitude as in other forms of pathology (brain tissue subjected to trauma, infarction, or infection; Figs. 5, 6). This was also pointed out by Heiser and Quinn (1966) who recentlycompared the brain scan pattern of ischemic infarcts and gliomas by using technetium 99m pertechnetate as a tracer. No significant difference was found between the intensity or homogeneity of the uptake in the two categories. 4. There is no true selectivity in the uptake; none of the agents studied, so far, concentrated in brain tumors to a greater extent than in some other organs of the body. Neither did the tumor concentration ever exceed the initial blood level. 5. Generally speaking, the highest concentration of isotopes is found in nonneurogenic brain tumors; these include a great variety of neoplasms from slow groRcJercnr.e.r f f . 336-339

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wing, benign meningiomas to rapidly expanding, malignant metastases from other organs. Gliomas, the truly characteristic tumors of the central nervous system, do not behave uniformly. Accumulation of tracer substances is most commonly observed in glioblastomas. The uptake rate by astrocytomas, which have a tissue structure that closely resembles that of normal brain, is usually only slightly higher or not higher at all than that of normal brain tissue. The inherent implication of this observation is that really meaningful statements concerning the neoplastic changes of the bloodbrain barrier should be limited to tumors of the glioma group since tumors that are not linked genetically to elements of the nervous tissue could not necessarily share in its organization which includes the blood-brain barrier. ACKNOWLEDGEMENT

The author’s own investigations were supported by research grant NB 03754 from the National Institute of Neurological Diseases and Blindness, United States Public Health Service. REFERENCES R. AND TERRY, R. D. (1963) Fine structure and electrolyte analysis of cerebral ALEU,F. P., KATZMAN, edema induced by alkyl tin intoxication. J. Nerrropathol. Exptl. Neurol., 22, 403-41 3. BAKAY,L. (1955) Studies on blood-brain barrier with radioactive phosphate. V. Effect of cerebral injuries and infarction on the barrier. Arch. Neurol. Psychiat., 73, 2-12. -, (1956) The blood-brain barrier with special regard to the use of radioactive isotopes. Charles C. Thomas, Publisher, Springfield, Illinois. -, (1960) Studies in sodium exchange. Experiments with plasma, cerebrospinal fluid, and normal, injured, and embryonic brain tissue. Neurol., 10, 564-571. -, (1965) Morphological and chemical studies in cerebral edema. Triethyl tin-induced edema. J. Neurol. Sci., 2, 52-67. BAKAY,L., BALLANTINE, JR., H. T. A N D BELL,H. (1959) srP uptake by normal and ultrasonically irradiated brain tissue from cerebrospinal fluid. Arch. Neurol., 1, 59-67. BAKAY,L. AND BENDIXEN, H. H. (1963) Central nervous system vulnerability in hypoxic states: Isotope uptake studies. In: “Selective Vulnerability of the Cerrtral Nervous Systeni in Hypoxaemia”. W. H. McMenemey and J. P. Schade (Eds.). Oxford, Blackwell Scientific Publishers, 63-78. BAKAY, L. AND UL HAQUE,I. (1964) Morphological and chemkal studies in cerebral edema. 1. Cold induced edema. J. Neuropathol. Exprl. Neurol., 23, 393418. BAKAY, L., HUETER, T. F., BALLANTINE, H. T.AND SOSA,D. (1956) Ultrasonically producedchanges in the blood-brain barrier. Arch. Neurol. Psychiar., 76,457461. BAKAY, L. A N D LEE,J. C. (1965) Cerebral Edema. Charles C. Thomas, Publisher, Springfield, Illinois. -, (To be published): Ultrastructural changes in the edematous central net voiis systeni. IV. Hypoxia and hypercapnia. -, (To be published): Ultrastructural changes it1 rlre edenrafous central nervous systenr. V. Peritumoral edema. BECKER, H. ANDQUADBECK, G. (1952)Untersuchungen uber Funktionsstorungen der Blut-Hirnschranke bei Sauerstoffmangel und Kohlenoxidvergiftung mit dem neuen Schrankenindicator Astraviolett FF. Z. Naturforsch. (B), 7, 498-500. BLANC,W. A. AND JOHNSON, L. (1959) Studies on kernicterus. Relationship with sulfonamide intoxication, report on kernicterus in rats with glucuronyl transferase deficiency, and review of pathogenesis. J . Neuropathol. Exptl. Neurol., 18, 165-189. BRIERLEY, J. B. (1952) The penetration of 32Pinto the nervous tissue of the rabbit. J . Pliysiol., 116, 24-25. BROMAN, T. (1949) The Permeability of’Cerebrospinal Vessels in Normal and Pa thological Cotrditions. Copenhagen, Einar Munksgaard. BROMAN, T., EDSTROM, R. AND STEINWALL, 0.(1961) Technical aspects on dyes and radiotracers in the determination of blood-brain barrier damage. Acta Psychiat. Neurol. Scand., 36, 69-75.

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CASSEN, B. mi) NEFF,R. (1960) Blood-brain barrier behavior during temporary concussion. Amer. J. Physiol., 198, 1296-1 298.

CHEN,H., LIN, C. S. AND LEIN,1. N. (1966) Ultrastructural studies in experimental kernicterus. Amer. J. Pathol., 48, 683-698. CLASEN, R. A., COOKE,P. M., PANDOLFI, s., BOYD,D. A N D RAIMONDI A. J. (1962) Experimental cerebral edema pradiiced by focal freezing. I. An anatomical study utilizing vital dye techniques. J. Neuropatliol. Exptl. Neurol., 21, 579-596. CLEMEDSON, c. J., HARTELIUS, H. AND HOLMBERG, G . (1957) Carbon dioxide and the blood-brain barrier. Acta Plrysiol. Scand., 42, (Siippl. 145), 30-31. CUTLER,R. W. P., WATTERS,G. V. A N D BARLOW,C . F. (1964) 1125-labeledprotein in experimental brain edema. Arch. Nerirol., 11, 225-238. DAY,R. L. (1956) Kernicterus: Further observations on toxicity of bile pigments. Pediat., 17,925-928. DIAMOND, I. AND SCHMID,R. (1966) Experiment11 bilirubin encephalopathy. The mode of entry of bilirubin-’T into the central nervous system. J. C h i . Invest., 45, 678-689. DOBBING, J . (1961) The blood-brain barrier. Physiol. Rev., 41, 130-188. EDSTROM, R.(1961) Citrate-buffered WrC13 - an indicator of blood-brain barrier damage - experimentally compared with IHSA. Acta Psychiat. Neurol. Scand., 36, 11 1-1 18. L. AND ZETTERSTROM, R. (1957) Experimental studies on pathogenesis of kernicERNSTER, L., HERLIN, terus. Pediat., 20, 641-652. GALAMBOS, J. T. A N D ROSENBERG, D. G. (1959) Cerebrospinal fluid in jaundiced patients. Clin. Res., 7, 168-169.

GXNsHIRT, H. AND T ~ ~ N NW. I S ,(1956) Durchblutung und Sauerstoffverbrauch des Hirns bei intrakraniellen Tumoren. Deli/. Z. Nervenheilk., 174, 305-330. GOLDBERG, M. A., BARLOW, C. F. A N D ROTH,L. J. (1961) The effects of carbon dioxide on the entry and accumulation of drugs in the central nervous system. J. Pharmacol. Exptl. Therap., 131, 308-3 18.

-, (1963) Abnormal brain permeability on COs narcosis. Arch. Neurol., 9, 498-507. GOLDMAN, E. E. ( I 91 3) Vitalfiivung am Zentralnerve~~syste~~. Berlin, Eimer. S. (1963) Ultrastructure of inflammation with edeGONATAS, N. K., Z i M M E R M A N , H. M. AND LEVINE, ma in the rat brain. Anrer. J . Pathol., 42,455-469. GROWOFT,0. (1 954) Intracranial Haeniorrhage and Blood- Brain Barrier Problenis in the Newborn. Copenhagen, Einar Munksgaard. GUNN,C. K. (1938) Hereditary acholuricjaundice in new mutant strain of rats. J. Hered., 29,137-139. HAGERSTRAND, I. (1961) Vascular changes in cerebral metastases. Acta Parhol. Microbiol. Scand., 51, 63-71.

HARRIS,R. C., LUCEY,J. F. AND MACLEAN, J. R. (1958) Kernicterus in premature infants associated with low concentration of bilirubin in plasma. Pediar., 21, 875-884. HAUSER, H. M., SVIEN, H. J., MCKENZIE,B. F., MCGUCKIN,W. F. AND GOLDSTEIN, N. P. (1963) A study of cerebral protein and polysaccharide in the dog. 111. Albumin changes in experimental cerebral edema. Nnrrol., 13, 915-952. HEISER, W. J. AND @INN, J. J. (1966) Analysis of brain scan patterns in cerebral ischemia and astrocytoma. Arch. Neurol., 15, 125-128. HESS, A. (1955) Blood-brain barrier and ground substance of central nervous system. Effect of brain wounds. Arch. Neurol. Psychiai., 74, 149-1 57. HODGES,P. c.,SELL~RS,R. D.. STORY, J., STANLEY, P. H., TORRES, F. A N D LILLEHEI, c. w.(1958) The effects of total cardiopulmonary bypass procedures upon cerebral function evaluated by the electroencephalogram and blood-brain barrier test; a clinical and experimental investigation. In: “Extracorporeal Circrila/ion”, J. G . Allen (Ed.). Springfield, Illinois, Charles C. Thomas Publisher, 219-294.

HUGH-JONES, K., SLACK,J., SIMPSON,K., GROSSMAN, A. AND HSIA,D. Y.(1960) Clinical course of hyperbilirubinemia in premature infants. A preliminary report. New Engl. J. Med., 263, 1225-1229. I s H i I , S., HAYNER, R., KELLY,W. A. AND EVAN::, J. P. (1959) Studies of cerebral swelling. 11. Experimental cerebral swelling produced by supratentorial extradural compression. J . Neurosurg., 16, 152-166.

KALSBECK, J. E.

AND

CUMINGS, J. N. (1962) Experimental edema in the rat and cat brain. J. Neuro-

pathol. Exptl. Neurol., 22, 237-247.

KATZMAN, R., ALEU,F. Neurol., 9, 178-187.

AND

WILSON,C. (1963) Further observations o n triethyl tin edema. Arch.

338

L. B A K A Y

KATZMAN, R., GONATAS, N. K. AND LEVINE,S. (1964) Electrolytes and fluids in experimental focal leukoencephalopathy. Arch. Neurol., 10, 58-65. KLATZO,I., AND MIQUELJ. (1960) Observations on pinocytosis in nervous tissue. J. Neuropathol. Exptl. Neurol., 19., 475487. KLATZO,I., MIQUEL, J. AND OTENASEK, R. (1962)Theapplication of fluorescein labelled serum proteins (FLSP) to the study of vascular permeability in the brain. Acta Neuropathol., 2, 144-160. H. A N D SMITH,D. E. (1965) Observations on penetration of serum proteins KLATZO,I., WISNIEWSKY, into the central nervous system. In: “Biology of Neuroglia”, (Progress in Brain Research, Volume 15), E. D. P. De Robertis and R. Carrea (Eds.). Elsevier Publishing Company, Amsterdam, 73-88. LEE,J. C. AND BAKAY, L. (1965) Ultrastructural changes in the edematous central nervous system. I. Triethyl tin edema. Arch. Neutol., 13, 48-57. J. (1959) Permeability of cerebral blood vessels in healing of brain wounds. LEE,J. C. AND OLSZEWSKI, Neurol., 9, 7-14. LONG,R.J., MACAFEE, J. G. A N D WINKELMAN, J. (1963) Evaluation of radioactivecompounds for the external detection of brain tumors. Cancer Res., 23, 98-108. LUCEY,J. F., HIBBARD, E., BEHRMAN, R. E., ESQUIVEL DE GALLARDO, F. 0. AND WINDLE, W. F. (1964) Kernicterus in asphyxiated newborn rhesus monkeys. Exptl. Neurol., 9, 43-58. MAGEE,P. N., STONER,H. B. AND BARNES, J. M. (1957)The experimental production of edema in the central nervous system of the rat by triethyl tin compounds. J. Pathol. Bacteriol., 73, 107-124. MALAMUD, N. (1963) Pattern of CNS vulnerability in neonatal hypoxaemia. In : “Selective vulnerability ofthe brain in hypoxaemia”, J. P. Schade and W. H. McMenemey (Eds.). Blackwell Scientific Publishing Company, Oxford, 21 1-225. G . (1963) A study of the relative value of radioactive substances MATTHEWS, C. M. E. AND MOLINARO, used for brain tumour localization and of the mechanism of tumour:brain concentration. Uptake in transplantable fibrosarcoma, brain and other organs in the rat. Brit. J. Exptl. Pathol., 44,260-277. MENKEN, M., BARRETT, P. V. D., SWARM, R. L. AND BERLIN, N. I. (1966) Kernicterus. Development of an experimental model using bilirubin 14C.Arch. Neurol., 15, 68-73. MILLEN,J. W. AND HESS,A. (1958) The blood-brain barrier: An experimental study with vital dyes. Brain, 81, 248-257. MUNDINGER, F. (1965) The biological bases of the utilization of radioisotopes for gamma-encephalography. In: Radio-lsotopes et Affections du Systeme Nerveux Central” T. Planiol (Ed.). Masson and Cie, Paris, 73-87. NICHOLLS, J. G. AND KUFFLER, S. W. (1964) Extracellular space as a pathway for exchange between blood and neurons in the central nervous system of the leech: Ionic composition of glial cells and neurons. J. Neurophysiol., 27, 64-671. S. (1960)Pathological changes in blood vessels of human glioblastoma multiforme. ComNYSTROM, parative studies using plastic casting, angiography, light microscopy, and electron microscopy, and with reference to some other brain tumours. Acta Pathol. Microbiol. Scund., 49, (Suppl. 137). ODELL,G. B. (1959) Studies in Kernicterus. I. The protein binding of bilirubin. J. C h i . lnvest., 38, 823-833. PLUM,F., POSNER, J. B. AND ALVORD,E. C. (1963) Edema and necrosis in experimental cerebral infarction. Arch. Neurol., 9, 563-570. POLANI,P. E. (1954) Experimental haemolytic anaemia in the albino rat; neurological aspects. J . Pathol. Bacteriol., 68, 109-1 20. PRADOS, M.,STROWGER, B. A N D FEINDEL, W. (1945) Studies on cerebral edema. 11. Reaction of the brain to exposure to air; physiologic changes. Arch. Neurol. Psychiat., 54, 290-300. RAIMONDI, A. 5. (1964) Localization of radio-iodinated serum albumin in human glioma. An electronmicroscopic study. Arch. Neurol., 11, 173-184. RAIMONDI,A. J., MULLAN, S. AND EVANS,J. P. (1962) Human brain tumors: An electron-microscopic study. J. Neurosurg., 19, 731-753. ROZDILSKY, B. (1966) Kittens as experimental model for study of kernicterus. Amer. J. Dis. Childh., 111, 161-165. ROZDILSKY, B. AND OLSZEWSKI, J. (1957) Permeability of cerebral blood vessels studied by radioactive iodinated bovine albumin. Neurol., 7, 270-219. -, (1961) Experimental study of the toxicity of bilirubin in newborn animals. J. Neuropathol. Exptl. Neurol., 20, 193-206. SAMOJARSKI, T. AND MOODY,R.A. (1957) Changes in the blood-brain barrier after exposure of the brain. Arch. Neurol. Psychiat., 78, 369-376.

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SASS-KORTSAK, A. (1961) Kertiicterus. University of Toronto Press, Toronto. SCHENKER, S., MCCANDLESS, D. W. A N D ZOLLMAN, P. E. (1966) Studies of cellular toxicity of unconjugated bilirubin in kernicteric brain. J. Cliu. hivest., 45, 1213-1220. SELVERSTONE, B. AND MOULTON, M. J. (1957) The phosphorus metabolism of gliomas: A study with radioactive isotopes. Brain, 80, 362-370. SPERL, JR., M. P., SVIEN, J. J., GOLSDTEIN, N. P., KERNOHAN, J. W. A N D GRINDLAY, J . H. (1957) Experimental production of local cerebral edema by a n expanding intracerebral mass. Proc. Muyo Clinic, 32, 744-749.

STEMPFEL, R. (1955) Serum and cerebrospinal fluid bilirubin in hemolytic disease of the newborn. Acta Puecliat., 41, 502-5 10. TANI, E. AND Istiii, S. (1963) Ontogenic studies on the rat brain capillaries in relation to the human brain tumor vessels. Acra Neuropathol., 2, 253-270. TATOR,C. H., EVANS,J. R. A N D OLSZEWSKI, J. (1966) Tracers for the detection of brain tumors. Evaluation of radioiodinated human serum albumin and radioiodinated fatti acid. Neu, ol., 16, 650-66 I . TATOR,C. H., MORLEY, T. P. A N D OLSZEWSKI, I. (1965) A study of the factors responsible for the accumulation of radioactive iodinated human serum albumin (RIHSA) by intracranial tumors and other lesions. J. Neurosurg., 22, 60-76. TORACK, R. M . , (1961) Ultrastructure of capillary reaction to brain tumors. Arch. Neurol., 5,416428. TSCHIRGI, R.D. (1950) Protein complexes and the impermeability of the blood-brain barrier to dyes. Amer. J . Plipsiol., 163, 756.

VANVLIET, P. D., TAUXE, W. N., SVIEN,H. J. AND JENKINS,D. A. (1965) The effect of craniotomy on the brain scan. J . Neurosrtrg., 23,425-430. WATERS, W. I.,AND BOWEN W. R. (1955) Bilirubin encephalopathy: Preliminary studies related to production. Pediur., 15, 45-18. WRIGHT, R.L. (1963) Neovascularization in experimental gliomas. J. Neuropathol. Exptl. Neurol., 22, 435-445.

ZIJELZEK, W. W.

AND

BROWN, A. K. (1961) Neonatal jaundice. Amer. J. Dis. Childh., 101, 87-127.

DISCUSSION

D. B. TOWER: I would like to say a word of caution about the presence of ruptured membranes seen in electronmicrographs. When a cell is swollen, one can not necessarily attribute a ruptured membrane to the fact that the cell was swollen beyond its elastic capacity or to other properties of the membrane, because a knife also has had to pass across the section. Certainly, in light-microscopy, knives have been known to create such artifacts. Therefore, I would object to anybody’s being dogmatic about these ruptured membranes. Furthermore, I have a feeling that cells can withstand a tremendous amount of swelling without rupturing the membrane. 1 would also like to suggest that we be cautious about saying that spaces never increase or decrease in size, because I doubt if we understand all the factors influencing these spaces between the time when we sample the tissue and the time when we are able to look at it with the electronmicroscope. I am also disturbed about the apparent constancy which is insisted upon for the size of the spaces between cells. This does not seem quite reasonable from a biological point of view. Finally, I would be cautious concerning ruptured membranes in such conditions as edema, particularly in attempting to interpret where a marker like ferritin might logically be. Dr. Brightman, I remember, called attention to the fact that you can very easily translocate or dislocate ferritin from one place to another if you are not careful with your preparation. I don’t mean this in terms of criticism of Dr. Bakay, or anybody else, but we must be very careful in making statements about these thingswhich are too dogmatic.

L. B A K A Y

L. BAKAY: I can answer this simply. First of all, I don’t think that the rupture of the cell membrane is of any importance. The point remains that the extracellular space increases in size morphologically in edema in the white matter, whether there is a rupture or not, and it does not increase in the cortex where, incidentally, we never see rupture. As far as the artificial nature of the rupture of the cell is concerned, we never see it in unswollen cells, and you don’t see a ruptured cell membrane even if you use a magnification of 10,000 times with electronmicroscopy. It just simply cannot be seen, ruling out the possibility that this is a random artifact. It might be an artifact associated with swelling, but it is an artifact that is never seen in unswollen cells. As far as the size of the space is concerned, when 1 say that there is no evidence of any increase of the extracellular space in the cortex, even in conditions of edema, and there is one seen in the white matter, 1 mean that purely on the ultrastructural morphological basis. This has no bearing on whatever we mean by a functional extracellular space, and if we assume that some of the extracellular space is intracellular as far as the glia is concerned, that is all very well; but we do not see any evidence of an increase in cortical extracellular space size by electronmicroscopical methods. As far as ferritin is concerned, I know about Dr. Brightman’s work, and I fully agree with much of what he said. But ifit would be artificially translocated in these specimens, wouldn’t you expect that it would be translocated from this extremely narrow extracellular space between these enormously swollen cells? If ferritin would be all over the place, you could say that this has been transferred as an artifact. But if you see two extremely large cells and the narrow cleft inbetween, and the material does not go into the very largely hydrophilic cells on both sides, this is not likely to be an artifact. 0. STEINWALL: I would like to comment on the problem of the neonatal brain barr;er and kernicterus. mentioned by Dr. Bakay. The unconjugated bilirubin is very apt to be bound to protein in the blood and there is reason to believe that, even in the embryo, the blood-brain barrier to protein-bound material is effective. This can be illustrated by a quite recent investigation (together with Klatzo, Olsson and Sourander) on newborn rats and embryos (2 weeks or longer gestation) injected with fluorescein-labeled albumin. As far as could be observed by fluorescence microscopy, there was no cerebral extravasation of the tracer. There might exist conditions, however, in which the proteinbinding of bilirubin is loosened. Being a lipophilic compound the free bilirubin would then pass freely into the brain. I wonder if Dr. Bakay would comment on Grontofts work on human fetuses, where he concluded that the cerebral vessels were impermeable under normal conditions while anoxic fetuses showed signs of blood-brain barrier damage. L. BAKAY:I know about Grontoft’s work, and he emphasizes strongly that it is necessary for the brain to be anoxic to permit a trypan blue infiltration into the brain tissue. The difficulty is that in adult animals you can do the most drastic kind of anoxic and asphyxic experiments, but you always will find tissue necrosis, which there should not be in anoxic as versus ischemic anoxia. I don’t know what kind of damage those infants have been exposed to in addition to anoxia. K. NEAME:I would like to ask Dr. Bakay about the effects of pathological states as seen by brain scanning. Is this a change in the so called blood-brain barrier, or is it a change seen in tissues in general? L. BAKAY: That is a very hard one to answer, because it depends on the tissue. One thing is for certain: that there is no selectivity in the increased uptake in a brain scan due to any condition. The amount of maximal concentration you can get in almost any lesion in the brain corresponds to what you get in normal tissue elsewhere. There is no selective concentration. This answers your question: there is no specific change; in other tissues (muscle, liver or spleen), whether tissue is normal or abnormal, it has about the same concentration, although there could be more of course in the case of a pathological process than there would normally be. What you see as a positive lesion in a brain scan is not a selective concentration but a relatively free exchange of the substance with the blood stream, as occurs in other organs, and it becomes selective only because the suirounding brain does not have completely free exchange. How much of this is an intracellular, how much an extracellular process is a long story which I would like to go into, but do not have the time.

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341

J. DOBEING: About the spread of edema throughout the white matter of the hemisphere which is being damaged at a focal site in the cortex, I want to ask Dr. Bakay if he thinks that this has any lessons for experimenters who routinely mutilate the brain in the course of the experiments. The question - which is impossible to answer - is, what size of lesion, what extent of trauma is necessary to produce the effect throughout the white matter of the hemisphere? Would ventricular perfusion do it? Would needles, passing through thecerebrum into a ventricle (in most oftheselaboratory animals it is only a potential space anyway) cause such edema? Therefore, the evidence that one is in the ventricle, at least in a small animal, is likely to be at the expense of at least some trauma to the ependyma as well as throughout the tract of the needle? Certainly pharmacological intracerebral injections, which are horrible from the point of view of structures and edema, but will probably suit the purpose of the pharmacologist, would be likely to do this. The question is as to what extent can you inflict damage to the brain before it becomes necessary (particularly when investigating blood-brain barrier effects with drugs and substances concerned with edema) to provide controls rather better than with trqpan blue to discover whether you have this spreading edema from the trauma of your procedure or from the test substances employed.

L. BAKAY: I shall give you an extremely dogmatic answer to this. I don’t know about ventricular perfusion. If it is done without changing tht. pressure conditions and if the solute is not very different in osmolarity or very definitely toxic, I suppose a careful perfusion might not cause any damage, at least on theoretical grounds, in the surrounding brain. Whatever is introduced into the brain causes edema: how much is unpredictable. But you cannot put a lesion in the brain and you cannot put even a simple needle tract in the brain without having a certain amount of edema around it. A. LOWENTHAL: You said that there are two types of edema: one type with an increased protein content and another type with a less increased or with a reduced protein content. Could you say which are those two types?

L. BAKAY:There might be a great number of different types of edema. For example, we don’t know what category anoxia would fit in. The two fairly well defined edemas so far are: ( I ) the traumatic inflammatory peritumoral focal type of edema which seems to be characterized by a fairly free exchange of serum albumin and an obviously greatly disturbed capillary permeability, and (2) the type, which is really only characteristic of traumatic edema and seems to be an intramyelinic accumulation of protein-free plasma ultra-filtrate with no evidence of capillary damage. These are the two extremes, and we don’t really have any other categories as yet.

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343

Clinical Importance of Alterations in Barrier G .QUADBECK Heidelberg (Germany)

Before discussing the problem of alterations of the blood-brain barrier, I have to define what I mean by the term blood-brain barrier (bbb). Anatomically, the bbb includes all structures between the capillary lumen and the central nervous tissue: the endothelium of the capillaries, the basement membrane, and the surrounding glial foot processes. Functionally, the bbb should be regarded as a system limiting the free exchange between blood and brain; it further exerts its function on transport processes essential for the nutrition of the brain and on those in the opposite direction, from the brain into the blood. The bbb is also effective as an intermediate system in humoral regulations of peripheral vegetative functions. Alterations of the bbb with increased permeability cause enhanced exchange between blood and brain which is dependent on the intensity of the disturbance; by this means the penetration of viruses and bacteria from the blood into the brain may be facilitated. This increased permeability may be followed by cerebral edema and by an increased predisposition for epileptic seizures. In his paper Dr. Bakay has amply demonstrated the increased permeability of the bbb in pathological conditions. Therefore, I will mostly discuss pathological conditions involving a reduced exchange between blood and brain. Under normal conditions glucose is the most important if not the only source of energy for the brain. Because glucose cannot enter the brain from the blood by a process of diffusion, glucose uptake is independent of the glucose level of the blood, over a wide range. The nutrition of the brain depends on a transport mechanism specific for glucose in the bbb system. The glucose uptake of the brain can be measured in man by the method of Kety and Schmidt (1948). With this method, one may determine the cerebral blood flow, the cerebral uptake of glucose, oxygen, and amino acids; and at the same time the output of COz, lactic acid, pyruvic acid, and other metabolic products of the brain. A reduced glucose uptake by the brain can be brought about by the following mechanisms : (a) reduced blood flow caused by disturbances of the blood circulation. (b) reduced glucose transport across the bbb. (c) reduced cerebral metabolic activity. (d) markedly reduced glucose levels in the blood. In my opinion, only point b is related to the bbb system. With reduced cerebral blood Rrferences p . 347

G. Q U A D B E C K

344

TABLE I CEREBRAL BLOOD FLOW

( C B F ) A N D C E R E B R A L G L U C O S E U P T A K E ( C G L U ) IN E L D E R L Y M E N M E A S U R E D BY D I F F E R E N T G R O U P S

( % of the normal value)

Gottstein et a/., 1962 Gottstein et a/., 1964 Dastur et a/., 1965 Becker and Hoyer, 1966

N = 13 N = 21 N = 17 N = 26* N = 18

CBF

CGIU

95 953 70 93 105

71 80 62

I7 63

* Normal elderly men without mental disturbances flow, the brain has the possibility of increasing the glucose uptake from the blood. Erbsloh et al. (1958) found, however, that with blood glucose levels ranging between 56 mg % and 25 I mg % there was no difference in the uptake of glucose by the human brain. It is usually assumed that in cerebral arteriosclerosis the reduced bloodflow is the cause for the mental disturbances seen clinically. However, at autopsy the clinical diagnosis of cerebral arteriosclerosis often cannot be confirmed. There seems to be no correlation between the autopsy findings on the brain vessels and the intensity of the mental disturbance in older men. Table I shows the results of measurements of cerebral metabolism in patients with mental disturbances and in older men without great mental disturbance. All groups had the same results. The glucose uptake in older men more frequently showed the greater alterations in metabolic values than in bloodflow and oxygen uptake. Many studies by Erbsloh have indicated that the lactic acid output by the brain is not increased in patients with reduced oxygen uptake. This would be expected if the oxidative metabolism of glucose by the brain were inhibited and the glucose metabolized by the glycolytic pathway. Such an increase of lactic acid production by the brain may occur in brain tumors, as we found, where the values were 20 times the normal output. In patients with failure of the metabolic processes of the brain, one may differentiate between the pathological mechanisms and arrive at a correct diagnosis by means of measurement of cerebral metabolism. It has been found by Becker and Hoyer (1966) that a reduced cerebral glucose uptake can be found not only in older mentally disturbed patients but also in younger patients with mental or neurological disorders. In earlier experiments we were able to demonstrate that some drugs used in geriatric patients influenced the glucose transport across the bbb by increasing the cerebral uptake. In normal animals (mice) this can be demonstrated only in very high doses, since the glucose uptake by the brain is an excellently regulated mechanism which can be affected only by extremes. For example, drugs effective in animal experiments were nicotinic acid, centrophenoxine, and pyrithioxine,

345

A L T E R A T I O N S IN B L O O D - B R A I N B A R R I E R

Pyrithioxine

The latter was given to patients with reduced glucose uptake by the brain in daily doses up to 1200 mg. Under this treatment the cerebral glucose uptake rose together with a clinical improvement of the neurological or psychic condition (Table 11). The time needed for the measurement of all the data in one patient by this method is so great that only two patients can be measured each day by one person in the laboratory. We, therefore, looked for a simpler screening method to diagnose disturbances of the bbb permeability, and remembered a study of L. Doust published some years ago. Doust (1960) found different frequencies of the rate of vegetative regulations in some patients. In epileptics the frequency increased as compared with the normal, whereas in schizophrenics a marked reduction of this frequency was found. TABLE I 1 C H A N G E S O F CE RE B RAL METABOLISM U N D E R TR EA TMEN T W I T H P Y R I T H I O X I N E

(from Becker and Hoyer) a: Man ( 5 1 ) with clinical diagnosis: cerebral arteriosclerosis before treatment

33 days treatnient

62 % 29 7” 64 7;

98 % 120% 84 %

Blood flow Glucose uptake 0 2 uptake

b: Man (35) with clinical diagnosis: Jackson seizures be$ treatin. 12 days treatni. Blood flow 244 74 133% Glucose uptake 36 % 86 % 02 uptake 173 83 %



Acids

Bases .

- ._ - -.

Glucose (nutrients)

Blood

BBB

--

+ c-? - Indicator --

4 KIDNEY:

Biood

Tub. ce!ls

Urine

The initial working hypothesis, discussed earlier together with some experimental results (Steinwall, 1961), is represented by the diagram in Fig. 1. It proposes a system of transport mechanisms within the frame of the BBB, selectively conveying different categories of substances inward or outward. In teleological terms these mechanisms would serve for the uptake of nutrients and the excretion of metabolic waste products through a system of membranes otherwise practically impermeable to lipid-insoluble material. Table I shows these ideas inserted in a simplified outline of transfer and barrier conditions with respect to differences in physicochemical and biological characteristics (Steinwall, 1964). The hypothesis has been approached experimentally along two main lines. Of these the attempts to prove that a “counter”

* Head professor Tore Broman. References

p . 363-364

358

0. S T E I N W A L L

TABLE I SIMPLIFIED I N T E R P R E T A T I O N OF B L O O D - B R A I N P A S S A G E A N D BARRIER EFFECTS I N RELAT I O N TO P R O P O S E D T R A N S P O R T M E C H A N I S M S

Passive diffusion

No barrier effect

I . “Nutrients” (glucose, amino acids, etc).

Specific transport* blood CNS

2. “Waste products”, organic ions (conjugates, indicator dyes, etc.)

Specific transport* blood + CNS

3. Nonmetabolizable and

No transfer

Barrier effect above max. transport capacity Barrier effect Partly due to the “counter” transport Barrier effect “passive”

1. LIPOPHILIC SUBSTANCES

11. HYDROPHILIC SUBSTANCES

--f

noncharged solutes, (inulin, sucrose, mannitol, etc.)

* Carrier mediated - limited transport capacity; inhibited by competitive overloading or by toxic influences. transport from brain to blood might be involved in the barring of acid dyes and other organic ionized material have not led to conclusive results. More convincing findings have been obtained in the experiments designed to determine if decreased brain uptake of nutrient tracers can occur in a BBB damage commonly interpreted only in terms of abnormal extravasation of acid dyes. EXPERIMENTAL TECHNIQUE

The basic experimental procedure employed in the studies discussed here was designed to meet the following conditions: (a) Injection of noxious agents within the cerebral vessels in living animals with satisfactory control of concentration in loco and of application time. This allows for the determination of critical levels for bringing about barrier dysfunctions. (b) Restriction of the damaging influence to one hemisphere in order to get a control hemisphere for direct comparison in each experiment. (c) Avoidance of circulatory disturbances after the application of the injurious agent by adequate choice of chemical agent and concentration. The injection of the noxious solutions was performed according to a technique developed by Broman and Olsson (1948, 1955) and modified by Steinwall (1958). Rabbits (under nembutal and/or urethane anaesthesia) were used because their cerebrovascular supply make them particularly suited for this experimental procedure as they have no rete mirabile anastomosis between the internal and external carotid arteries. The various steps of a standard experiment are illustrated in Fig. 2. The solution was injected through a plastic catheter secured in one common carotid artery after ligation of its proximal part and external trunk as well as detectable extracerebral branches from the internal carotid artery. By keeping the injection pressure at the blood pres-

UNILATERAL DAMAGE OF BLOOD-BRAIN BARRIER

359

Fig. 2. Main steps in the experimental procedure for unilateral perfusion with barrier-damaging agents and subsequent demonstration of the effects by means of intravenously given tracers. (EEG records from a study by Flodmark and Steinwall, 1963a.)

sure level the solution displaces the blood of the ipsilateral hemisphere as controlled by inspection of the pia, vessels through the intact transparent dura in a trephine opening. If the injection (application) time is kept within narrow limits (generally about 30 sec), the concentration will be decisive for the effect of each chemical agent. Afterwards the injected hemisphere was recirculated via the circle of Willis, which in rabbit is supplied with blood predominantly from the vertebral arteries. As tested in some studies on brain uptake of nutrients the blood flow in the experimental hemisphere after mildchemical injury was practically equal to that in the control (see also Flodmark and Steinwall, 1963a). For the detection of the barrier dysfunctions induced by the unilateral perfusion, suitable tracers were given intravenously and comparatively determined in the two hemispheres. The presence of staining was estimated by visual inspection and radioactivity assayed by conventional methods for counting or by autoradiography. Organic ion transport

The proposed transport from brain to blood of ionized “waste” material, which was also thought to impede the extravasation of blood-borne organic ions (e.g. acidic or basic indicators of BBB damage), has as yet but fragmentary support from experimental observations. Data in favour of this theory are summarized in the following statements: 1. Different types of noxious agents which are known to inhibit the renal tubular transport also cause BBB dysfunction (Steinwall, 1961). 2. In adequate concentrations a number of organic acids give rise to reversible BBB “inhibition” as tested with acid dyes. The barrier inhibiting potency roughly parallels References p. 363-364

360

0. S T E I N W A L L

their respective tubular Tm, i.e., their ability to occupy maximum transport capacity (Steinwall, 1961). 3. Acid tracers, such as sodium fluorescein, which have permeated into the brain in reversible BBB impairment are eliminated within a few hours of restored function of the BBB (Flodmark and Steinwall, 1963 b). A corresponding phenomenon was recently reported by Werdinius (1966) who found that acid monoamine metabolites, produced within the brain, appeared to be secreted into the blood. It is, however, still an unsettled problem if the organic ions are eliminated directly through the bloodbrain interphase or have to make a detour via the choroid plexus, where such transport events are convincingly shown to take place. Obviously, the hypothesis of a brainblood counter transport of organic ions as contributor to the BBB effect needs more experimental substantiation. Brain uptake of nutrients in BBB damage

In earlier investigations mercuric ions in low concentrations (of the order of 10 pM) were found to impair the blood-brain barring of ionized organic acids or bases (Steinwall, 1961; Hansson and Steinwall, 1962). From studies on other membraneous systems it was known that this agent could be a potent inhibitor of the specific transfer of glucose and other nutrients (Passow et al., 1961). It was therefore used in the experiments aimed at bringing about a BBB damage characterized by simultaneous decrease in blood-brain transfer of tracers of the nutrient type and extravasation of normally barred organic ions such as acid dyes. All these experiments were of short duration (5-15 min) in order to minimize the possible influence of unequal metabolic events in the damaged and control hemisphere. The following nutrient representatives were studied separately : [14C]glucose, [14C]methyl-O-glucose, [14C]cycloleucine and [75Se]selenomethionine. In the first study [14C]glucosewas used and assayed by counting of plated trichloroacetic acid extracts of brain (Steinwall, unpubl. data). The results (Table 11) suggested TABLE I1 U P T A K E O F [ ' 4 c ] G L UCO S E FROM B L O O D I N U N I L A T E R A L B B B D A M A G E C A U S E D BY MERC U R I C IONS. CO M P ARAT I VE ASSAY O F ''CACTIVlTY I N EX P ER IMEN TA L A N D C O N T R O L HEMISPHERE. DE G RE E OF S T A I N I N G W I T H A C I D D Y E D ETER MIN ED BY GR OS S I N S P E C T I O N

No.

HgClz conc. pcM 50 50 50 40

80 80 Saline

Staining of experimental hemisphere

14Cratio experinlentat

weak weak weak weak medium strong none

0.8 0.7 0.7 0.6

/control

1.o 1.7 1 .o

UNILATERAL DAMAGE O F BLOOD-BRAIN BARRIER

36 1

that an inhibition of glucose uptake occurred and that it was best detected in mild injuries, while in more severe barrier damage with strong staining from extravasated dye the experimental hemisphere seemed to contain equal or higher amount of 14Cactivity than the control. This tendency illustrated a diffuculty implicit in the experimental situation. Evidently, the diminished [ “T]glucose uptake caused by inhibition of a specific transport mechanism might be countered and overpowered by a passive leakage in more pronounced BBB damages. Later experiments confirmed that the most distinct indication of blocked nutrient uptake was achieved in very weak BBB impairments with respect to extravasation of acid dyes. In another series (4 experiments) with [ 14C]glucose, sections from brain slices, frozen immediately after removal, were used for autoradiography (Steinwall and Hansson unpubl. data). In two of these brains the decreased uptake in the hemisphere perfused with HgC12 (20 pM)was quite evident (Fig. 3) and the counting assay showed an experimental/control ratio of 0.61 to 0.65. These studies on [14C]glucose did not include any attempt to identify the radioactive material within the hemispheres. Thus, the possibility that hemispheric dif-

Fig. 3. Autoradiogram of cross sections of a rabbit brain (14Cactivity white). The left hemisphere was perfused with 20 /tM HgClz (40seconds) and [‘4C]glucose and an acid dye (Prontosil soluble) given intravenously. The left-sided BBB dysfunction was indicated both by extravasation of the dye and by decreased amount of I4Cactivityas compared with the control hemisphere. References p . 363-364

362

0. S T E I N W A L L

ferences in glucose metabolism influenced the results could not be ruled out, even if the short duration of the experiments made it less likely. By replacing glucose with [14C]methyl-O-glucosean attempt was made to refine the situatiton with regard to the transport problem. According to studies on other organs this glucose analogue is transferred by a specific glucose transport mechanism without being further metabolized (Csaky, 1958, Jorgensen et al., 1961). In the experiments with this tracer, a method was employed which allowed a sensitive fluore-photographic image to be made of extravasated fluorescein-Na in the same sections that were used for autoradiography. As reported in detail elsewhere (Steinwall and Klatzo, 1966) there was in 2 of the 4 animals a markedly reduced uptake of [14C]methyl-O-glucose in the experimental hemisphere in regions with scarcely visible fluorescence, while an accumulation of radioactivity appeared in small spots which in the photography showed a rather bright fluorescence. The studies with [“C]cycloleucine (Steinwall and Snyder, to be publ.) were also based on the assumption that this amino acid shared the transport meachanism of its natural relative leucine (Christensen, 1962; Lajtha et al., 1963). In series of 4 experiments all brains showed a considerably diminished uptake of the amino acid in the perfused hemisphere (ratios of 0.29 to 0.71). Chromatographic analysis verified that the radioactivity in the brain was derived solely from unchanged cycloleucine. In 3 similar experiments the blood flow factor was tested by applying the rapidly permeating compound [14C]penthothal, which was recovered in equal amounts from the two hemispheres in each brain. In the last series [75Se]L-selenomethionine was used as nutrient tracer. According to Blau and collaborators (see Blau, 1964) this amino acid behaves biologically like the natural L-methionine with regard to transport in the intestines as well as in uptake in the pancreas. In these experiments a direct control of circulatory symmetry could be achieved by administering to some of the animals both the amino acid and the blood flow tracer, [131I]antipyrine (Oldendorf and Kitano, 1965). The differences in half life of these 2 gamma emitters, conveniently assayed by well scintillation counting, permit the deduction

-

[ESejL selenomethionine

pI]

4-iodoantipyrine

11 11 ‘I1

Fig. 4. Brain uptake of [7Y3e]~-selenomethionineand concurrent blood flow test with [1311]4-iodoantipyrine in unilateral mercurial BBB damage. Left column : experimental hemisphere. Righr colitnm : control hemisphere (arbitrary unit in each pair). Decreased amount of 75Se activity in the experimental hemisphere is apparent also when the blood flow tracer shows a practically symmetrical deposition.

UNILATERAL DAMAGE OF BLOOD-BRAIN BARRIER

363

of the activity emitted by each of them. From this series, still in progress, results are given in Fig. 4, showing a markedly diminished uptake of the amino acid in the barrier damaged hemisphere without significant asymmetry of the blood flow. CONCLUSION

The presented experiments, although crude and at best semiquantitative, were able to illustrate a BBB damage in which an abnormal ouflow of conventional tracers like acid dyes might occur concomitant with a decreased transfer of other tracers such as glucose or amino acids. With regard to the extravasation of acid dyesitisan open question whether or not this reflects in part the blockage of a counter transport mechanism for organic ions, or if it is solely due to increased passive permeability. In any case, the latter factor does not explain the diminished uptake of the nutrient tracers, a finding that can be postulated to reflect an inhibition of specific transport mechanisms. An increasing number of data from other investigations corroborate that such mechanisms operate in the blood-brain interphase (for reviews, see Lajtha, 1962; Crone 1965; a.0.). In the general discussion of BBB damage the pathogenic significance of transport inhibition might deserve more attention than usually given.

REFERENCES

L. AND LEE,J. C. (1965) Cerebral Edema. Charles C. Thomas Springfield, Illinois. BAKAY, BLAU,M. (1964) Pancreas scanning with 75Se-Selenomethionine.Medical Radioisotope Scanning Vol. 11. International Atomic Energy Agency, Vienna (p. 275). BROMAN, T. AND OLSSON, 0. (1948) The Tolerance of Cerebral Blood Vessels to a Contrast Medium of the Diodrast Group. Acta Radiol., 30, 326-342. -, (1956) Technique for the Pharmaco-dynamic Investigation of Contrast Media for Cerebral Angiography. Acfa Radiol., 45, 96-100. CHRISTENSEN, H. N. (1962) Biological Transport. W. A. Benjamin, Inc. New York. (p. 54). CRONE, C. (1965) Facilitated Transfer of Glucose from Blood to Brain. J. Physiol. (London), 181, 103-113.

CSAKY, T. Z. (1958) Active Intestinal Transport of 3-0-Methylglucose. In: 4th International Congress of Biochemistry. Infern. Abstr. Biol. Sci., Supp. p. 80. Vienna. FLODMARK, S . AND STEINWALL, 0. (1963a) Differentiated Effects on Certain Blood-Brain Barrier Phenomena and on the EEG Produced by Means of Intracarotidally Applied Mercuric Dichloride. Acra physiol. Scand., 51, 446453. -, (1963b) Reversible Blood-Brain Barrier Alteration Induced by Certain Organic Acids and Indicated by Means of EEG and Dye Tests. Acta Physiol. Scand., 58, 368-375. HANSSON, E. AND STEINWALL, 0. ( I 962) Abnormal Blood-Brain Passageof aQuarternary Phenothiazine Derivative (S35-labelled AprobitR) Induced by Chemical Agents. Acta physiol. Scand., 54, 339-345.

J ~ R G E N SC. E NE., , LANDAU, B. R. AND WILSON, T. H. (1961) A Common Pathway for Sugar Transport in Hamster Interstine. Amer. J. Physiol., 200, 1 1 1-1 16. KLATZO, I., STEINWALL, 0. A N D STREICHER, E. (1967) Dynamics of cold injury edema. Proc. Symp. on Brain Edema, Vienna, Sepf. 1965. I. Klatzo and F. Seitelberger (Eds.), Springer-Verlag, WienNew York. LAJTHA, A. (1962) The Brain Barrier System. Neurochemistry. Thomas Springfield, Illinois. (p. 399). LAJTHA,A., LAHIRI,S. AND TOTH,J. (1963) The Brain Barrier System-IV. Cerebral Amino Acid Uptake in Different Classes. J. Neurochem., 10, 765-773. OLDENDORF, W. H. A N D KITANO, M. (1965) The Symmetry of P1 4-i~doantipyrineUptake by Brain after Intravenous Injection. Neurol., 15,994-999.

364

0.S T E I N W A L L

PASSOW,H., ROTHSTEIN, A. AND CLARKSON, T. W.(1961) The General Pharmacology of the Heavy Metals. Pharmacol., Rev., 13, 185-224. 0. (1958) An Improved Technique for Testing the Effect of Contrast Media and Other STEINWALL, Substances on the Blood-Brain Barrier. Acta Radiol., 49, 281-284. -, (1961) Transport Mechanisms in Certain Blood-Brain Barrier Phenomena. - A Hypothesis. Acta psychiat. neurol. Scand., 36, Suppl. 150, 314318. -, (1964) Blood-Brain Barrier Dysfunction: Some Theoretical Aspects. A c f a nertrol. Scmid., 40, SUPPI. 10, 25-29. STEINWALL, 0 .AND KLATZO, I. (1966) Selective Vunerability of the Blood-Brain Barrier in Chemically Induced Lesions. J. Neuropathol. Exptl. Neurol., 25, 542-559. WERDINIUS, B. (1968) Effect of Probenecid on the Levels of Monoamine Metabolites in the Rat Brain. Acta pharmacol. toxicol., 25, 18-23.

DISCUSSION T. Z. CSAKY:Dr. Steinwall, with regard to your 3-methyl-glucose experiments, I would like to ask if you followed this experiment up with a quantitative determination of 3-methyl-glucose, and what did you find? 0. STEINWALL: This has not been done.

T. Z. CSAKY:Where is the label in your 3-methyl-glucose? 0. STEINWALL: In the methoxyl group.

T. Z. CSAKY:Is it in the methoxyl group? This is very important, because there are two preparations available: one is prepared from uniformly C-14 labeled glucose which is methylated. This is always contaminated with radioactive glucose. The other is prepared from inert glucose by methylation with radioactive methyliodide. The reason for my asking about this is that some five years ago we published experiments on the distribution of 3-methyl-glucose in rats. If the methyl-glucose was injected into nephrectomized animals, after about two hours, equilibrium was reached in the body. If we then determined the relative concentrations of 3-methyl-glucose in the various organs, the brain always contained very low amounts. With cardiac muscle and liver we obtained an almost complete equilibrium between the organs and the blood. The muscle contained less water and in the brain the ratio was less than 1 : 10. Moreover, if radioactively labeled 3-methyl-glucose was injected into a rat, it was excreted in the urine. After 48 hours we still found some radioactivity in the brain, which means that the exchange between the blood and the brain is extremely slow in the case of 3-methyl-glucose. 0. STEINWALL: As I said, we did not d o quantitative estimations; however, the brain uptake of our methyl glucose was very convincing as judged from the autoradiographic findings in comparison with such carbohydrates as mannitol and sucrose, studied under corresponding conditions. T. Z. CSAKY:But mannitol probably does not penetrate the brain at all. 3-methyl-glucose does, but at an extremely slow rate, so it certainly does not behave the same way as glucose. 0. STEINWALL: One would not expect that. But there may be enough uptake under normal conditions to reveal this inhibition under the abnormal conditions that we created in the left hemisphere.

H. M. ADAM:If you damage the blood-brain barrier with mercuric ions. then can you reverse the process? 0. STEINWALL: That might perhaps be possible by special means. The spontaneous course of this mercurial barrier damage, however, seems to be of long duration and progressive rather than regressive in nature (Flodmark & Steinwall, Acta physiol. scand., 1963, 57, 446453). The mercuric ions are probably tightly fixed to their site of action.

U N I L A T E R A L D A M A G E OF B L O O D - B R A I N B A R R I E R

365

H. M. ADAM:Did you estimate the amount of mercuric ions that was captured by the cerebral tissue? 0. STEINWALL: No. A. LAJTHA: I would just like to make some general philosophical remarks for the concern of the

morning session. Perhaps every scientist should be entitled to his own concept of a blood-brain barrier and perhaps one of the functions of a meeting such as this could be to simplify somewhat the matter. I t needs to be emphasized that this is a living brain with more than just one blood-brain barrier. We could classify, as the first step, the test-substances into at least three classes: (1) substances that do not penetrate membranes; (2) substances that penetrate membranes by diffusing through membrane constituents, and (3) substances that penetrate membranes by carrier or mediated mechanisms. Now, if we realize that one may effect one kind of passage (such as mediated transport) without effecting another (such as ditfusion) we may perhaps use the term blood-brain barrier somewhat more selectively. Definitions of the blood-brain barrier would also depend on how we define the brain. May I add one difinition? My own is that the brain is the nucleus of the neuron. This is of course said only as a joke, but with this I would like to emphasize that between the blood and the brain there are many more membranes than the capillary membrane; that these would include the neuronal membranes and perhaps even the mitochondria1 membranes. In fact the cell membrane by altering the concentration in the cytoplasnia, must have an important influence on the concentration of substances in the nucleus.

This Page Intentionally Left Blank

367

Changes in Blood-Brain Permeability during Pharmacologically induced Convulsions ROBERT W. P. CUTLER, A N T O N I O V. L O R E N Z O

AND

C H A R L E S F. BARLOW

Neurology Service of the Childreds Hospital Medical Center, Peter Bent Brigham Hospital, and the Department of Neurology, Harvard Medical School, Boston, Massachusetts, U.S.A.

INTRODUCTION

Disturbances of the normal functions of the “blood-brain barrier” are well known following a large variety of pathological stresses. Relatively less attention has been directed towards the transient alterations in solute exchange between blood and brain that may accompany less destructive stimuli. Such alterations may be significant in the pathogenesis of certain disorders of central nervous system function. In the experiments to be reported, temporary alterations in the entry of [35S]sulfate into brain were found to accompany pharmacologically-induced convulsions. Marked concomitant changes in brain surface pH, oxygen and carbon dioxide tensions were recorded by direct measurement during the convulsion. As a result of these observations, the influence of hypercapnia and acidosis on brain vascular permeability was studied in some detail. [35S]sulfateand [125l]albumin were employed as indicators. The former solute is a diffusible anion with an intermediate rate of entry into brain. A steady concentration in all regions of brain is reached one to two hours following intravenous injection. While sulfate is predominantly confined to the extracellular compartment of brain, a portion is incorporated into metabolites, permitting an evaluation of alterations in sulfate metabolism on brain uptake. Such factors as enlargement of the sulfate space, enhanced metabolism of sulfate, expanded capillary surface area from vasodilatation, or increased cerebral blood flow may contribute to an accelerated early entry of [35S]sulfateinto brain. By contrast, iodinated albumin is confined to the brain vasculature, with no evidence of entry into brain under normal conditions (Barlow et al., 1958). Therefore, extravasation of albumin may be considered to reflect a pathological change in barrier mechanisms. A . The efect of pharmacologically induced convulsions on the penetration of [35S]sulfate into cat brain 1. General methodology. Adult cats were anesthetized locally with xylocaine, immobilized with gallamine, and artificially respired at carefully controlled rates and minute References p . 378

368

R. w. P. C U T L E R et al.

volumes. The convulsant drug was administered intravenously. Following the appearance of electroencephalographic seizure activity, carrier free [35S]sodium sulfate, 1 mC/kg, was given intravenously. After a five minute circulation period, the animals were anesthetized with pentobarbital, heparinized, and were then perfused through an aortic cannula with 200 ml isotonic saline. The brain was removed, frozen, and sliced freehand for gross autoradiography. Fifteen regions of brain, CSF, and plasma were assayed for 35s radioactivity. [3jS]metabolites were separated by paper electrophoresis (Goldberg et al., 1963) to permit expression of the data as a tissue to plasma ratio for [35S]sulfate. The experimental procedures will be published in detail elsewhere (Lorenzo et al., 1967). Control animals were treated in a fashion comparable to the experimental animals, except that the convulsant agent was omitted. The results of this series of experiments are presented in Fig. 1 and Table I.

Fig. 1. 35Sautoradiograms made from frozen coronal slices of cat brain at the level of thalamus and medulla. [35S]sulfatewas injected intravenously five minutes before sacrifice.

2. Pentylenetetrazol (metrazol) in a dose of 30 mg/kg produced increases in the fiveminute uptake of [35S]sulfate generally throughout the brain. However, distinct regional accentuation of sulfate uptake was observed, with an approximate 12-fold increase in the thalamus and lateral geniculate ganglion, an 8-fold increase in the medial geniculate ganglion, and a 4-fold increase in the cerebral cortex and white matter. There was no increase in penetration of sulfate into the cisternal cerebrospinal fluid. In addition, the percentage of 35s incorporated into metabolites was not significantly increased. When metrazol was given to animals that had received [35S]sulfate after ureter ligation six hours previously, no change in the steady-state concentration of [35S]sulfate was observed. 3. The eflect of strychnine (0.5 mg/kg) on sulfate uptake, while less pronounced than metrazol, was nonetheless distinctly regional. The highest uptake, approximately four times control, was observed in the lateral geniculate body, followed in order

369

BLOOD-BRAIN PERMEABILITY

TABLE I cpm/gm wet tissue 35S04 Uptake by Brain

~

-

-

_x 100

_

J

\ cpm/ml plasma Region

Cerebral cortex Cerebral white Thalamus Lateral geniculate Medial geniculate Caudate Hippocampus lnferior colliculus Cerebellar cortex (paleo) Cerebellar cortex (neo) Cerebellar white Central medulla Cervical cord CS F

Coritrol

Metrazol

Strychnine

( 9 )*

(5 )

(10)

Methionine Sulfoximine (6)

0.38 f 0.02 0.11 0.02 0.18 i 0.02 0.19 f 0.01 0.18 i 0.02 0.23 t 0.03 0.22 1 0 . 0 3 0.28 f 0.01 0.35 f 0.02 0.35 f 0.02 0.10 0.01 0.14 0.01 0.45 10.05 I S O & 0.20

1.61 0.34 0.49 f 0.09 2.19 0.45 2.34 f 0.67 1.43 i 0.26 1.37 f 0.17 0.86 & 0.16 1.02 & 0.13 1.02 & 0.14

0.55 f 0.05 0.16 f 0.01 0.44 f 0.03 0.79 f 0.08 0.35 f 0.02 0.27 f 0.03 0.34 f 0.04 0.31 f 0.03 0.80 f 0.12 0.49 f 0.06 0.16 f 0.02 0.43 f 0.04 0.74 f 0.10 1.80 f 0.20

1.10 f 0.18 0.34 f 0.06 0.69 f 0.16 0.96 & 0.24 0.70 f 0.13 0.54 f 0.09 0.78 f 0.20 0.69 f 0.09 0.49 f 0.05 0.55 f 0.04 0.27 f 0.04 0.38 rt 0.10 1.02 f 0.14 1.33 f 0.18

*

*+

_

_

0.28 :k 0.03 0.59 & 0.12 0.96 f 0.21 1.89 0.45

~ _ _ _

*

Number of animals

by the thalamus, the cerebellar paleocortex, and the central medulla. Less, but significant increases in sulfate uptake were observed in most other regions, while the cerebrospinal fluid showed no change (Lorenzo and Barlow, 1967).

4. The data for a series of cats convulsed with methionine sulfoxamine are tabulated in the last column of Table I. A dose of 5-20 mg/kg was given twenty-four hours prior to the onset of the study. This drug produces a seizure disorder characterized by both psychic and motor activity. Upon the appearance of a clinical seizure, the five minute sulfate uptake was measured as described. A more generalized increase in sulfate penetration was found, particularly prominent in the cerebral and cerebellar cortex and hippocampus. In addition, autoradiography revealed a pronounced uptake in the substantia nigra (arrow, Fig. l), an area ordinarily inaccessible to direct radioassay. While detailed comparisons were lacking for methionine sulfoximine, the regions of increased sulfate penetration during metrazol and strychnine convulsions were comparable to the regions which exhibit enhanced electrical excitation in depth electrode studies (Starzl et al., 1953; Johnson, 1955). It seemed reasonable to propose that the enhanced regional uptake of anion in these drug-induced convulsions was a consequence of heightened neuronal activity, rather than of direct alterations in permeability produced by the drugs. Several observations are consistent with this hypothesis. Previous studies (Lorenzo et al., 1965) have demonstrated specific increases in sulfate uptake in the visual and auditory systems upon photic and acoustic stimulation, indicating that neuronal activation by more physiological stimuli may be accompanied Rcfivriiri~sp. 378

370

R.

w.

P. C U T L E R et

al.

by alterations in [35S]sulfate exchange. In addition, sub-threshold doses of the drugs employed produced no changes in sulfate penetration. Furthermore, when blindfolded cats were convulsed with strychnine, the tissue-plasma ratio for [35S]sulfate was 0.61 f 0.03, as compared to 0.19 for control animals. When the eyes were exposed to laboratory illumination during the strychnine convulsion, however, the [35S]sulfate tissue-plasma ratio in the lateral geniculate increased significantly to 0.91 f 0.1 I . This observation was consistent with the recognized ability of strychnine to facilitate neuronal activation by an afferent stimulus. The alterations in sulfate penetration induced by metrazol and strychnine convulsions were rapidly reversible. In experiments in which the sulfate was injected one hour after the cessation of seizure activity, brain [35S]sulfate uptake had returned to control values. In the case of metrazol, convulsions were stopped by the intravenous injection of trimethadione. With strychnine, convulsions were abolished with methocarbamol and trimethadione. Such rapid reversibility lends additional support to the concept of heightened neuronal metabolic activity as a factor in the production of permeability changes. B. The effect of metrazol on the penetration of['3lI]albumin into cat brain In this series of experiments, cats were convulsed with metrazol for five, fifteen, thirty and sixty minutes, and the uptake of [131'l]albumin (100 pC/kg) during the seizure was studied. There was no detectable tissue penetration of iodinated albumin in control animals, nor in those convulsed for five minutes. After fifteen minutes of seizure activity, patchy extravasation of protein was observed in the thalamus, which by thirty minutes became extensive and confluent (Fig. 2). By one hour, the amount of

Fig. 2.

1311

autoradiogram made from frozen coronal slice of cat brain at the level of the thalamus, following a thirty-minute metrazol convulsion.

albumin in extravascular compartments of the thalamus was five to six times the amount normally contained in the blood vessels of that region, while the cortex and white matter remained relatively impermeable, as illustrated.

37 1

BLOOD-BRAIN PERMEABILITY

C. Additional physiologicaI alterations accompanying metrazol convulsions Blood pressure, blood pH, p0z and pC0z were recorded during a five minute pentylenetetrazol convulsion. Cortical pH, oxygen tension, carbon dioxide tension, and the electroencephalogram were recorded from electrodes on the exposed cortical surface. Cortical temperature (cerebral blood flow) was measured by a thermistor. Craniec-

/

I

...

Wood pC0,

Fig. 3. Continuous in vivo recording of the physiological parameters indicated prior to and during a live-minute metrazol convulsion. The interruption of the time pulse recording at the top of the polygraph signifies the period of injection of rnetrazol (from Lorenzo et a/., 1967). Refiriwws p . 378

372

R. w. P. C U T L E R et al.

tomy and electrode placement was performed under ether anesthesia, and recordings were begun thirty to sixty minutes after anesthesia was discontinued. The results of a representative experiment are illustrated in Fig. 3. Thirty seconds following the intravenous administration of metrazol, seizure discharges appeared on the electroencephalogram, accompanied by tachycardia and systemic hypertension. Cortical temperature rose promptly and remained elevated during the entire convulsion. There were no significant alterations in blood gases or hydrogen ion concentration. By contrast, changes on the cortical surface were quite marked. Brain oxygen tension fell from 16 mm Hg to 0 mm Hg, while carbon dioxide tension rose from a control value of 22 mm Hg to 85 mm Hg. The pH fell from 7.30 to 7.10. It was apparent that the state of increased neuronal metabolism during a convulsion was sufficient locally to generate excessive quantities of carbon dioxide, with a concomitant fall in tissue pH. D . The effect of hypercapnia on the penetration of sulfate andalbumin into the brain 1. I n a series of cats artificially respired with 20

% COz : 80 % 0 2 , marked alterations

in the five-minute uptake of [35S]sulfatewere observed. While somewhat greater variability was noted in the hypercapnic animals than in the convulsed animals, the most significant increases in uptake occurred in the thalamus and brain stem (Fig. 4). As

Fig. 4. The influence of hypercapnia on [35S]sulfatepenetration into cat brain.

shown previously (Goldberg et al., 1963), hypercapnia affected the rate of entry of sulfate without altering the steady state tissue-plasma ratio or the degree of metabolic incorporation.

2. A more detailed study of the effects of hypercarbia on brain vascular permeability was carried out in guinea pigs, employing [125I]albumin as a tracer (Cutler and Barlow, 1966). Because serum albumin enters brain to a very limited extent, if at all, the ratio of brain tissue~[125l]albuminto plasma [125l]albumin serves as a measure of brain plasma volume in the normal animal, while extravascular penetration of radioiodinated albumin into brain under experimental circumstances reflects an alteration in brain vascular permeability. The methods employed in this series of experiments were as follows: anesthetized

373

BLOOD- BRA1 N P E R M E A B I L I T Y

guinea pigs were artificially respired with room air or various mixtures of carbon dioxide : oxygen. In the first series of animals, [1251]human albumin was injected intravenously, and circulated for one hour. Animals were sacrificed at the end of one hour either by decapitation with rapid immersion of the head in solid carbon dioxide: acetone at -6O"C, or by vascular perfusion with 100 ml isotonic saline. Frozen brains were dissected into nine regions and assayed with plasma for 1251 radioactivity. pH and p C 0 z were measured in samples of jugular venous blood drawn from a cannula inserted cranially. Histological autoradiograms were prepared from fixed, paraffin embedded tissue sections dipped in liquid photographic emulsion. T A B L E 11 METHOD O F DETERMINING ['"I]ALBUMIN

( A ) C02 Decapitate

(B ) Control Decapitate ( C ) COZPerfuse

*

A - [B I C]

=

D I S T R I B U T I O N I N BRAIN

Nornial Vascular Volutne

Exfravascular Volume

Change In Vascular Volume*

+ +

+

t

0

0

-t

0 0

Change in Vascular Volume.

By separate analysis of experiments terminated by either vascular perfusion or rapid freezing, the iodinated albumin activity in the brain may be partitioned into normal vascular, dilated vascular, or extravascular compartments, as summarized in Table 11. In order to equate tissue plasma volume with tissue vascular volume, it was necessary to assume that regional changes in hematocrit did not occur under conditions of hypercapnia. This assumption was supported by the lack of change ofjugular venous blood hematocrit in hypercapnic animals as compared with controls. Penetration of serum albumin into the guinea pig brain under normal conditions was not detectable. The slight amount of radioactivity remaining in the normal brain after perfusion may be identified within vessels by histoautoradiography. Upon exposure to high concentrations of carbon dioxide, marked changes in vascular permeability were observed. There was a consistent extravasation of albumin into the thalamus, hypothalamus, mid-brain, medulla and spinal cord. By contrast, there was little evidence of abnormal permeability in the cerebral cortex, cerebral white, caudate nucleus or cerebellum, as illustrated in Fig. 5, and in column C,Table 111. During more prolonged exposure to carbon dioxide, for periods up to eight hours, protein extravasation continued at the same rate in vulnerable regions, while the cortex and white matter remained resistent to permeability alteration. It was evident that the state of altered vascular permeability persisted as long as the animal remained hypercarbic, and that no adaptation to this stress occurred, at least during an eight-hour interval of time. However, the changes in permeability were rapidly and completely reversible. When animals which were exposed to twenty-five percent COz for one hour and then room air for ten minutes were subsequently injected with radioiodinated Refirc~rtri~s p. 378

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Fig. 5. 1251 autoradiograms of frozen slices of guinea pig brain following a one-hour exposure to twenty-five percent COZ.(a) mid-sagittal plane; (b) coronal, mid-thalamus; (c) coronal, mesencephalon; (d) coronal. medulla. (From Cutler and Barlow, 1966). T A B L E 111 ONE-HOUR BRAIN-PLASMA RATIOS*

C COz-Perfitse

(5)t

B ControlDecapitate (6)

5.40 f 0.30 2.86 f 0.20 4.09 f 0.44 5.58 f 0.77 6.70 f 0.80 4.60 f 0.61

4.00 f 0.17 1.83 f 0.34 2.85 f 0.27 3.52 f 0.13 4.80 f 0.46 2.86 f 0.34

0.34 f 0.06 0.22 0.02 0.37 0.06 2.25 f 0.65 2.44 f 0.55 2.03 f 0.50

A COz-Decapifate

Cortex White Matter Caudate Thalamus Colliculus Medulla

*

t

(6)

A Vascular Volume

( A -[ B t C l ) -I 1.06 0.81 0.87 -0.19 i-0.06 -0.29

+ +

CPM/mg dry tissue

x 100 CPM/,uI plasma number of animals.

albumin, no penetration of albumin into the brain was found after a one hour circulation. With prolonged hypercapnia (eight hours), relatively large quantities of plasma albumin escaped into the tissues. In the thalamus and medulla, this amounted to seven times the normal quantity contained in the vasculature of these tissues. While the

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Fig. 6 . [1251]alburninhistoautoradiogram showing high silver grain density overlying a neuron in the reticular formation of the medulla. Hemdtoxyhn. X 560. resolution of light microscopy was insufficient to permit precise localization of the extravasated albumin by autoradiography, it was apparent that some of the protein had entered cells, notably into large neurons of the reticular formation (Fig. 6). By ultracentrifugation it was found that approximately thirty percent of the [125I]radioactivity was sedimented with subcellular particles. In animals exposed to twenty-five percent COZ for one hour, it was possible to determine the amount of /125I]albumin within the brain vasculature, as well as in extravascular compartments, as outlined in Table 11. The results of this analysis for six brain regions are presented in Table 111. I n this table, valuesincolumn Crepresent the amount of extravascular protein, while the values in the last column are a measure of expansion or constriction of the vascular compartment. It was apparent that the greatest permeability changes occurred in regions in which the vascular volume remained unaltered. Conversely, enlargement of the vascular compartment, either by vasodilatation or by opening new vascular channels was not associatedlwith pathological permeability. When extravascular protein concentration was expressed as a percentageof the normal vascular concentration andplottedlagainst changes in vascular volume, a consistent inverse relationship was found for the nine regions of brain assayed (Fig. 7). Additional experiments were performed to assess the effects of lower concentrations of carbon dioxide and of metabolic acidosis. With ten percent carbon dioxide Rcfcrmri~rp. 378

376

w. P.

R.

w z

3

9

50

40 30

p

20

u)

w +I0 z

(3

z

40 .

et al.

60

3 3 0

CUTLER

o

- 10 s

0

0

0 0 0 0 0 0 fi

1

10 20 30 40 50 60 70

RELATIVE PROTEIN PENETRATION

Fig. 7. The relationship between protein permeability and vascular volume changes in hypercapnic guinea pigs. Each point represents one of nine brain regions sampled. (From Cutler and Barlow, 1966).

exposure, the jugular venouspCO2 rose from 48 mm Hg to 79 mm Hg, but no protein extravasation was found. Similarly, in animals made acidotic by HCI infusion (mean pH 7.02, mean pC0z 53 mm Hg), there were no alterations in protein permeability. It was apparent that the relatively high levels of carbon dioxide tension produced by exposure to twenty-five percent COz (mean pH 6.94, mean pC0z 136 mm Hg) were required to produce pathological protein permeability. DISCUSSION

Alterations in [35S]sulfate penetration into the brain during convulsions have followed a distinct pattern for each of the pharmacological agents studied. The observation of enhanced sulfate uptake in regions shown to be electrically activated during metrazol (Starzl et al., 1953) and strychnine (Johnson, 1955) convulsions provides evidence that local acceleration of neuronal metabolism may be accompanied by heightened solute exchange. Neither the degree of metabolic incorporation of 35s nor the size of the steady state sulfate pool were altered by the convulsions. Theenhanced [35S]sulfate uptake may be considered as a partial consequence of increased delivery of the anion to the tissues secondary to capillary vasodilatation or increased blood flow. In support of this thesis is the close correlation of enhanced regional sulfate penetration (Lorenzo et al., 1965) and increased regional cerebral blood flow (Sokoloff, 1957) in visual centers of photically stimulated cats. However, as sulfate enters the brain relatively slowly (one to two hours for maximum levels to be reached following intravenous injection in the cat), its rate of entry cannot be closely regulated by regional blood flow. Alternatively, or in addition to change in blood flow, the enhanced penetration of [3%] sulfate may be a more direct consequence of regional changes in vascular permeability to sulfate. The finding of regional entry of iodinated albumin during more prolonged convulsions is strongly in support of this argument. Because this tracer is normally excluded from the brain even after long circulation times, its- entry under these circumstances must imply altered vascular permeability.

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Changes in arterial blood pH, oxygen tension, and carbon dioxide tension were not observed during a brief metrazol convulsion, while the same recorded values from the brain cortical surface were markedly altered. Carbon dioxide is one of the most active agents in the regulation of cerebrovascular tone and cerebral blood flow (Sokoloff, 1959). Its influence on brain vascular permeability was first demonstrated by Clemedson et al. (1958) who found the brains of hypercapnic animals stained by trypan blue. In the experiments reported here, hypercarbia had a profound effect on vascular permeability in some regions of the brain while sparing others. There were no demonstrable defects in vessel walls on light microscopy, nor other neuropathological change. Diapedesis of erythrocytes was not an associated feature in our studies as in the experiments of Clemedson et al. (1958). From direct measurement of vascular volumes, we have concluded that vasodilatation was not a necessary factor in producing increased permeability. While the selective vulnerability of brain vessels to a systemically administered agent was emphasized by these results, the reasons for the selectivity were not forthcoming. It is of interest that during metrazol convulsions, increased entry of sulfate was observed in a number of regions of the cat brain, while protein penetration was predominantly restricted to the thalamus. Similar observations were made by Lee and Olszewski (1961) following repeated electroshock convulsions in the rabbit. It is reasonable to expect that a continuous rapid production of metabolic COZby excited neurons occurs during more prolonged convulsions. Regional increases in carbon dioxide tension of sufficient magnitude to produce protein extravasation would be anticipated. However, protein penetration would be detected only in those regions which both generate large amounts of carbon dioxide and are susceptible to the effects of hypercapnia. Therefore, in the case of metrazol, while both the thalamus and cortex are under excitation, the pathological permeability changes to albumin would be expected only in the thalamus. The techniques of autoradiography and quantitative regional tissue assay used in this study may provide a general method for the identification of physiologically or pharmacologically activated neuronal groups which are functionally interrelated. In this regard, attention is called to the autoradiograms depicting methionine sulfoximine convulsions in Fig. I . As indicated by the arrow, there is an apparent high uptake of 3% in the region of the substantia nigra, which could be followed in serial sections to its caudal extent. This region of brain is not ordinarily sampled for direct radioassay or probed by the recording electrode. The finding of a high autoradiographic density in these studies should provide a stimulus for investigating this nucleus with electrophysiological techniques. ACKNOWLEDGEMENT

This work was supported in part by grant NB-05172 of the National Institute of Neurological Diseases and Blindness. Rcfcrcnws p. 378

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al.

REFERENCES BARLOW, C. F., SCHOOLAR, J. C. AND ROTH,L. J. (1958) An autoradiographic demonstration of the relative vascularity of the central nervous system of the cat with iodine 131-labeled serum albumin. J. Neuropathol. Exptl. Neurol., 17, 191-198. CLEMEDSON, C. J., HARTELIUS, H. AND HOLMBERG, G. (1958) The influence of carbon dioxide inhalation on the cerebrovascular permeability to trypan blue (the blood-brain barrier). Acta Pathol. Microbiol. Scand., 42, 137. CUTLER,R. W. P. AND BARLOW,C. F. (1966) The effect of hypercapnia on brain permeability to protein. Arch. Neurol., 14, 54-63. GOLDBERG, M. A., BARLOW,C. F. AND ROTH, L. J. (1963) Abnormal brain permeability in C02 narcosis. Arch. Neurol., 9, 498-507. JOHNSON, B. (1955) Strychnine psroxysms in brain stem. I. Anatomical distribution. J . Neurophysiol., 18, 189-199.

LEE,J. C. AND OLSZEWSKI, J. (1961) Increased cerebrovascular permeability after repeated electroshocks. Neurol., 11, 515-519. LORENZO, A. V., FERNANDEZ, C. AND ROTH,L. J. (1965) Physiologically induced alteration of sulfate penetration into brain. Arch. Neurol., 12, 128-132. LORENZO, A. V., BARLOW, C. F. AND ROTH, L. J. (1967) Effect of metrazol convulsions on S-35 entry into cat nervous system. Amer. J. Physiol., 212, 1277-1287. LORENZO, A. V. AND BARLOW, C. W. (1967) Effect of strychinine convulsions upon the entrv of V5 sulfate into the cat central nervous system. J. Pharmacol. Exptl. Thrrup. (in press). SOKOLOFF, L. (1957) Local blood,flow in neural fissues. New Research Techniques of Neuroanatomy. W. F. Windle, Editor. Springfield, Charles C. Thomas (p. 51). - (1959) The action of drugs on the cerebral circulation. Pharmacol. Revs., 11, 1-85. STARZL,T. E., NIEMER,W. T., DELL,M. AND FORGRAVE, P. R. (1953) Cortical and subcortical electrical activity in experimental seizures induced by metrazol. J. Neuropathol. Exptl. Neurol., 12, 262-276.

DISCUSSION

D. B. TOWER: Two things, Dr. Cutler. First of all I would like to make a plea in regard to methionine sulfoximine. It is nice to see people using this agent. This providesavery interesting seizure preparation because it is somewhat more chronic than many of the convulsant agents that one normally uses. When you give a convulsant dose of metrazol it generally gives a completely generalized kind of acute convulsion unless you fractionate the dose rather carefully. But in the case of methionine sulfoximine, the development is, as you indicated, over quite a period of time unless you inject it directly into the central nervous system. Furthermore, you tend to see more different forms of seizures (which one would like to relate by analogy to clinical situations in patients) in that you may have partial seizures that may develop into a generalized convulsion. The running behavior, the salivation and chewing movements are characteristics of four-footed animals and can be seen in other types of seizures, and are not peculiar to methionine sulfoximine at all. It is just that here is an agent that is showing many more manifestations because of the longer period over which they develop, and is in fact a sort of a semi-chronic affair. The one question I had in mind regards your perfused preparation: How much of a factor in analyses of the type you are doing is an asphyxia1 change in the cells of the central nervous system during this time? In other words: if you alter the inflow of nutrients, oxygen and glucose and so on o the nervous system, does it take very long for some of the cells to swell, as many people have shown. R. CUTLER:There was no demonstrable protein in control animals perfused in the same manner. One comment I might make about the methionine sulfoximine: agree that one cannot call this a perfect model of a temporal lobe seizure.

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W. W. TOURTELLOTTE: I would like to speak about perfitsion of the bruin to remove blood “coniaminants.” Perfusion, while it removes all contaminating blood, may introduce complications. For example, published data (Hogeboom, G . H., Schneider, W. C., and Stribbich, M. J.: J. Biol. Chem. 196, I 1 I , 1952; and Luch, J. M.: J. Biol. Chem. 115: 491, 1936) indicate that as much as 20-30% of liver nitrogen is removed by perfusion. On the other hand, estimations of the vascular space in the rat liver (ml of bloodikilogram of tissue) can vary from 99 to 178 to 270 depending on whether the rat was sacrificed by “slow” exsanguination (Caster. W. O., Simon, A. B., and Armstron, W. D.: Am. J. Physiol. 183: 317, 1955). sacrificed by ether (Friedman, J. J.: Am. J . Physiol. 196: 420, 1959) or sacrificed by rapid immersion in liquid nitrogen (Gibson, J. G., Sebgman, A. M., Peacock, W. C., Aub, J. C., Fine, 5.. and Evans, R. D.: J. C/in. Invest. 25: 848, 1946). Since the concentration of nitrogen in whole blood is very close to that of liver, the large nitrogen losses as a result of perfusion suggest considerable losses from extravascular liver substance. The best solution to these difficulties would seem to lie in a method for the determination of whole blood in fresh tissue rather than in any attempt to remove or stabilize this variable “contaminant.” To check this possibility in regards to brain, we have studied (unpublished observations) immunoglobulin-G (IgG) concentration (see chapter by Tourtellotte for discussion of the technical aspects of the immunochemical IgG assay) before and after perfusion of the guinea pig brain with 100 ml of 0.1 5 M sodium chloride. Furthermore, the blood content of the brains was determined (see Chapter by Tourtellotte for discussion of the technical aspects of brain hemoglobin determination); hence, it was possible to calculate the IgG concentration contributed by blood in the brain as well as the extravascular brain values. It was found that the IgG concentration, corrected for residual blood IgG concentration of the brain after 30 minutes of pentobarbital anesthesia (70 mg/kg, intraperitoneally) and quick removal of the brain after guillotining, was 0.78 f 0.04 gm/kg of brain (five guinea pigs). After perfusion with 100 ml of isotonic saline in 10 minutes, the brain IgG content, corrected for residual blood IgG, was 0.1 I t 0.01. Therefore, perfusion of the cerebral vessels under the conditions mentioned above removed 86 percent of the extravascular brain IgG. Since our results suggest that perfusion with isotonic saline of the brain immediately after death can reduce the concentration of rather large molecules, such as immunoglobulin-(?, would it not be more accurate to include in your experiments a determination of the amount of blood in the brain, (rather than to try to remove the blood “contaminant” by perfusion) and make a calculated correction for the extravascular concentration of the tagged substance you are studying? The volume of the blood in the brain and its “contaminating” substance can be estimated rather simply and precisely (Tourtellotte, W.W., and Parker, J. A.: Science 154: 1044, 1966). R. CUTLER: This whole problem of perfusion is a difficult one to know the real answer to. In answer to the last question: in that study we were dealing with sulphate, which has an extremely high concentration in the blood after five minutes, relative to the concentration in the tissue. If we don’t perfuse out the blood we won’t see any changes. With regard to the technique of determining haemoglobin, it seems much easier and more accurate for us to use a radioactive labeled protein that is excluded from the brain during this time period.

Did the [1311]albuminmove into the brain and hence interfere with your brain W. W. TOURTELLOTTE: blood volume determination?

R. CUTLER: Not in five minutes, though. Our method for determining blood flow per capillary surface area is to give an injection of iodinated albumin, determine the cardiac output in an individual animal, and let the albumin circulate for five minutes. Then we give an injection of 1311-labelled antipyrine, let that circulate for one minute, chop off the head and fix the vascular contents for dissections at -30”. The tissue is assayed for its iodinated antipyrine content, which can then be related to the cardiac output. This gives you a direct measurement of milliliters per gram of blood flow in an area. In the normal animal one can calculate the capillary volume; from the volume determine the capillary length, assuming a mean capillary diameter; then in the experimental situation further assume that the capillary length does not change, but that the capillary diameter will be the parameter that will change, and then see what the flow-surface re1ationship:is. W. W. TOURTELLOTTE: The only substance for sure that does not moveout ofthevascular space into the brain is hemoglobin. Hence, it would appear that the measurement of hemoglobulin to determine the extent of the contribution of blood “contamination” in the brain is on sound grounds.

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L.BAKAY:I would just like to make one statement as far as determination of the blood content is concerned. The determination of blood volume in any small area of brain tissue by determining haemoglobin is very difficult, because many of the small vessels of the white matter for example are very small indeed, In a cerebral capillary the haematocrit is onlyabout 16 to 18%. In otherwords, 70 to 8076 of the blood in the small vessels is plasma. This means that the determination of plasma proteins or any other plasma constituent is far superior to determination of the haemoglobin, which reflects directly only on the number of the blood cells which is identically distributed in the small capillary vessels. J. P. S C H A D ~I :would like to make a short remark about methionine sulfoximine. We have been studying with the light microscope the effect of methionine sulfoximine on the dendritic organization. Even after 24 hours, a severe degeneration in both cerebral and cerebellar cortex was found, resulting in a reduction of the dendritic plexus.

D. B. TOWER: You can get severe lesions from methionine sulfoximine, but you can also have a long period of seizures in which you cannot demonstrate any pathological change under the light-microscope by the usual neuropathological procedures. Therefore, I think that this is a question which depends apparently on the severity of the seizures, since it is well known that changes secondary t o the seizures themselves can occur after very severe seizure manifestation. I would like to make one comment in relation to what Dr. Bakay has just said: He has reminded me of one of the things which has not been mentioned here which is perhaps something that neurosurgeons see from time to time. This is a sort of “bloodless” capillary. When the cortex is exposed and proximal flow is temporarily occluded or sluggish, you can watch a capillary fill up with fluid in which there is absolutely no blood at all. D. P. RALL:I would like to make two points. One is just to get the record straight; I think Dr. Cutler well recognizes that you cannot really equate a p H of 7 caused by an increase in COZwith a pH of 7 caused by infusion of hydrochloric acid. With the COz-induced pH, your intracellular and cerebrospinal fluid-pH will be the same as your plasma-pH. This is a very different story from the situation in which you have induced this acidosis by HCI. In that instance the CSF-pH will be, if anything, higher than normal, and the intracellular pH will probable not have changed at all. This can lead to a very different set of circumstances. Secondly, a number of people have mentioned the use of iodoantipyrine as a method of following body water. I do not know the reference, but it is clear that iodoantipyrine does not act like nonlabeled iodopyrine. It is more ionized and less soluble, and furthermore, it can be metabolized; so as a convenient agent for tracing body water iodopyrine with its problems of determination is very good, but iodoantipyrine, I am afraid, is not. GENERAL DISCUSSION

I just wanted to make a comment on the remarks of both Dr. Bakay and Dr. Dobbing. R. KATZMAN: I quite agree with them that in so-called physiological measurements in ventriculo-cisternal perfusions one really has to consider the possibility of artifacts such as edema occurring. The only evidence in this regard is the fact that in chronic repeated perfusions, animals d o quite well, and d o not have any increase in intracranial pressure. However, I am not aware of any specific studies as to the existence of a minimal amount of cerebral edema in association with such perfusions, and it certainly ought to be studied. One thing that is quite clear, however, is that many movements of substances from blood to brain are controlled by COZ,and perhaps the experimenters who do ventriculocisternal perfusions should be monitoring the COz-level of the blood in their animals. L. BAKAY:I believe that changes occur in perfusion only if the pressure is drastically changed, or if the tonicity of the perfusate is way off, or when it is toxic. 1 don’t assume that there are changes under other circumstances than that. R. V. COXON:What do you mean, Dr. Bakay, by “pure hypoxia”? Would this mean that the animals were breathing a low oxygen concentration in nitrogen? Because if that is the case, they would also be hypocapnic.

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38 1

L. BAKAY: They were not hypocapnic. As far as this situation is concerned, when I spoke about pure hypoxia, I meant just that. In other words: the animals were breathing a mixture low in oxygen, but with a normal minute volume, or a slightly increased minute volume occasionally. There was only a very slight drop in the pH.

M. BRIGHTMAN: I cannot share Dr. Bakay’s unconcern about the presence of broken membranes or even an absence of membranes in the electronmicrograph if that micrograph is used as a basis for ascertaining whether edema has occurred or not. In order to determine whether the compartment is intracellular or extracellular, the least you must have is a membrane separating the two compartments. It is even more difficult to say whether a protein marker has got there through a membrane, or whether it was simply an artifact. The effect of fixation on undamaged tissues is difficult enough to ascertain, especially if there are any signs that damage has occurred during the process of fixation. But when chemical fixation is superimposed upon some sort of damage before that, it is cven more difficult. I fully concur with that. The fact remains, that when it comes to edema ofthe white matter, L. BAKAY: you do occasionally see ruptured cell membranes. I d o not believe that that necessarily is the sole reason, or perhaps even the principal reason for the fact that the extracellular space in the white matter expands.

D. P. RALL:I would like to come back to Dr. Dobbing’s question. You could perhaps suggest that there might be two problems with the perfusion of the ventricular system. One is related to the introduction of the needle, which I will not comment on. The second concerns what the fluid does to the brain, and I can comment on this. We have put indwelling capillaries into patients to treat malignant processes within the brain, and we have perfused these patients repeatedly. We use a solution, called Elliot’s B-solution. These patients suffer no ill effects. They do not have headaches, and they are not disturbed in any way. If you use the living human brain as an indicator of the state of function of that brain, eight hours of perfusion at normal pressures has no deleterious effect. P. MANDEL: Is there a physiological control too?

D. P. RALL:Yes, rather simple EEG’S, and they are normal. L. BAKAY: Everybody with a modicum of arterial hypertension has a measurable amount of cerebral edema, without having any ill effect, or any headache, or anything whatsoever. So the fact that a person is perfused and is able to talk and does not have headache does not necessarily mean that he does not have a slight amount of edema. We have been doing ventriculo-cisternal perfusions in rats. In a situation where D. M. WOODBURY: we measured thc water content in the brain during various times of perfusion, there was no change at all worth mentioning, and there was no cerebral edema. As far as inulin movement was concerned: we sampled the brain at some points where the perfusions were made and as well as in other portions of the brain. There was very little difference in the content of inulin in these different areas.

K. A. C. ELLIOTT: Long ago I did a series of experiments to comparethemetabolismofvariousexcised tissues. I t was pointed out that brain and testes were more alike than any other tissues. I would like to ask Dr. Bakay a question. In his remarks on kernicterus, he mentioned that any relation to it with the undeveloped brain-barrier system could not be dismissed, but he did not give us a reason why. L. BAKAY: The reason why most people dismiss the theory in terms of kernicterus is because there is a striking discrepancy between the high incidence of kernicterus in premature babies with erythroblastosis fetalis, and a very low incidence, or almost absence of the same condition in premature infants, who have icterus of another nature. This strongly suggests that there is something specific about the unconjugated bilirubin in erythroblastosis fetalis. If this were simply a matter of membrane or capillary permeability, there should be more of a similarity in these various forms of icterus in the newborn babies. J. FOLCH-PI:In the literature it is mentioned that there is a kind of blood-brain barrier system in the testes quite similar to that for the brain.

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GENERAL DISCUSSION

L. BAKAY:I did not want to insinuate that this was a statement of generalized implication. I personally d o believe that the barrier, whatever it is, whether in the form of increased membrane permeability, or in the form of an increased size of extracellular space, is different in newborn and that it develops as the time goes on. It is just in relationship to kernicterus that we don’t understand what we see. Here is a point which is so difficult: One can equally well reason theoretically that kernicterus occiirs in the basal ganglia and not in the rest of the brain because of differences in vascularity or metabolism or in other ways. One point is very striking. This is that the region in which kernicterus develops is also where the anoxic damage in carbon monoxide poisoning occurs. However, whether this is due to a matter of membrane permeability or to a special metabolic factor, 1 don’t know.

J. FOLCH-PI:The blood-brain barrier has definite regional differences and is essentially a dynamic situation. A dynamic situation requires an input of energy which is probably controlled in some way or other feed-back information on the needs of the tissue. Another point is that weallshould remember that the barrier does not necessarily work in only one way. L. BAKAY:I really can’t answer most of the remarks that Dr. Folch made. But one answer, which is also pertinent to what Dr. Lajtha said, is that 1 applied a great variety of markers or tracers, some of them for the sake of technical convenience in pathological conditions. It was not elaborated which of them was metabolically active and which was not, and which might go into the brain by passive diffusion and which by a carrier mediated active transport. The fact remains, that when it comes to a pathological condition, there is hardly any difference in the majority of cases, as to whether the tracer is an inactive structure or one which is a highly metabolically active agent. There is very little selection in the distribution between metabolically active or inactive or even small or large particle-sized tracers between what we consider normal and abnormal brain tissue. R. CUTLER: Just a short comment. It is the unconjugated non-protein bound moiety of the bilirubin that enters the brain. This never occurs in an adult because one never reaches such high levels of unconjugated non-protein bound bilirubin as occur in the newborn infant. But, if you infuse Cl4--labeled unconjugated bilirubin into an adult animal, it will enter the brain and cause kernicterus.

L. BAKAY:I happened to have cases with hepatitis with a very high concentration of unconjugated bilirubin in the human adult. And while it did stain the lesion it did not seem to go into the normal white matter brain tissue. A. LAITHA:I just want to add a very short remark to what Dr. Folch said. We have some evidence that developmental differences also exist as far as active processes in the barrier systems are concerned. The pumping-out mechanism, although not completely absent, is not quite as well developed in the early stages, so that this may also be an explanation.

D. B. TOWER:I would just like to add a comment to what Dr. Bakay said about how in various kinds of pathological conditions the brain responds in the same way. We have to recognize that from a neuropathological point of view the range of reactions is extraordinarily small. Many different conditions produce the same end-result, at least in a gross sense. Perhaps, looking a t this philosophically, it has some relevance to the subject we are discussing here. Normally the brain is, so to speak, protected from many insults and therefore has not been stimulated to develop a versatility for reacting to insults. W. W. TOURTELLOTTE: It is rather striking to see at postmortem examination of the brain an acute cerebral infarct in a jaundiced patient. The infarct stains yellow and the surrounding “normal” appearing tissue is unstained. Furthermore, with formalin fixation the bilirubin is oxidized and the infarct turns green. L. BAKAY: You misunderstood me. In the person in whom I found a high unconjugated bilirubin and who died, there was an infarct that was stained, but the surrounding brain was not stained. But, to add more complications, I must add here one more factor to confuse us. A patient with severe long-lasting icterus, has bilirubin in his cerebrospinal fluid, that is low in protein. So, you can’t say that all the bilirubin is albumin-bound. The main problem is that here we have a patient with a high serum bilirubin, a high cerebrospinal fluid-bilirubin and an unstained brain, except for the ependymal area.

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D. H. FORD:I have a comment on this problem of the differential uptake of amino acids between brains of very young and adult animals. A point which is very seldom made in regard to this transfer of amino acids from blood to brain, is the tremendous differential in growth and synthesis of new protein in these young neurons which must increase in size a hundred-, two hundred-, or a thousandfold in some instances. If one then examines a group of rats injected intraperitoneally with [35S]cystine and determines the uptake or incorporation of cystine into the brain areas, e.g. at 5 weeks of age - a time when most people consider the blood-brain barrier to be more or less mature - one finds that the accumulation of cystine by various areas of the brain more closely resembles that occurring in young rats rather than in adults. Since growth of neurons is still progressing a t this time, it seems more likely that the higher than adult uptake of 35S-labeled compounds is more related to growth than to an incomplete formation of a blood-brain barrier. B. D. WYKE:Several papers during this session have emphasized the importance of monitoring the arterial P c o ~ or , the necessityfor it. lfwe slip needles into brains of patients or animals to take biopsies, we d o this under anaesthesia. Under general anaesthesiawith any agent, the arterial PcoZ is elevated, unless the animals or the patient is given a relaxant and ventilation is controlled. If you slip a needle into a brain of an animal with a normal arterial Pco,, there is not any spreading edema of the sort that Dr. Bakay described. We showed in our laboratory some years ago that if you combine hypercapnia with a small cerebral lesion, made either by repeated needling, or by biopsy, then you get a spreading edema through that hemisphere which develops quite quickly. T. Z. CSAKY:May I discuss the paper of Dr. Quadbeck in some detail? Three basic facts stand out: ( I ) There is a low capacity transport mechanism for glucose in the brain, yet, the need of the brain for glucose is large. (2) If a non-metabolized sugar is offered to the brain the rate of uptake is extremely low. (3) Certain drugs, as we have just learned, can increase the uptake of glucose by the brain. How can we reconcile these various findings? A partial answer may be offered in the following: When we talk about the uptake of glucose by the brain, this is not necessarily equivalent t o transport. Uptake in case of glucose means that the sugar is pouring into a very active metabolic sink. A relatively low capacity carrier will be sufficient to mediate this in-pour. If a sugar is offered which uses the same carrier but is not metabolized, the system will soon be “saturated”, an equilibrium reached, and the further uptake will slow down. If a drug is administered which increases the glucose metabolism of the brain cell this will simply increase the metabolic disappearance of the free glucose and hence facilitate the in-pouring of more sugar. I suspect that this is what Dr. Quadbeck’s drug does. What we measure in this case is really the clearing of glucose from the blood. So I think that we can reconcile these different facts by assuming a sugar carrier mechanism with relatively low capacity which carries glucose from the blood into the brain cell but, because of the rapid metabolic disappearance of the sugar, the reaction will proceed in the direction of uptake. As long as the metabolism of glucose is vigorous, a large amount of the sugar can be cleared from the blood.

G. QUADBECK: With metamphetamine it is possible t o stimulate cerebral metabolism. Metamphetamine in animal experimentswith oxygen deficiency, hohevcr, fails to improve the glucose supply to the brain. In patients with a reduced uptake of glucose the drug does not bring about an increase. Consequently, I have to believe that the effect of the drug we have observed is on the pump mechanism and not an expression of increased requirements of the brain for glucose.

D. B. TOWER: Dr. Quadbeck pointed out that this compound is structurally related to the pyridoxine group of vitamins. 1 would like to know from Dr. Quadbeck whether he has evaluated the effects of the 8-6 group of vitamins, and 1 say “group” because it is evident now that one cannot do this with pyridoxine alone. In certain circumstances you have to evaluate the whole group in order to make sure that you have not overlooked the effect of pyridoxal, or pyridoxal phosphate, and so on. It is conceivable, as Dr. Csaky has pointed out. that you are dealing with a situation here in which the cells are somehow unable to consume glucose, and that the effect of this drug is at a cellular level rather than at some other level which has to d o generally with the transport of glucose into the central nervous system. Pyridoxine has such myriad co-enzyme functions that such possibilities must be immediately suspect.

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G. QUADBECK: Pyridoxine and pyrithioxine differ markedly in their effects.The latter has novitamin effect. Ifweadminister large doses of pyridoxine to an animal, transport of phosphate into the brain is increased. With an equivalent dose of pyrithioxine, the phosphate transport is reduced. Consequently, I am sure these two compounds are not related in their biological actions.

D. B. TOWER: I don’t think that answers the question, Dr. Quadbeck. Because 1 am not quite sure that one can make a complete parallel here. So, I would like to know whether you have tried the effects of pyridoxine, pyridoxamine, pyridoxal, and pyridoxal phosphate on the apparent depression of glucose utilization in these patients. G. QUADBECK: We have not studied pyridoxine with the Schmitt-Kety method. We have never seen with pyridoxine, however, an effect in older patients which is comparable to animal experiments where a disturbance of the glucose transport had to be assumed.

C. CRONE:Before we enter into too much of a detailed discussion of the mechanism of action of this substance, I would like to ask Dr. Quadbeck a question on the accuracy of the methods of measurement. If you have a situation where the cerebral blood flow rises with a factor of 2.5, it means that if the cerebral glucose uptake stayed constant under these conditions, the normal arterial-venous difference of 10 mg per 100 ml would go down to 4 mg per 100 ml. If the glucose uptake were reduced further, as was shown on the slide, down to 36%, it would mean that you would now have a difference of about 1.2, or 3 mg %. I would hesitate very much to put too much weight on differences based on this sort of calculation. In order to put a concrete question to you, I would like to ask: How many times were the controls controlled before the substance was given? G. QUADBECK: In one patient we were able to make measurements a week apart without intervening therapy. No significant differences were noted in these measurements. If we determined the glucose uptake of the brain after treatment for several weeks, then we found a distinct effect. D. P. RALL:As far as Dr. Steinwall’s presentation is concerned, I want to reemphasize that if we allowed paraminohippurate to diffuse into the brain, it would be able to move in 10 mm, in a way consistent with passive diffusion. The concentration of paraminohippurate was very low, deep in the brain. Any active transport at the capillaries should not have severely altered the shape of this curve. I think this sort of evidence, almost virtually conclusive, suggests that there is no active transport of this sort of compound in the brain. 0. STEINWALL: I hope my presentation made it clear that I was not as pleased with my results on this point as with the hypothesis. R. V. COXON:I take it for granted that Dr. Rall does not claim that this is not active transport through this particular region? He would not deny that at least a transport against the concentration gradient could exist? D. P. RALL:At the choroid plexus, probably, yes. I would be in conflict with myself if I denied this. A. LAJTHA:May I ask Dr. Steinwall whether he investigated any compound that is not actively transported under the same conditions? I would almost expect an opposite effect. Perhaps this would be a nice demonstration that there is more than one kind of barrier. A metabolic inhibitor by decreasing available energy may inhibit active uptake or transport of substances. The decrease in energy may at the same time have an effect on the membrane itself which may increase the penetration of nutrients entering by diffusion alone.

0. STEINWALL: In many experiments we found a seemingly opposite effect, as there could be an extravasation of normally barred dyes such as sodium fluorescein simultaneously with the decreased uptake of the nutrient tracer in the hemisphere perfused with the mercuric solution. I think this indicates the coexistence of more than one kind of barrier function.

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The Effect of Hypothermia on Electric Impedance and Penetration of Substances from the CSF into the Periventricular Brain Tissue I G O R K L A T Z O , C H O H - L U H LI, D O N M. L O N G , A N T H O N Y F . B A K , M I R O S L A W J. M O S S A K O W S K I , L E V O N 0. P A R K E R A N D LOIS E. R A S M U S S E N Branch of Swgical Neurology, National Institute of Neurological Diseases and Blinclness, National Institutes of Health, Public Health Service, Departinen! of Health, Education and Weljare, Bethescla, Marylarrd 20014 ( U S A )

1NTRODUCTION

Lowering the temperature of the brain can affect it in either a beneficial or an adverse manner. The ability of mild hypothermia to reduce brain volume has been extensively used by neurosurgeons in alleviating increased intracranial pressure and cerebral edema. On the other hand, a number of adverse effects have been reported following cooling of the brain below 28°C. At these temperatures numerous clinical complications (such as cerebral edema or hypoxic brain injury) and experimental disturbances (such as a breakdown of the blood-brain barrier) have been reported (Brendel et al., 1966). A further elucidation of the basic changes due to lowering brain temperature appears to be of considerable importance. Hypothermia causes an increase in the electric impedance of brain tissue in the rabbit (Collewijn and SchadC, 1962, 1964). This increase was thought to be a result of changed physical properties of the tissue since a similar change was found in electrolyte solutions at corresponding temperatures (Collewijn and SchadC, 1962). Recently a series of experiments were designed in our laboratory (Li et al., 1966) for the study of physiological changes in the brain under hypothermia. These experiments show that impedance changes recorded from the grey matter at temperatures above 20°C were in accordance with temperature coefficients of the blood serum and NaCl solution. Below this temperature, the changes were greater than those recorded from serum and electrolyte fiuid. These observations probably reflect changes in the electrolyte and extracellular compartments, as has been reported to occur in asphyxia (Van Harreveld, 1957). It was then conjectured that alteration in such basic properties of nervous parenchyma should influence penetration of various substances from the cerebrospinal fluid (CSF) (Davson and Spaziani, 1962). After crossing the ependymal barrier the substances can migrate further in several ways. Generally, the inert, extracellular compounds, such as inulin, may spread primarily by passive diffusion through extraR I ~ PPII( I P B p. 396

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cellular spaces (Rall et a/., 1962), whereas, an active transport mechanism has been implicated in penetration of diverse substances such as histamine (Draskoci et al., 1960), amino acids(Lajtha, 1962; Levin et a/., 1966), and albumin (Klatzo etal., 1964). The purpose of the present study is to correlate some of the electrophysiological data obtained after brain cooling with observations on periventricular penetration of substances, selected for their association with either diffusion or active transport. Materials and Methods

For the study of electric impedance changes, 35 cats under Fluothane anesthesia and extracorporeal hypothermia were used. The temperature and the blood flow i n the brain were regulated by extracorporeal circuits connected with the common carotid arteries. This method is similar to that described by Kristiansen el a/. (1960) and will be reported in a separate communication (Ohta rt a / . , 1966). In brief, the blood flow through the carotid artery was constant at a rate of 7.3 ml/kg/min and the temperature of the cerebral hemisphere was gradually lowered to 13-12°C. The perfusion pressure was continuously measured at the entry of the extracorporeal catheter into the carotid and the general systemic blood pressure was taken from the abdominal aorta. Temperatures of various structures of the brain and of the rectum were likewise continuously recorded. Before, during, and towards the end of the cooling experiment blood samples were obtained from the abdominal aorta for determination of Pco,. PO, and pH. Square pulses of low intensity current were applied through two Ag-AgC1 electrodes across the brain while recordings were made with two microelectrodes in the tissue. The difference of potential recorded was taken as a measure of impedance between the two recording electrodes. These electrodes were also capable of recording membrane resting potentials and action potentials of the brain tissue. Impedance changes at various temperatures were also measured in 0.9% NaCl solution and cat’s blood serum. Observations on passage of various substances from the CSF into the periventricular brain tissue were carried out on 20 cats using ventriculo-cisternal perfusion. Palmer’s intraventricular cannula was introduced into the right lateral ventricle through an opening located 8 mm from the midline and 5 mm posterior to the fronto-parietal suture. An outflow needle was inserted into the cisterna magna. An adjustable flow rotary pump was used to regulate the rate of flow through a polyethylene tube connecting the intraventricular cannula with a reservoir of perfusing fluid. The latter consisted of Elliott’s fluid containing one of the radioactive substances and sodium fluorescein which was used as a standard marker for comparison. The radioactive compounds and their concentrations in the perfusing fluid were as follows: ( I ) 0.014 mM [14C]inulin1, specific activity 2.7 mC/gm ; (2) 0.201 mM [14C]sucrose2, specific with specific activity 34.1 mC/ activity 9.0 mC/mM; (3) 0.0009 mM [14C]~-leucine3 mM. In each case approximately 210,uC of the individual radioactive tracer in mixture

3

New England Nuclear Corp., Boston, Mass. Intern. Chem. and Nuclear Co., Los Angeles, Calif. Tracerlab, Waltham, Mass.

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with sodium fluorescein was perfused for 45 min at the rate of 0.15 ml/min. Following passage of the tracers the ventricular system was rinsed for 10 min at the same perfusion rate with Elliott's fluid after which the animals were sacrificed by rapid exsanguination. In hypothermic animals the ventriculo-cisternal perfusion was started about 30 min following the onset of cooling when the brain temperature had been lowered to approximately 15°C. In this hypothermic group, 5 cats were perfused with [14C]inulin, 3 with [14C]sucrose and 2 with [14C]~-leucine.An equal number of animals were perfused with the same tracers at the normal body temperature. Following the sacrifice, the brains were immediately removed and a standard coronal block at the level of lateral ventricles, corpus callosum and putamen was rapidly frozen on a metal holder. From this tissue l o p sections were cut in the cryostat. Using a previously described (Steinwall and Klatzo 1966) double tracer technique it was possible to compare individually in the same section the areas of penetration of sodium fluorescein and the distribution of the radioactive tracers by subjecting single sections consecutively to photography under the U.V. light and radioautography. For assessment of the ultrastructural effects of these manipulations the following procedure was utilized. Three cats underwent brain cooling to 15-12"C, ventricular perfusion with Elliott's solution for 55 min, and then were fixed by direct carotid perfusion with 1000 ml 2.5% glutaraldehyde in a 440 mM phosphate buffer at the same flow rate as utilized in the blood perfusion. Three animals were similarly fixed at normal body temperature after undergoing only ventricular perfusion. Following fixation the tissue was diced (1 x I mm), exposed to 1 % osmium in the same buffer for one hour, and then embedded in epon according to standard technique. RESULTS

The time course of the change in temperature of the cerebral hemisphere under investigation was almost identical in all experiments as shown in Fig. 1. In about 28 rnin the temperature dropped from 36-34°C to 15°C and, thereafter, was maintained at a level of 13-12°C. Measurements of perfusion pressure, systemic arterial pressure, PO,,PCO, and pH at various temperatures obtained from different animals were found to be similar. Fig. 2 shows the average values of these measurements. In all cases the oxygen tension in the blood was increased with a decrease of brain temperature. It is to be noted that the body temperature as taken from the rectum ranged between 31 and 30°C while the braintemperaturewas 15-12°C. If the increase in PO, is due to a decrease in oxygen consumption of the body tissue as a result of hypothermia, the decrease must have been more marked in the cooled cerebral tissue than in other tissues. In any case, there was no indication of hypoxia in any of these animals. During the cooling procedures resting membrane potentials were measured from 87 polarized elements in the cortex of the cooled hemisphere. At temperatures above ITC, 60% of these elements showed injury discharges with a sudden shift of potential to negativity. In the absence of injury discharges the negative potential may represent resting potentials recorded from glial elements (Li, 1955 and 1959). At temperatures RpJi,renci,s p. 396

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Fig. 1. Time course of change in brain temperature under extracorporeal hypothermia.Arrows indicate beginning and end of ventricular perfusion.

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Fig. 4. Impedance changes simultaneously recorded from the grey matter (first tracing) and white matter (second tracing) at various brain temperatures (indicated at right upper corner of each record)

below 17"C, none of the penetrated elements showed injury discharges. The mean value and the standard deviation of these measurements are shown in Fig. 3. Since there is no way of identifying neurons from neuroglia by electrophysiological methods at low temperatures, the values given in Fig. 3 must represent the mean values from Hejorrnccs p . 396

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Fig. 5. Impedance changes of the grey matter, white matter, blood serum and NaCl solution.

all polarized elements in the cortex. It follows that no change in the resting potential occurs in these polarized elements at temperatures between 30 and 11°C. Impedance changes recorded at low temperatures consistently revealed a discrepancy in the measurements from the grey matter and white matter. An example is given in Fig. 4, which shows a gradual increase in the electric resistivity of the cortex and little or no change in the white matter when the temperature of the hemisphere was lowered from 33 to 13°C. These measurements of impedance changes obtained from each experiment were converted into proportional values and the average values at a given temperature obtained from all experiments were plotted in Fig. 5. It can be seen that in the white matter there is very little change in the impedance at temperatures above 20°C; and a gradual increase to 10% at temperatures between 20 and 10°C. This is in contrast with the findings from the grey matter from which an increase of over 60 % at 15°C and 70 % at 12.6"C were recorded. Fig. 5 also shows the average values of impedance measurements recorded from cat's serum and NaCl solution. At temperatures above 20"C, these values are comparable to those recorded from the grey matter; but below 20°C they are significantly smaller. It also shows that these values are much higher than those recorded from the white matter throughout the entire range of temperatures tested. The penetration of various substances from the ventricles into the surrounding parenchyma revealed the following features. In animals subjected to selective cooling of the brain sodium fluorescein, ["C]inulin and [14C]sucrose showed a striking reduction of periventricular spreading in comparison with cats perfused with these substances at normal temperature. On the other hand, the effect of hypothermia on the passage of [W]~-leucineappeared to be rather insignificant. At normal temperatures all indicators extended for approximately 3-4 mm into the putamen, whereas the spread into the corpus callosum seemed to be more limited (Fig. 6a, b). At lower brain temperatures the reduction in penetration of the first three indicators was very conspicuous, and was usually more pronounced in the putamen where only a narrow rim of subependymal tissue showed the presence of

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Fig. 6. (a) Cat subjected to ventriculo-cisternal perfusion with sodium fluorescein and [14C]inulin at thc normal temperature. The 101‘ section photographed under the U.V. light. The fluorescence of sodium fluorescein is seen extending into brain tissue adjacent to the lateral ventricle; (b) Radioautograph showing the distribution of [14C]inulinon the same section. Fig. 7. (a) Ventriculo-cis‘ernal perfusion with sodium fluorescein and [“Tlinulin at 13°C. Thesame anatomical level as in Fig. 6. The penetration of sodium fluorescein from the lateral ventricle is very restricted, especially into the putamen; (b) Radioautograph showing the passage of [W]inulin on the same section.

the tracer (Fig. 7a, b). Although it appeared that the passage of [14C]~-leucine was also affected by lowering the temperature the reduction was small and sometimes even difficult to ascertain (Figs. 8, 9). Electron microscopy revealed no major abnormality in either normothermic or hypothermic brain. Preservation of tissue was adequate. An occasional pyknotic neuron was seen, but in general nerve cells were not abnormal. Definite astrocyte R&riwrca p . 396

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Fig. 8. (a) Ventriculo-cisternal perfusion with sodium fluorescein and [14C]~-leucine at the normal temperature. This section photographed under the U.V. light shows the distribution of sodium o n the same fluorescein; (b) Radioautograph showing the periventricular penetration of [14C]~-leucine section. Fig. 9. (a) Ventriculo-cisternal perfusion with sodium fluorescein and [14C]~-leucine at 14°C. Fluorescent photograph shows the reduction in passage of sodium fluorescein as compared with that at the normal temperature; (b) Radioautograph of the section shown in 9a. The reduction of L-leucinc passage into periventricular brain tissue appears to be insignificant when compared with Fig. 8b of a cat perfused at the normal temperature.

swelling or shrinkage was not seen, and there were no major changes in the extracellular space in grey or white matter (Fig. 10). There were no obvious differences between the two groups. These preliminary observations are not complete, however, and measurement analysis has not been applied to the tissue. While inspection reveals no obvious abnormalities which might explain the observed phenomena, it is not possible to compare exactly cellular, process, and space sizes in the two groups until quantification is complete.

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Fig. 10. Cat caudate nucleus fixed at 12°C by perfusion with 2.57; glutaraldehyde in PO4 buffer. A normal appearing neuron ( N U ) with a dendrite ( D )are shown. Another dendrite (D, right) crosses the field. No dramatic change in dendrite size. Cellular elements of neuropil (NP) and extracellular space do not appear different than normal. x 10 300.

DISCUSSION

Under normal brain temperature, the electric current applied to the brain for measurement of impedance changes is primarily carried by electrolytes in the extracellular space. From experiments on circulatory arrest (Van Harreveld and Ochs, 1956), it was estimated that the blood, which flows through the brain tissue, is responsible for about 1 I ofthe total conductivity of the brain. Thecellular elementscontributelittleornone to the change in brain conductivity owing to the high resistance of their limiting membrane. In the present investigation, impedance changes recorded from the grey matter at temperatures above 20°C were found to be comparable to those recorded from NaCl solution and blood serum. This can be attributed to the temperature coefficient of the electrolyte fluid in the extracellular space, which is in complete agreement with Rrfercnms p. 396

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Collewijn and SchadC (1962, 1964). However, at temperatures below 20°C the impedance changes of the grey matter became greater than those recorded from NaCl solution and serum, suggesting that impedance changes of the grey matter at temperatures below 20°C are not entirely determined by temperature coefficient of the extracellular fluid. One may argue that the excessive increase of grey matter impedance is due to a decrease in extracellular space as a result of an increase in the volume of the cell (Van Harreveld et al., 1965). It was shown (Reulen et al., 1966) that a significant increase in the uptake of sodium and water in brain tissue occurred at temperatures below 10°C. Above 10°C there was no significant increase. It is also known that cells in brain become swollen in experimental animals under asphyxia (Van Harreveld, 1957). I n the present investigation the brain temperature was reduced, but not below 11°C; and, in all instances, there was no indication of hypoxia. The measurements of resting membrane potential are comparable to those recorded from cortex of normothermic cats (Li, 1955 and 1959) and brain slices of the guinea pigs (Li and Mcllwain, 1957). The consistent values obtained at low temperatures are in agreement with the findings from single skeletal muscles of the hypothermic frogs (Li and Gouras, 1958) and rats (Li, 1958). The lack of change in resting membrane potential indicates not only an absence of disturbance in the differential distribution of ions between the extracellular and intracellular compartments but also an absence of excessive accumulation of water in the tissue. The indication that cell swelling did not occur is supported by the preliminary findings from electron microscopy. Despite the regulated blood flow in the common carotid artery of our experimental animal, the actual volume of blood circulating in the cooled hemisphere is not known and this variable remains to be determined. In experiments (Li et al., 1966) designed for the study of systemic blood pressure and brain impedance changes, it was found that the lower the blood pressure the lower is the tissue impedance. This observation implies that the impedance change of the grey matter at temperatures below 20°C should be lower than the change caused by the temperature coefficient of the extracellular fluid alone; but this is not the case. The above arguments leave an alternative that at temperatures below 20"C, the increased impedance recorded from the grey matter is due to a decrease of the extracellular space. This suggestion, however, cannot be applied to the changes recorded from the white matter of the brain. The conspicuous inhibition of the passage of sodium fluorescein, inulin, and sucrose may well be related to a reduction in extracellular space. Thedistributionof these tracers (especially the last two) is generally believed to be extracellular. Restriction of the space available for migration should inhibit their rate and extent of spread. Since temperature has a direct influence upon rate of diffusion, the inhibition of migration of inulin in our study was compared with the inhibition to be expected if the effect was simply one of temperature reduction on diffusion. The quantitative technique of Rall et al., (1962) was utilized for us by Dr. Fenstermaker of that laboratory. Preliminary results indicate the reduction in inulin migration from the ventricular surface

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markedly exceeds the predicted reduction if the results were related to temperature coefficients alone. Reduction of the extracellular space available for diffusion is a plausible explanation of this discrepancy. We expected that hypothermia would have a definite effect upon the active transport mechanism which supposedly is responsible for the movement of L-leucine from the CSF. While the results were not unequivocal, the reduction in passage of L-leucine was small at best, and difficult to ascertain. This is unexplained, and additional study of this phenomenon is planned. The ultrastructural analysis of the tissue is not complete. However, it can be stated that no obvious tissue damage or large differential increase or decrease in any tissue element occurred. I t is entirely possible that exact measurements may reveal a significant change in some volume component, however. This possibility cannot be fully evaluated until measurements are complete. At least there is no serious cellular damage or destruction of tissue which should markedly influence the results.

SUMMARY

Study of impedance changes and resting membrane potentials as well as the rate of passage of various substances from the cerebral ventricles to the brain tissue in hypothermic cats has led to the following results and suggestions: 1 . At brain temperatures above 20”C, impedance changes recorded from the grey matter were comparable with those recorded from blood serum and NaCl solution at corresponding temperatures. This can be attributed to temperature coefficient of the electrolyte fluid in the extracellular space. 2. At temperatures below 20”C, impedance changes recorded from the grey matter became greater than those from serum and NaCl solution. It is suggested that at these low temperatures there is a reduction of the extracellular space. 3. At temperatures above ITC, there was no indication of hypoxia and no change in the resting membrane potential of the cellular elements in the cerebral cortex. These findings imply that swelling of cells and disturbance of differential distribution of ions did not occur at these low temperatures. 4. Impedance changes recorded from the white matter were much smaller than those recorded from the grey matter. The mechanism by which this discrepancy exists remains to be determined. 5 . Using fluorescence and radioautography the effect of lowering the brain temperature on periventricular passage from CSF was assessed with regard to sodium fluorescein, [14C]inulin, [14C]sucrose and [“C]~-Ieucine.Sodium fluorescein, [“Wlinulin and [14C]sucrose showed a marked reduction of periventricular passage at 15-1 2”C, whereas only a small effect was observed with regard to [“C]~-leucine. 6. These data indicate the possibility that at 15-12°C there is a reduction in extracellular compartment without appreciable shift in electrolytes which may account for the increased impedance values in the grey matter and for the observed reduction in pasage of the mentioned compounds. R&wwc,s

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COLLEWIJN, H. AND S C H A DJ.~ ,P. (1962) Cerebral Impedance Changes in Hypothermia. Arch. Intern. Physiol. Biochem., 70, 20&2 10.

-, (1964) Chloride, Potassium and Water Content of Apical Dendrites and Their Changes after Circulatory Arrest at Body Temperatures from 37°C to 20°C. Arch. Intern. Physiol. Biochem., 72, 194-2 10.

-, (1964) Conductivity of the Cerebral Cortex after Circulatory Arrest from 37°C to 18°C. Arch. intern. Physiol. Biochem., 72, 181-193.

DAVSON,H. AND SPAZIANI, E. ( I 962) Effect of Hypothermia on Certain Aspects of the Cerebrospinal Fluid. Expt. Neurol., 6, 118-129. DRASKOCI, M. FELDBERG, W.,FLEISCHAUER, K. AND HARANATH, P. S. R. (1960) Absorption of Histamine into the Blood Stream on Perfusion of the Cerebral Ventricles and its Uptake by Brain Tissue. J. Physiol., 150, 50-72. KLATZO,I.. MIGUEL,J., FERRIS,P. J., PROKOP,J. 0.AND SMITH, D. E. (1964) Observations on the Passage of the Fluorescein Labeled Serum Proteins from the Cerebrospinal Fluid. J. Neuropathol. Exptl. Neurol., 23, 18-35. KRISTIANSEN, K., KROG.J. AND LUND,1. (1960) Experiences with Selective Cooling of the Brain. Acta Chirurg. Scand. Suppl., 253, 151-161.

LAJTHA,A. (1962) Amino Acid Transport in the Brain. In: Properties of Membranes and Diseases of the Nervous System. M. D. Yahr (Ed.). Springer Publishing Co., New York, pp. 43-54. LEVIN, E., NOGUEIRA, G. J. AND GARCIA ARGIZ,C. A. (1966) Ventriculo-Cisternal Perfusion of Amino Acids in Cat Brain. J. Neurochem., 13, 761-767. LI, C. L. (1955) Action and Resting Potentials of Cortical Neurones. J . Physiol., 130, 96-108. -, (1958) Effect of Cooling on Neuromuscular Transmission in the Rat. Amer. J. Physiol., 194, 200-206.

-, (1959) Cortical Intracellular Potentials and Their Response to Strychnine. J. Neurophysiol., 22, 436-450.

LI, C. L., BAK,A. F. A N D PARKER, L. 0. (1966) Some Electrophysiological Changes in the Brain under Hypothermia. (In preparation). Lr, C. L. AND GOURAS,P. (1958) Effect of Cooling on Neuromuscular Transmission in the Frog. Amer. J. Physiol., 192, 464-470. Lr, C. L. AND MCILWAIN, H. (1957) Maintenance of Resting Membrane Potentials in Slices of Mammalian Cerebral Cortex and Other Tissues I n Vitro. J . Physiol., 139, 178-190. OHTA,T., PARKER, L. 0. AND Lr, C. L. (1966) The Perfusion Pressure in Hemicooling of the Brain. (In preparation). C. F. (1962) Extracellular Space of Brain as Determined by RALL,D. P., OPPELT,W. W. AND PATLAK, Diffusion of Inulin from the Ventricular System. Life Sci., 2, 43-48. REULEN, H. J., AIGNER,P., BRENDEL, W., AND MESSMER, K. (1966) Elektrolytenveranderungen in tiefer Hypothermie. I. Die Wirkung akuter Auskuhlung bis 0°C und Wiedererwarmung. Pfliigers Arch., 228, 197-219.

STEINWALL, 0. AND KLATZO,I. (1966) Selective Vulnerability of the Blood-Brain Barrier in Chemically Induced Lesions. J. Neuropathol. Exptl. Neurol., 25, 542-559. VANHARREVELD, A. (1957) Changes in Volume of Cortical Neuronal Elements During Asphyxiation. Amer. J. Physiol., 191, 233-242. VAN HARREVELD, A., CROWELL, J. AND MALHOTRA, S. K. (1965) A study of Extracellular Space in Central Nervous Tissue by Freeze-Substitution. J . Cell Eiol., 25, 117-137. VAN HARREVELD, A., AND OCHSS. (1956) Cerebral Impedance Changes after Circulatory Arrest. Amer. J. Physiol., 189, 180-192.

HYPOTHERMIA AND

C S F - B R A I NB A R R I E R

397

DISCUSSION C. CRONE: I was very impressed by Dr. Klatzo’s contribution, and I have a few questions: I did not understand your argument when you said that the constancy of the membrane potential, particularly at decreasing temperatures, signified that the cells did not increase in size. If you accept the Goldnian equation, as describing the relevant factors which determine the membrane potential, I don’t see how you get from that to the size of the cell.

The other comment relates to the impedance studies you mentioned. If the impedance increases in a damaged brain, this may be due to a decrease in the extracellular space. One would expect an intercellular distance below 2008, under these conditions. As this seems not to be the case, the logical consequence is eidier that artefacts invariably occur when preparing brain tissue for electron microscopy, leaving an intercellular slit of 200 A, whatever the condition was in situ, or that the impedance studies do not inform us about the extracellular space. 1. KLATZO:With regard to my statement that the constancy of the membrane potential, particularly at decreasing temperature, signified lack of cellular swelling; what I had in mind was the fact that the swelling is usually related to intracellular penetration of sodium accompanied by water. In such swelling, as it occurs, e.g., in asphyxia, the translocation of sodium influences the resting membrane potential. It is true that in some instances, such as reported by Pappius in osmotically induced swelling of the brain in uremic dogs subjected to hemodialysis, the Na/K remains unchanged and intracellular swelling may occur without inflow of sodium. I would like to emphasize, however, that gross observations in our material were definitely against presence of swelling. The hypothermic brains appeared smaller and firmer to touch than normal controls. With regard to your second comment, I am wholeheartedly in agreement that in the living brain the intercellular spaces must be larger than 150-200 8, as suggested by some electronmicroscopists. Presently, there is data forthcoming indicating that this assumption may be wrong and may be related to artefacts of tissue processing. Physiological experiments, such as, e.g., Van Harreveld’s, elegantly demonstrate variations in extracellular compartment, whereas in many electronmicroscopic observations the distance between the cell membranes appears to be rigidly always the same, even in drastic conditions such as acute asphyxia or edema.

R. KATZMAN:I would like to raise for the record the question as to the interpretation of the impedance measurements. If indeed one is dealing (as was originally done on the red cell membranes) with a group of spherical cells with very high membrane resistances, suspended in an aqueous medium, then indeed one can use the impedance measurements to directly determine the size of the surrounding fluid compartment. When you deal with a very complex tissue, it becomes much more difficult. And if you use the analysis of Dr. Ranck (1963, Exprl. Neurol., 7: 153-174), who took into account the various components of this complex tissue, one comes up with quite a different value for the extracellular space than Van Harreveld estimates. Ranck calculated that the spaces were from 5 to 12% instead of Van Harreveld’s 25;& using essentially the same data with a different conceptual interpretation. There is one final point that I think ought to be emphasized, and that is that the cell membrane by itself does not have a low impedance in the dead state; in fact it has a very high impedance. A biomolecular lipid layer without any protein in it has an impedance of 10” Ohm/cm2, compared to the lo? of the neuron, and perhaps 100hm/cm2of the glial cell. In order to maintain the low impedance of the glial cell and of the neuron, vital processes are probably necessary. And one of the effects of fixation may be to destroy some of the mechanisms that underly the usual lower impedance of some cell membranes. J . DOBEING: It will be interesting to know people’s views on where Dr. Rall’s inulin is, if it is not in an extracellular space, and the inulin fills 12 to 15% of the total space.

398

I. K L A T Z O

et al.

D I S C U S S I O N O N T H E P E R M E A B I L I T Y OF T H E B L O O D - B R A I N B A R R I E R

The field of pathology can furnish material that permits under certain conditions, the study of problems related to the permeability of the blood-brain barrier. Two examples are given here: ( I ) the results obtained studying the changes in the composition of the central nervous system during cerebral edema, and (2) the results showing possible relationships between the free amino acid pools of blood, or cerebrospinal fluid, and urine in certain congenital disturbances of metabolism. 1. In cerebral edema there is an increase in water content of the nervous tissue and an increase in sodium; in addition, there is an increase of a rapidly moving protein fraction on the electropherogram. These proteins migrate on agar gel to the level of albumin or fast a-globulins (Fig. I), and can

Fig. 1. Agar gel electropherogram of water-soluble proteins from edematous cerebral tissue. Note the presence of a significant rapidly migrating fraction.

be isolated after passing through columns of Sephadex and DEAE cellulose. The protein responsible for the increase in content of rapidly migrating material could be shown (Karcher and Lowenthal) to be albumin, although it differed slightly from serum albumin with respect to molecular weight and sensitivity to the action of tryptic digestion, and also in its immunological characteristics. 11. The investigation of changes in the concentration of free amino acids in serum, cerebrospinal fluid, and urine in patients with congenital disturbances of amino acid metabolism shows that the changes are not parallel in all three pools. For example, in phenylketonurics on a phenylalaninedeficient diet, the drop in phenylalanine concentration is not parallel in all fluids (Fig. 2). The variations in phenylalanine concentrations (Mardens and collaborators) are inversely proportional to variations in the concentrations of other amino acids in the serum (Fig. 3) while this is not the case for cerebrospinal fluid.

Len ... Guy

E 8 300c v1

-

s t i200-

al

'\ \

Mois

Fig. 2. Changes in serum and CSF phenylalanine concentrations in a phenylketonuric on a phenylalanine-deficient diet. Note that the changes in concentration are not parallel in serum and CSF.

HYPOTHERMIA AND

CSF-BRAIN BARRIER

399

'f

R&mc

Guy Len

c .PHE SERIQUE p m o l e s / l O O m l *--a AMINOACIDEMIE, PHE non compr lse + + PHE L c R pm oles /10 0 ml x-

- -x

AMINOACIDORACHIE, PHE non comprise

Fig. 3. Concentrations of phenylalanine and other amino acids in the serum of phenylketonurics on a phenylalanine-deficient diet. Note that the concentration of amino acids increases as the phenylalanine concentration decreases during the course of treatment, and vice-versa.

These data would point to the usefulness of human material in observing the permeability of the blood-brain barrier. A. Lowenthal

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40 I

Changes in Brain Accumulation of Amino Acids and Adenine Associated with Changes in the Physiologic State DONALD H. F O R D

State University of New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York (U.S.A.)

The observations by Ehrlich (1885, 1887) that certain aniline dyes stained all tissues rapidly when injected into the blood stream, except the brain, was undoubtedly the first step in the creation of a concept of a blood-brain barrier. Confirmation of this concept has been provided by many investigators (Bakay, 1956; Broman, 1955; Goldman, 1913; Lewandowsky, 1900; Roux and Borrel, 1898) using a wide number of techniques. Attempts to localize the site of the barrier to a specific anatomical structure (Barrnett, 1963; Dempsey and Wislocki, 1955; De Robertis and Gerschenfeld, 1961; Donahue et al., 1961 ; Farquahar and Hartman, 1957; Hess, 1953, 1955; Maynard et a]., 1957; Rodriguez, 1955; Tschirgi, 1962; Wislocki and Leduc, 1952) have finally led to the conclusion that every membrane existing between the blood stream and neuronal cytoplasm may possibly contribute. The absence of perivascular and perineuronalspaces (De Robertis, 1962; Maynard et al., 1957) as well as the particular interdigitated organization of brain capillary endothelium (Maynard et al., 1957) may also be important in influencing rates of exchange of solutes between the blood and brain parenchyma. Reasons for exclusion of materials from the brain have included binding to plasma proteins, molecular charge, lipid solubility, aqueous solubility and the electrical charge of a molecule (Garoutte and Aird, 1955; Goldworthy et al., 1954; Krogh, 1946; Robbins and Rall, 1960; Review by Bakay, 1956). The restriction on penetration of metabolic substrates into the nervous system was at one time considered as being so stringent that it was suggested that the only amino acid capable of reaching the neurons was glutamine. Glucose, oxygen, carbon dioxide, lipid soluble materials and water appeared to move freely into brain parenchyma, while inorganic ions and other highly dissociated ions equilibrated between brain and blood more slowly (Tschirgi, 1962). More recently, the investigations of Waelsch and Lajtha (1961), Lajtha and Toth (1963), Gaitonde and Richter (1955), Guroff and Udenfriend (1962), Chirigos et al. (1960), Ford et al. (1965), as well as many others, have demonstrated that most amino acids move freely into the brain. However, the rapid influx of amino acids seems well balanced by a rapid efflux, and both depend upon specific transport systems (Lajtha, 1964; Levi and Lajtha, 1965a and b; Blasberg and Lajtha, 1965). Considering these rapid transport mediated movements of amino acids in and out of brain parenchyma, Rc,ferences p. 411413

402

D. H. F O R D

one might readily suppose that the entrapment of amino acids into neurons might be influenced by the metabolic activity within the neuron and that the over-all metabolic turnover rates within neurons might be influenced by the over-all metabolic activity of the body. This suggestion that uptake of amino acids into neurons might be influenced by the metabolic demands can in part at least be attributed to Dobbing (1961). If increases in amino acid uptake into neurons can be associated with the metabolic activity of neurons, it would appear that the membranes which constitute what is considered the blood-brain barrier may be facilitative as well as restrictive in function. Uptake of triiodothyronine in brain compared with muscle Before proceeding to changes in internal or external environmental states which may influence brain metabolism in such a way as to appear to influence neuronal amino acid accumulation, I would like to point out that CNS accumulation of some compounds, while exceeded by many organs, is paralleled by a comparable uptake in muscle (Fig. I). Such a similarity in uptake between brain and muscle (Ford, 1965)

Fig. 1. A comparison of the percent of an injected doseof 131 I present per gram of tissue in the pineal, plasma, cerebral grey matter, striated muscle and adrenal gland at various time intervals after the I.V. injection of 1311-labeledtriiodothyronine (0.5 ,ug/kg) into normal male rats (Ford, 1965).

is well illustrated by triiodothyronine (T3). In this instance a dose of 0.5 ,ug/kg of ['3lI]T3 was given intravenously and animals killed at varying times after injection. The amount of the injected dose of 1311 present in the tissue was calculated on the basis of the percent of the injected dose present/g. Most of the tissue radioactivity was shown to be triiodothyronine by paper chromatography (Ford, 1965). It is clear, that while pineal and adrenal uptakes and plasma levels of radioactivity are higher than that seen in cerebral grey matter, muscle levels of radioactivity were essentially the

CHANGES IN BRAIN ACCUMULATION

403

.

Fig. 2 Autoradiograms of ventral horn neurons from the cervical spinal cord of a normal rat (right) and a rat exercised in an activity wheel for 2 h (25 feet/min) and injected during exercise with [3H]leucine (Courtesy of Altman, 1964).

same as those demonstrated in the cerebral grey matter. This similarity in accumulation might mean that there is a blood-muscle barrier to triiodothyronine, which seems unlikely. One may add parenthetically that the percent of an injected dose of radioR[~i~re.nw p.s411-413

404

D. H. F O R D

activity present in the brain and muscle after intravenous injection of labeled lysine, tyrosine and glutamine is also quite comparable. Effect of exercise on the accumulation of [3H]leucine by neurons In 1964,Altman noted that rats run in an exercise wheel at a rate of 25 feet/min had an increased amount of radioactivity in neurons and surrounding neuropil if the labeled amino acid was injected during the exercise period. The animals were killed after an exchange period of one hour and the uptake determined by radioautography (Fig. 2). The increases in the amount of radioactivity present occurred in a wide variety of neurons as well as in the neuropil of the exercising rats, which Altman felt was more marked in such of the motor regions as the motor cortex, cervical cord neuropil, and ventral horn cells and in the visual cortex than in some non-motor areas, such as the hippocampus, cochlear nucleus and choroid plexus. Injections of labeled amino acid after the exercise were associated with increased uptakes of radioactivity which were much less marked and in which there were no distinctive differences between “motor” and “non-motor” neurons. The radioautographic illustration also demonstrates that the neuronal uptake of amino acid as compared with the uptake in the surrounding tissue was quite high. Since the volume of grey matter occupied by neurons is relatively small (Brizzee er al., 1964; SchadC, et al., 1964), it is apparent that studies on brain accumulation of labeled materials using whole tissue slices or blocks will be likely to provide data suggesting that over-all incorporation is low due to the very low amount of the labeled materials present in the tissue termed neuropil. This heterogeneity of uptake complicates interpretation of blood-brain barrier influences. Effect of neuron regeneration Complicated changes in neuronal protein and RNA are known to occur following nerve section or crush, as determined by biochemical methods (Brattgard et al., 1957). These changes can also be demonstrated radioautographically (Rhodes et a/., 1964). In a large series of rats the hypoglossal nerve was sectioned. Two weeks later the animals received a dose of [3H]lysine (593 pg/kg) intravenously. A half hour later the animals were killed by intracardiac perfusion and the tissues processed for radioautographic analysis. The uptake of radioactivity was compared in the normal and regenerating hypoglossal neurons in the same animal by grain counting procedures (Table 1). Ascending paper chromatographic analysis of brains from other animals injected with lysine demonstrated that the main labeled compound present in the brain was lysine. Inasmuch as the presence of labeled amino acid in cells following the usual histological procedures utilized with radioautographic studies is believed to be directly related to protein synthesis (Leblond, 1965), the increased grain count over the regenerating neurons suggests an accelerated protein synthesis in these cells (Column A). This was also apparent in animals which were either hypo- or hyperthyroid in metabolic activity. Cell size was also appreciably increased in regenerating

405

CHANGES IN B R A I N ACCUMULATION

TABLE I C O M P A R I S O N OF U P T A K E O F [:'H]LYSlNE OF T H E HYPOGLOSSAL

IN NORMAL A N D REGENERATING NERVE CELLS

NUCLEUS AS AFFECTED

Euthvro id A. Grain counts/cell Regenerating Normal B. Cell size (mg of paper/cell outline, reflecting cell area) Regenerating Normal C. Grain count/mg of paper (reflects cell area) Regenerating Normal

BY V A R Y I N G T H Y R O I D

Hypothyroid

+

32.210 4.592" 19.095 & 1.714

50.098 -+ 3.914bC 26.102 1.886

28.464 1.984d 18.883 I 1.502

28.205 16.924

1.110 *0.037 1.034 i 0.097

STATES

Hyperthyroid

36.337 f 2.035b 21.711 f 2.139

+ 1.208e + 1.718

26.683 f 1.090e 17.781 f 1.296

1.7758 f 0.1131 1.5769 & 0.096'

1.3680 f0.0759 1.2154 & 0.059

Significantly greater than normal cells ( p , : 0.02, 0.05). Significantly greater than non-regenerating cells ( p < 0.001). c Significantly greater than euthyroid cells ( p < 0.02, 'i 0.01). d Significantly larger than non-regenerating cells ( p > 0.001, < 0.01). e Significantly larger than non-regenerating cells ( p i 0.001). Significantly greater than euthyroid and hyperthyroid cells ( p > 0.001, < 0.01). g Significantly greater than normal and regenerating euthyroid cells ( p > 0.001, < 0.01). (From Rhodes el a/., 1964.) a

K :

b

neurons (Column B). The net result of the increases in grain count and in cell size was that the concentration of labeled lysine in regenerating cells remained close to what it was in the normal cells of euthyroid animals (Column C ) . However, to maintain this normal concentration in the regenerating enlarged hypoglossal neurons, a net increase in total amount of labeled amino acid present was necessary. Both changes in thyroid state (Column C) increased amino acid uptake in regenerating cells in a manner comparable to that seen in euthyroid rats. Moreover, both changes in thyroid state were associated with increased neuronal accumulation of labeled material which was not relative to regeneration. While it seems likely that the increased incorporation of labeled amino acid in neurons from hypo- and hyperthyroid states may be caused by dissimilar reasons, an explanation for this observation is not clear at this time. It may be pointed out, however, that a change in the metabolic requirements of the neuron, in this instance a need for increased amounts of amino acid for the synthesis of new protein in a regenerating cell, was associated with an increased incorporation of labeled material. The extent to which the labeled amino acid was accumulated by either normal or regenerating neurons also seemed to be influenced by the thyroid state of the animal. A similar radioautographic experiment was performed with regenerating hypoglossal neurons, using 13Hladenine as the test material. It seemed that it might be useful to use this purine for what was virtually the same experiment because so much of a dose of exogenously provided adenine becomes associated with AMP, ADP and ATP (Pakkenberg et al., 1965). The effect of dysthyroidal states was also included in Refcrcnrrs p. 411-413

406

D. H. F O R D

TABLE I1 C O M P A R I S O N OF U P T A K E O F [ 3 H ] A D E N l N E I N N O R M A L A N D R E G E N E R A T I N G N E R V E C E L L S O F T H E H Y P O G L O S S A L N U C L E U S AS A F F E C T E D B Y V A R Y I N G T H Y R O I D S T A T E S

Grain Counts Neuropil areas equivalent to neurons

Control (11)* Hypothyroid (1 1)8 Hyperthyroid (1 3p

130 & 12.70+ 326 & 52.68*+ 174 & 13.48*+

Nerve cells, Normal Xllth Nucleus

**

53 14.16 73 9.94 73 i 12.61

Nerve cells, regenerating Xllth Nucleus 33 f 12.33 52 & 18.62 60 10.65

*

Significantly different from neuropil counts in euthyroid rats ( p < 0.02). Significantly higher than the count from normal and regenerating hypoglossal nucleus cells (on an equivalent area basis) ( p < 0.001). * Number of animals studied. (From Pakkenberg et al., 1965). +

this investigation on uptake of intravenously injected adenine by normal and regenerating neurons. The dose of adenine given was 104.0 pg/kg, and the animals were killed by intracardiac perfusion one-half hour after injection. In this study, the regenerating cells had a lower radioautographic level of activity than did the control cells of the adjacent nucleus (Table 2). Again, both dysthyroidal states influenced cell accumulation of radioactivity and in the same direction with comparable effects being seen in both normal and regenerating cells. The comparison of cell-neuropil levels of radioactivity following r3HIadenine injection was the opposite from what was observed following [3H]lysine injection (Table 1). Thus, with the injection of adenine, neuropil was observed to have higher levels of activity than did the neurons 1/2 h after injection. This neuropil associated radioactivity was significantly higher than that of the normal neurons for both the euthyroid and dysthyroidal groups, and the level of radioactivity in the neuropil around regenerating neurons was, like the activity of the neurons, lower than on the control side. Thus, it appears thatuptake of adeninein the neuropil and neurons in the regenerating hypoglossal nucleus is depressed by the processes associated with regeneration. However, this may be an illusion, since the adenine taken up into neural tissue appears primarily associated with the synthesis of the adenine nucleotides, which may be more rapidly expended in the synthesis of new protein and tissue respiration in regenerating than in normal cells. The high concentration of radioactivity in the neuropil after adenine injection is maintained for only a short period (Fig. 3), for when one examines the levels of radioactivity in neuropil and a variety of neurons at later time intervals after injection by radioautography, neuronal uptake clearly exceeded that of the neuropil,

CHANGES IN BRAIN A C C U M U L A T I O N

407

8ol

xl\L . t,

Fig. 3. A grain count analysis of radioautograms of the amount of 3H accumulated in various neuron types at various intervals after I.V. injection of [3H]adenine. Grain count is based on total counts from areas encompassed by 20 neurons for each neuron type and compared with the grain count from an equivalent area of neuropil. Data for the f hour period was available for only hypoglossal (XII) motor neurons and neuropil, which is continued at subsequent periods with data from ventral horn motor neurons and neuropil. The dose of adenine given was 969 pg/kg (314 /tC/kg).

Amino acid uptake into grey matter and neurons as influenced by thyroid state Theeffect ofdysthyroidal states on uptake of amino acids into brain grey matter and ventral horn motor neurons following intravenous injection of labeled amino acids may also be analyzed with liquid scintillation counting techniques (Ford et al., 1965) utilizing a Nuclear Chicago liquid scintillation counting system. In this study a dose of 590 to 600 pg of [ 3 H ] ~ ~ - l y s iwas n e injected intravenously/kg into male rats, which were then killed by intracardiac perfusion with citrated saline at various time intervals after the injection. Accumulation of radioactivity was determined on the basis of the percent of the injected dose present/g of tissue, which was then plotted against time. Ventral horn motor neurons and spinal cord grey matter will be the only types of neuronal tissue considered. The spinal cord grey samples were prepared by removing the white matter from the region to be analyzed. Ventral horn motor neurons were dissected with fine needles from a comparable region free hand, using a dissecting microscope with a magnification of 130 x . For details of the procedure see Ford et al. (1965). In the spinal cord grey, the amount of radioactivity in euthyroid rats was intermediate between that seen in the hypo- and hyperthyroid animals (Fig. 4). The attainment of a maximal concentration occurred earlier in the hyperthyroid group and latest in the hypothyroid group, which suggests that turnover in the hyperthyroid group is somewhat faster. Such a faster turnover of the labeled amino acid contained in a larger non-labeled pool of lysine could readily account for the lower amount of labeled material present in tissue samples taken from the hyperthyroid group, while a slower Rrfermces p . 411-413

408

D. H. F O R D

0

6.0[

U C

0 0.3:: c

-:

.-

I'

Ventral horn neurones

1: ;

:

I

Spinal cord grey /

O . hypo

/-

Fig. 4. A comparison of the amount of 3Hpresent in ventral horn motor neurons and spinal cord grey matter in euthyroid and dysthyroidal male rats at various time intervals aftes I.V. injection of [3H]DL-lysine (Ford et a/., 1965).

turnover of labeled lysine in the hypothyroid rats could reasonably explain the higher accumulations which occur in this group. The amount of radioactivity in the ventral horn motor neurons was significantly higher than that of the spinal cord grey for all three groups. Furthermore, ventral horn cell radioactivity on a percent dose per gram basis exceeded even that of the liver. There was again a tendency for the radioactivity in the neurons from hyperthyroid rats to be lower than normal, while that entering the neurons from hypothyroid rats was above normal. The pattern of the distribution of radioactivity at different time intervals after injections was not one, however, which would permit one to arrive at the same conclusions regarding turnover rates as were made for the spinal cord grey, which illustrates in part the difficulties inherent in metabolic studies with tissues as heterogeneous in nature as brain grey matter. A similar investigation on the effect of dysthyroidal states on accumulation of labeled amino acids was done with [14C]~-glutaminein which a dose of 1.44 mg/kg of glutamine was given intravenously (Ford and Rhines, 1967). The amount of radioactivity present in ventral horn motor neurons and spinal cord grey were compared (Fig. 5 ) in the same manner as after injection with [3H]lysine. Again the ventral horn cell levels of radioactive material were appreciably higher than those seen in the spinal cord grey, and again both dysthyroidal states appeared to have some influence on the levels of radioactivity present in both tissues. This was most noticeable in the hyper-

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409

Fig. 5. A comparison of the amount of I4C present in ventral horn motor neurons and spinal cord grey matter in euthyroid and dysthyroidal male rats at various time intervals after I.V. injection of [14C]glutamine(Ford and Rhines, 1967).

thyroid neurons which not only contained a higher level of radioactivity, but attained maximal concentration during the experimental period. (Chromatographic analysis indicated that most of the 14C in the brain was associated with glutamine or glutamic acid.) A final example wherein CNS and neuronal uptake of labeled amino acid may be influenced by a change in the environmental state of an animal may be observed in animals subjected to a condition in which they are respiring pure oxygen at a pressure of 3 atmospheres (Fig. 6). [3H]Lysine was given intravenously at a dose level of 190.0 pg/kg and the animals were killed by intracardiac perfiision at various time intervals after iiijection. The amount of radioactivity present in the various tissue samples was expressed in terms of the percent of the injected dose present/g. Again, the level of radioactivity in the ventral horn cells was significantly higher than that determined in spinal cord grey matter. The effect of respiring pure oxygen at a pressure of 3 atmospheres was to significantly depress activity levels in both the ventral horn neurons and the spinal cord grey, on the basis of group analysis. This depression in uptake may be related to interference in the activity of various enzymes (Chance et al., 1965; Dickens, 1962; Jacobsen et al., 1964; Kety and Schmidt, 1948; Thomas et al., 1963; Wood et al., 1963, 1964) or to the ischemia known to occur in the CNS under such conditions. It might be noted parenthetically that the changes in uptake of amino acid occur much sooner than do the convulsions which are induced by high pressure oxygen, and which require subjecting the animals to higher pressures. Riferences p. 411413

410

D. H. F O R D

1

0 High

Control

pressure oxygen

0.7E

/-----Ventral horn cells O ,

,

.-I

, -

Spinal cord grey

n

z

0

10

20

30

60min

Fig. 6. A comparison of the amount of 3H present in ventral horn motor neurons and spinal cord grey matter in male rats at normal atmospheric pressure and in rats subjected to hyperbaric oxygen (3 atmospheres) after I.V. injection of [3H]lysine (Ford and Rhines, 1967).

CONCLUSION

That some mechanism exists in the brain which appears to restrict entry of many compounds, as compared with other tissues, has been accepted for many years. A variety of anatomical, physiological and biochemical explanations have been introduced to describe this phenomenon. However, this barrier phenomenon does not appear to hold true in all instances. Certainly, if one compares the amount of labeled triiodothyronine or amino acid in brain and muscle after intravenous injection, one finds that the amount of labeled material present in the two tissues is rather comparable, as is the turnover. If one then compares the distribution of various labeled amino acids and adenine between neurons and the surrounding neuropil radioautographically (Altman, 1964; Rhodes et al., 1964) (Figs. 2 and 5), one finds that neuronal uptake is considerably higher than that of neuropil. In other words, the low concentration of a physiologic substrate incorporated into a large mass of neuropil may mask the relatively high neuronal levels when one does total tissue slice or block analysis. The high amino acid accumulation by neurons as compared to neuropil was then confirmed by the application of liquid scintillation counting techniques to determine the extent to which accumulation occurs in neurons as compared with blocks of tissue containing both neurons and neuropil. An increase in neuronal physiologic activity induced by exercise and regeneration of neurons were both shown to increase incorporation of labeled amino acid in neurons. Adenine uptake was depressed in regenerating cells, possibly because of an increased utilization rate of adenine associated with the ATP utilized during the increased protein synthesis which occurs in neuron regeneration. Changes in thyroid

CHANGES IN B R A I N ACCUMULATION

41 1

function (hypo- or hyperthyroidism) were both associated with changes in accumulation of amino acid or adenine into normal or regenerating neurons or into neuropil. Finally, an increase in the amount of oxygen in the respired gas in association with an increase in pressure to three atmospheres significantly depressed amino acid accumulation by neural tissues. Thus, the uptake or incorporation of amino acids or adenine into neural grey matter or into neurons appears to be easily and readily altered by internal or external environmental factors, which would appear more likely to exert their influences on transport systems than on particular anatomical subunits existing between the blood stream and the neuronal cytoplasm. The term “barrier” seems somewhat inappropriate in relation to amino acid uptake into neurons, since the uptake appears to occur easily and to be readily influenced by changes in cellular environment in a dynamic fashion. ACKNOWLEDGEMENTS

Supported in part by a U.S.P.H.S. Research Grant (NB-04568-03) from the National Institute of Neurological Diseases and Blindness, Public Health Service, and in part by a Research Grant from the Physiology Branch (NONR 4018(00)), Office of Naval Research. The author wishes to express his appreciation for the assistance provided by Mr. Ralph Rhines, Mrs. M. Buschke and Mrs. Gloria Cohan.

REFERENCES ALTMAN, J. ( I 964) The use of fine-resolution autoradiography in neurological and psychobiological research. Response ofthe Nervous Systeni to Ionizinr Radiation, 2nd Ed. T. J. Haley and R . S. Snider (Eds.). Boston, Little, Brown and Co., (p. 336). BAKAY, L. (1956) The Bloo(l-Brain Barrier: With Special Regard to the Use of Radioactive Isotopes. Springfield, Illinois, Thomas. BARRNETT, R. J. (1963) Fine structure and function of the neurone. Trans. Anier. Neurol. Ass., pp. 123-126. BLASBERG, R. A N D LAJTHA,A. (1965) Substrate specificity of steady-state amino acid transport on mouse brain slices. Arch. Biocheni. Biophys., 112. 361-377. BRATTGARD, S.-O., EDSTROM, J.-E. A N D HYDEN,H. (1957) The chemical changes in regenerating neurons. J. Neurocheni., I, 3 16-325. BRIZZEE, K. R., VOGT,J. A N D KHARETSCHKO, X. (1964) Postnatal changes in glia/neuron index with a comparison of methods of cell enumeration in the white rat. Growth and Maturation of the Brain, D. P. Purpura and J. P. SchadC (Eds.). Progress in Brain Research, vol. 4, Amsterdam, Elsevier, (p. 136). BROMAN, T. (1955) On basic aspects of the blood-brain barrier. Acta Psychiat. Scand., 30, 115-124. CHANCE,B., JAMiEsoN, D AND COLES,H (1965) Energy-linked pyridine nucleotide reduction. Inhibitory effects of hyperbaric oxygen in vitro ‘and in vivo. Nature, 206, 257-263 CHwmos, M A , GREENGARD, P A N D UDENFRIEND, S (1960) Uptake of tyrosine by rat brain in vivo. J. B i d . Cheni., 235, 2075-2079. DEMPSEY, E. W. A N D WisLocKi. G. B. (1955) An electron microscopic study of the blood-brain barrier in the rat, employing silver nitrate as a vital stain. J. Biophys. Biocheni. Cytol., 1, 245-256. DE ROBERTIS, E. (1962) 1. Some old and new concepts of brain structure. World Neiirol., 3, 98-1 11.

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DE ROBERTIS, E. AND GERSCHENFELD, H. M. (1961) Submicroscopic morphology and function of glial cells. Intern. Rev. Neurobiol., 3, 1-65. DICKENS, F. (1962) The toxic effects of oxygen in nervous tissue. Neurochemistry, 2nd. ed., Elliott, K. A. C., Page, I. H., Quastel, 5. H. Editors, Springfield, Illinois, C. C. Thomas (p. 851). DOBBING, J. (1961) The blood-brain barrier. Physiol. Rev., 41, 130-188. DONAHUE, S. AND PAPPAS, G. D. (1961) The fine structure of capillaries in the cerebral cortex of the rat at various stages of development. Amer. J. Anaf., 108, 331-348. EHRLICH, P. (1885) Das Sauerstoff-Bediirfnisdes Organismus. Eine Farbenanalytische Studie, Berlin, A. Hirschwald, (p. 69-72). -, ( I 887) Zur Therapeutischen Bedeutung der Substituerenden Schwefelsaure-Gruppe. Therap. Monatsh., 1, 88-90. FARQUAHAR, M. G. AND HARTMAN, J. F. (1957) Neuroglial structure and relationships as revealed by electron microscopy. J. Neuropafhol. Exptl. Neurol., 16, 1 8-39. FORD,D. H. (1965) Uptake of 1131-labeled triiodothyronine in the pineal body as compared with the cerebral grey and other tissues of the rat. Structure and Function of the Epiphysis Cerebri, J. A. Kappers and J. P. Schadk (Eds.). Progress in Brain Research, vol. 10, Amsterdam, Elsfvier (p. 530). FORD,D. H. AND RHINES,R. (1967) Uptake of C14 into the brain and other tissues of normal and dysthyroidal male rats after injection of C14-~-glutamine.Acta Neurol. Scand., 43, 3 3 4 7 . FORD,D. H., WINES,R., HARTSTEIN, M. AND RHODES, A. (1965) The uptake of DL-Lysine-H3 into the nervous system as compared with other tissues in euthyroid and dysthyroidal male rats. Acfa Neurol. Scand., 41, 21 5-232. GAITONDE, M. K. AND RICHTER, D. (1955) The metabolic activity of the proteins of the brain. Proc. Roy. SOC.Brit., 145, 83-99. GAROULTE, B. AND AIRD,R.B. (1955) Diffusion of sodium ions from cerebral tissue in vifro. Science, 122, 333-334.

GOLDMAN, E. E. (191 3) Vitalfarbung am Zentral Nervensystem. Beitrag zur Physiologie der Plexus Choroideus und der Hirnhaute. Berlin, G. Eimer. R. A. (1954) The blood-brain barrier: The effect of GOLDWORTHY, P. D., AIRD,R. B. AND BECKER, acid dissociation constant on the permeability of certain sulfonamides in the brain. J. Cell. Comp. Physiol., 44, 519-526. GUROFF, G. AND UDENFRIEND, S. (1962) Studies on aromatic amino acid uptake by rat brain in vivo. J . Biol. Chem., 237, 803-806. HESS,A. (1953) The ground substance of the central nervous system revealed by histochemical staining. J. Comp. Neurol., 98, 69-92. -, (1955) The relation of the ground substance of the central nervous system to the blood-brain barrier. Nature (Lond.), 175, 387-388. JACOBSON, I., HARPER, A. M. AND MCDOWALL, D. G. (1964) The effects of oxygen at 1 and 2 atmospheres on the blood flowand oxygen uptake of the cerebral cortex. Surg. Cynecol. Ohstet., 119, 7 3 7-742.

KETY,S . S. AND SCHMIDT, C. F. (1948) The effects of altered arterial tensions of carbon dioxide and oxygen on cerebral blood flow and cerebral oxygen consumption of normal young men. J . Clin. Invest., 27, 484-492. KROGH,A. (1946) The active and passive exchange of inorganic ions through the surfaces of living cells and through living membranes generally. Proc. Roy. SOC.(London), B., 133, 140-200. LAJTHA, A. (1964) Protein metabolism of the nervous system. Infer. Rev, Neurobiol., 6, 1-97. LAJTHA, A. AND TOTH,J. (1963) The Brain-Barrier System. V. Stereospecificityof amino acid uptake, exchange and efflux. J . Neurochem., 10, 909-920. LEBLOND, C. P. (1965) What radioautography has added to protein lore. The Use of Radioautography in Investigating Protein Synthesis. C. P. Leblond and K. B. Warren, Editors, New York, Academic Press, (p. 321). LEVI,G. AND LAJTHA,A. (1965a) Cerebral amino acid transport in vitro. 11. Regional differences in amino acid uptake by slices from the central nervous system of the rat. J. Neurochem., 12,639-648. LEVI,G., CHERAYIL, A. AND LAJTHA, A. (196513) Cerebral amino acid transport in vitro. 111. Heterogenecity of exit. J . Neurochem., 12, 757-770. LEWANDOWSKY, M. (1900) Zur Lehre von der Cerebrospinal-Flussigkeit.Z . Klin. Med., 40,480494. MAYNARD, E., SCHULTZ, R. L. A N D PEASE, D. C. (1957) Electron microscopy of the vascular bed of rat cerebral cortex. Amer. J . Anat., 100, 409434.

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PAKKENRERG, H., FORD,D. H., RHINES, R. AND ISRAELY, R. A. (1965) Adenine H3-uptake in nervous tissue including regenerating nerve cells, as compared with other tissues in euthyroid, hypo and hyperthyroid male rats. Actu Nerirol. Scand., 41, 497-512. RHODES, A., FORD,D. H. A N D RHINES, R. (1964) Comparative uptake of ~L-Lysine-H~ by normal and regenerative hypoglossal nerve cells in euthyroid, hypothyroid and hyperthyroid male rats. Exprl. Neurol., 10, 251-263. ROBEINS, J. AND RAI.L,J. E. (1960) Proteins associated with the thyroid hormone. Physiol. Rev., 40, 41 5 4 8 9 . RODRIGUEZ, L. A. (1955) Experiments on the histologic locus of the hematoencephalic barrier. J. Conip. Ncrirol., 102, 2145. Roux, E. AND BORKtL, A. (1898) Tetanos cerebral et immunite contre le tetanos. Ann. Inst. Pusteur (Paris), 12, 225-239. SCHADE, J. P., VAN BACHER, H. A N D COLON, E. (1964) Quantitative analysisofneuronalparameters in maturing cerebral cortex. Gron~tliand Mcitrirarion ofthe Brain, D. P. Purpura and J. P Schade (Eds.). Progress in Brain Research, vol. 4, Amsterdam, Elsevier (p. 150). TtioMAs, J. J. JR., NEPTUNE, E. M. JR. AND SUDDUTH,H. C. (1963) Toxic effects of oxygen at high pressure in the metabolism of D-glucose by dispersions of rat brain. Biochem. J., 88, 31-45. TSCHIRGI, R. D. (1962) Blood-brain barrier; fact or fancy? Fed. Proc., 21, 665-671. WAELSCH, H. A N D LAJTHA,A. (1961) Protein metabolism in the nervous system. Pltysiol. Rev., 41, 709-736. WISLOCKI, G. B. A N D LEDUC,E. H. (1952) Vital staining of the hematoencephalic barrier by nitrate and trypan blue, and cytological comparisons of the neurohypophysis, pineal body, area postrema, intercolumnar tubercle and supraoptic crest. J. Comp. Neurol., 96, 3 7 1 4 5 . WOOD,J. D. AND WATSON,W. J. (1964) The effect of oxygen on glutamic acid decarboxylase and gamma-amino butyric acid-alpha-ketoglutaric acid transaminase activities in rat brain homogenates. Canad. J. Pliysiol. Pharmzcol., 42, 277-279. WOOD,J. D., WATSON,W. J. AND CLYDESDALE, F. M. (1963) Gamma-amino butyric acid and oxygen poisoning. J. Neiirochern., 10, 625-633.

DISCUSSION P. MANUEL:I should like to make some general remarks. The first one concerns the incorporation of amino acids into tissue using radioautography. When you look for the incorporation of amino acids into proteins in vifro and you precipitate the proteins with TCA, you may have a high degree of incorporation. However, to determine if there has been real incorporation of amino acids into proteins, one must treat the preparation with TCA at 90°C because when you provide exogenous basic or acidic amino acids they may simply become attached to another amino acid in a protein chainzwithout actually becoming incorporated into the proteins. I think that many mistakes might result from this kind of phenomena in experiments with radioautography. During perfusion you have a free exchange between free and bound amino acids, because you are doing two kinds of experiments. One is called a “chase-experiment”. When this is done at 10°C there can still be an exchange with a peptide chain. With water perfusion there may also be an exchange with the amino acids which are attached to the peptide chains and which are not incorporated in the-chains. Thus, the problem remains as to whether the incorporation of amino acid represents a protein synthesis or not. I think that the control experiments which you did with the scintillation counting after the radioautography are very useful, but it seems to me that it will also be useful to do this after treatment by TCA, before the counting. The second remark concerns the nucleotide pool. We have observed an increase of nucleotide synthesis following diffcrcnt forms of stimulation. There was namely an increase in the turnover of ATP or in ATP-synthesis. If one looks for RNA synthesis and finds an increase in the specific radioactivity of a purine or pyrimidine base in RNA, it might be difficult to determine if R N A synthesis occurred because of an increase in the specific activity of the precursor pool of bases.

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D. H. FORD: This might be true for RNA or ATP. P. MANDEL:It is believed that there is an increase of the RNA synthesis in stimulated neurons. As far as the ATP-synthesis-increase is concerned, we have experiments with amphetamine, wherein examination of the y-phosphate or the u-phosphate demonstrates a good correlation between the increase of functional activity and nucleotide synthesis. The last point concerns the different salts which one uses. We found some years ago that there are really differences after injection of a precursor. There are differences in the radioactivity of the free nucleotides in different salts, which agrees well with what you found.

D. H. FORD:The problem of interpreting radioautography is very complex, since most people rarely determine the chemical nature of the labeled material producing the radioautograph. In our findings with the dissected neuron preparations from perfused animals, there may be activity in the cells which are being monitored, which is not associated with protein, but which is associated with a carrier or some other marker, or which may be a lipoprotein. When [3H]lysine was injected, the major labeled material in the neurons was lysine as determined chromatographically. With adenine injected rats, tissue samples were hydrolyzed with KOH which was followed by perchloric acid precipitation. This provides quite different results than those obtained by acid hydrolysis and precipitating with the base. The labeled molecules are not completely split t o liberate the adenine base itself. Thus, one ends up with literally all of the possible compounds that adenine can be associated with, including the break-down products of adenine (representing perhaps about 30% of the total pool). Most of these were found in the hydrolyzed brain preparations. K. D. NEAME: I would like to bring up a point about the terminology. Dr. Ford used the word upl(ikP, implying it with reference to incorporation of amino acids into protein. “Uptake” is also used to refer to the movement of free amino acids. I think one must definitely distinguish between these two types of movement, one: incorporation into protein, and the other: movement into the cell, but not into the protein. D. H. FORD:Actually, if one could determine the total amount of labeled material taken up into the cell, the values would probably be higher. However, with the process used, these small amino acids, free bases and perhaps peptide units as well are by and large removed. So in our preparation one is literally left with only what has been incorporated or bound in some fashion within the cell if not actually into protein. However, you are quite right in cautioning against the rather loose use of the term “uptake”. P. G. SCHOLEFIELD: Did I understand you, Dr. Ford, to say that most of the tritiated adenine went into the nucleotides when you measured incorporation? D. H. FORD:The greatest fraction of activity which we have found is in nuclcotides. There were other smaller fractions, which we have not been able to identify.

P. G. SCHOLEFIELD: It seems to me that in other studies with tritiated adenine, certainly in in vitro studies, it disappears within a matter of minutes. As 1 understand it, previous irr vivo work on incorporation of adenine indicates that it disappears very quickly, but in some of your slides you showed that adenine was going into the cell, and being incorporated into nucleotides even after 48 hours. How do you explain this?

D. H. FORD : We have performed chromatographic analysis on animals killed 24 hours or 48 hours after the injection with labeled adenine. ADP and AMP could be seen as identifiable compounds, plus a very small amount of adenosine, and traces of activity which migrated chromatographically like adenine. It would seem, therefore, that most of the adenine has disappeared and become incorporated into nucleotides. Of course, if one hydrolyzes with perchloric acid in the usual fashion, over 60% of the activity is present as ade nine.

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P. G . SCHOLEFIELD: Yes, that is normal, because if you hydrolyze it you liberate the purines. Could you hydrolyze the R N A for example with KOH? D. H . FORD:We have done some preliminary salt extractions of brain for RNA, and the total amount of activity taken up into the RNA fraction as compared to the rest of the activity in the sample is so much lower, that I distrust the figures. In liver, I have more confidence in the results, since the extracted fraction has a U V absorption comparable to that of RNA. Therefore, the radioactivity present in this fraction would seem to be, in part at least, RNA. However, in brain the technique which we used does not seem to be adequate. P. G. ScHoLEwLD: Just one more inquiry, if I may. Did you identify this ATP? Would not you have thought that the alkali would have hydrolyzed most of the ATP? P. MANDEL:In the normal hydrolysis you need barium hydroxide at loo". You cannot hydrolyze in the same condition as you hydrolyze RNA. A. LAJTHA: The changes in the label in the tissue may be caused either by changes in the transport of the labeled precursor or by changes in the rate of incorporation of this precursor into an acidinsoluble product. I wonder which one you think is affected by hypothyroidism. If I remember well, Sokoloff showed an increase in protein formation in in virro systems.

D. H. FORD:In young animals? A. LAJTHA: Only in young animals.

D. H. FORD:We have been interested in this, because we think, looking at our data from adult animals, that a point of maximal concentration of activity occurs earlier in hyperthyroid rats than normally observed. This suggests that there is actually a slight acceleration of incorporation of labeled material into the protein in the hyperthyroid state. We observed while doing amino-nitrogen determinations, that the amino-nitrogen levels of normal and the hyperthyroid rats was essentially the same. Thus, if the total amount of amino nitrogen is relatively constant, but there is an acceleration of turn-over of protein in neurons, one might logically expect the time needed for attaining maximal concentration of an amino acid might be altered, reflecting the change in metabolic rate, which might well occur without changing the amount of label present in a protein at any given time. H. KOENIG: You referred to Dr. Altnian's work in which he demonstrated an increased incorporation in the protein of ['JCIleucine in the nerve cell in exercised animals. He also observed an increase of a similar magnitude into the choroidal plexus, which he did not discuss. So one is left with the question: what was the meaning of this increased incorporation? The problem of radioautography is that while it tells you where the incorporated label is located, it tells you nothing about penetration since the acid soluble precursor pool is eluted out. In studies of this kind, it would be well to obtain measurements on the acid soluble pool from fresh tissue adjacent to that being radioautographed to gain some idea of penetration as well as incorporation.

D. H. FORD:We also have information on the distribution of lysine in the various pools which, as you have indicated, is very helpful in understanding the radioautograph. P. MANDEL:It is quite sure that in 30 minutes after injection there is very little incorporation of adenine into RNA: it is mainly present in the acid soluble pool.

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417

The Development of the Blood-Brain Barrier JOHN DOBBING Department of Physiology, London Hospital Medical College, London (England)

Classical views on the blood-brain barrier have usually included the idea that it is not present in early life, but develops gradually as the organism develops, to become fully operative only when the animal achieves maturity. I n the first decades of this century, during the period when the “barrier” was considered exclusively in terms of dyestuffs or other histologically identifiable substances, it was widely claimed and believed that trypan blue entered the foetal and newborn brain readily from the blood stream, and was only excluded in the matureindividual (Behnsen, 1927; Stern and Peyrot, 1927). When radioactive isotopes became available for biological research, it was quickly found that such non-specific markers as 32P appeared to be less restricted in their entry into neonatal brain than into adult (Fries e t a / . , 1940; Bakay, 1953), and this was naturally interpreted as a further manifestation of the “developing blood-brain barrier”. Since this time, the concept has been further extended. Analagous to the alleged behaviour of dyestuffs, it has been held to account for the phenomenon of kernicterus, a condition confined to the neonatal period in humans in which unconjugated bilirubin from the plasma enters certain areas of the brains of jaundiced babies. Also, as an extension of the early findings with 32P many other types of metabolic materials have been found to enter the immature brain more readily than the adult, in accordance with the much greater activity of many metabolic processes at this time. One of the more recent examples was reported for inulin (Woodbury, 1967). In many quarters the belief dies hard that trypan blue enters immature brain freely. Nevertheless, for several years now, experimentalists in the field have known that however immature the brain, dyestuffs like trypan blue do not enter it any more readily from the blood than in the adult state (Millen and Hess, 1958). More recently it has been shown that bilirubin will not enter the normal newborn brain (Lucey, Hibbard, Behrman, de Gallardo and Windle, 1964) unless it is present in sufficient quantity to have exceeded the capacity of the plasma albumin to bind it (Diamond and Schmid, 1966). The extreme rarity of an unconjugated hyperbilirubinaemia of this magnitude in adults accounts for kernicterus being confined to the neonatal period in humans. However, in the Gunn strain of rats in whom there can be a high plasma level of such pigment due to a conjugation defect, kernicterus does occur in adults provided the form and physical state of the plasma bilirubin is suitable. The operative factor is in the plasma, not in the maturity of the brain. RpJ’fiwnci.rp. 424-425

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For those whose concept of the blood-brain barrier is limited to the behaviour of dyestuffs across the blood-brain interface, the idea of a developing barrier of increasing impermeability with age, must therefore be abandoned. Most experimenters, however, are concerned with the blood-brain relationships of other classes of substances, and it must be agreed that something like this older concept still seems to be valid for many metabolic materials as well as in cases like that of inulin mentioned above. The best methods of investigation of the blood-brain barrier involve direct measurements of the rates of entry of substances from blood to brain. In the developing brhin these methods can perhaps be supplemented by observations on the rate of accumulation of brain constituents during the normal growth period of the organ. With certain reservations these rates of accumulation must represent their rates of arrival (or those of some of their precursors) within the brain from the blood. In most species the brain undergoes a period of maximum growth (measured in increments of weight) earlier than the corresponding period for the rest of the body. During this “growth spurt” it is to be presumed that the rate of entry of many substances must be greater than at any other time. The brain “growth spurts’’ of several species are portrayed in Fig. 1 which illustrates two points: that there are important species

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Fig. 1 . The timing of brain growth in different species in relation to birth (Davison and Dobbing 1966). Curves of the rate of brain growth in different species are expressed as weight increments (percentage of adult wet weight of brain) per unit period of time. Brain-weight data are taken from the following sources: man (Spector, 1956); rat (Dobbing, unpublished); dog (Himwich and Petersen, 1959); pig (Dickerson and Dobbing, 1967); guinea pig (Dobbing, 1968). The time scale in each case has been adjusted according to the life span of the species, on the quite arbitrary assumption that brain development may be related to life span (Donaldson, 191 1).

differences in the timing of the brain “growth spurt” in relation to birth; and that the periods of maximum rate of accumulation of brain substance is preceded as well as succeeded by a time of slow, or even negligible rate of accumulation. Because of the species differences it is evident that to speak of the neonatal brain, or even of the foetal brain without a knowledge of the particular species intended, can be misleading.

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The brain of the guinea pig foetus three-quarters of the way through gestation is of a similar state of maturity to that of a rat towards the end of its second postnatal weeks, and can presumably be expected to have a similar metabolic performance or “bloodbrain barrier”. Is it possible that our impression of a more permeable blood-brain barrier to metabolic substances in early life, may simply be a reflection of the enhanced activity associated with the “growth spurt”? If so, it should be possible, by selecting the correct timing for a given species to demonstrate the “pre-growth-spurt” period of slow entry into the brain (as well as the “growth spurt” itself and its subsequent decline) by direct experiment. We would still be left with the almost philosophical question of whether changing “blood-brain barrier” permeability dictates the changing growth rate, or whether the rate of entry is merely a reflection of the characteristics of brain growth; but at least we should be faced with a developing “blood-brain barrier” whose characteristics were not simply those of gradually increasing permeability with age.

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Fig. 2 represents the rate of accumulation of DNA-P and cholesterol in the brain of the developing pig. The velocity curve of increasing weight is occupied first by a period of rapidly increasing numbers of cells, followed by a period of lipid deposition. These broadly represent the two phases of myelination: a phase of oligodendroglial proliferation followed by the manufacture of lipid-containing myelin sheaths by the established oligodendroglia. It is known that both these substances are mainly concerned with brain structures of very slow (if any) turnover (Davison and Dobbing, 1961). Is i t valid to equate the rate of access of a brain constituent or its precursors to the growing brain with the observed rate of accumulation of the product? Provided that the constituent has a sufficiently slow turnover once incorporated into the brain, it is probably reasonable to assume that the two observations are related. That is to say if a brain constituent, on incorporation into the developing brain structure, is subsequently metabolically inert, or nearly so, then its rate of accumulation is likely to References p. 424-425

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resemble its rate of entry. In practice most brain constituents are synthesized in situ, and therefore it is the rate of access of their precursors which is likely to be represented by the velocity curve of accumulation of inert product. In order to demonstrate this association directly, the entry rate of labelled cholesterol into developing rat brain has been compared with the velocity curve of its normal accumulation during growth (Dobbing, 1963b). Although most brain cholesterol is synthesized within the developing tissue, a measurable proportion is derived as the preformed molecule from the blood and this can be demonstrated by injecting [4-“C]cholesterol intraperitoneally and finding some of it unchanged in the brain. Fig. 3 showed the result of such an experiment, from which it can be seen that the relative specific activity a short time after injection into rats of different ages closely follows the velocity curve of its accumulation. In other words the peak rate of entry is preceded as well as succeeded by conditions in which the rate of entry is slow. It may well be asked which of the two factors normal growth or “blood-brain barrier” - controls or limits the other?

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Fig. 3. Pattern of entry of [4-14C]cholesterol into rat brain at different body weights compared with a relative specific activity of brain cholesterol rate curve of brain cholesterol accumulation. 0-0 1 day after injection. 0-0 increments in whole brain cholesterol for each 5 g gain in body weight. (Dobbing, 1963b).

Fig. 4 shows velocity curves for DNA-P, weight and cholesterol in rats, and it can again be seen how the one for DNA-P is declining rapidly at a time when that for cholesterol is rising fast. Any “developing blood-brain barrier” hypothesis must therefore include a different timing for each substance as well as the “stop-go-stop” characteristics outlined earlier. Fig. 5 shows a similar pattern of events in the developing guinea pig brain. The use of 32P as a label for investigating the “blood-brain barrier” has many critics on account of the variety of metabolic events which must influence its rate of entry. A great deal of the phosphate entering the brain is concerned, particularly during myelination with phospholipid metabolism, and a peak period of incorporation has often been demonstrated, particularly into myelin lipids at the time of

THE DEVELOPMENT OF THE BLOOD-BRAIN BARRIER

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Fig. 5. Rate curves of the increase in fresh weight and amounts of cholesterol and DNA-P in the whole brain of the developing guinea pig plotted as increments per one day interval. All values calculated as a percentage of the mature value. -brain weight; whole brain cholesterol; DNA-P. (Dobbing, 1968).

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Fig. 6. Rate curve of the increase. in the amount of cholesterol in the whole brain of the developing rabbit, plotted as increments per 5 day interval. All values calculated as a percentage of the mature value. (calculated from Davison and Wajda, 1959).

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myelination (McMurray, 1964). Most of the evidence has naturally come from in vitro experiments, or following intracerebral injection, techniques which are not applicable to the direct investigation of the “blood-brain barrier” in its usual sense. Recently it has been claimed on the basis of in vivo experiments, that the newborn rabbit brain admits 32P no more readily than the adult, when proper regard is had to different conditions such as vascularity (Grontoft, 1965). Fig. 6 shows the rate curve for the accumulation of cholesterol into rabbit brain of different ages. This closely follows the entry characteristics of the other lipids, particularly the myelin lipids, and the same features of a transient period of increased entry can be seen. Is it possible, in selecting the two ages (newborn and adult), that this intermediate period has been missed?

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2s

~

Fig. 8. Rate curves of the increase in amounts of DNA-P in the forebrain, cerebellum and spinal cord of the developing pig, plotted as increments per 2 week interval. All values calculated as a percentage of the mature value. ___ cerebellum; .. .. . .. forebrain; ------ cord (Dickerson and Dobbing, 1967).

.

Fig. 9. Rate curves of the increase in amounts of cholesterol in the forebrain, cerebellum and spinal cord of the developing pig, plotted as increments per 2 week interval. All values calculated as a percentage of the mature value. ___ cerebellum; ...... forebrain; ------ cord (Dickerson and Dobbing, 1967).

.

.

One of the findings in this very careful study by Grontoft was that the newborn cerebellum took up 32P more readily than the adult. The reverse was true of the cerebrum, and in the brain stem uptake was equal at both ages. It is, of course, well known that there are regional differences in the metabolic behaviour of the brain. In the same way different regions develop at different relative rates, and the cerebellum develops relatively earlier and more quickly than other parts (Dickerson and Dobbing, 1966). As far as is known no figures are available for the rabbit, but in the human and in the Re-fcrences p. 424-425

424

J. D O B B I N G

pig, it is documented and there is no reason to believe this is not a common mammalian phenomenon. Fig. 7 shows velocity curves of weight increase in cerebellum, whole brain and cord. In each case these can be divided into an earlier period of cellular proliferation (Fig. 8) and a later one of lipid deposition (Fig. 9). It would seem, therefore, that the findings reported by Grontoft could well correspond with the characteristics of normal regional growth. It has often been suggested (Dobbing, 1961, 1963a) that the “blood-brain barrier” for metabolic substances can be accounted for on the basis of known metabolic behaviour. In the developing brain it does indeed still seem that the so-called barrier may in large measure be simply a reflection of some of the characteristics of brain growth. ACKNOWLEDGEMENTS

I wish to thank the Multiple Sclerosis Society (of Great Britain and Northern Ireland) and the Pig Industry Development Authority for supporting this work.

REFERENCES BAKAY,L. (1953) Studies on blood-brain barrier with radioactive phosphorus. 111. Embryonic development of the barrier. Amer. Med. Ass. Arch. Neurol. Psychiat., 70, 30-39. BEHNSEN, G. (1927) Zellforsch., 4, 515. DAVISON, A. N. AND DOBEING, J. (1961) Metabolic stability of body constituents. Nature, 191, 844848.

-, (1966) Myelination as a vulnerable period in brain development. Brit. Med. Bull., 22, 40-44. DAVISON,A. N. AND WAJDA,M. (1959) Metabolism of myelin lipids: Estimation and separation of brain lipids in the developing rabbit. J. Neurochem., 4, 353-359. DIAMOND, I. AND SCHMID,R. (1966) Experimental bilirubin encephalopathy. The mode of entry of bilirubin - 14C into the cextral nervous system. J . Clin. Invest., 45, 678-689. DICKERSON, J. W. T. AND DOBEING, J. (1966) Peculiarities of Cerebellar Growth. Proc. Roy. SOC. Med., 59, 1088. -, (1967) Prenatal and postnatal growth and development of the central nervous system of the pig. Proc. Roy. Ser. B, 166: 384-395. DOEBING, J. (1961) The blood-brain barrier. Physiol. Rev., 41, 130-188. -, (1963a) The blo3d-brain barrier: some recent developments. Guy’s Hosp. Repts., 112, 267-286. -, (1963b) The entry of cholesterol into rat brain during development. J. Neurochem., 10, 739-742. -, (1968) Prenatal and postnatal growth and development of the central nervous system of the guinea pig (in preparation). DONALDSON, H. H. (1908) A comparison of the albino rat with man in respect to the growth of the brain and of the spinal cord. J. comp. Neurol. Psycho/., 18, 345-389. FRIES, B. A., CHANGUS, G. W. AND CHAIKOFF, I. L. (1940) The influence of age o n the phospholipid metabolism of various parts of the central nervous system of the rat. J. Biol. Chem., 132, 23. GRONTOFT, 0. (1965) The permeability to P32 in different regions of the brain of new-born and adult rabbits. Acta pathol. microbiol. Scand., 63, 481-492. HIMWICH, W. A. AND PETERSEN. J. C. (1959) In: Messerman, J. H. edition Biological Psychiatry, p. 2. New York. Grune and Stratton. LUCEY, J. F., HIBBARD, E., BEHRMAN, R. E., ESQUIVEL DE GALLARDO, F. 0.AND WINDLE, w.F. (1964) Kernicterus in asphyxiated newborn rhesus monkeys. Exptl. Neurol., 9,43-58. MCMURRAY, W.C. (1964) Metabolism of phosphatides in developing rat brain - 1. Incorporation of radioactive precursors. J. Neurochem., 11, 287-299,

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MILLEN,J. W. AND HESS,A. (1958)The blood-brain barrier: an experimental study with vital dyes. Brain, 81, 248-257. SPECTOR, W. S. (1956) Hatidbook of Biological Data. Philadelphia. Saunders. STERN,L. A N D PEYROT,R. (1927) Le fonctionnement de la barriere hematoenckphalique aux divers stades de development chez les diversesesptces animales. C.R. SPances SOC.Biol. Fil., 96, 1124. WOODBURY, D. M. (1967) This volume pp. 297-314.

DISCUSSION L. BAKAY:I wanted to ask you one question, and I want to make a small comment. There must be a species difference as far as the immature or infantile barrier is concerned. Previously, the ground substance, which was supposed to be a polysaccharide, was thought to fill an extensive area. Now it is delegated to be nothing more than the capillary wall. I understand that in guinea pig it is already present at birth, while in other animals it is not. This region was supposed to be an area where the barrier is already well established at the moment of birth as far as the guinea pig is concerned, while in other animals it is still undergoing “maturation.” As far as bilirubin is concerned, injecting large amounts of bilirubin in some species of animals could produce bilirubin staining of the brain (Rozdilsky and Olzewski). The same amount injected into other animals (dog, cat or rabbit), appears very different under the same experimental circumstances. The question I wanted to ask you, pertains to Barlow’s [35S]-studiesand why it is that we think that there is an increased permeability of the barrier at the time of birth which provides for the uptake of large amounts of sulphate. Is the inorganic sulphate space an extracellular space and is it larger in the newborn and does it become smaller as the animal grows older? What do you think about this? J. DOBBING: I don’t know. However, it should not be difficult to investigate the rates of entry in vivo, although determining the in vivo entry rates is more difficult than it would first appear. If results were obtained which represented an entry rate, then it might be considerably influenced by the rate of sulphatide accumulation during this period. Now to refer to the point that you made about experimental kernicterus in different species, there are very definite species differences in the accumulation of all of these things which we have measured.

T. Z. C S ~ K YWith : regard to the suggestion that unconjugated bilirubin actually saturates all the protein binding receptors, do you recall what was the technique used for this study? Should this really occur, it would be very interesting to determine if phenylbutazone will cause kernicterus. Phenylbutazone is one of those very rare substances which can saturate with great avidity all the protein receptors, and can actually displace other drugs after they have been bound to proteins. If this occurs, then a large dose of phenylbutazone could cause kern-icterus in many animals, even in adults. Phenylbutazone will also dislodge gantrisin and cause a larger extracellular distribution. J. DOBBING: I think the fact that this will occur with gantrisin and produce kernicterus in babies at a very low level of plasma bilirubin puts the etiology of kernicterus in the plasma rather more than on the age of the brain. J. FOLCH-PI:The difficulty with which we are dealing is that the developing brain is the same organ as the adult brain in name only. The rapid changes which occur in the cell population and the shift in the position and perhaps the size of the spaces means that one cannot really study steady states, except in a very limited period of time. This is further complicated by the increases in weight as cells grow and fibers become myelinated. Since the brain may increase in weight as much as 10% in one day, it is very difficult to really draw valuable comparisons unless you do a longitudinal study, a

426

J. D O B B I N G

complete profile of the brain of one animal. Once you have that profile, which has to be completed with a full awareness of the whole anatomy and physiology and biochemistry of the brain, it is only then that you are in a position to compare between one species and the other. Also, we place too much value on a new point of view, which may permit the entrance of the uninformed into a field, which has happened many times in science. This has often been a source of completely absurd experiments. Now I think that we neurochemists are very well aware that we are finally getting data and the man who educated us was Flexner. He was a man who demonstrated a beautiful multidisciplinary approach to science which is classic. V. TENNYSON: I just want to say that as far as spaces are concerned, there is a very little more space in the embryo than we find in the adult. The only difference that I can see in the embryo in the blood vessels is that the basement membrane is not as well developed. J. FOLCH-PI:On the other hand, Cr. Tennyson, in in vivo chemical spaces, such as the sodium and chloride spaces, there are very marked and well documented differences which really illustrate how cautious one has to be in correlating or trying to make equivalents between in vivo anatomical spaces and in vivo chemical compounds. V. TENNYSON: But as one sees in the electronmicroscope there seem to benolargespacesin theembryo.

D. B. TOWER: Could I ask you to tell us in some sort of relative terms what is the difference between “somewhat bigger” and “not very much bigger”? V. TENNYSON: 1 really would hesitate to say that there is any. Once in a while one sees cell processes rounding off, and you get a more triangular space rather than a space between perfectly parallel walls of cells.

D. B. TOWER: We are not really talking about inches or feet. A few A difference may make quite a lot of difference volumetrically. 0. STEINWALL: Dr. Woodbury, a short question about inulin: Was it possible that it entered the brain by first entering the cerebrospinal fluid? D. M. WCODEURY:Our data, based on calculation of the concentrations in the two compartments, indicate that inulin en ters the brain first, then the cerebrospinal fluid next and not vice versa. A. LAJTHA: Let me mention amino acids here, although one can’t generalize from the behavior of one type of metabolite to that of another. In measuring the exchange of amino acids between plasma and brain, we found a very high rate for most amino acids, with a half-life in brain pools in minutes. This rate of exchange is probably unrelated to the rate of metabolism of the amino acids and it certainly is at least one order of magnitude larger than metabolic rates. Cholesterol may be an entirely different case, where the influx of cholesterol increases when the highest rate of deposition of this compound occurs, which may be a turning-on and off kind of transport phenomenon. An alternate explanation would be that there is a constant rate of exchange of cholesterol, but you find significant counts in the brain only when significant amounts are deposited.

DOBEING: It is of course, a problem of semantics to argue whether it is a change in the permeability of the system which is controlling growth, or that it is reflected by changes in what we have called the blood-brain barrier. I would not claim to know what controls growth and I think we ought to maintain an open mind about what is found in the literature of our colleagues on the subject.

J.

There may be a small free cholesterol pool in constant equilibrium with plasma which A. LAJTHA: may be too small to detect. This pool. however, can contribute significant labeling to the myelin during myelination. If this is the case, the label will measure not the transport but the incorporation of the molecule into myelin.

THE DEVELOPMENT OF THE BLOOD-BRAIN BARRIER

421

J. DOBBING: We happen to have evidence with the materials we use, that the very molecules which get into the brain stay for a considerable period after they have arrived there, in some cases for several years.

P. MANDEL:Of course it has not been proven that there is not a free cholesterol pool, J. DOBBING: I am sorry, no, it has not indeed, and my data for rates of entry are very inferential

...

I agree.

P. MANDEL:I think that the experiments concerning growth are still significant if we keep in mind that any kind of cell has its own rate of uptake and synthesis. This is all that we can say, and in this way it is useful. J. FOLCH-PI:I have not said it is not useful, but I have always maintained that a single isolated point may mean very little. You need a whole thesis-point, you need a time sequence, a longitudinal study in the way the physiologists are already working in behavior. J. DOBBING: For meaningful time curves, you need a great many observations. P. MANDEL:It depends on the kind of cells with which one is working. H . M . ADAM:How many points did in fact attribute to your growth rate curves? J. DOBBING: In the case of the pigs, there were animals at 40 different ages from the last 50 or 60 days of gestation to the first two or three months of life. The animals were not equally spaced in age and tended to be further apart at the older ages. With that number, it was a relatively simple analysis with these substances, but we have done more complicated things. With the relatively simple substances used, it is possible without any very great exercise of judgement to draw these kinds of rate curves.

H. M . ADAM:Would there be any objection against using a logarithmic scale for the time sequence?

J . DOBBING: No, the original intention of the work was to test the hypothesis that at the peak period of activity this might be a particularly vulnerable period to such restricted influences as malnutrition or anoxic conditions.

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429

Intrinsic* Amino Acid Levels and the Blood - Brain Barrier C L A U D E F. BAXTER Neurochemistry Laboratories, Veterans Administration Hospital, Sepulveda, California; Department of Biochemistry City, of Hope Medical Center, Duarte, California; and Department of Physiology, UCLA School of Medicine, Los Angeles, California.

The instantaneous substrate level of a compound at a given site in the nervous system reflects the interaction of at least five dynamic processes: synthesis, degradation, uptake, extrusion and exchange. Since the latter three processes define the kinetic interrelationship between the blood and the brain, it is apparent that substrate levels not only are affected by but will themselves affect this interrelationship. A. I N T R I N S I C A N D E X T R I N S I C AMINO A C I D P O O L S

In recent years several laboratories have studied the effect of changes in the substrate levels of amino acids upon uptake, extrusion and exchange phenomena in nervous tissues, both in vitro and in vivo (Abadom and Scholefield, 1962; Blasberg and Lajtha, 1965; Lajtha et al., 1963; Lajtha, 1964; Levi et al., 1966; Levi et al., 1965; Nakamura and Nagayama, 1966; Neame and Smith, 1965; Tsukada et al., 1963; Wiechert, 1963). Substrate level equilibria were altered by utilizing extrinsically added amino acids. In many experiments the movement of a specific, isotopically labeled amino acid was charted. Results from these studies fail to distinguish whether an amino acid is taken up, extruded or exchanged from only one specific compartment in the tissue, or if the movement of the amino acid reflects an involvement of the total tissue pool. All available evidence shows that an extrinsically added amino acid, such as y-aminobutyric acid (GABA), even when added to a subcellular fraction of brain tissue in vitro, equilibrates within a reasonable period of time with only a part of the intra-particulate pool (Weinstein et al., 1965). In vivo studies have indicated a similar situation (Roberts et al., 1958). So-called “free” amino acids in brain tissue appear to be contained in several compartments, and labeled amino acids, injected into the brain, may become distributed rapidly in only a few of the many compartments of the total substrate pool. Berl, Lajtha and Waelsch (I96 I ) found that [U-l4C]glutamate injected intracerebrally gave rise to glutamine with a higher specific activity than that of glutamate. This result, which has been confirmed repeatedly (Berl et al., 1962; Baxter, 1963; Gaitonde, 1965; Berl and Pur-

* The term intrinsic has been used to designate “located within”. The evidence contained in this paper suggests that most oft he intrinsic amino acids in brain also must beendogenous“originating internally”. Rrfcrrnces p. 441-444

C. F. B A X T E R

430

TABLE I C O M P A R T M E N T A L I Z A T I O N OF G L U T A M I C A C I D A N D G L U T A M I N E I N R A T B R A I N

Time after Compound injected

injection (niin)

[ I -14C]glutamic acid [U-14C]glutamic acid [I -14C]y-aminobutyric acid [U-14C]glucose

Activity Ctutamine Glutamic acid (cts/niin/100 mg brain tissue)

Ratio of relative specific activity*

GMEIGA*Z

40 40

6270 3840

4600 1997

2.7 3.8

40 32

3827 380

2230 1025

3.4 0.7

* Based upon a glutamine pool which is f the size of the glutamate pool.

**

GME = glutamine: GA

=

glutamic acid.

One to two microcuriesof W-labelled metabolite was injected stereotaxically into the lateral ven'ricle of fed female Sprague Dawley rats weighing 200 to 220 g. The injected dose was contained in 1OA of a neutral solution. Rats were lightly anesthetized with Nembutal (8 to 9 mg/200 g rat) prior to placement into the stereotaxic instrument. Glutamic acid and glutamine were isolated from tissue extracts by two dimensional high voltage electrophoresis. Radioactivity was determined as described by Baxter and Senoner (1964).

pura, 1966), is the same irrespective ofwhether [l-"C]- or [U-"T]glutamic acid is injected into the ventricle of the rat brain (see Table 1). The widely accepted interpretation of these results holds that the 14C-labeled glutamic acid did not become distributed evenly in all of theglutamic acid pool of brain tissue, but only in a specific compartment which happens to be in rapid equilibrium with the major portion of the glutamine pool. Thus, there must be at least two glutamate compartments in brain. In more recent experiments [I-WIGABA was injected intracerebrally and again it was found that, within minutes, the glutamine had acquired a higher specific activity than the glutamic acid. The relationship, 40 minutes after the injection, is recorded in Table 1. Since GABA cannot be carboxylated to glutamic acid, all 14Clabeling from GABA must have passed through the intermediates of the tricarboxylic acid cycle before reaching glutamate or glutamine. The results suggest therefore that all of the tricarboxylic acid cycle intermediates derived from [1-l4C]GABA were in metabolic pools which did not equilibrate with a major compartment of the glutamic acid pool, within the time period of the experiment. A similar conclusion can be drawn from the experiments of Roberts and Morelos (1965), who found that glutamine acquired a higher specific activity than glutamic acid, after [U-"C]leucine had been injected intravenously into rats. Even the intracerebral administration of [I-"C]- or [2-W]-acetateto mice resulted in a higher specific activity in glutamine (Van den Berg et al., 1966). By contrast, the injection of 1%-labeled glucose has consistently led to a higher specific activity in glutamate than in glutamine (see ratio GME/GA Table I), a result which is in agreement with those published by Cremer (1964); Gaitonde et al. (1965); Van den Berg et al. (1966); and by Lindsay and Bachelard (1966). Presumably,

I N T R I N S I C A M I N O A C I D LEVELS

43 1

these experiments show that [“W]glucose can label a glutamate pool in brain tissue which was not labeled by the direct intracerebral injection of [14C]glutamate, [“TIGABA, or [14C]acetate. It is not within the scope of this paper to speculate about the interpretation of these results. The data are presented only to illustrate that amino acids added extrinsically to a biological system are not necessarily equivalent kinetically to an elevation of intrinsic levels of the same amino acids. In the past, the effects of changed amino acid levels upon their uptake, extrusion or exchange across brain barriers have been investigated almost exclusively through the addition of extrinsic amino acids to biological systems. It seemed desirable, therefore, to study these phenomena also with a system in which intrinsic levels of amino acids were changed. Intrinsic levels of “free” amino acids in brain tissue are extremely stable under a variety of physiological conditions (Roberts and Simonsen, 1962). We know of only some acute vitamin deficiency studies and one nutritional study in which a few select amino acid levels were altered by a synthetic diet (Roberts, 1963). No normal physiological conditions have been described which have altered, dramatically, overall amino acid levels in the central nervous system. The object of this paper is to describe a vertebrate system in which intrinsic levels of amino acids of brain tissue are changed drastically in response to a normal physiological stress and to explore in a preliminary way the possible involvement of brain barriers in these changes. B. ENVIRONMENTAL EFFECTS ON AMINO ACID LEVELS IN EEL AND TOAD BRAINS

Aquatic species found in or near an euryhaline environment can be divided into two groups : osmoregulators and osmoconformers. Whereas the former group can maintain a sizable osmotic gradient between the external environment and the internal tissue environment, the latter group adapts to brackish or ocean water by establishing an iso-osmotic equilibrium between external salt concentrations and the body tissues. Many marine invertebrates are osmoconformers and it is well established that amino acids play a role in their internal osmotic regulation (Florkin and Schoffeniels, 1965). Similar changes in free amino acids which parallel alterations in environmental salinity have been described in muscle tissue and plasma of some amphibians (Gordon, 1965; Tercafs and Schoffeniels, 1962), and in teleosts such as the flounder and the three-spined stickleback (Lange and Fugelli, 1965). The regulation of intracellular amino acid concentrations appears to be the only mechanism employed by the sipunculid marine worm (Golfingia gouldii) to counteract a decrease in external salinity (Virkar, 1966). Changes in free amino acids in the central nervous system in response to osmotic stress have not been described previously. Studies were conducted with two species comparing GABA levels in brain tissues of individuals found in fresh water with those adapted to an euryhaline environment.The first study used a teleost, the silver eel (Anguilla anguilla) which during its life cycle migrates from ocean water to fresh RrJivenres p. 441444

432

C. F. B A X T E R

water and back to ocean water. Fresh water eels were collected in the Zuider Zee in the Netherlands and salt water eels in the Atlantic Ocean off the Netherlands coast. For the second study, the Western toad (Bufo boreas) was chosen because it is a dweller in mud caves near fresh and brackish water. These toads were adapted in the laboratory to a brackish environment. GABA was chosen as a representative amino acid for the central nervous system since, in vertebrates, it is found almost exclusively in this organ system. TABLE I1 Y - A M I N O B U T Y R I C A C I D I N B R A I N T I S S U E S O F S I L V E R E E L (AnEuilla anguilla) A N D W E S T E R N T O A D (Bufo boreas) I N F R E S H A N D O C E A N W A T E R E N V I R O N M E N T

Environment

Fresh water Ocean water

Change in GABA level

Silver Eel* GABA (pnoleslg)

Westerit Toad* * GABA (pnoleslg)

3.8 ( & 0.7)8 4.2 (rt 0.5)b 3.3 (f0.8)c 4.5 (f0.6)d

25.8 (3 0.9)

N.S.

-I-32 %

33.8 ( * 1.0)

* Eels caught in fresh water were predominantly females, those from ocean water were predominantly males. Each group value represents a n average of 6 t o 12 eel brains. Standard deviations are shown in parenthesis. Eels were designated as: (a) immature, (b) mature, (c) 39 to 41 cm long, and (d) larger than 50 cm in length. ** Western toads ranged in weight from 25 to 108 g, with an average body weight in each group of 44g. Each group consisted of 24 toads. Four brains were pooled for every GABA determination. Ocean water toads were adapted for 2 days to a n environment containing 20% ocean water and 2 days t o 40% ocean water. Toads were kept in aquaria inclined at a n angle so that f the floor area was covered by aqueous phase while the other half remained dry. Results in Table I1 show that in the eel, which is an osmoregulator, no statistically significant difference in levels of GABA in the brain of ocean water and fresh water specimens could be detected. This finding is in agreement with the observations by Boucher-Firly (1935), that in the silver eel the osmolality of body fluids increased by less than 15 % when these fish migrated from fresh to ocean water. Large female eels in fresh water or salt water showed no significant differences at all in the osmolality of their body fluids. Under similar conditions, only small changes in Naf concentration of eel serum have been reported by Sharrett, Jones and Bellamy (1964), whose investigations essentially confirm the earlier osmolality measurements. By contrast, the toad (Bufo boreas) is an osmoadapter and in the laboratory adjusts readily to an environment containing up to 50 % ocean water and 50 % fresh water. A significant increase in levels of GABA in brain tissue was found when these amphibians were placed for two days in a mixture consisting of 20 % ocean water and 80 % fresh water and two days in 40 % ocean water and 60 % fresh water. The results, shown in Table 11, were the same for males, females and hermaphrodites (for details see Baxter and Ortiz, 1966). Amino acid levels in the brains of toads from any single

433

INTRINSIC AMINO ACID LEVELS

shipment were quite uniform, as was the biochemical response to ocean water adaptation. There appeared to be, however, seasonal variations, both in normal levels of amino acids and i n the amount of change induced by the brackish water environment. Thus, levels of GABA in osmotically stressed toads were elevated in some experiments by only 30 while in others the increase was as high as 80 %. Seasonal variations in metabolism and metabolites of amphibians have been observed by other investigators (Mizell, 1965).

x,

T A B L E I11 MAJOR C O M P O S I T I O N A L C H A N G E S I N N I T R O G E N O U S C O N S T I T U E N T S O F T O A D B R A I N A S A RESULT O F A D A P T A T I O N T O BRACKISH WATER

(From Baxter and Ortiz, 1966) Atiiino Acid

Aspartic acid Alanine Glycine Glutamic acid y-Aniinobutyric acid Glutarnine $- asparagine Urea Ethanolamine

Fresh water 40u/, Ocean water (pnioleslg tissue, wet weight)

0.94 0.37 0.87 5.5

2.7 3.5 15.8 1.7

2.4 0.87 1.6 9.1 4.0 4.8

31.8 0.8

Change ( %I

4-149 +135 84 65 48 37 139 - 55

+ + + + +

Alcoholic tissue extracts were prepared and processed as previously described (Baxter, 1961). Ninhydrin positive compounds were measured using a Technicon amino acid analyzer system connected to a Gilford Model 2000 spectrophotometer and recorder. Results obtained by column separation were verified for glutarnic acid, glycine, y-aminobutyric acid and urea wing established enzymatic assay techniques (Graham and Aprison, 1966; Aprison and Werman, 1965; Baxter, 1961; Bernt and Bergrneyer, 1963).

In Table I l l are shown the changes of some amino acids found in brain tissue which were observed when the toads were adapted to 40 % ocean water. Although aspartic acid showed the greatest increase on a percentage basis, the change in glutamic acid level was quantitatively the most significant. On a molar basis, increases in aspartic acid and GABA were the same. The increase in urea concentration by 22 pmoles per gram of brain tissue (wet weight) was the largest change found for any nitrogenous compound in brackish water toads. This change was reflected also in muscle and plasma and doubtlessly helped to maintain osmotic equilibrium. The very high levels of urea in the tissues of Bufo boreas are noteworthy. In a fresh water environment, urea levels in the brain of this species were higher than those reported for any other vertebrate known to us and three times higher than the levels in frog brain as measured by Buniatian and Davitian (1966). These results fit the correlation which Schoffeniels* has made between the ability of animals to adapt to an euryhaline environment and the level of urea in their tissues.

*

Schofleniels, E., personal communication.

Rcfcrmws p. 4 4 1-444

433

C. F. BAXTER

It is apparent from the results in Table 111 that osmotic stress primarily increased the concentration of those amino acids in toad brain which are closely related metabolically to the tricarboxylic acid cycle. Only minor elevations in the concentration of threonine, valine, tyrosine, phosphoethanolamine and phosphoserine were observed. No significant changes in levels of creatine, taurine and ammonia were detected. Of the other amino acids found in brain extracts, leucine increased and lysine decreased in concentration, but the molar changes were extremely small (see Table VII for leucine). It is probable that minor elevations in amino acid concentrations, expressed as a function of wet tissue weight, reflect only the dehydration of brain tissue in osmotically stressed toads. It was found that under our experimental conditions the water content of toad brain, usually about 85 % of the wet weight, decreased by a maximum of 1.8 % corresponding to a maximum increase in dry weight of 12 % over control values. Therefore it is clear that the major changes shown in Table 111 are not the result of dehydration. C. MECHANISMS O F A M I N O A C I D A C C U M U L A T I O N I N T O A D B R A I N

There is some evidence to suggest that the elevated levels of urea and amino acids in muscle tissue of euryhaline adapted amphibians can be accounted for, in part, by an enhanced degradation of larger molecules (Tercafs and Schoffeniels, 1962). A decrease in the rate at which amino acids are metabolized has also been postulated (Florkin et al., 1964). However, the detailed mechanisms by which intracellular amino acid levels are altered in response to a brackish environment are not clearly understood. Studies by Schoffeniels and Gilles (1963), on glutamic dehydrogenase in aquatic invertebrates suggest activation of this enzyme system by cationic constituents whereas Chaplin et al. (1965) favor a mechanism involving anions. Activation of other

Fig. I . Toad brain amino acids. All salt solutions and sucrose were prepared to represent an osmotic concentration which was equivalent to a brackish water mixture containing 40% ocean water and 60% fresh water. These solutions were 410 10 mOs. Toads were adapted to salt solutions and sucrose for 2 days at f strength and 2 days at full strength in inclined aquaria as described in Legend for Table 11. Control animals were maintained in salt free water.

435

INTRINSIC AMINO A C I D LEVELS

enzyme systems related to amino acid and energy metabolism by ionic constituents is well documented and could play a role in modifying amino acid levels in the central nervous sytem of Bilfo boreas. When toads were adapted to environments containing specific salt solutions, equivalent in osmolality to 40 % ocean water, changes in amino acids of brain tissue were observed. Results for glutamic acid and GABA shown in Fig. 1 confirm preliminary data (Baxter, 1966). Solutions of sodiumsalts proved to be the most effective environment for elevating glutamic acid and GABA levels in brain. They exceeded in effectiveness ocean water of equivalent osmolality without causing any additional water loss from the brain tissue. Potassium chloride had no effect on levels of amino acids in brain tissue, despite the fact that it was the most toxic environment used. Presumably these effe,nts of sucrose, brackish water and sodium salt solutions are mediated through ionic changes in the internal environment of the toad. The plasma i n fresh water (control) toads ranged from 235 to 265 milliosmolal, but increased by 60 to 70 percent when these amphibians were adapted to brackish water or equivalent sodium salt solutions (Table V). Invariably the elevated levels of amino acids in brain tissue of these toads were accompanied by raised levels of Na+ both in brain tissues and blood plasma. Preliminary data indicate, that the Na+/K+ ratio in brain tissue was also increased (Table IV). T A B L E 1V SODIUM A N D POTASSIUM ION C O N C E N T R A T I O N IN T O A D B R A I N E X T R A C T S -.

Brairi coriiposiriori ~~

B i virorinicnf

Water ( 1/,)

.

Naf

Kf

Nal K

(rwquiv.lkg)

(niequiv./kg)

ratio

85 98 114 107 85

0.69 0.81 0.86 0.67 0.82

Fresh Water

83.6

40 :‘c Ocean Waler

82.0

59 80

NaCl KCI Sucrose

81.6 83.1 81.9

72 70

98

~~~

Sodium and potassium were determined by flame photometry. For details of salt concentrations and environmental conditions, see Legends of Table I 1 and Fig. I .

Toads in the potassium chloride and sucrose solutions lost weight rapidly because of dehydration. However, dehydration of brain tissue in these animals was no greater than that observed in toads living in a brackish or salt water environment. All of these results indicate a well-developed mechanism i n toad brain by which solute concentrations are regulated so as to minimize hydration changes in the cerebral tissues. The effect of a saline environment upon plasma osmolality, Na+ plus K+ concentration and the molarity of glutamic acid and urea, is shown in Table V. Our results for changes in cationic constituents of plasma in brackish water toads are in overall agreement with those reported by Gordon (1965). Heferencrs p, 441-444

436

C. F. BAXTER

TABLE V EFFECT O F E X T E R N A L S A L I N E E N V I R O N M E N T U P O N SOME T O A D P L A S M C O N S T I T U E N T S

Eiivirotinient

Fresh water 40;( Ocean water NaCl Solution (420 mOs)

Na+ plirs K+ (iiieyuiv.lliter) *

Clutaiiiic Acid

(niOs/kg)

256 422

127 173

0.04 0.20

19.8 42.1

435

184

0.21

48.9

Osinolality

Urea

(liiiioles/iiil plasma)

* The K t concentration of plasma was extremely low and the increase observed was due primarily to Na+. In order to eliminate the addition of Na+ with the anticoagulant, ammonium citrate or citric acid was added to those aliquots of blood in whichNa+and K ' xere determined by flame photometry. Plasma for amino acid and urea determinations was obtained from heparinized blood. All results are based upon pooled samples. Measurements were made on blood obtained by heart puncture as described in text. Red cells were removed by centrifugal precipitation in cold, within 30 min after collection of sample. Levels of urea and amino acids were elevated not only in brain tissue, but also in the plasma of toads in a sodium salt environment. Since amino acid levels in toad plasma are normally very low, the proportionally large changes observed after osmotic stress represent only an insignificant fraction of the total osmolal effect in the plasma. In the case of urea, the degree of elevation is similar in both brain and plasma and represents, on a molar basis, the largest change of all of the nitrogenous substances. Although many of the enzymes of the urea cycle have been found in brain tissues (see Kemp and Woodbury, 1965 for review), one of the essential ones, carbamyl phosphate synthetase, appears to be present only at rather low levels (Jones et al., 1955). In the liver, on the other hand, carbamyl phosphate synthetase is quite plentiful. Recent experiments have shown that the activity of this enzyme in the liver of the dogfish can be considerably increased by placing this species into dilute ocean water (Watts and Watts, 1966). Since the blood-brain barrier does not exclude the net entry of urea from the blood (Kleeman et al., 1962), all of the above evidence suggests that a major portion of the urea found in brain tissue may have originated in other organs and was carried into the brain via the plasma. D . B L O O D - B R A I N B A R R I E R A N D I N T R I N S I C A M I N O A C I D LEVELS

The foregoing results have shown that the levels of nitrogenous solutes in brain and plasma of the toad (Bufo boreas) are elevated when this species is placed in an aqueous environment containing a sodium salt concentration which is equivalent in osmolality to that of 40 % ocean water. Changes in Na+ concentration and gradients between blood and brain also have been observed. The origin of the amino acids which accumulate in the brain tissue of osmotically stressed toads is a matter for conjecture. Since there is restricted net transport of most amino acids from blood into the adult brain of vertebrates, both mammalian and non mammalian (Wiechert and Knaape, 1963; Lajtha et al., 1963; Guroff and Udenfriend,

INTRINSIC AMINO A C I D L E V E L S

43 7

1962; Kuttner et al., 1961 ; Neame, 1961 ; Dingman and Sporn, 1959; Lajtha et a]., 1957; Schwerin et a / . , 1950), the most likely sources for many of the amino acids in brain are the metabolic cycles within the brain tissue itself. Vidaver (1964a, 1964b) has shown that the net direction of a mediated transport for glycine in the pigeon erythrocyte depends upon the direction of the Na+ gradient between the exterior and interior of the cell. Experiments by Christensen's group and by others (Wheeler e t a / . , 1965; Finerman and Rosenberg, 1966; Fox et a / . , 1964) confirm that the involvement of Na+ in transport mechanisms for amino acids is a widespread phenomenon. In a situation of osmotic stress with altered Na+ gradients, it might be possible, therefore, that some amino acids would transfer more readily between plasma and brain tissue. Under the same conditions it is equally possible that the blood-brain barrier is altered as a function of changed intrinsic amino acid levels. The experimental results recorded below represent a preliminary effort to test the feasibility of the latter two possibilities. Evidence for a change in the blood-brain barrier was sought by measuring the uptake of 1Clabeled amino acids from plasma into the brain of toads kept in a fresh water environment and toads adapted to an aqueous environment containing a salt concentration of 420 milliosmolal NaCI. Toads were adaoted to their environment as previously described (Baxter and Ortiz, 1966).

Fig. 2. The heart was exposed by making a longitudinal incision through the skin and pectoral girdle with a pair of scissors. The incision was made along the mid-ventral line extending approximately from 1.5 cm below to 1.5 crn ahove the xiphisternurn. A segment of the xiphisternum was removed and a small hole cut into the pcricardial sack. By applying slight pressure to the abdomen of the toad the heart was pushed through the opening. Isotope injections were made with a size k'27 needleand blood was withdrawn using a size ,# 18 needle and a heparinized syringe. References p . 4 4 1 4 4 4

438

C.

F. B A X T E R

Intravenous injections or blood sampling proved to be a problem. However, by externalizing the toad's heart (Fig. 2) it was possible to make all injections into, and to withdraw blood from, the ventricles of this organ. Animals with externalized hearts appeared normal, hopped around, ate and survived for many days without loss of weight. Death occurred only when the exposed pericardium became infected. In preliminary experiments, plasma space in brain was measured in toads both from fresh water and saline water environments. Two microcuries of 125I-labeIed albumin were injected and after 5 min blood was collected and the toads sacrificed. Brains were excised rapidly, blotted lightly and weighed. Both 0.1 ml plasma aliquots and brain were counted directly in a well counter. [125I]albumin is retained for considerable periods of time in the circulation without penetrating appreciably into tissue spaces (Von Lombard0 and Tamburino, 1963; Love, O'Meallie, Lemmon and Burch, 1962). Assuming that five minutes is sufficient time to permit complete mixing of injected albumin with the total plasma pool of toads at 21"C, the ratio of specific TABLE V l C H A N G E S I N [L"I]ALBUMIN

Brain

Environment

Fresh water control NaCl Solution (410 mOs)

(crslmitilg)

SPACE OF TOAD BRAIN

Pla.srna

(ct.s/niiti/ni/)

x 10-1

x 10-1

445 913

46 650 51 500

Plarrna space (ndlg) 0.0095 0.01 77

For details of environmental conditions, see Legends of Table I1 and Figure I

activity in brain tissue versus plasma is proportional to the plasma space in the brain tissue. Results are shown in Table VI. Plasma space, expressed as ml plasma/g of brain tissue, is calculated from the ratio: counts/min/g of brain tissue counts/min/ml blood plasma The plasma space of brain almost doubled in toads adapted to a saline environment. Corrections for differences in plasma space were made in TablesVII and VIII. I t can be seen in Table VII that the correction factors did not significantly influence the results in which [I-"W]I-leucine uptake by the toad brain was measured in vivo. The distribution of [ I-l4C]leucine between plasma and brain tissue was determined 30 and 60 minutes after the isotope was injected into the blood stream. The results show that the brain/plasma ratio of isotope concentration in toads adapted to the NaCl environment was twice as great as that found in controls. Expressed as a function of plasma concentration, the level of [ I-14C]leucineretained in brain tissue was doubled by osmotic stress. At the same time, however, the total leucine pool was more than doubled. It seems probable that both of these phenomena involve intracellular as well as extracellular spaces in the brain tissue.

+ z

TABLE VII

%

c1

2 P

UPTAKE OF

[I-“c]/-LEUCINE

9

2

9

2

Group

1

2 3 4

Owironnient

H2O Control NaCl Solution HzO Control NaCl Solution

Time after 1-1~CCJ’leuririe itijectiori ( m i t i ) .r

30 30 60 60

BY TOAD BRAIN

Brain Leircitie

(.rcl,,oles/gl 0.067 0.137 0.067 0.137

Plasma

Toral cis Plast,ia space Net crs (cts/niiri/g wet 10) ,: 10-2 734 1301 532 1323

30 45 18

40

704 1256 514 1283

10-2

Ruiio brain/ plasma

3171 2525 1877 2236

0.22 0.50 0.27 0.57

(ct”t’lit’/‘’il) *\

NaCl adapted toads were kept in an aqueous environment containing salt at the level of 400 mOs/liter. Each group represents four or more toads. 0.75 pc of [l-14C]leucine,together with 0.015 pnoles of I-leucine carrier, was injected in a 0.1 ml neutral soli*tiondirectly into the left ventriclc of control and saline adapted toads. The dose injected into each toad was the same, but as the weight of individual toads differed the counts/min/ml of plasma also differed considerably. Ratios of counts brainiplasma were, however, extremely consistent for each group. Results above are the average for each group.

P

w

\o

440

C. F. B A X T E R

Lajtha and Mela (1961) have shown that exchange rates of isotopically labeled lysine between plasma and brain of the rat are increased if extrinsic [12C]lysine is injected subarachnoidally into cerebral tissues. Thus, the results in Table VII could be interpreted as showing that an increase in the size of the intrinsic leucine pool in toad brain affected ["Tlleucine uptake and exchange from the plasma in a manner analogous to the one described by Lajtha and Mela (1961) with extrinsic lysine in the rat. As long as the source of elevated intrinsic leucine in toad brain remains unknown, it is possible only to conclude that under conditions of osmotic stress a new equilibrium ratio between the pools of leucine in plasma and brain is obtained. In the case of a-aminoisobutyric acid (Table VIII) no known pool of this amino acid exists in brain or plasma. It has been used extensively as a model amino acid to study uptake phenomena in various systems (Christensen and Riggs, 1956; Akedo and Christensen, 1962). When [ I-"T]a-aminoisobutyric acid was injected into the circulation of toads at room temperature (21"C), the rate of uptake by brain tissue was extremely slow. As in the case of [I-"C]leucine, the brains of osmotically stressed toads accumulated [ 1 -14C]a-aminoisobutyric acid more rapidly than the brains of controls. TABLE VIII U P T A K E O F [I-'4C]U-AMtNOtSOBUTYRtC A C I D B Y T O A D B R A I N

Group

Environment

Time after injection (min)

Brain (cts/nrin/g*)

Plasnia (cts/min/tnl)

Ratio brain/ p,asr,la

965

21 130

0.046

1 2

HzO control NaCI** solution

60 60

1563

12 900

0.121

3 4

HzO control NaCI** solution

90

1058

20 880

0.05I

90

1479

10 760

0.139

* corrected for "Plasma Space" in brain. ** 400 mOs/l NaCl for 2 days.

Approximately 0.5 pc and 3.1 pmoles of carrier u-aminoisobutyric acid were injected as a dose of0.2 rnl directly into the heart of each control and saline adapted toad. Each group represents4or more oads. Results above represent averages.

The brain/plasma ratio of isotope concentration was almost three times greater. These data, too, must be interpreted with caution. The transport of a-aminoisobutyric acid in and out of the brain tissues appears to be mediated through carrier mechanisms which are used also for the transport of naturally occurring, neutral amino acids such as alanine, glycine and serine. Competition for the carrier mechanisms may affect both rates of entry and exit in brain slices in vitro (Levi, Blasberg and Lajtha, 1966; Blasberg and Lajtha, 1965; Blasberg and Lajtha, 1966). As can be seen in Table 111, the pool of both alanine and glycine in brain approximately doubled when toads were adapted to a saline environment. It is possible, therefore, that the effect recorded

INTRINSIC A M I N O A C I D LEVELS

44 1

in Table VlII does not reflect an alteration in the blood-brain barrier but is once again an expression of the increased amino acid pool in the brains of osmotically stressed toads. SUMMARY

The influence of intrinsic amino acid levels in brain tissue upon amino acid movements from blood to brain has been studied. 1 . Isotopically labeled amino acids, administered to rats intracerebrally, had ready access to only a portion of the amino acid pools of brain tissues. Thus, there is a kinetic difference in brain tissues, between intrinsic amino acids and those applied extrinsically. 2 . The intrinsic levels of many amino acids and of urea in brain tissue of the toad (Bufo boreas) were elevated dramatically when these animals were exposed to an environment containing 40 percent ocean water or osmotically equivalent salt solutions containing Naf. Levels of aspartic acid, alanine and urea were raised by more than 100 percent and those ofglutamic acid, glycine and GABA by 49 percent or more. Osmotically equivalent solutions of sucrose had a lesser effect and of K+ had no effect. 3. All elevations in amino acid levels of brain were correlated with increased levels of Na+ in blood plasma and an elevated Na+/K+ ratio in brain tissue extracts. 4. There is limited evidence to suggest that the unusually high levels of urea in toad brain originate, in part, outside of the brain. 5 . The relationship of blood amino acids to brain aniino acids was altered as toads became adapted to a saline environment. This was shown by an increased uptake by the brain of isotopically labeled amino acids from the blood. These findings are not necessarily indications of a changed blood-brain barrier, but may be the result of the enlarged intrinsic amino acid pools in brain. ACKNOWLEDGEMENTS

This research was supported by Grant #NB-03743 from the National Institutes of Health, Institute for Neurological Diseases and Blindness, U.S. Public Health Service. I am greatly indebted to Dr. C. L. Deelder and his colleagues at the Rijksinstituut voor Visserij Onderzoek in IJmuiden, The Netherlands, who made this study feasible by collecting eels from fresh and ocean water and sending the weighed brains to me, pickled in alcohol for analysis in California.

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442

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DISCUSSION R. KATZMAN: I just want to ask Dr. Baxter if he can give the average osmolarity of the total blood in the normal state and in the sodium-chloride stressed state.

C. F. BAXTER:In the normal state it is around 230 milli-osmols; in the stressed state it is slightly above that of the environment, that is about 420 milli-osmols.

I should like to make two comments. First of all I don’t agree that a substrate may have a P. MANDEL: regulatory influence on the metabolism of a cell, unless its amount is limiting. The most important fact we know in molecular biology is that the syntheses are dominated by some form of RNAsynthesis which depends on DNA or some inducers, repressors or depressors, but not on the substrate. The main experiment performed by Kornberg was that he got a primer-like D N A with several different pools of free nucleotides. It is the same for RNA, so 1 think that first of all we should admit that the substrate has not a very important role in the metabolism of a cell rrnless there is a limiting factor. The second comment is that you have a tremendous increase of glutamic acid over the glutamine. D o you have some data concerning the turn-over of the proteins in relation to the high pool ot glutamic acid or glutamine? C. F. BAXTER: To discuss the first question: I think I would have to disagree with you that substrate levels are not important in regulating the metabolism of the cell. I don’t think very many enzymologists hold the view that we have complete saturation of most enzymes with substrate in a normal metabolic system. I don’t know either, for that matter, if in any one transport system the carrier is fully saturated at any one time. If you are talking about adaptation and adaptive enzyme synthesis, this is a different matter altogether. But in the case of just normal metabolism, I believe substrate levels often are rate limiting to an enzyme system. Your second question concerned the protein turn-over. I do not have any data concerning this. You may have noticed the fact that the urea content in the saline adapted toad brain was extremely high, and this may indicate that there is quite a bit of protein break-down. There are also some data for invertebrates which ‘‘seem” to indicate that during osmotic stress there may be an increased break-down of protein contributing to the amino acids in muscle tissue.

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445

G . Ltvi: I think that the higher exchange rates of labeled amino acids that you find has an explanation in the fact that thc cerebral concentration of these amino acids in the brain is increased. Higher exchange rates of amino acids have also been shown by Dr. Lajtha when the amino acid level in the brain is artificially increased. I agree with you for leucine, but for ci-amino iso-butyric acid there is no pool. C. F. BAXTER: G . LEVI:But there is a pool of amino acids of similar structure and charge (such as alanine), with which (1-amino isobutyric acid could easily exchange.

K. A. C. ELLIOTT: Could Dr. Baxter speculate on the teleology of these changes, or in more orthodox terms: what is the survival value of thesechanges? C. F. BAXTER: Well, 1 think it is fairly obvious that if these toads could not adapt to their normal environment they would not survive for very long. K. A . C. ELLIOTT: But the nature of the changes, how d o they contribute to this survival? By changing the osniolarity of the tissue; at least that is all we know of to date. C. F. BAXTER:

K. A. C. ELLIOTT: So it is sufficient to make an appreciably large contribution to the new osmolarity? C. F. BAXTER:Yes, I think the nitrogenous compounds which we have measured so far make a ontribution of about 37 milli-osmols. K. A. C. ELLIOTT: And you have got to make up about 150? C. F. BAXTER: This is correct. I would agree with you that it is only a portion of the total, but 37 milliosnioles is a noteworthy contribution - about 20 plus percent of the total requirement. R. CUTLER: In your last slide you showed a tissue plasma ratio about twice as high for the sea water animals. But the plasma counts were about half those of the control animals. 1 wondered if therc was a difference in dose, or how you could account for the difference.

C. F. BAXTER: We injected the same dose into both groups of animals. You were asking why plasma counts are so much lower in the sodium chloride stressed toads? Presumably in these animals, the labelcd (1-aniinoisobutyric acid has also penetrated more rapidly into tissues other than brain, and therefore the plasma counts would be lower than those in fresh water toad plasma after a very short period of time. R. CUTLER: Wouldn't this be sufficient to account for your higher ratio in the brain? C. F. BAXTER: Yes, it probably would be. However, the brain is in contact directly or indirectly with the plasma, and not with other tissues. Therefore, the comparison we have to make is hetween plasma level and brain level. So I think thc significance of the ratio should still apply. D. M. WOODBURY: Is there any evidence that the salt stress increased in some way the number of polypeptide or smaller molecules in the cell and thereby increased osmolality by increasing the total number of particles in the cell? C. F. BAXTER: I have no such evidence. D. M. WOODBURY: And one other question. Were there any pH changes produced which could increase the total charge on the protein molecules and thereby allow an increased number of osmotic particles to exist in the cells? C. F. BAXTER: There is a change in plasma pH. Normal heparinized toad plasma has a pH of abou 7.9 which is about 0.3 pH units higher than the plasma of toads in 40% ocean water.

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P. G. SCHOLEFIELD: Dr. Baxter, I just wonder how long you have to incubate your toads in saline to get this effect? I remember that toads produce a steroid analogue, and 1 thought about ouabain being a steroid analogue. In this respect I wondered how much you are altering the control of the sodium movement, e.g., through the change in production of steroids, and this led me to wonder just how the toads produced these effects. C. F. BAXTER:We can get this change without any difficulty by placing the toads into 40”/, ocean water for one day. However, in practice we have adapted them more gradually to their saline environment. We have not done a systematic study with steroids.

GENERAL DISCUSSION

D. B. TOWER:Many substances have been discussed here in the last few days, for most of which someone has suggested a transport system in one way or the other. However, inulin seems to stand alone in being excluded by many membrane systems. 1 just wonder how we are going to consider inulin in the context of this meeting. D. M. WOODBURY: I think I can explain the distribution of inulin, at least its movement out of the CSF, and its low levels in the CSF and brain, merely by flow in the CSF-sink, coupled with slow entrance across the endothelial barrier between the brain capillaries and the interstitial space of the brain. lnulin follows the same sort of behavior as radioiodinated serum albumin or any other large molecule. As Dr. Rall and others including Dr. Reed and 1 have shown, all such non transported molecules leave the CSF at the same rate by bulk flow. D. B. TOWER:Yes, but why is it excluded from corning in?

D. M. WOODBURY: It isn’t excluded, it just enters more slowly than most substances. The rate of entrance of such water soluble substances across the endothelial barrier appears to be merely a matter of their size. Thus, sucrose enters more rapidly than inulin, but at a lower rate than the smaller molecule. mannitol. D. B. TOWER: I am just asking this for the sake of clarification. D. M. WOODBURY: I don’t think there is any question that there is a barrier between the endotheliuni and the interstitial space of the brain for inulin, and related substances since their rate of entrance is extremely slow.

A. LAJTHA: We have some information on inulin flux in brain slices, and may 1 mention that brain slices are, in some respects, an altered system as compared to the living brain. If the influx of inulin is measured, there is a definite break. One of the possibilities, as mentioned before, would be that inulin penetrates slowly into damaged ctlls.

D. M. WOODBURY: May I discuss that? The only experience I have had in this respect is with some studies on the thyroid gland. We were interested in measuring the extracellular space of the thyroid to see if the inulin entered just the stromal space or whether it also penetrated into the luminal space. Dr. Chow and 1, therefore, performed some radioautographic studies with tritiated inulin. The inulin was found only in the stromal (interstitial) space outside the follicle. We also measurtd the inulin space of the thyroid by detrrmination of the uptake curve and compared the spaces at various times with radioautographs determined at one and 24 hours after administration of inulin. During the initial phase (1 hour) when the inulin was equilibrated, it was only in the stromal (interstitial) space by radioautography and nowhere else in the thyroid. After 24 hours the inulin space of the thyroid had increased to quite an extent; at this time the radioautographs showed that it was still in thc interstitial space and had not penetrated the cells or into the lumen, but that it was now beginning t o accumulate in clumps in the interstitial space, probably due to phagocytic activity by macrophages. The increase in these “clumps” corresponded to the increase in the space. Thus, during the early uptake phase, inulin measures the extracellular space of the thyroid, but after that, due to the accumulation in macrophages it no longer measures the volume of this compartment.

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A number of years agoCotlove also showed that inulin penetration into muscle involved two phases: a rapid phase and a very slow phase that took 24 hours for equilibration. He showed that the slow phase involved penetration into the connective tissue of the muscle. This also is probably due to accumulation in macrophages. Thus, the inulin space, after the initial rapid phase of equilibration, is not a valid measure of extracellular space in skeletal muscle. It is evident, therefore, that in both these experiments inulin remains in the extracellular space although secondary accumulation occurs. Our data for the inulin space of the brain in young rats also strongly suggest an extracellular distribution only. A. LAJTHA: Was this measured after several days?

No, only up to 24 hours after injection. D. M.WOODBURY: K . A. C. ELLIOTT: Is it in order to go back to Dr. Ford’s paper? If you give an animal radioactive glutamate, a lot of it will be rapidly metabolized and the radioactivity will be distributed all over the place. However, some of the labeled material will presumably be incorporated into protein. If you give the animal radioactive lysine which is not rapidly metabolized, some goes into protein. Now I am wondering: will the protein which incorporates the lysine be the same in kind and amount as the protein that is formed from the glutamate. In fact would it be possible to do the same experiment with glutamate as with lysine, by eliminating the metabolites and studying only the substances incorporated into protein? If the protein does get labeled in this way, this is partial evidence of turnover, then there should be a disappearance of the label from these neurons after a certain length of time. I wonder how soon this disappearance of the label occurs. 1 seem to be asking two questions: Does glutamate give you a similar degree or rate of labeling of protein as lysine, even though glutamate is being metabolized in another way? Does labeling of the protein in either way demonstrate that it is being turned over?

D. H. FORD:I think Dr. Lajtha has more information actually on this than I do. As 1 recall he has inforniation which suggests that there is a family of proteins present in differing amounts which would lead one to think that the turn-over rates or half-lives of any amino acid that could go into different proteins could have difference half-lives. He has one experiment in particular with young animals which 1 think is pertinent here. A. LAJTHA:May I mention a pertinent point here form the results of experiments that we recently published, where we fed mice with food containing labeled lysine with constant specific activity. The labeled diet was fed before the animals became pregnant, and the same labeled diet was continned during pregnancy and during subsequent growth until adult age. With this procedure we were sure that every protein molecule in the brain was labeled, since throughout the development the animal had only labeled lysine with constant specific activity available. When the animals were adult, we withdrew the labeled food and substituted unlabeled food. Within five months most of the label disappeared, showing that at least 95 per cent of the cerebral protein in adults is in a dynamic state. Then we analyzed lysine itself, and this was, of course, metabolized. In the long time experiment we found that about half of the label in brain proteins was in compounds other than lysine; one third was in glutamic acid. The incorporation of glutamic acid as compared to lysine was fairly similar, although we did not measure this very carefully. May 1 also mention that the average rate of turnover, calculated from this experiment, was rather similar to what we found when we calculated it from initial rates of incorporation. In mice, the average half-life was about 14 days. I don’t think there is very good evidence at the present time showing that there is an exchange of a single amino acid in cerebral proteins without a complete turnover of the whole protein molecule. Not many people measured this, and so, although we don’t have too much evidence against it, we certainly have no evidence that is convincing for it.

C. F. BAXTER:I wonder whether it would be in order to go back to Dr. Tower’s question to Dr. Quadbeck about the effect ofvitamin Bti and its analogues on the blood-brain barrier, or penetration rate: The similarity in structure between pyrithioxine and vitamin Bti has already been pointed out. However, Dr. Quadbeck stated that vitamin B6 had no effect upon the blood-brain barrier criteria which he has measured.

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Preliminary observations in our laboratory might lead to an opposite conclusion and we would suggest that, under certain conditions, vitamin Bemight play a role in blood-brain barrier mechanisms. Some years ago we were engaged in experiments with rats, in which thiosemicarbazide (TSC) was injected intraperitoneally and convulsions prevented, by the administration of pyridoxine o r pyridoxal some time later (Baxter and Roberts, 1962). Measurements were made of glutamic acid decarboxylase (GAD), GABA lcvels and y-aminobutyric-a-keto-glutaric transaminase (GABA-T) in brain tissues of the treated rats. G A D and yABA-T activities were measured in virro without the addition of pyridoxal phosphate. While TSC administered in vivo decreased lcvels of GABA in brain and inhibited GAD, it had no apparent effect on GABA-T. When TSC treated animals were injected with pyridoxal, the levels of GABA in the brain continued to decline while G A D activity appeared reactivated and GABA-T remained unchanged. There were various ways to interpret these results. It had been reported in the literature (Gammon er al., 1960) that the blood-brain barrier to GABA was altered in rats injected with methoxypyridoxine. It seemed possible that in our experiment a similar alteration in the bloodbrain barrier had occurred as a result of TSC plus pyridoxal treatment. The continued decrease in cerebral levels of GABA at a time when GABA synthesis was accelerated, would then be explainLd by a leakage of GABA out of brain tissues into the rest of the body. This hypothesis was tested in a preliminary way using 3 groups of 4 adult male Swiss mice. At zero time of the experiment, groups 2 and 3 received TSC intraperitoneally at a level of 12 nig/kg body weight. At I5 minutes, group 3 micl received an anticonvulsant dose of pyridoxal HCI (40 mg/kg). At 30 minutes, 101of2 [I4C]-GABA(containing about 2pC) was injected into the left brain ventricle of each mouse. Fifteen minutes after the isotope injection (and before the TSC treattd animals could convulsr) all mice were quick-frozen in liquid nitrogen. Whole body sections were prepared and radioautographs made using the techniques pioneered by Ullberg and his colleagues (Ullberg, 1954).

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Although the number of animals tested precludes a conclusion and quantitative data are yet to be obtained, these preliminary experiments suggest that the administration of pyridoxal to TSC treated mice increaspd the outflow of isotopically labeled material from the ventricle or brain tissue into other tissues of the body. This increased flux then might reflect a change in permeability of the brainblood barrier i n response to vitamin BS administration. BAXTER, C. F. A N D ROBERTS, E. (1962)Effect of 4-MethoxyniethylpyridoxineandCarbonyl-Trapping Agents on Amino Acid Content of Mammalian Brain and Other Tissues. In Amino Acid Pools, J. T. Holden (Ed.), pp. 499-508. Elsevier, Amsterdam, London and New York. GAMMON, G. D., GUMPUIT, P. rt a/. (1960)The Effect of Convulsant Doses of Analeptic Agents upon The Concentration of Amino Acids in Brain Tissue. In Irihibifiotrin the Nervous System and Gamma Arniiiohrityrir Acid, E. Roberts e f a/.(Ed.), pp. 328-330, Pergamon Press. U L L H t w , S. (1959)Autoradiographic Studies on the Distribution of Labeled Drugs in the Body. Progress if1 Nuclear Energy - Series 6, Vol. 22 - Biological Sciences, pp. 29-35, Pergamon Press, London, Oxford, New York and Paris. G. QUADBECK: I don't think that this has anything to d o with pyrithioxine, because pyrithioxine has no vitamin Be like-effect. It is also ineffective in the intoxication with thiosemicarbazide compound. P. MANDLL: I should like to come back to the problem of substrate metabolism. We agree that the level of substrate has an influence on the level of hydrolytic enzymes; for example, if we inject more arginine, more argenase is found in the liver. I agree that there is a feed-back control for the synthesis of amino acids. But when we deal with the uptake of amino acids, we are concerned mainly with protein synthesis. I don't believe that there is any evidence that change in the conccntration of amino acids (unless an amino acid is limiting), has any influence on the protein synthesis. Moreover, if we admit that the pool of amino acids could have any influence on the protein synthesis in a cell and that the genetic control of protein synthesis is not constant, we should know what to expect; because as far as the distribution of amino acids in the system is concerned, the kind of synthesis which depends on the RNA which is present in the cell or on the DNA is always the same. Therefore, the problem of the substrate level of amino acids in view of protein synthesis cannot play a role unless it is a limiting factor. A. LAJTHA: The effect of changes in amino acid pool on cerebral metabolism is very important, but unfortunately only a few experiments have investigated this point. Dr. Sidney Roberts did some vcry interesting experiments in which he found that omitting phenylalanine from the diet changes cerebral amino acid pools. When he measured the transport and the metabolism of free leucine and the incorporation of leucine into proteins in the brain, he found that all threc of these processes were significantly altered, showing that a change in the pool may affect both amino acid and protein metabolism.

P. MANDEL: That is entirely different. He removed from the medium an amino acid which is limiting, because it is an essential amino acid, without which protein synthesis is not possible. Under these conditions you have protein break-down. A. LAJTHA: No. Phenylalanine did not disappear from the brain; however, the altered diet changed, among other things, the level of leucine in the brain. I don't know what all the changes were; perhaps they were manifold. One of the possibilities would be that changes in the ribosomal structure caused by alterations in the pool are then reflected in the synthetic activity of the ribosomes. P. MANDEL: But if you remove phenylalanine from the diet the synthesis of proteins is disturbed. A. LAJTHA:No, the changes were in the structure of the ribosomes, because of the diet. But somehow the ribosomes reacted to the pool size and their synthetic avidity was altered. I can't believe that the structure of the ribosomes change when you remove an amino acid. P. MANDEL: The evidence which we have was that even removing whole proteins from the diet did not alter the pattern of proteins in the ribosomes. So, what occurs is that there are fewer ribosomes and there is a break-down of proteins in liver and muscle providing amino acids to produce the proteins. The kind

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of proteins YOU produce depends on the information which comes from DNA or messenger-RNA. You cannot do anything to change it. If vou remove an essential amino acid from the diet, what you produce is a decrease of the synthesis of messenger-RNA by a phenomenon which would take too long to explain. Thus, in a deficient diet there would be fewer polysomes which would decrease the synthesis of protein and the incorporation of [14C]lysine. The synthesis of protein depends only on the genetic information and not on the available substrate.

C. F. BAXTER: There is plenty of evidence in the literature that ions can stimulate the rate of an existing enzyme. And in the particular case of the osmotically stressed toads we have ionic changes, so we may have changes in rate of enzyme activity, because of the ion stimulation. Would you accept that? P. MANDEL: Yes, but the synthesis of protein does not depend on the substrate concentration. It depends on whether you induce a new enzyme, e.g. Under these conditions there is a synthesis of an enzyme from the pool of amino acids. However, I think it would be difficult to obtain any evidence that if you stimulate the synthesis of an enzyme, that it depends (again, it is the same problem) on the substrate level, unless there is an amino acid which is limiting. What we can say is that glutaniic acid in brain is not a limiting factor. C. F. BAXTER:But I don’t think we have to stipulate that there is an increase in enzyme synthesis under the conditions that I showed. In an increased level of amino acids in the brains of those animals, 1 don’t think that is necessary.

D. P. RALL:May I change the subject? It occurs to me along the same lines of your paper, Dr. Baxter, that there is another thing that you can d o to these toads. You can functionally dehydrate them the way seawater does, by barring their access to water, either fresh water or sea water. If you have done this, what happened? C. F. BAXTER: We have done this, and it has no appreciable effect. D. P. RALL:Do toads drink sea water? C. F. BAXTER: In the case of Bufo boreas, I do not know, but they well might do so. We have increased internal osmotic concentrations in these toads by keeping them on moist blotting paper. In this way they cannot excrete any of their urine, and the urea level in the plasma goes way up. You can also dehydrate them. The brain is the last organ that will dehydrate to any significant degree.

N. M. VANGELDER: What is the inhibition of blotting paper? C. F. BAXTER: I don’t know. That is the way Dr. Malcolm Gordon at UCLA found he could prevent toads from urinating.

Spaces in the CNS

Introduction to Session on Brain Spaces K. A. C . ELLIOTT Departnient of Biocheniistry and Montreal Neurological Institute, McGiN University, Montreal (Canada)

I was very glad to be invited to this interesting symposium but I was embarrassed by the fact that for the last few years 1 have been primarily an office worker and steadily getting out of date. So, I invited myself to be chairman of this session. In this position, and as probably the oldest member of the symposium, I felt I could sermonize a little without greatly hindering the progress of knowledge. I have been interested in cerebral edema and in brain spaces since I was put on to the study of cerebral edema with Herbert Jasper in relation to war surgery when I first went to the Montreal Neurological Institute towards the end of the last war. This interest was accentuated by my observation of the way brain tissue slices swell when immersed in media resembling cerebrospinal fluid. My early work on this subject seems to have more or less missed the point, as has been shown largely by later work by Dr. Pappius. Perhaps my missing the point has a moral. Perhaps I am not the only one who must be prepared to find his work lead in different directions from what he originally expected. In the work with Dr. Jasper (Elliott and Jasper, 1949) we were watching’and trying to measure cerebral edema in rabbits. One of the most striking observations in rabbits with their brains exposed was the sporadic, quite sudden, bulging of the brain. The literature showed that this occurred on unaccountable occassions in various animals and, in fact, it is a known trouble in human brain surgery. The only people I could find who seemed to have considered it experimentally were Obrador and Pi-Suner (1943) in Mexico, who described sudden inflation of exposed brain on production of lesions near the fourth ventricle. We found that the bulge collapsed when the animal was decapitated and we could find no evidence of extra fluid in the tissue. So we called the phenomenon “inflation” and blamed it on vascular dilatation and perhaps extra cerebrospinal fluid production. Perhaps increased intracranial pressure due to a tendency to “inflation” is a part of the trouble when symptoms ascribed to “edema” are observed. Cannon had postulated a metabolic-osmotic mechanism of brain swelling and Jasper and I produced real brain swelling, or shrinkage, by hypoosmotic or hyper-osmotic (25 per cent glucose) intravenous infusions. Dr. Pappius, however, has indicated that osmotic swelling is not the common feature of cerebral edema though osmotic swelling may occur during drastic changes in plasma osmolarity, as during use of the artificial kidney (Pappius ef al., 1967). The water content of the brain as a whole, grey and white matter, can be reduced osmotically by hypertonic iieferences p . 454

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urea injections but the edema near lesions is not affected (Pappius and Dayes, 1965). The problem of the swelling of slices has been energetically worked out by Dr. Pappius and finally, through collaboration with Dr. Klatzo, she has proved that the main swelling in slices in vitro is in a space that is extracellular, in free communication with the surrounding medium, and which occurs appreciably only af the edges of slices where there is the mechanical damage of cutting (Pappius et af., 1962). So it is very doubtful that this kind of swelling is the main swelling of cerebral edema. In fact it is almost certainly not, since Dr. Pappius has shown, as surgeons know from experience, that cerebral edema occurs in white matter, scarcely at all in grey (Pappius and Gulati, 1963), whereas the swelling of slices due to the damage caused in their preparation occurs with both grey and white slices and it was slices of grey matterthat we studied particularly. Still this swelling at the site of damage does indicate the existence of a potential space. Evidently certain structures normally keep brain tissue properly compressed and when these are damaged the tissue can swell up like a sponge. I don’t think we should forget this potential space though it is not a normal space. In fact it seems that a space of this kind, the fluid in which seems to be extracellular and in communication with plasma, opens up under conditions that produce cerebral edema and is actually the main space of cerebral edema. But why this space opens up in white matter, and not in grey matter, remains to be figured out. Histological considerations might explain this. The neuropil feltwork of grey matter may bind this tissue so tightly that spaces cannot open unless there is mechanical damage to the feltwork as in cut slices. Our early studies did show that there is also the possibility, under certain abnormal conditions, of a change in an infra-cellular space. This occurs, for instance, during hypoxia or in the presence of high extracellular glutamate or potassium. This may be the glial swelling that has been observed with the electron microscope in swollen slices. Such intracellular changes seem too small to account for the volume of cerebral edema but they do require us to think carefully in other directions. For instance, there was the discovery by Krebs and his coworkers that, when glutamate is present in the surrounding medium, brain slices take up both extra glutamate and potassium. But consideration of intracellular space changes show that there is no concentration of potassium (Pappius and Elliott, 1956). Actually the potassium concentration in the increased intracellular space is not higher than normal. In Table I, I have summarized our thinking on this subject. Theconsideration of spaces in the brain, or in any tissue, is really rather complicated. You start with three apparently obvious spaces: the blood vessels, an extracellular space and an intracellular space. But then these, at least the last two, are not so obvious. We have a flat contradiction between the quite large extracellular space in brain if this is equated with sodium and chloride spaces or, at least in slices, with thiocyanate or sucrose spaces, and the smaller spaces apparently seen by the electron microscope. But the electron microscope space agrees approximately with the chemically determined inulin or protein spaces of Dr. Pappius (Pappius et al., 1962). But what do we mean by intra- and extra-cellular spaces? Does the extracellular space extend into the innerds of the cell via the endoplasmic reticulum? And if so, how

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TABLE I T H E S E S CONCERNING C E R E B R A L E D E M A

I . “Inflation” can be confused with edema. 2. Brain swelling (and shrinkage) can be produced experimentally by osmotic action. but 3a. Traumatic edema is not osmotic swelling. b. Traumatic edema is rnnstly an uptake of fluid into an extracellular space. This may be only a potential space in normal tissue. (The fluid apparently enters this space from defective blood vessels) c. Traumatic edema occurs in white matter, very little in grey matter. 4. Trauma nevertheless produces some generalized change in grey and white matter though only the white swells grossly. 5. Direct damage to brain, as at edges of cuttissue,allows local uptake of fluid into a potential space in grey or white tissue. 6. Unfavourable metabolic conditions can produce mine intracellular swelling, at least in vifro. 7. Osmotic swelling of white and grey matter can occur when there is rapid change of concentration of a plasma solute as in the use of the artificial kidney. (Osmotic swelling is, of course, intracellular) 8. Osmotic reduction of intracranial pressure can be achieved with hypertonic urea (or other substances). This involves general reduction of brain volume, not reduction of edema.

f a t will proteins, metabolites and electrolytes diffuse in and out of this tortuous space? That is, though it may be connected with the really external space, will it behave as extracellular space for all solutes? Once we concede that we are in the intracellular space, we find we are merely reaching further complexity. We have spaces : intramitochondrial, intra-lysosomal, intraGolgi apparatus, intra-nucleus, intra-nucleolus and intra-subdivisions of these. And presumably we have a little fluid that is plain intra-cellular, bathing all these organelles and interacting with extracellular fluid osmotically, through simple diffusion and through various kinds of pumps and active transport systems. Most probably such pumps and systems also apply to interactions between the various subcellular spaces. But even the commonly used term “space” is ambiguous. We have an example in our own experience. GABA exists in brain in at least three different states (see e.g. Elliott 1965; Varon et al., 1965). One is in free solution, one is occluded in particles that are obtained in sucrose suspension and is completely separated from free GABA. Labelled free GABA does not mix with it. The third exists when sodium is present, and its presence is inhibited by cold. It is completely exchangeable with free GABA. Now the free and the occluded GABA may be regarded as being in two particular spaces. But what about the third state? It is presumably adsorbed in some way and not occupying a “space” as we commonly mean the word. How often do we talk of a “space” which isn’t really a space? The substance whose space we are determining may not all be in ordinary solution but may be partly absorbed or adsorbed to a solid surface or to a substance in solution, as in enzyme-substrate, antigen-antibody, lipidlipid, or electrostatic combinations, or in micellar or clathrate occlusions. And even when we speak of a space, meaning a volume of solution containing the substance in question, are we sure we know what we mean? The solvent may in many cases not be water as we know it in bulk. Water in narrow spaces, the walls of which Rrferrncrs p . 454

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are full of ionized groups and hydrogen bonding potentialities, is probably structuredice-like according to Szent-Gyorgyi. We could hardly expect solutes to behave in such matter as though they were in free solution. I am inclined to think that this kind of condition applies not only in intracellular spaces but also to some of the narrowly confined extracellular space. So we could have to consider several different kinds of extracellular fluid spaces according to the precise location and state of the fluid. There would be, for instance, the truly external fluid and perhaps fluid within connecting endoplasmic reticular tubules, and there would be the normal water and the structured water. Finally, we should remember that these structures and fluids are not all necessarily stationary. We should keep in mind old observations on protoplasmic streaming and the more recent evidence of axoplasmic flow in neurons and the pulsations and gyrations of cultured glial cells that we used to see in the movies of Dr. Pomerat. I have now, I believe thoroughly scrambled the issue and I shall hope that our main speakers and the general discussion will straighten things out. Dr. Pappius has shown that tangible observations can be made in spite of all these complexities. I call on her to speak now.

REFERENCES

ELLIOTT, K. A. C. (1965) y-Aminobutyric acid and other inhibitory substances. Br. Med. Bull., 21, 70-75; see also Strasberg, P. and Elliott, K.A.C. (1967) Can. J. Biochem., 45, 1795-1807. ELLiOTT, K. A. C. AND JASPER, H. H. (1949) Measurement of experimentally induced brain swelling and shrinkage. Amer. J. Physiol., 177, 122-129. OBRADOR, S. AND PI-SUNER, J. (1943) Experimental swelling of the brain. Arch. Neurol. Psychiat., 49, 826-830.

PAPPIUS, H. M. AND DAYES, L. A. (1965) Hypertonic urea. Its effect on the distribution of water and electrolytes in normal and edematous brain tissue. Arch. Neurol., 13, 395402. PAPPIUS, H. M. AND ELLIOTT, K. A. C. (1956) Factors affecting the potassium content of incubated brain slices. Canad. J. Biochem. Physiol., 34, 1053-1067. PAPPIUS, H. M. A ~ GULATI. D R. (1963) Water andelectrolytecontent ofcerebral tissues in experimentally induced edema. Acta Neuropathol., 2,451460. PAPPIUS, H. M., KLATZO, I. AND ELLIOTT, K. A. C. (1962) Further studies on swelling of brain slices. Canad. J. Biochem. Physiol., 40,885-898. PAPPIUS, H. M., OH, J. H. AND DOSSETOR, J. B. (1967) Effect of rapid hemodialysis on brain tissues and cerebrospinal fluid of dogs. Canad. J. Physiol. Pharmacol., In press. VARON,S., WEINSTEIN, H., KAKEFUDA, T., AND ROBERTS, E. (1965) Sodium-dependent binding of y-aminobutyric acid by*.morphologically characterized subcellular brain particles. Biochenr. Pharmacol., 14, 1213-1224.

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Spaces in Brain Tissue in vdro and in viva H A N N A M. PAPPIUS The Dorurer Lahoratory of Experinletrial Neurocheniisiry. Montreal Neurological Insiiiuie and the Deparinrerri of Neurology and Neurosurgery, McCill Uiriversiiy, Montreal (Canada)

Several years ago, Dr. Elliott and I studied in detail the distribution in incubated cerebral cortex of substances thought to equilibrate with extracellular tissue water. We used thiocyanate, sucrose, inulin and labeled protein (Pappius and Elliott, 1956; Pappius et a/., 1962; Pappius, 1965). In our experiments, rat cerebral cortex slices about 0.5 mm thick were weighed and incubated in Ringer-bicarbonate-glucose medium containing one of the ‘markers’. After incubation, the slices were drained, reweighed and suitably extracted for chemical analysis of the ‘marker’ substance under study. The ‘marker’ space in the tissue in each case was taken as equal to that volume of the medium which would contain the same amount of the ‘marker’ as was found in the slice. I would like to stress that when discussing spaces I am referring to the volume of distribution of a particular substance when steady-state conditions exist. Such conditions are fairly easily established in vifro. In vivo serious problems are encountered in attaining equilibration of the ‘marker’ substance between the various compartments available to it. FLUID COMPARTMENTS I N CEREBRAL CORTEX INCUBATED AEROBICALLY FOR 60 M I N .

c

0

20

60 80 100 Milligrams o r Microliters

40

Dry weight Tissue water not equilibrated with Inulin, Protein, Sucrose and Thiocyanate

I 120

140

Tissue water equilibrated with Sucrose and Thiocyanate, but not with lnulin and Protein Tissue water equilibrated with Inulin, Protein, Sucrose and Thiocyanate

0

Fig. 1. Fluid compartments in cerebral cortex incubated aerobically for 60 min. Composite summary of data. For details see text.

Fig. 1 is a composite summary of data obtained in many experiments in which the ‘marker’ spaces were determined separately. The results are expressed on the basis of 100 mg or microliters of the original tissue. The total length of the bar represents the average weight or volume of the slice after incubation, the portion to the right of the Ri~feriwcap. 460

456

H. M. P A P P I U S

broken vertical line representing the extent of swelling. The solid portion represents dry weight. We found that all the ‘markers’ equilibrated with the fluid taken up on incubation, about 30 pl in a 100-mg slice. Inulin and protein equilibrated also with about 20 pl of the original tissue water, giving a total inulin-protein space in the swollen slice of about 50 pl. Sucrose and thiocyanate equilibrated with a significantly greater portion oftheoriginaltissue water-about 55pI-so that the total sucrose-thiocyanate space in the swollen slice was 85 pl. These results show that three distinct fluid compartments exist in incubated cerebral cortex slices : ( I ) the non-thiocyanate, non-sucrose space, (2) the inulin-protein space and (3) the compartment which is penetrated by sucrose and thiocyanate but not by inulin and protein. With the help of Dr. Klatzo we were able to visualize microscopically the tissue water compartment equilibrated with protein labeled with a fluorescent dye (Pappius et al., 1962). The bulk of the protein could be seen in a zone near the edge of the slice which consists of tissue damaged during its preparation. On the basis of this finding, we were able to conclude that protein and, by inference, inulin equilibrate with the fluid in damaged regions of the slice where the bulk of the in vitro swelling has occurred. On the basis of other experiments (Pappius et al., 1962; Pappius, 1965) which I have no time to describe, we know that under our standard aerobic experimental conditions intracellular swelling, associated mainly with anaerobic conditions, has been kept at a minimum. Thus the 2 0 4 of original tissue water which equilibrates with inulin in vitro must include the damaged areas of the slice and therefore represents an overestimate of the true extracellular space of brain. On the other hand, the non-inulin space of the slice can be regarded as the intracellular space of grossly undamaged cells. We interpret the fact that thiocyanate and sucrose equilibrate with a significant fraction of the non-inulin compartment of tissue water as evidence that they have someintracellular distribution and we have postulated that it may be intraglial (Pappius et al., 1962; Pappius, 1965). In fact, the distribution of sucrose in incubated tissue is not quite the same as that of thiocyanate. In recent experiments cat cerebral cortex slices were incubated in media containing thiocyanate and trace amounts of either [14C]sucrose or [“Tlinulin. Thus two spaces were determined simultaneously in each tissue sample. The results of these experiments are summarized in Fig. 2. It will be seen that under aerobic conditions the thiocyanate space was on the average 7 pl greater than the sucrose space in the same slice. This difference was quite consistent and statistically highly significant (p < 0.01). When sucrose and thiocyanate spaces were measured in the presence of 1 % sucrose, the highest concentration of sucrose in our original experiments on sucrose distribution (Pappius and Elliott, 1956), both the non-sucrose and non-thiocyanate spaces were diminished t o about the same extent, presumably due to osmotic dehydration. The agreement between the non-thiocyanate space in one case and the non-sucrose space in the other is thus coincidental. Since there is experimental evidence that thiocyanate, like chloride, crosses neuronal membranes (Coombs et a/., 1955) and thus must be distributed approximately according to the Nernst equation, the small dis-

SPACES IN B R A I N TISSUE

in VitrO A N D in

457

ViVO

Distribution of Inulin, Sucrose and Thiocyonote in Ctrebral Cortex Slices of Cat Incubated for 60 min. Aerobic

,

Aerobic; 1% sucrose in medium

I

(4) 1

d

20

40

60

80

100

120

140

Id0

Milligrams or Microliters

Fig. 2. Distribution of inulin, sucrose and thiocyanate in cerebral cortex slices of cat incubated for 60 min. Number of experiments in brackets. Short markers indicate standard deviation when six experiments were averaged and range of results in case of four experiments.

crepancy between sucrose and thiocyanate distribution does not necessarily invalidate our suggestion that both penetrate the glial space (see Discussion, Pappius and Elliott, 1956). The question arose whether the apparent intracellular distribution of sucrose and thiocyanate is associated with the in vitro situation and reflects partial damage, or whether, in fact, certain cellular elements in cerebral tissue are permeable to thiocyanate and sucrose. In an effort to answer thisquestion, thiocyanate distribution in cerebral tissues of cat was studied in vivo under conditions where equilibrium between the blood, the brain tissues and the cerebrospinal fluid, if not fully achieved was at least closely approached. In these experiments twice isotonic (2.5 %) sodium thiocyanate solution was slowly infused intravenously into cats over 30 to 40 min to give final blood concentration of about 10 mM. This relatively high concentration of thiocyanate was chosen in the hope of saturating the mechanisms which are known to actively transport thiocyanate from the cerebrospinal fluid to blood (Pollay and Davson, 1963). The animals, anesthetized with Nembutal, did not show any obvious signs of respiratory difficulties or other toxic effects of thiocyanate. Their blood remained well oxygenated with spontaneous breathing. The thiocyanate content of the blood was found to remainconstant throughout the experimental period. The animals were killed 2,4, 6 or 10 h after the infusion and the water and thiocyanate contents of the cerebral tissues and of the cerebrospinal fluid were determined. In vitro dialysis experiments showed that at 10 mM thiocyanate concentration in the blood 83 % of the thiocyanate was in free solution. In Fig. 3 the thiocyanate contents of the tissue water and of the cerebrospinal fluid are expressed as percent of the free thiocyanate concentration in the blood. Each point represents the average of results from three animals except at 10 h where five animals were used. The range of results is also indicated. With reference to the paper presented by Dr. French, at two hours after infusion there was indeed a suggestion of slight dehydration of the brain. The dry weight of both cerebral tissues was found to be at the higher border of the normal range. At later time intervals the dry weight was within normal limits (see Table I). Rrfercncrs p . 460

H. M. P A P P I U S

458

TABLE I EFFECT O F I N F U S I O N O F T H I O C Y A N A T E O N T H E D R Y W E I G H T O F C E R E B R A L TISSUES O F CAT

Time in hours after start of insusion of thiocvanatel

Dry weight mg % fresh weight of tissue Cerebral cortex Subcortical white matter

Number of atiinials

-

19.2 40.7 20.5 19.3 19.1 19.1 0.4

2 4 6 10

31.8 f 1.2 33.7 32.5 31.2 32.05 k 1.5

Averages -fr standard deviation where 5 or more animals used. 1 2.504 sodium thiocyanate was infused slowly over 30 to 40 min. Final concentration of thiocyanate in the plasma 11.3 3: 2.4 mM (14 animals).

I

0

I

‘

2

I

hrs.

nma afta hlurin of nbeynati

I

Ib

.

A W.T O U hrrmu I1 4 , I I Mill] W. ha 0C Er*d Y cam, 0 u n p c d .ku Mtfl(

Fig. 3. Percent of cerebral tissue water and cerebrospinal fluid water equilibrated with free thiocyanate in blood. For details see text.

Results presented in Fig. 3 show that the thiocyanate content of the CSF rose slowly and even 10 h after the thiocyanate infusion it had not come to an equilibrium with the free thiocyanate in the blood. At 10 h the average concentration ofthiocyanate in the CSF was 75 % of that in blood. The apparent thiocyanate concentration in the water of cerebral cortex and white matter increased slowly up to 6 h at which time it reached a plateau. At 10 h the concentration of thiocyanate in the water of both cerebral tissues amounted to 35 % of that in blood. When expressing the thiocyanate content of cerebral tissues in vivo in terms of thiocyanate space, it can be assumed either that the tissue thiocyanate has come to an equilibrium with thiocyanate in the blood or that it has equilibrated with the thiocyanate in the CSF. Since it is difficult to decide which of the two assumptions is the

SPACES I N B R A I N TISSUE

in vitro

AND

in vivo

459

Thioqanate Spate in Cerebral Tissurs of Cat in Vivo lllod l b l q l w ( ~11.3 f 2.4 mM 110I CEREBRAL CORTEX

Equilibrated with

2 hrs.

BLOOD

(3)

Clf

6 his. l.OOD csi

(3)

SUBCORTICAL WHITE MATTER 2 hrr.

6 hrs BLOOD

(3)

(Sf

1

0

10

40

b0

80

I 100

Milligrams or Microliters

Fig. 4. Thiocyanate space in cerebral tissues of cat in v i v a Number of animals in brackets. Short markers indicate range of results. For other details see text.

correct one, in Fig. 4 thiocyanate space in the upper portion of each bar was calculated assuming equilibration with the blood and in the lower portion assuming equilibration with the CSF. At 2 h after infusion when steady state conditions have not been achieved, it is clear that expressing the thiocyanate space in terms of the concentration of thiocyanate in the blood gives an underestimate of the total space in vivo available to thiocyanate while in terms of thiocyanate in the CSF on overestimate of the tissue space is obtained. By 10 h when the difference between the blood and the CSF thiocyanate content has greatly diminished, the two estimates of thiocyanate space were considerably closer. It can be concluded that the thiocyanate space in cerebral cortex of cat in vivo when equilibrium between all water compartments has been achieved must be within the limits of 29-38 %, probably closer to the former figure. This, by the way, agrees fairly well with the figure of 30% for iodide space under conditions when iodide transport out of the CSF was inhibited, as reported at this conference by Dr. Woodbury. Fig. 5 is a comparison of the distribution of inulin and thiocyanate in vivo and in vitro. For the purpose of this discussion, I have assumed an in vivo inulin space of 10 3%, basing this estimate on the published inulin space figures of Rall and his colleagues (Rall et al., 1962). This is not too far from the figure of 13.5%inulin space mentioned here by Dr. Woodbury and 15 % sulfate space referred to by Dr. Cutler. Dr. Tower has reported considerably higher values for inulin space in vivo (Bourke et al., 1965), but it is unlikely that under his conditions even very careful experimentation can overcome the basic problem that equilibration between the various water compartments was not established. While both in vivo and in vitro existence of three

H. M. P A P P I U S

460

Fluid Conportmtntr in Cerebrol Cortex Tissue in Vivo ond i n Vitro Blood Thiocyanate 9.8 m M . 10 hrs. after lhiocvanats inlurion.

60 rnin. aerobic incubation

I

"

0

"

'

20

I

1

40

b0

I

I

10

1

1

100

8

110

1

1

140

Milligrams or Microlilsrs .Dry

l i ~ ~ woler u s .quilibmIsd with l h i o r y a n o h only

w.qhl

lillu. w ~ l m n d .qucl,brai.d a w i l h ei1h.r lnulin or I h i o < y m o ! .

Tinu.

u,,,h

r01.r

.quilibrc.l.dwiih

lnYlinand Ihiolyanol.

Fig. 5 . Fluid compartments in cerebral cortex tissue in vivoand in vitro. Number of animals in brackets. Short markers indicate range of results for in vivo experiments and standard deviation for in vitro experiments.

fluid compartments can be demonstrated in terms of the distribution of inulin and thiocyanate, it is quite clear that quantitative relationships are rather different under the two sets of conditions. We know that the increased inulin space in vitro is associated with swelling in the damaged areas of theslice. However, thiocyanatedistribution relative to inulin distribution is also increased in v i m . Thiocyanate equilibrated with approximately 30 % of the non-inulin space in vivo and nearly 60 % of the non-inulin space in vitro. Further experiments will show, I am sure, whether this increase in the space available for thiocyanate distribution is an artefact of damage involved in the in vitro preparation or whether it is another demonstration of the restricted permeability into cerebral tissues in vivo, the so-called blood-brain barrier.

REFERENCES E. S. AND TOWER, D. B. (1965) Variation of cerebral cortex fluid spaces BOURKE, R. S., GREENBERG, in vivo as a function of species brain size. Amer. J. Physiol., 208, 682-692. COOMBS, J. S., ECCLES, J. C. A N D FAW,P. (1955) The specific ionic conductances and the ionic movements across the motoneuronal membrane that produce the inhibitory post-synaptic potential. J . Physiol., 130, 326-373. PAPPIUS, H. M. (1965) The distribution of water in brain tissues swollen in vitro and in vivo. Biology of Neiiroglia. E. D. P. De Robertis and R. Carrea (Eds.), Progress in Brain Research, vol. 15 (p. 135-154). PAPPIUS, H. M. AND ELLIOW,K. A. C. (1956) Water distribution in incubated slices of brain and other tissues. Canad. J. Biochern. Physiol., 34, 1007-1022. H. M., KLATZO, I. AND ELLIOTT, K. A. C. (1962) Further studies on swelling of brain slices. PAPPIUS, Canad. J. Biochem. Physiol., 40, 885-898. POLLAY, M. AND DAVSON, H. (1963) The passage of certain substances out of the cerebrospinal fluid. Brain, 86, 137-1 50. C. S. (1962) Extracellular space of brain as determined by RALL,D. P., OPPELT,W. W. A N D PATLAK, diffusion of inulin from the ventricular system. LifeSci., 2, 43-48.

SPACES IN B R A I N TISSUE

in

VitVO A N D

in ViVO

46 1

DISCUSSION D. B. TOWER:I would like to say that the areas of agreement between Dr. Pappius and myself are vast compared to the areas of disagreement, and I would like to support what Dr. Pappius has said. In other tissues (e.g., liver, kidney, etc.), not only is there a disproportion between the space accessible to inulin and that accessible to thiocyanate, but also to that accessible to sucrose. Sucrose occupies almost the same-sized spaces as thiocyanate or chloride in the liver and kidney. In the white matter there is a real problem. I think Dr. Pappius must be much more skillful than I am at cutting slices of white matter, because I could not get slices to work, and finally resorted to a preparation of what might be called “hemi” corpus callosum, which in the cat, fortunately, is of appropriate dimensions for incubation (less than 0.5 mm thick). One can simply cut it free at the two sides and have a very small cut surface relative to the total surface area. In contrast, if you are not careful in s!icing white matter thz tissue imbibes too much water and disintegrates. As far as the difference in results for the cortex is concerned, it occurs to me that this may be partly a species difference with in vitro preparations. Since I have not done any of these experiments with the rat, I can’t say definitely, but under our conditions there is a very distinct difference between the size of the fluid spaces accessible to chloride and the size of those accessible to sucrose (for rabbit, cat, monkey cortex). There is a further complication, which I did not originally intend to bring up here, but which, I think, may be important for our discussion as it has developed during this session. There is a difference in the size of the sucrose (inulin) spaces in incubated slices of cat cerebral cortex, depending upon when one adds the indicator solute and depending upon the time of incubation. This is illustrated in summary form in the following table.

TABLE I SLICE SUCROSE SPACES A S FUNCTIONS OF T I M E S OF SOLUTE ADDITION A N D OF SLICE INCUBATION

lncubatim Time (miti)

Time Sucrose Present ( m i n )

Sucrose Space (%)

65 125

65 125

42.3 41.9

65 125 125 I25 125

20 20 40 60 90

29.4 29.7 30.5 35.4 39.1

Slices of cat cerebral cortex were incubated aerobically at 31°C for the times indicated in bicarbonatesaline-glucose media containing 27 mM K+.Trace amounts of [14C]sucrosewere added to be present for the incubation periods specified. Slice swelling and chloride spaces were in all cases essentially similar (C’ Table VI of TOWER:this Conference). Values are means of 4 or more experiments; those above the dashed line differ significantly ( P < 0.01) from the values below the line except in the case of the final entry (incubation 125 min; sucrose present 90 min). Taken from BOURKE AND TOWER (1966a). If the sucrose or inulin is added at the beginning (when the slice is immersed in the incubation medium), there is a space of about 42%whether the slices are then incubated for one hour or for two hours. However, if the sucrose or inulin is added later on, after incubation is under way (in this example up to 20 minutes before the end of incubation), there is a significantly smaller space, and in this situation the longer the period of incubation after addition of solute the larger the mace becomes, so that eventually (90 minutes after addition) it reaches the same size as that observed wh n the solute is added initially.

462

H. M. P A P P I U S

Now I don’t know the explanation for this phenomenon, but I suspect it may be as Dr. Elliott suggested, namely that some of the channels between cells are very, very small, and these size differences may reflect some sort of diffusion problem for the solute. Perhaps Dr. Rall may have a comment on this point. But one is faced with several possible explanations. (I may point out that if we select the smaller values, then we are in even better agreement with Dr. Pappius than before.) One could also suggest that the difference between the two extremes has something to do with cut surfaces of slices, although the mechanisms involved are a little more difficult to work out. H. M. PAPPIUS:First of all, Imamrelieved that Dr. Tower thinks that the differences between his results and mine are not as striking as they seemed on paper. The point of definite disagreement is still the fact that in vitro 1find a distribution of sucrose closer to that of thiocyanate than 01 inulin, while Dr. Tower, in the cortex at least, finds that the inulin and sucrose distributions are of the same order of magnitude. I have measured the distribution of these markers in white matter also, and found the same difference between the sucrose and the inulin spaces as in cortex tissue. However, the thiocyanate space in white matter became progressively smaller with time of immersion, because I think there was relatively more tissue damage and the thiocyanate was slowly penetrating into the open ends of fibers. This can be shown quite easily with nerve preparations, where the thiocyanate will equilibrate with all of the watir if you wait long enough, or increase the extent of damage by cutting the nerve. It is also interesting to point out that if, as we suggest, thiocyanate penetrates into the glial space, this does not make glia particularly different from other cells. We found in kidney or liver slices incubated in vitro that a definite non-inulin space could be demonstrated, but that thiocyanate equilibrated with all of the tissue water. In muscle, thiocyanate and inulin had the same distribution

K. A. C. ELLIOTT: We used sucrose and thiocyanate in our early experiments, because one is a charged particle and the other is not. My picture of thiocyanate is that it is a very freely permeable ion which goes into the same places as chloride. If you calculate either the chloride or the thiocyanate space from the ordinary transmembrane potential, assuming these ions are distributed only according to the potentials, the ratio of extracellular to intracellular concentration should be 1 : 16. Supposing, contrary to my own remarks, that we think of the cell as a bag, the thiocyanate inside should be 1/16 of what it is outside. Thus, the main bulk of the thiocyanate would be extracellular. That could explain why thiocyanate occupies a little greater space than sucrose, which all stays out of the cells. If you make a slice anoxic, or maltreat it in other ways, you destroy the transmembrane potential; the thiocyanate will then enter the intracellular space in much higher concentration and you will find, as demonstrated by Dr. Pappius, that the thiocyanate space steadily increases in the anoxic slice. 1 think then that these extended thiocyanate spaces are dependent on the metabolic condition of the slice and on the extent to which the membranes are damaged. There must be cells in which the thiocyanate is distributed according to the Nernst equation. There may be cells in which there is no such distribution, whose cell membrane is freely permeable to thiocyanate, permitting a lOOu/, exchange between the ions inside and outside the cell. The two cell types together make up the apparent difference in the space. The most remarkable difference, I think, between the findings of Dr. Pappius and Dr. Tower is the observation, in vivo at any rate, that the inulin and the sucrose spaces are the same. Do I understand that you find the same also in your slices? D. B. TOWER: Yes. K. A. C. ELLIOTT: This is astonishing.

I have mentioned at the beginning of my talk that when measuring spaces in v i m H. M. PAPPIUS: it is essential to establish that equilibration has been reached between the given marker in the medium and in the water compartment in the tissue available to it. 1 have found that under aerobic conditions, provided sufficient time is allowed for the marker to diffuse into the tissue, any further increase in the duration of incubation with the marker does not affect its distribution. The relevant experimental data for the distribution of sucrose, inulin, and thiocyanate are summarized in Fig. I . When cerebral cortex slices were incubated in the presence of oxygen for 120 min, the distribution of sucrose was the same whether the sucrose was present in the medium for the last 60 min of the incubation or throughout the experimental period. If, however, the sucrose had been added only 15 or 30 min

SPACES I N B R A I N TISSUE

INCUBATED 120 MIN

AEROBIC

,/Y////+j

in V i t r O

AND

-I

in ViVO

463 TIME W I T H SUCROSE MIN 60

I ,

SUCROSE I

I*

SUCROSE

I20

TIME W I T H INULIN

120

INULlN

ANAEROBIC

INCUBATED

120 MIN.

I ,

'

INULIN

. I

INULIN

ANAEROBIC

!

INCUBATED 60 MIN THIOCYANATE

I

20

40 60 80 MILLIGRAMS OR MICROLITERS

I

100

-I

60 120

MIN.

I'

10

60

THIOCYANATE I

'

TIME W I T H THIOCYANATE

I

0

1

I

I

120

140

160

Fig. 1. Spaces and swelling of incubated cerebral cortex slices of rat. Total length of each bar represents the average weight of slices after incubation, per 100 mg of initial weight. The portion to the right of the vertical broken line indicates the extent of swelling. The black portion represents dry weight and the open portion represents the marker (sucrose, inulin, or thiocyanate) space. The shaded portion, obtained by difference, gives the respective non-marker space. Six o r four determinations were averaged in each case. The short marks on either side of divisions show the widest range of individual determinations. before the end of the incubation, the sucrose space would have been significantly smaller in the presence of the same amount of swelling. Under anaerobic conditions the swelling was greater and equilibration did not appear to be complete even at 60 min, although the results may also indicate slow penetration of the marker into a compartment not available to it under aerobic conditions. Similar results were obtained with inulin. Aerobically, 60 min incubation with inulin was sufficient to obtain equilibration, while after 30 min a smaller inulin space was obtained (not shown on figure). Thiocyanate can be shown to equilibrate with the compartment available to it aerobically within 10 t o 15 min, and longer incubation does not affect its distribution. Results presented in the figure show, in contrast, that under anaerobic conditions thiocyanate slowly penetrates into all of the tissue water. Thus in slices incubated for 60 min, thiocyanate space increased steadily with duration of incubation in the presence of this marker. From the foregoing it isclear that it is very important to keep tissueslices well oxygenated throughout the experimental period, as both the swelling and the distribution of markers is changed under anaerobic conditions. In our initial work on spaces in v i m , we placed the slices in the medium and gassed the incubation vessels at room temperature. We consistently obtained swelling of 40%. Subsequently, we made sure that the preparation procedure was as quick as possible and that the slices were kept cold until full oxygenation of the medium was achieved. This resulted in a decrease of 10% in the swelling and a corresponding decrease in the non-inulin space (see Fig. 2). In other words, intracel-

464

H. M. P A P P I U S

;

Preparation at room temperature

Preparation in the cold

0

20

40

60

80

100

120

140

Milligrams or microliters Dry weigh!

Tissue w a t e r equilibrated with thiocyanate only

Tissue water not equilibrated with either inulin or thiocyonate

Tissue water equilibrated with both inulin and thiocyanate

Fig. 2. Effect of initial cooling on fluid compartments in cerebral cortex slices of rat incubated aerobically for 60 min.

M a r swelling occurs in tissue which is not well oxygenated and appears as increased non-inulin space. In connection with species differences reported for spaces in vivo by Dr. Tower, it may be of interest that, under optimal experimental conditions, we found that 20% of the original tissue volume equilibrated with inulin in v i m in the case of rat cerebral cortex slices, while in cat cerebral cortex slices this figure was 29%. These were two different groups of experiments, but they may indicate that species differences also exist in spaces in v i m .

H. MCILWAIN: The question of membrane potential has been raised in discussing the distribution of electrolytes and sucrose. With our measurement, both in vivo and in vi/ro, it can be shown that two clear compartments exist in cerebral tissue: extracellular and intracellular, with a difference in potential of about 60 mV between the inside and outside. From the analysis for chloride, which again is simple to determine in vivo and in vitro, one can come to some fairly direct conclusions about the size of the two compartments. I n vivo, it does give the information that there is a 26-27% extracellular space in grey matter while in the incubated tissue the value for the space is about I5 ”/, greater. One can also weigh one’s slices before and after, and we do in fact find that the weight will increase about 15 %, so that this seems a quite simple situation.

465

Delineation of Fluid Compartmentation in Cerebral Tissues D O N A L D B. T O W E R Laboratory of Neuroclietnistry, National Institute of Neurological Diseases and Blindness, Bethesda Maryland ( U.S.A.)

INTRODUCTION

Much of the preceding discussion at this Conference has dealt with barrier and transport mechanisms. Such mechanisms usually cannot be described quantitatively or meaningfully without knowledge of the relevant compartments into or out of which transport takes place and between which “barriers” to some or all solutes appear to exist. Hence I wish to consider briefly some of the important problems encountered i n attempting to delineate these compartments in terms of size and locus. I shall deal with two general situations, studies in vivo and studies in vitro, and with factors of species differences, ontogeny, areal differences and the like. If some points have been presented in earlier contributions, the repetition will hopefully have the virtue of emphasizing the importance of the problems. I would be remiss if I failed to acknowledge here our debt to the late Heinrich Waelsch, who started us thinking again about compartmentation in the nervous system in the course of a discussion at a meeting in Amsterdam almost exactly 7 years ago (Waelsch, 1960). Studies in vivo

Turning first to studies in vivo, there are several problems peculiar to the central nervous system. Solutes when introduced directly into the cerebrospinal fluid (CSF) exhibit a distinctly non-homogeneous distribution. This behavior is illustrated for trace amounts of I “C]sucrose after intracisternal injection into monkey CSF (Fig. I). Note that levels of the solute in lumbar CSF are rapidly established at 4 to 6 times comparable levels in cisternal CSF and that within several hours after injection levels of the solute in subarachnoid CSF (over surfaces of the cerebral hemispheres) are twice those in cisternal CSF (Bourke et a/., 1965). Thus, there would be considerable error introduced by referring the solute content of a cerebral cortex tissue sample to lumbar or cisternal CSF concentrations for purposes of calculating tissue spaces accessible to the solute (Table I). Even greater complexity is imposed by delivery of the solute indicator via the cerebral circulation, particularly if tissue spaces are calculated with reference to plasma rather than CSF solute concentrations. Solute distribution is importantly influenced by factors of solute diffusion and/or Ri,/l.renres p . 478-480

D. B. TOWER

466

10:'

.h -

E

.

10':

10'

Cistarnal Lul

05

I

naurs

5

.

I

.

50

10

Fig. 1. Distribution and levels (in cpm/ml.) of UL-[13C]sucrose(S.A. 3 ,/rc/,/cM) in monkey CSF (ordinate, log scale) as a function of time after intracisternal injection of 1 pc (abscissa, log scale). Individual observations are plotted except at 6 h for which means (iS.D.) of 3 or more samples are given. The slopes have been fitted by the method of least squares. (Reproduced from TOWER1965 Fig. I.)

TABLE I F L U I D S P A C E S I N C E R E B R A L C O R T E X it1 V i V O : F A C T O R O F R E F E R E N C E P O I N T FOR C A L C U L A -

TIONS

Tiitie after i.c. injectioii

Sucrose spaces ( O4) calctrlatedfrotn siicrose coticetiti atiotir in :

of [ ' T ] s i ~ r o s e

Si~barachrioirl (adjacent) CSF

Cistertial C SF

Luiiihar C SF

Ih 6h 22 h

32.5 34.1 33.9

3.25 134.5 67.5

1.35 32.4 13.3

Derived from studies on monkeys (Fig. I ) repotted by BOURKEet a/. (1965).

transport. We need not dwell on this point but merely recall, for example, the distribution data for [Wlthiocyanate under conditions identical to those for sucrose in Fig. I . Cisternal levels of thiocyanate decrease much more rapidly after injection, primarily as a consequence of the transport system mediating its efflux from the central nervous system (Pollay, 1966; Pollay and Davson, 1963; Streicher et a]., 1964; Welch, 1962). Thus thiocyanate levels in cisternal CSF are less than half those found for sucrose at 5 to 6 h after injections of identical amounts, and very different ratios of solute levels in lumbar CSF (0.3-0.4) or subarachnoid CSF (0.6-0.9) to those in cisternal CSF are found (Bourke et a/., 1965). The two factors just cited emphasize the importance of choosing the proper point of reference, i.e., which value of extracellular fluid solute concentration is to be used for

DELINEATION O F

CSF

467

COMPARTMENTS

TABLE I1 F L U I D S P A C E S I N C E R E B R A L C O R T E X it2 ViVO: F A C T O R O F S P E C I E S D I F F E R E N C E S ~

Brain Weigh1 (g)

Species Rabbit Chimpanzee

10

380

Relative Neurotr Detrsity

Chloride Spaces

44 13

30 45

I 70)

Sucrose Spaces

f %) 23 40

-

Data taken from TOWER(1954) and BOURKEet al. (1965). All differences shown are statistically significant ( P . 0.01 or better). '

calculation of the tissue spaces accessible to the solute. The principles here involve not only the question of CSF VS. plasma but the proximity of the CSF sample to the tissue sample taken for analysis and a knowledge of the behavior of the solute after administration. I t has only recently become apparent that for cerebral cortex, at least, the sizes of tissue fluid compartments delineated by chloride, thiocyanate, inulin or sucrose vary as a direct function of species brain size. Details of these correlations have been reported by us elsewhere (Bourke et a/., 1965), so that I will simply show an illustrative example, comparing data for rabbit with those for chimpanzee(TableI1). The point to be emphasized here is that one cannot generalize about cerebral compartments but should specify and consider species individually. The factor of species differences may have a bearing on conclusions derived from electron microscopy as discussed below. Tissue heterogeneity is obviously a much more serious problem in neural tissues than i n most other body tissues, such as liver or muscle, upon which studies of fluid compartmentation have beenconducted. There are not only gross areal differences (cortical gray, subcortical white, cerebellar cortex, etc.), as discussed below, but probably also more subtle differences between various anatomically or physiologically defined areas within the same gross subdivision. In this context the differences in size of chloride space in the various layers of allocortex in the region of Ammon's horn (Lowry et al., 1954) provide an illustrative example (Table 111). T A B L E 111 C H L O R I D E S P A C E S IN C O R T I C A L L A Y E R S OF A M M O N ' S HORN ( R A B B I T ) - ~-

Layer

Morphology

CI Space ( oh)

Thicktress ( p )

Dry Wt. ( %)

200

20.6

30.6

I50 400

27.1 23.3

38.5 39.0

50 250 200

17.0 20.4 30.3

31.3 29.9 45.8

-~

Molecularis Lacunosum Radiata Pyramidalis Oriens Alveus

Terminal dendrite and axon arborization; pial vessels Dendrites; rnyelinated fibers Dendrites - closely picked Cell bodies (closely picked), giving origin to fibers of other layers Non-rnyelinated axons; dendrites Myelinated fibers

Taken from data reported by LOWRYer al. (1954). Chloride spaces have been calculated assuming an extracellular CI- (CSF) of 12'3 pequiv./ml (BOURKEel a / . , 1965). Xcqrrcmes p. 478-480

D. B. TOWER

468

Swelling

CI Space

(70)

(701

K

Na

(peq./g.)

Fig. 2. Fluids and electrolytes of cat cerebral cortex in samples biopsied immediately (open bars) or 30 min (solid bars) following circulatory arrest. All values (ordinare) are referred to fresh weight of tissue and areexpressedinthe units specified beneath each set of bar graphs. Means of 2 or more determinations are given and except for those for CI space, differences shown are statistically significant (P < 0.01 or better). (Derived from data reported by TOWER,1967.)

Finally, we should not overlook pathological factors and the artifacts which they may superimpose on such underlying complexities. Consider, for example, the effects of circulatory arrest for 30 min before sampling brain tissue and adjacent CSF (Fig. 2). Such a period of ischemia is not unusual for a tissue sample being excised incident to a neurosurgical procedure and is certainly brief compared to most intervals before postmortem sampling. The critical consequences in the tissue are intracellular (nonchloride space) edema, loss of K+ and gain of Na+, changes which are reflected by a pronounced rise of CSF K+ (from the normal 3.8 to 29.5 pequiv./ml) and a persistence of tissue swelling and abnormal electrolyte distribution during subsequent studies in vitro (Tower, 1967). The elevation of CSF K+ in the cat under these conditions is of the same order of magnitude as has been reported for human CSF postmortem (Mason et ul., 1951 ; Naumann, 1958).The edema observed here is only one of several types of edema, each with rather characteristic sites of predilection, which may be encountered (Table IV). The foregoing examples of problems associated with delineation of fluid compartments in neural tissues in vivo do not represent a complete listing, but they should suffice to stimulate thinking and discussion about these and other problems at the in vivo level. Studies in vitro

Turning now to in vitro approaches to the delineation of compartments in neural tissues, one would expect to have the opportunity to study compartments and factors affecting them under more precisely defined and more readily controlled conditions. Basically such is the case, but these advantages are countered to some extent by new problems peculiar to the in vitro situation. Such studies generally involve the use of

DELINEATION O F

CSF

469

COMPARTMENTS

T A B L E IV SOME TYPES OF C E R E B R A L E D E M A ENCOUNTERED

in ViVO

Dry Weighr (04) Cerebral Cortex Subcortical White

Species aiid Pathology

Man: Tumorn

C E Rabbit: Alkyl tin” C E Cat: Cold lesionC C E Car: Circulatory arrest“ C E

17.4 16.9 20.2 20.0 17.9 18.5 16.0 13.5

Degree of Swelling ( 74)

30.7 17.4 30.8 22.9 31.2 22. I 29.6

-

C: controls; E : experimental (italicized values differ significantly from respective controls); w: white matter swelling; c: cortical swelling. a STEWART-WALLACE (1939); ALEUer at. (1963); PAPPIUSand GULATI (1963); TOWER (1967) I ” “ ” ’ “ “ “ ‘

40

i

E.. 0,

b

4

,’

a

.-

,

I

.

Ouabain

20Control

,

0

,

I

I

0.5

,

,

I

t

I

incubation time

L

U

2

i

Ihrs.)

Fig. 3. Swellingor imbibition ofextra fluid in slices of cat cerebral cortex (ordinate) as a function of time after immersion (abscissa) in bicarbonate-saline-glucose incubation media containing 5 m M K+. Mean values ( & S.D.) for 4 or more determinations are plotted and are all referred t o initial fresh weights of tissue. Swelling attributable to “adherent” medium or of the “preparatory” type (see text) is denoted by x . Data on slices incubated at 37”are denoted by 0 for control slices incubated aerobically; by 0 for slices incubated anaerobically; and by A for slices incubated aerobically in the presence of M ouabain. (Derived from data reported by BOURKEand TOWER, 1966a.)

tissue slices (in which cellular integrity and tissue architecture are largely preserved) incubated under optimal basal or carefully specified experimental conditions. Most of the examples to follow are taken from our own studies on incubated slices of cat cerebral cortex (Bourke and Tower, 1966a; b), unless otherwise specified. When carrying out such incubations in vitro, one immediately appreciates the potentialities of neural tissues for swelling or imbibition of additional fluid. Fig. 3 summarizes the unavoidable in vitro artifacts so well investigated by McIlwain and colleagues (Varon and McIlwain, 1961 ; Keesey et al., 1965). A portion of the total swelling of inReferences p. 478480

470

D. B. T O W E R

Lc

0

I

1

20

40

I

60

Medium K’

I

I

80

100

,

120

I

140

I

(mM/L)

Fig. 4. Swelling in incubated slices of cat cerebral cortex (ordinate)as a function of K +concentration in the incubation medium (abscissa). Mean values (k S.D.)for 4 or more determinations are plotted and are all referred to initial fresh weights of tissue. All incubations were carried out aerobically at 37” for 1 hr in bicarbonate-s3line-glucose media containing as principal anion either 125 mM chloride (0) or 125 mM isethionate (0).(Reproduced from BOURKEand TOWER 1966a. Fig. 4.)

cubated slices is attributable to incubation medium “adherent” to cut surfaces of the slices-a portion which is demonstrable within seconds after immersion of fresh slices in incubation medium, which is accessible to all indicator solutes including inulin, and which amounts to about 8-10 per cent of the initial fresh-tissue weight even with greatest care of slicing. A second portion of the total slice swelling is associated with “preparatory” procedures prior to institution of optimal incubation conditions - a type of swelling which is progressive with time, which is inaccessible to inulin but is accessible to chloride,andwhich can be minimized (as in Fig. 3) to about 5-10 per cent of the initial fresh-tissue weight by rapid, careful preparation of slices. Thus, with care the total swelling or excess fluid uptake (“adherent” medium plus “preparatory” swel1ing)can be held to about 12-16 per cent of the initial freshweight of cortical slices (Keesey ef al., 1965; Bourke and Tower, 1966a). During subsequent incubation under the usual optimal conditions, little if any further swelling occurs (control, Fig. 3), but swelling of or analogous to the “preparatory” type can increase to major proportions if suboptimal conditions persist, as illustrated here by anoxic conditions (N2, Fig. 3) or conditions which interfere directly with cellular cation transport systems (ouabain, Fig. 3). Superimposable upon the foregoing is additional swelling of slices dependent upon the K+ concentration of the incubation medium and the type o f principal anion therein (Fig. 4). Note the degree o f slice swelling associated with K + concentrations of 100 or more ,uequiv./ml of incubation medium. Such high K + media have been used by some investigators to study various aspects of neural tissue metabolism, and one wonders about the significance of such data in view of this degree of fluid imbibition. The substitution of a relatively non-diffusible anion (isethionate = 2-hydroxyethane sulfonate) for the usual chloride of incubation media clearly prevents the development of such K+-dependent swelling of cortical slices (Bourke and Tower, 1966a).

DELINEATION OF

CSF

i

27mM K

Chloride; SCN

0

Sucrose; lnulin In vivo- spaces-

47 1

COMPARTMENTS

Chloride; SCN In Vitro

Sucrose; lnulin

0 Swsliinp

Fig. 5 . Comparison of fluid spaces in vivo with spaces and swelling irr vitro in slices of cat cerebral cortex. Mean values (ordinate) of 4 or more observations are expressed in per cent of initial fresh weight of tissue for biopsied slices (open bars) and for slices incubated aerobically at 37" for 1 h in bicarbonate-saline-glucose media containing either 5 mM K+ (data on left) or 27 mM K+ (data on right). Fluid spaces in incubated slices are denoted by the hatched bars and slice swelling is denoted by the solid bars, which are positioned to indicate the extra fluid added to that originally measured by chloride or sucrose irr vivo for comparsion with chloride or sucrose spaces measured in vitro. The horizontal lines through the bars denote the levels of values obtained for slices incubated under the same conditions in media containing 125 rnM isethionate instead of chloride. All differences shown are statistically significant ( P . 0.01 or better). (Derived from data reported by BOURKE ef a/., 1965 and BOURKE and TOWER, 1966a.)

I20

I

-I

125 mM K

40

20

Chloride

Sucrose

Fig. 6. Same depiction as Fig. 5 for slices of cat cerebral cortex incubated in bicarbonate-salineglucose media containing 125 mM K + .See legend for Fig. 5 for details.

Relationships between the fluid of swelling and spaces of incubated slices accessible to various indicator solutes are summarized in Figs. 5 and 6 . In the case of slices incubated in a 125 m M Ki- medium (Fig. 6), note the discrepancy in size between tissue spaces delineated by sucrose and those delineated by chloride and how these spaces in vitro relate to those measured irt vivo and to the swelling associated with incubation R&rcnrrs

p. 4711-480

D. B. TOWER

472

c 0

ICP

10''

10.'

10-8

10.'

Ovaboin (MI

Fig. 7. Contents of K+ (a),Na+ (0) and CI- (A)in pequiv./g and the swelling ( x ) in per cent of initial fresh weights of tissue (ordinate) for slices of cat cerebral cortex as functions of the molar concentrations of ouabain in the incubation media (abscissa).Mean values ( f S.D.) for 4 or more observations are plotted. All incubations were carried out aerobically at 37" for 1 h in bicarbonate-salineglucose media containing 5 mM K+. The arrows near the righthand ordinate scale denote correspondand ing mean concentrations of Na+, K+ and CI - in the incubation media. (Reproduced from BOURKE TOWER 1966b, Fig. 2A.)

TABLE V in vitro:

FLUID SPACES A N D ELECTROLYTES OF CEREBRAL CORTEX SLICES INCUBATED

EFFECTS OF A D D I T I O N S TO THE I N C U B A T I O N MEDIUM

Conditions

Control ( 5 mM K+) 10 mM glutamate* 10-5 M ouabain Anaerobic 27 mM K+

+ +

Swelling ( %)

16.8 27.8 30. I 44.4 32.7

Spaces ( %) CISucrose

62.2 59.4 104.3 107.2 64.1

47.5 47.5 46.0 46.6 42.2

(pequiv.ld

K+

Nu+

74.9 74.0 28.4 27.1

95.4 113.4 132.8 166.3 75.3

99.9

Incubation 1 h at 37" aerobically (except as noted) in bicarbonate-saline-glucose media. Italicized mean values differ significantly (P < 0.01 or better) from control mean values (from BOURKEand TOWER, 1966a, b). * Final tissue glutamate level was 29.2 pM/g compared to a mean control value of 9.55 and to a final concentration in the incubation medium of these experiments of 4.7 pM/ml (TOWER, 1962).

in vitro. Similar but less extreme examples are provided by the observations on slices incubated in media containing 5 or 27 mM Kf (Fig. 5). If during incubation the K + concentration of the incubation medium is changed from 5 to 27 mM or vice versa, no change in size of slice chloride spaces and only minimal changes in size of slice sucrose spaces occur, yet there is a significant change of slice swelling, implying shifts of fluid into (5 + 27 K) or out of (27 + 5 K) intracellular spaces of the slices (Bourkeand Tower, 1966a, b). The fact that chloride (and thiocyanate) spaces in virro are considerably

DELINEATION O F

CSF

473

COMPARTMENTS

T A B L E VI F L U I D S P A C E S A N D E L E C T R O L Y T E S O F C E R E B R A L C O R T E X SLICES I N C U B A T E D EFFECTS OF V A R I O U S A L T E R A T I O N S O F I N C U B A T I O N M E D I A

Conditions

Swelling ( %)

Spaces ( %) CISucrose

K+

NU+

Control (27 mM K) 98 mM Li/Na

32.7 36.5

64.1

42.3

99.9

75.3

84.8

58.8

59.3

19.3

98 mM Choline/Na

24. I

70.5

36.6

83.3

20.I

27 mM Rb/K

33.3

56.5

43.3

17.2

79.4

125 mM Isethionate/CI

14.4

56.2*

36.6

105.5

61.2

M Ouabain

45.1

115.2

45.9

46.1

127.2

in vitro:

f ,wuiv./d Other

117.4 (Li) 75 (Choline) 84.6 (Rb) 35 (Iseth.)

-

N

-

Incubation I h at 37” aerobically in bicarbonate-saline-glucose media. Jtalicized mean values differ significantly ( P < 0.01 or better) from control mean values. * Determined with SCN- (from BOURKEand TOWER,1966a, b).

larger than those in vivo raises the possibility that there may be a mechanism operating in vivo to exclude (“pump” out) chloride from some compartment (? glial) and that under in vitro conditions this mechanism fails. Effects related to specific transport systems are illustrated in Fig. 7 and Tables V and VI. Ouabain in concentrations which completely inhibit Na+ extrusion and K+ accumulation by incubated slices exerts comparatively much less effect on slice swelling or on slice spaces accessible to sucrose (Fig. 7; Bourke and Tower, 1966b). Addition of glutamate to the incubation medium is associated with a significant degree of intracellular (non-chloride space) swelling (Table V), which presumably reflects transport into and concentration within cells of glutamate so that it can function there as additional intracellular anion (Pappius and Elliott, 1956; Tower, 1962; Bourke and Tower, 1966a, b). When other ions are substituted for normal components of the incubation media, striking effects on swelling, spaces and electrolytes of incubated slices may ensue (Table VI). The outstanding feature of this last set of observations is the disparity or lack of parallelism between changes in slice swelling and changes in accessibility of tissue spaces to chloride or to sucrose. Thus, increased (or decreased) swelling is not necessarily accompanied by increase (or decrease) of chloride spaces, and the latter may expand markedly without any change in swelling and/or sucrose spaces. The socalled Na+ substitutes, Li+ or choline+, not only interfere with monovalent cation transport but also affect fluid and solute distribution in such slices (Bourke and Tower, 1966a, b). It would be of considerable interest to be able to examine the role of water itself in the various shifts of fluid which are encountered. In studies on plants, the use of D2O in place of H2O has been informative in this context. For example, segments of apical stem from etiolated pea seedlings normally elongate and gain weight during incubation under optimal in vitro conditions. This “growth” is almost entirely attributable to inReferences p . 478-480

474

D. B. T O W E R I

I

CI

Iseth

Swelling (%)

CI

Sucrose

Spaces 1%)

K Na lpeq./g )

Fig. 8. Comparison of fluid spaces and electrolytes in slices of cat cerebral cortex incubated in media employing HzO (open b m ) or DaO (hatched bars) as solvent. All values (ordinate) are means of 4 or more determinations referred to initial fresh weights of tissue and are expressed in the units denoted beneath each set of bar graphs. Only the differences in swelling and in K t content are statistically significant ( P :. 0.01 or better). All incubations were carried out aerobically at 37" for 1 h in bicarbonate-saline-glucose m-dia containing 27 mM K+.(From unpublished studies by TOWER, D. B. and D. A.) TOWER,

creased water content and fails to occur when the incubations are conducted in DzO media (Tower, D. B. and Tower, D. A. - unpublished). Analogous experiments with incubated slices of cat cerebral cortex yield quite different results (Fig. 8). In D20 media additional slice swelling occurs- swelling which is independent of K+concentration or anion species in the medium which is not paralleled by changes in slice spaces accessible to chloride or sucrose, but which is associated with a significant increase of tissue K + content. The import of such findings remains to be elucidated. The relevance of the foregoing examples to the problems of delineating compartments in slices of neural tissue incubated in vitro should be immediately evident. It is seldom possible or permissible to suggest direct correlations between such experiments in vitro and apparently analogous situations in vivo. Yet certain of these observations may be relevant to studies in vivo under experimentally altered conditions. The use of perfusion fluids of abnormal composition and the addition of amino acid (iontophoretically or in bulk) or extra Ki or ouabain represent a few examples ofrecorded studies where such correlations deserve consideration. Comparative and Ontogenetic Aspects

Some obvious differences of fluid compartmentation in corpus callosum of cat brain compared to cat cerebral cortex are illustrated in Fig. 9. Data on cell densities of the two tissues (Heller and Elliott, 1954) indicate that the total number of non-neuronal cells (mostly glia) per unit volume is about equal in cerebral cortex and corpus callosum. If, as has been suggested by a number of investigators, some of the swelling and some of the spaces accessible to chloride in cerebral cortex are identified with glia (? astrocytes), then glial cells of the subcortical white matter (corpus callosum) must be-

DELINEATION O F

CSF

COMPARTMENTS

47 5

I 0 White Gray

80

““i 40

20

Dry Wt.

(%I

Swelling

(%I

CI

lnulin Spaces (%I

K No (peq./g.)

Fig. 9. Comparison of fluid spaces and electrolytes in incubated slices of cat cerebral cortex (solid bars) with those of cat corpus callosum (open bars). All values (ordinate) are means of 2 or more determinations referred to initial fresh weights of tissue and are expressed in the units denoted beneath each set of bar graphs. All differences shown between cortex and corpus callosum (except for N a content) are htatistically significant ( P .: 0.01 or better). The horizontal lines across the bar graphs denote the levels of values observed for biopsy samples in vivo, which with the exception of values for dry weight ’ or better). All incubations were and for K ‘ content differ significantly from in vi/ro values (P -0.01 carried out aerobically at 37“ for I h in bicarbonate-saline-glucose media containing 27 mM K+. (Derived from data by TOWER and BOURKE,1966.)

have quite differently under comparable conditions in vitro. This deduction should not come as a surprise since morphologists have long ago recognized a number of differences for glia in these two locations, but again the differences emphasize the principle of not generalizing about brain but referring more specifically to the area or subdivision in question. I t is interesting that in vitro it is cerebral cortex samples that swell so readily (Fig. 9), whereas ill vivo it is the subcortical white matter which seems most prone to edema (Table IV). Also, in contrast to cerebral cortex where both in vivo and it1 vitro sucrose spaces = inulin spaces and both are chloride spaces, in corpus callosum in vitro chloride spaces = sucrose spaces and both are > inulin spaces (Tower and Bourke, 1966). What the morphological counterpart is for the space in corpus callosum accessible to chloride and sucrose but inaccessible to inulin remains to be determined. Ontogenetic studies, particularly in a species like the cat, may be very helpful in localizing more precisely some of the phenomena referred to above. Kittens are especially useful because brain maturation involves 3 postnatal months and henceevents are sufficiently spread out in time that some tentative correlations become possible, as discussed in details elsewhere (Tower and Bourke, 1966). Some of the data obtainable in this way for kitten cerebral cortex are illustrated in Fig. 10. By one month postnatal age, maturation of cortical neurons is complete (Noback and Purpura, 1961 ; Voeller et a / . , 1963; Purpura et a/., 1964). Coincident with this stage one can demonstrate for the first time K i -dependent swelling of incubated cortical slices and the establishment of adult levels of tissue K+ and Na+. Association of these biochemical parameters with mature cortical neurons is thus strongly suggested. Similarly the onset of additional s’

R ~ ~ f i i pw. 478-480 i ~ ~

476

D. B. TOWER

Fig. 10. Changes in the extent of swelling (Sw), sucrose spaces (Sucr) and chloride spaces (CI) in incubated slices of kitten cerebral cortex as functions of postnatal age (abscissa). Values for fluid compartments (ordinate) are means ( & S.D.) or 4 of more observations expressed as per cent of initial fresh weights of tissue. Values which differ significantly (P < 0.01 or better) from the value immediately preceding are indicated by solid symbols. Values for mature cat cerebral cortex are plotted for age 12 (+) months. All slices were incubated aerobically at 37" for 1 h in bicarbonate-saline-glucose media containing 27 m M K'. See text for discussion of morphological correlations. (Derived from data reported by TOWERand BOURKE,1966.)

slice swelling and larger chloride spaces is first demonstrable at about 3 months postnatal age well after completion of cortical myelination (Noback and Purpura, 1961 ; Tower and Bourke, 1966) but coincident with the period of glial proliferation in kitten cortex(Brizee and Jacobs, 1959a,b). Thus it is possible that the biochemical characteristics which become demonstrable at this stage may be associated with glial (? astrocytic) cells. The examples considered briefly here illustrate how comparative and ontogenetic approaches may be helpful in elucidating the significance or nature of various observations obtained on adult tissues. CONCLUDING R E M A R K S

In all that has been said here and by others at this Conference there has been little appraisal of the many intracellular compartments which are lumped together by our relatively crude space measurements into the non-inulin or non-chloride spaces of neural tissues. This complex category includes at least six different cell types (neurons, astrocytes, oligodendroglia, microglia, ependymal cells and endothelial cells), and within each cell there are a number of additional compartments (nuclei, mitochondria, endoplasmic reticulum, etc.). All these compartments have obvious relevance to problems of solute transport and the barrier systems imposed thereby. For example, various studies with radiosodium and radiopotassium indicate that only about 80-90 per cent of the cortical Na+ and Kf is rapidly exchangeable, so that the residual portion may be contained within an intracellular compartment with rather distinct permeability characteristics (Keesey and Wallgren, 1965; Bourke and Tower, 1966b).

DELINEATION O F

CSF

COMPARTMENTS

471

Recent investigations of ionic transport into and out of mitochondria clearly indicate the importance of this intracellular compartment (Ernster, 1965; Rasmussen et al., 1965; Chance and Mela, 1966; Tager et al., 1966). Although most such studies have been carried out on mitochondria from non-neural tissues, the observations by Rossi and Lehninger (1963) indicate that brain mitochondria can beexpected to conform to the general pattern for other tissues in this respect. The apparent discrepancy between sizes of fluid spaces discussed here and the much smaller sizes proposed by most electron microscopists requires a brief comment. Since this problem has been considered at length elsewhere (Bourke et al., 1965), I shall confine my remarks to three points. Firstly, the factor of species differences (v.s., Table 11) has an obvious bearing here, since most observations by electron microscopy have been carried out on rodent species with small brains, which from our investigations would be expected to exhibit relatively small interstitial spaces, whereas many of the estimates of such spaces based upon physiological (impedance) and biochemical (solute indicators) methods have involved species with larger brains for which larger interstitial spaces would be expected. Secondly, there is the problem of artifact in electron micrographs, a factor which has largely been ignored or overlooked by electron microscopers. The changes which specimens of neural tissue undergo during fixation and dehydration for embedding and examination by electron microscopy are summarized in Table VII (Bourke, R. S . , Wanko, T. and Tower, D. B. - unpublished). As originally pointed out by Bahr e t a / . (1957), these changes imply gross distortions of the tissue samples and presumably of the cells therein during the process of fixation and dehydration, so that any interpretation of the original geometry of the sections becomes very difficult. Since much of the swelling maybe intracellular, either on an asphyxial(Van Harreveld e?a/., 1965, 1966a) or fixation (Bahr et a/., 1958; Tooze, 1964) basis, extracellular spaces of neural tissue samples could be partially or completely obliterated during processing of the tissue sections. Thirdly, the dimensions involved should be considered. Recent studies demonstrate that molecules the size of ferritin can penetrate through interstitial spaces quite readily (Brightman, 1965) and that such relatively narrow clefts of interstitial fluid are entirely adequate to support neuronal activity (Nichols and Kuffler, 1964). It would not take much artifactual swelling of adjacent cells to narrow the interveningextracellular space to one-half or one-third its normal width or volume. The differences between interstitial spaces 150 A wide and those 300 A wide may not be very striking in an electron micrograph but for a cuboidal space the volume would be doubled (for example, from 10 to 20 per cent) and for a cylindrical space the volume would be tripled (for example, from 10 to over 30 per cent). That such possibilities are real has been nicely demonstrated by Rall et a / . , (1962) and by Van Harreveld et a/., (1965; 1966a), who showed the prompt and striking decrease of extracellular space postmortem or with asphyxia. Undoubtedly part of the confusion over interpretation of electron micrographs purporting to show very small (< 5 per cent) interstitial spaces arose because of apparent agreement of such estimates with space measurements in vivo based on such solutes as thiocyanate and sulfate before it was realized that the latter when referred to Rcfiwnces p . 478480

478

D. B. TOWER

T A B L E V11 E F F E C T S O F F I X A T I O N A N D D E H Y D R A T I O N O N S L I C E S OF C A T C E R E B R A L CORTEX

Change in total Slice solids weight of slice ( “/o of unfixed wt) ( 0; of fresh wt)

Conditions

Unfixed Fixed 30 min in I Osoc Fixed 5 min in I