Brain Mapping and Diseases

NEUROSCIENCE RESEARCH PROGRESS

BRAIN MAPPING AND DISEASES No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

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NEUROSCIENCE RESEARCH PROGRESS

BRAIN MAPPING AND DISEASES

DIANE E. SPINELLE EDITOR

Nova Science Publishers, Inc. New York

Copyright © 2011 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Brain mapping and diseases / editor, Diane E. Spinelle. p. ; cm. Includes bibliographical references and index. ISBN 978-1-61122-623-2 (eBook) 1. Brain--Diseases. 2. Brain mapping. I. Spinelle, Diane E. [DNLM: 1. Brain Mapping. 2. Brain Diseases. WL 335] RC386.B727 2010 612.8'2--dc22 2010036427

Published by Nova Science Publishers, Inc. † New York

CONTENTS vii 

Preface Chapter 1

Hallmarks of Apoptotic-Like Cell Death in Response to Hypoxic Injury in Various Developmental Models are Closely Related to Brain Immaturity Jean-Luc Daval and Christiane Charriaut-Marlangue 

Chapter 2

DNA Damage Response and Apoptosis of Postmitotic Neurons Inna I. Kruman and Elena I. Schwartz 

Chapter 3

Tissue-, Period- and Site-Specificity of Somatic DNA Recombination in the Genomic Region, BC-1. Toyoki Maeda, Ryuzo Mizuno, Saburo Sakoda, Tomokazu Suzuki and Naoki Makino 

Chapter 4

Highlights in Understanding White Matter Ischemia James J.P. Alix and Michael G. Salter 

Chapter 5

Applications of Diffusion Tractography to the Study of Human Cognitive Functions Emi Takahashi 

1  19 

43 

61 

81 

Chapter 6

Effects of COX-2 Inhibitors on Brain Diseases Takako Takemiya and Kanato Yamagata 

101 

Chapter 7

Neuro-Physiological Studies in Creutzfeldt-Jakob’s Disease J. J. Ortega-Albás and A. L. Serrano-García 

131 

Chapter 8

Neurofilament Proteins in Brain Diseases Olivier Braissant 

153 

Chapter 9

Segmentation Propagation from Deformable Atlases for Brain Mapping and Analysis Marius George Linguraru, Tom Vercauteren,  Mauricio Reyes-Aguirre, Miguel Ángel González Ballester and Nicholas Ayache 

179 

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Contents

Chapter 10

Brain Mapping Alterations in Strabismus Martín Gallegos-Duarte, Héctor F. Rubio-Chevannier and  Jorge Mendiola-Santibañez 

Chapter 11

Endovascular Brain Mapping: A Strategy for Intraoperative Visualization of Brain Parenchyma Functionality H. Charles Manning, Sheila D. Shay, Erich O. Richter, Swadeshmukul Santra and Robert A. Mericle 

Chapter 12

The Brain’s Neuroprotective and Proapoptotic Effects of Aspirin A Review Yair Lampl 

197 

249 

267 

Chapter 13

Regional Differences in Neonatal Sleep Electroencephalogram Karel Paul, Vladimír Krajča, Zdeněk Roth, Jan Melichar and Svojmil Petránek 

Chapter 14

Handedness of Children Determines Preferential Facial and Eye Movements Related to Hemispheric Specialization Carmina Arteaga and Adrián Poblano 

313 

Intact Environmental Habituation and Epinephrine-Induced Enhancement of Memory Consolidation for a Novel Object Recognition Task in Pre-Weanling Sprague-Dawley Rats Robert W. Flint, Shelby Hickey and Maryann Dobrowolski  Reviewed by Matthew Anderson 

323 

Chapter 15

Index

301 

339 

PREFACE Chapter 1 - With regard to the specificity of the developing brain, a better understanding of cellular mechanisms involved in perinatal hypoxic-ischemic injury would help to prevent neurological impairments. The authors therefore examined temporal features of brain injury in three different developmental models of oxygen deprivation capable of inducing apoptotic cell death. Nuclear staining by DAPI (4,6-diamidino-2-phenylindole) was used to identify healthy, apoptotic and necrotic nuclei as well as for cell counting in cultures and brain sections. DNA fragmentation was monitored by in situ terminal dUTP nick end labeling (TUNEL) and electrophoresis on agarose gels. Also, the expression profile of apoptosisrelated proteins Bax and Bcl-2 was studied by immunohistochemistry. In all cases, oxygen deprivation induced significant delayed cell death with morphological features of apoptosis and a progressive increase in the Bax/Bcl-2 protein ratio, except in the penumbra of the ischemic infarct where Bcl-2 remained predominant. As in the control newborn brain that still exhibited physiological death, hypoxia-associated DNA breakdown led to small fragments of ~200 bp in the cortex of hypoxic rat pups. Ladder pattern and TUNEL-positive cells exhibiting apoptotic bodies were only present in the penumbra of 7 day-old ischemic rats. These data indicate that hallmarks of hypoxia-induced apoptosis may vary according to brain maturity, possibly through specific nuclease activities. While retaining a part of the developmental death program, the newborn brain seems to be prone to an apoptotic-like response that resembles physiological programmed death. Chapter 2 - Programmed cell death or apoptosis is a relevant process in the physiology and pathology of the nervous system. Apoptosis is an organized form of cell death which is triggered by different factors including DNA damage. A growing body of evidence suggests that DNA damage and genomic instability are involved in neuronal abnormalities and may play a central role in neurodegeneration. DNA strand breaks and DNA lesions have been reported in Parkinson's and Alzheimer's diseases and as an early event after reperfusion of ischemic brain. DNA damage has been found to activate a cell death program in terminally differentiated postmitotic neurons. Since the genome is continuously damaged by a variety of endogenous and exogenous agents and the majority of DNA damage is produced by oxyradicals, generated by normal aerobic metabolism, neurons are particularly susceptible to DNA damage due to the high rate of oxidative metabolism. To maintain genomic integrity, cells are equipped with special defense mechanism, DNA damage response, to remove DNA damage by DNA repair pathways or eliminate damaged cells via apoptosis. Generally, differentiated cells, like neurons, are deficient in DNA repair and more vulnerable to DNA

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damage-initiated apoptosis. For example, neurons are more vulnerable than astrocytes to DNA-damaging conditions such as ionizing radiation. Breast cancer patients receiving hemotherapy commonly experience long-lasting cognitive impairment. It is known that radiotherapy may cause CNS toxicity. DNA damaging agents including γ-irradiation induce neuronal apoptosis in vitro, suggesting the direct adverse effect of these DNA damaging agents on neurons. The importance of DNA repair for neuronal survival is illustrated by disorders observed in patients with hereditary DNA repair abnormalities. These disorders combine the predisposition to cancer with progressive neurodegeneration. Although indirect evidence suggests that DNA damage and repair mechanisms play critical roles in neuronal survival, the pathways involved are poorly understood. Recently, the authors have found that cell cycle activation is essential for DNA damage-induced neuronal apoptosis which suggests that the cell cycle machinery is a critical element of the DNA damage response not only in cycling but also in quiescent cells. Here, the authors discuss the DNA damage response in postmitotic neurons and possible mechanisms by which neurons are forced to apoptosis versus DNA repair thereby controlling cell fate. Elucidation of these mechanisms promises to provide multiple points of therapeutic intervention in neurodegenerative diseases. Chapter 3 - The nuclear circular DNA population has been analyzed in mouse brain cells. The brain is active in producing extrachromosomal nuclear circular DNA during the embryonic and newborn neonatal stage. One circular DNA, BC-1,1 was cloned from a mouse embryonic circular DNA library. The genomic region containing the BC-1 DNA sequence was shown to undergo somatic DNA recombination yielding a DNA deletion and circular DNA in mouse embryonic brain. The genomic BC-1 region is also active in DNA recombination in non-brain organ tissue such as the ocular lens and spleen. Although the BC1 region contains an evolutionally conservative DNA sequence homologous to the DNA sequence on human chromosome 3, the BC-1 does not contain any conventional exon and intron structure. The physiological significance and the molecular mechanism of the BC-1 DNA recombination and the BC-1 RNA expression are not clear. In this study, the DNA sequence surrounding the BC-1 region and BC-1 RNA expression are further analyzed as a first step in order to explain for the mechanism of the somatic BC-1 DNA recombinational events. Chapter 4 - The pathophysiology underlying the ischemic injury of white matter has, in recent times, been under intense investigation. As a result, significant inroads have been made in elucidating the mechanisms of injury that lead to pathology observed throughout life, from periventricular leukomalacia (PVL) in the neonate, to stroke in adulthood. To the surprise of many working in the field there are both remarkable similarities and important differences between the ischemic injury of the more classically studied grey matter and its white matter counterpart. In the mature CNS early studies using isolated white matter tracts first demonstrated the importance of the Na+-Ca2+ exchange protein in mediating a toxic Ca2+ influx. Ca2+ channels have also been implicated, by both providing the conduit for Ca2+ entry and mobilising Ca2+ from internal stores. More recently, NMDA and AMPA receptors have been shown to play important roles in the development of irreversible white matter injury, both in mature white matter and during an important developmental window. With regard to development, injury to white matter in the form of PVL is the primary pathology associated with the most common human birth disorder, cerebral palsy. Oligodendrocytes, the myelin forming cells of the central nervous system, have been a primary focus of research in this field and their progenitors have been shown to be especially susceptible to ischemic injury. A

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sound understanding of such pathways will be essential if successful therapeutic strategies are to be developed. Here, we review the remarkable progress made in what may still be viewed as a developing field, as researchers work towards unravelling the physiology behind the pathology. Chapter 5 - Functional neuroimaging studies have significantly advanced our understanding of human cognitive functions. However, much less is known about the anatomical connections underlying higher cognitive processes in humans. One of the reasons why anatomical studies have lagged behind functional studies is that there are methodological limitations on studying anatomical connections of the human brain in vivo. There are numerous detailed anatomical studies of non-human primates that serve as the basis of our understandings of connections in the brain. However, those techniques are not feasible in humans. Diffusion imaging is a new technique based on detecting the diffusion of water molecules from magnetic resonance images. Diffusion imaging allows non-invasive mapping of anatomical connections and gives a comprehensive picture of connectivity throughout the brain, but there are still numerous technical issues to be addressed. Here, I introduce our recent studies on large-scale anatomical connections underlying episodic memory in humans. We studied an entire network based on some episodic memory tasks, and applied several new approaches to assess our tractography results. Our main finding was that encoding-related areas in the left dorsolateral prefrontal cortex and the left ventrolateral prefrontal cortex connect with another encoding-related area in the left temporal cortex. This suggests that there are two pathways between prefrontal cortex and temporal cortex related to encoding processes in episodic memory. Further, I discuss future applications of diffusion imaging in the study of the human memory system. Chapter 6 - Cyclooxygenase-2COX-2expression is induced in the brain in various pathological conditions, such as fever, pain, and neurological disorders related to neuroinflammation. Therefore, it is important to elucidate the roles of COX-2 and the effects of COX-2 inhibitors in the central nervous system. Here, we review the modulatory roles of COX-2 and its product, prostaglandinE2 (PGE2, in fever and pain, and discuss the effects of COX-2 inhibitors. In addition, we will review the latest findings regarding the neuroprotective effects of COX-2 inhibitors on neuronal loss regarding neuroinflammation associated with brain diseases, including epilepsy, ischemia, amyotrophic lateral sclerosis, Parkinson’s disease, multiple sclerosis, and Alzheimer’s disease. We also discuss the roles of non-steroidal anti-inflammatory drugs (NSAIDs, such as COX inhibitors and peroxisome proliferator-activated receptor-γ PPAR-γ agonists. Brain diseases have neuroinflammatory aspects involving the activation of microglia related to neuronal loss, and PPAR-γ agonists have been shown to inhibit the activation of microglia. Furthermore, we address two common points concerning various diseases. We discuss the clinical application of selective COX-2 inhibitors to neuronal death induced by epilepsy and ischemia. The short-term and sub-acute cure achieved using selective COX-2 inhibitors matching the elevation of PGE2 is expected for treatment after onset of neuronal excitatory diseases to prevent neuronal loss. We also discuss the responses in vascular endothelial cells related to fever and epilepsy. In the endothelial cells, mPGES-1 is colocalized with COX-2, suggesting that the two enzymes are functionally linked and that brain endothelial cells play an essential role in PGE2 production during fever and epilepsy.

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Further analysis of COX-2 inhibitors may provide a better understanding of the process of neuropathological disorders, as well as facilitate the development of new treatment regimens. Chapter 7 - Neurophysiological studies in Creutzfeldt-Jakob Disease (CJD) are mostly centred on the appearance, during development of the disease, of an electroencephalogram (EEG) called “typical”, which converts the clinical suspicion into a likely diagnosis. Early diagnosis avoids another series of unnecessary procedures, prevents iatrogenic transmission and recognises the invariably fatal prognosis. The EEG as a diagnostic tool is based on interpreting a series of graphical elements that express the brain’s bio-electrical activity as a particular form of language. Its conventional meaning, mainly based on practical aspects, has allowed a series of basic electroencephalogram patterns to be defined. Performing an EEG comprises three major stages: the first is the detailed analysis of the graphical elements of which it is composed; the second, matching it with one of the defined patterns and, lastly, the identification of EEG patterns with a sociological value, i.e., trying to establish the appropriate electro-clinical correlation. Within the basic EEG pattern catalogue, the typical findings seen in the course of CJD are included in a large group of periodic activities. Nevertheless, two important aspects must be considered: on the one hand the fact that the EEG is a dynamic test that presents wide variations in the evolution of the disease and, on the other hand, the lack of typicality in the new variant of the disease (vCJD) and in the genetic subtypes that lead us to seek new ways of trying to establish a neurophysiological characterisation of the disease. In this chapter, in the first place, we will discuss the EEG findings in the course of the evolution of sporadic CJD and the aspects differentiating them from other phenotypes before explaining the current status and future prospects for neurophysiological studies. Chapter 8 - Neurofilaments are the main components of intermediate filaments in neurons, and are expressed under three different subunit proteins, NFL, NFM and NFH. Neurofilaments act with microtubules and microfilaments to form and maintain the neuronal structure and cell shape. Phosphorylation is the main post-translational modification of neurofilaments, which influences their polymerization and depolymerization, and is responsible for their correct assembly, transport, organization and function in the neuronal process. In particular, phosphorylation is essential for the repulsion of the neurofilament polymers in axons, which determines the axonal diameter and the velocity of electrical conduction. The phosphorylation state of neurofilaments is regulated in a complex manner, including interactions with the neighbouring glial cells. Abnormal expression, accumulation or post-translational modifications of neurofilament proteins are found in an increasing number of described neurological diseases, such as amyotrophic lateral sclerosis, Parkinson’s, Alzheimer’s and Charcot-Marie-Tooth diseases, or giant axonal neuropathy. Some of these diseases are associated with mutations discovered in the neurofilament genes. Recently, altered expression and phosphorylation states of neurofilament proteins have also been shown in metabolic diseases affecting the central nervous system either during development or in adulthood, such as hepatic encephalopathy due to hyperammonemia, methylmalonic and propionic acidemias, and diabetic neuropathy. Finally, accumulation of neurofilament proteins in the cerebrospinal fluid has been described as discriminating marker for patients with multiple sclerosis, and as predictor of long-term

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outcome after cardiac arrest. This review will focus on the most recent investigations on neurofilament proteins in neurodegenerative, neurodevelopmental and metabolic diseases, as well as on the use of neurofilaments as markers of diseases. Chapter 9 – Magnetic resonance imaging (MRI) is commonly employed for the depiction of soft tissues, most notably the human brain. Computer-aided image analysis techniques lead to image enhancement and automatic detection of anatomical structures. However, the intensity information contained in images does not often offer enough contrast to robustly obtain a good detection of all internal brain structures, not least the deep gray matter nuclei. We propose digital atlases that deform to fit the image data to be analyzed. In this application, deformable atlases are employed for the detection and segmentation of brain nuclei, to allow analysis of brain structures. Our fully automatic technique is based on a combination of rigid, affine and non-linear registration, a priori information on key anatomical landmarks and propagation of the information of the atlas. The Internet Brain Segmentation Repository (IBSR) data provide manually segmented brain data. Using prior anatomical knowledge in local brain areas from a randomly chosen brain scan (atlas), a first estimation of the deformation fields is calculated by affine registration. The image alignment is refined through a non-linear transformation to correct the segmentation of nuclei. The local segmentation results are greatly improved. They are robust over the patient data and in accordance with the clinical ground truth. Validation of results is assessed by comparing the automatic segmentation of deep gray nuclei by the proposed method with manual segmentation. The technique offers the accurate segmentation of difficultly identifiable brain structures in conjuncture with deformable atlases. Such automated processes allow the study of large image databases and provide consistent measurements over the data. The method has a wide range of clinical applications of high impact that span from size and intensity quantification to comprehensive (anatomical, functional, dynamic) analysis of internal brain structures. Chapter 10 - Congenital strabismus affects 3% of world population. Millions of persons suffer this condition, but still its origin or the reasons why not all patients respond to the traditional treatment are unknown. Until very recently, it was believed that congenital strabismus had no relation to cortical alterations; therefore, neuroimaging studies were only required when strabismus was present in premature infants or when brain damage was suspected. A preliminary study on strabismal patients in 1968 provided some insight into the incidence of the different presentations of strabismus in our institution, as well as the correlation among the various clinical signs. Based on this experience we decided to enlarge our sample. Using conventional EEG and digitized brain mapping (DBM) methods, we analyzed 195 young patients with clinical diagnosis of congenital strabismus –111 females (56.92%) and 84 males (43.08%); the age range was from 2 to 14 years. The DBM approach was done in real time. Given its low cost, security and availability, DBM turned to be a useful tool to evince some alterations in cerebral cortex related to congenital strabismus, especially dissociated strabismus. We also employed complementary neuroimaging methods for research purposes. From 195 DBM images, 56.4% exhibited various neuroelectric alterations, whereas 43.6% were considered normal. Abnormal DBM were more frequent in the dissociated strabismus group (64.95%) than in non-dissociated strabismus patients (42.6%); the rate of altered DBM images was higher in horizontal dissociated deviation cases (73.3%). Based on

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these findings, we recommend the use of DBM in patients with dissociated strabismus, and in some cases the treatment must go beyond surgery and glasses. Some of our patients were subjected to different neuroimaging methods, such as single Photon emission tomography (SPECT), magnetic resonance imaging (MRI), granulometry, and proton nuclear magnetic resonance spectroscopy (1H NMRS) with the aim of correlating this data and gain further understanding on the origin of congenital strabismus, particularly dissociated strabismus cases. This chapter addresses aspects of congenital strabismus, as well as some of its cortical implications –neuroelectric, neurometabolic and morphometric. The illustrations are meant to make this interesting and scarcely-explored topic more accessible. Chapter 11 - Within the field of cognitive neuroscience, brain mapping strategies aim to localize neurological function within specific regions of the human brain. The burgeoning fields of functional magnetic resonance imaging (fMRI) and functional electrophysiology seek to map the human brain with ever-improving resolution. However, these functional strategies do not enable real-time, intraoperative discrimination of functional and nonfunctional brain parenchyma with precise, well-defined margins, as are necessary for surgical guidance and resection. To address the need for an intraoperative brain mapping strategy aimed specifically at neurosurgical guidance at resection, we have developed a novel brain mapping technique that we term preoperative endovascular brain mapping (PEBM). PEBM combines a super-selective, intraarterial approach with the delivery of visually detectable contrast agents to identify specific regions of functional and non-functional brain before and during craniotomy for brain resections. Our novel approach aims to avoid additional postoperative neurological deficits which would occur if functional brain parenchyma is inadvertently injured during an aggressive resection. Endovascular brain mapping aims to preserve brain function by providing a means of direct volumetric surgical guidance in realtime, whereby non-functional tissues are delineated by sharp, visible margins and can therefore be safely resected. The successful implementation of PEBM is highly dependent upon the proper selection and use of imaging probes, and we have developed a number of novel multimodal chemistries specifically aimed at PEBM. In this chapter, we will describe the PEBM technique in detail by highlighting its use in various small animal models, as well as our ongoing development of novel imaging probes suitable for PEBM. Chapter 12 - The investigation of techniques for neuroprotection plays a key role in brain research which involves finding a protective method for acute or chronic destruction of brain tissue. These methods are aimed either toward the necrotic pathway or the apoptotic one. The ability of acetylsalicylic acid (aspirin) to alleviate both destructive pathways is increasingly being recognized, as well as there being indirect evidence for its effective use in the attenuation of severity of neurologic diseases. The relation between the neuroprotective effects and the dosage of aspirin are not yet in agreement. The rationale of action appears to be aspirin’s direct and indirect specific effect on the nuclear factor Kappa β (NF Kappa-). Other targets of aspirin activity are the mitogen activated protein kinase (MAPK), the nitro oxide synthase (NOS) and the adenosine triphosphate (ATP). The protective effect of aspirin was studied in hypoxic damage, cerebral infarction, degenerative brain disease and epilepsy. An aspirin-induced apoptotic phenomenon was documented in gastric colon, lung and cervical cancer. Evidence of the same mechanism was shown also in brain malignant glioblastoma cells. The antiapoptotic and antitumoral effects are mediated by the Bcl-2 and

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caspase-3 pathways, as well as the mitochondrial permeability transfer mechanism. The proand anti-apoptotic mechanisms studied in regards to brain ischemic events are still unresolved issues. However, data from direct and indirect in vitro and in vivo studies, as well as epidemiological studies, lead to the assumption that aspirin probably does have an in vivo protective effect in humans. The promising data from these experimental studies bode well for an optimistic view for the possibility of aspirin’s therapeutic use as a neuroprotective agent in human diseases of the central nervous system. Chapter 13 - Background and purpose: While EEG features of the maturation level and behavioral states are visually well distinguishable in fullterm newborns, the topographic differentiation of the EEG activity is mostly unclear in this age. The aim of the study was to find out wether the applied method of automatic analysis is capable of descerning topographic particulaities of the neonatal EEG. A quantitative description of the EEG signal can contribute to objective assessment of the functional condition of a neonatal brain and to rafinement of diagnostics of cerebral dysfunctions manifesting itself as “dysrhytmia”, “dysmaturity” or “disorganization”. Subjects and methods: We examined polygraphically 21 healthy, full-term newborns during sleep. From each EEG record, two five-minute samples were subject to off-line analysis and were described by 13 variables: spectral measures and features describing shape and variability of the signal. The data from individual infants were averaged and the number of variables was reduced by factor analysis. Results: All factors identified by factor analysis were statistically significantly influenced by the location of derivation. A large number of statistically significant differences was also found when comparing the data describing the activities from different regions of the same hemisphere. The data from the posterior-medial regions differed significantly from the other studied regions: They exhibited higher values of spectral features and notably higher variability. When comparing data from homotopic regions of the opposite hemispheres, we only established significant differences between the activities of the anterior-medial regions: The values of spectral features were higher on the right than on the left side. The activities from other homotopic regions did not differ significantly. Conclusion: The applied method of automatic analysis is capable of discerning differences in the sleep EEG activities from the individual regions of the neonatal brain. Significance: The capability of the used method to discriminate regional differences of the neonatal EEG represents a promise for their application in clinical practice. Chapter 14 - Background: Despite repeated demonstrations of asymmetries in several brain functions, the biological bases of such asymmetries have remained obscure The objective of study was to investigate development of lateralized facial and eye movements evoked by hemispheric stimulation in right-handed and left-handed children. Methods: Fifty children were tested according to handedness by four tests: I. Monosyllabic non-sense words, II. Tri-syllabic sense words, III. Visual field occlusion by black wall, and presentation of geometric objects to both hands separatelly, IV. Left eye and the temporal half visual field of the right eye occlusion with special goggles, afterwards asking children to assemble a three-piece puzzle; same tasks were performed contralaterally. Results: Right-handed children showed higher percentage of eye movements to right side when stimulated by tri-syllabic words, while left-handed children shown higher percentages of eyes movements to left side when stimulated by tri-syllabic words. Left-handed children spent more time in recognizing mono-syllabic words. Hand laterality correlated with tri-

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syllabic word recognition performance. Age contributed to laterality development in nearly all cases, except in second test. Conclusions: Eye and facial movements were found to be related to left- and right-hand preference and specialization for language development, as well as visual, haptic perception and recognition in an age-dependent fashion in a very complex process. Chapter 15 - Within- and between-session environmental habituation were examined in infant rats on postnatal days 14 and 15 in an open field. Using a computerized animal tracking system, rats showed decreases in the total distance traveled (m) and overall average speed (m/s) across 6 30-sec time blocks each day and from day 1 to day 2 of testing. Although the literature is inconclusive regarding the ontogeny of environmental habituation, these results provide clear evidence of both within-session and between-session habituation. On postnatal day 16, animals were returned to the open field with 2 identical objects for novel object recognition training. Animals were either tested immediately after training or were given a subcutaneous injection of saline or .01 mg/kg of epinephrine, followed by testing 2-hrs later. For testing animals were placed into the open field with one familiar and one novel object and the number of object explorations and the time spent exploring each object were recorded by the animal tracking system. From these measures the absolute mean preference for novelty and relative percent preference for novelty were computed. Only the relative percent preference for novelty based on the time spent exploring each object revealed significant differences among the groups. Post-hoc pair-wise comparisons indicated that saline animals tested 2-hrs after training performed significantly worse than epinephrine animals and worse than those tested immediately after training. This indicates a rapid rate of forgetting for object recognition memory which is effectively attenuated with post-training epinephrine. Versions of these chapters were also published in Brain Research Journal Volume 1, Numbers 1-4, edited by Frank Columbus, published by Nova Science Publishers, Inc. They were submitted for appropriate modifications in an effort to encourage wider dissemination of research.

In: Brain Mapping and Diseases Ed: Diane E. Spinelle

ISBN: 978-1-61122-065-0 © 2011 Nova Science Publishers, Inc.

Chapter 1

HALLMARKS OF APOPTOTIC-LIKE CELL DEATH IN RESPONSE TO HYPOXIC INJURY IN VARIOUS DEVELOPMENTAL MODELS ARE CLOSELY RELATED TO BRAIN IMMATURITY Jean-Luc Daval1,∗ and Christiane Charriaut-Marlangue2 1

INSERM U.724, Université Henri Poincaré, Faculté de Médecine, 9 avenue de la Forêt de Haye, F-54500 Vandoeuvre-lès-Nancy, France; 2 Groupe Hypoxie et Ischémie du Cerveau en Développement, Université Pierre et Marie Curie-Paris6, UMR-CNRS 7102, 9 quai St-Bernard, Paris, F-75005 France.

ABSTRACT With regard to the specificity of the developing brain, a better understanding of cellular mechanisms involved in perinatal hypoxic-ischemic injury would help to prevent neurological impairments. We therefore examined temporal features of brain injury in three different developmental models of oxygen deprivation capable of inducing apoptotic cell death. Nuclear staining by DAPI (4,6-diamidino-2-phenylindole) was used to identify healthy, apoptotic and necrotic nuclei as well as for cell counting in cultures and brain sections. DNA fragmentation was monitored by in situ terminal dUTP nick end labeling (TUNEL) and electrophoresis on agarose gels. Also, the expression profile of apoptosis-related proteins Bax and Bcl-2 was studied by immunohistochemistry. In all cases, oxygen deprivation induced significant delayed cell death with morphological features of apoptosis and a progressive increase in the Bax/Bcl-2 protein ratio, except in the penumbra of the ischemic infarct where Bcl-2 remained predominant. As in the control newborn brain that still exhibited physiological death, hypoxia-associated DNA breakdown led to small fragments of ~200 bp in the cortex of hypoxic rat pups. Ladder pattern and TUNEL-positive cells exhibiting apoptotic bodies were only present in the ∗

Correspondence concerning this article should be addressed to Dr. Jean-Luc Daval, INSERM U.724, Université Henri Poincaré, Faculté de Médecine, 9 avenue de la Forêt de Haye, F-54500 Vandoeuvre-lèsNancy, France. E-mail: [email protected].

2

Jean-Luc Daval and Christiane Charriaut-Marlangue penumbra of 7 day-old ischemic rats. These data indicate that hallmarks of hypoxiainduced apoptosis may vary according to brain maturity, possibly through specific nuclease activities. While retaining a part of the developmental death program, the newborn brain seems to be prone to an apoptotic-like response that resembles physiological programmed death.

INTRODUCTION Apoptosis is considered as the ubiquitous form of naturally occurring cell death that plays a fundamental role in brain development, as about half of the cells in the immature brain are eliminated by apoptosis (Oppenheim, 1991). This physiological process needs the activation of an intrinsic ‘death program’ requiring time and energy (Richter et al., 1996) as well as gene transcription and translation (Pittman et al., 1993). Indeed, the apoptotic cascade involves the participation of ‘killer’ proteins (e.g., Bax) generally present constitutively in the cell but normally repressed by their survival counterparts (e.g., Bcl-2). Morphologically, apoptosis is typified by cell shrinkage, chromatin condensation and subsequent nucleus fragmentation, with a specific pattern of DNA breakdown leading usually to multiple segments of approximately 200 bp in length. By contrast, the second form of cell death, namely necrosis, which has been implicated in cell destruction consecutive to severe trauma, induces cellular swelling, membrane disruption with leakage of cell contents to the extracellular space, and leads to random DNA degradation (Wyllie, 1981). In response to a variety of insults, all cells seem to be able to undergo apoptosis but there is compelling evidence to suggest that developing brain cells are more prone to programmed death by retaining a part of the developmental cell death program (Blaschke et al., 1996; Sidhu et al., 1997). Perinatal hypoxic-ischemic injury remains a major cause of mortality and cerebral morbidity, susceptible to generate permanent neurological sequelae (Volpe, 1987; Berger and Garnier, 1999). Whereas necrotic cell damage was first considered prevalent in response to hypoxia-ischemia, the participation of apoptosis has been largely documented, including in the developing brain (Beilharz et al., 1995; Charriaut-Marlangue et al., 1996; Bossenmeyer et al., 1998, Bossenmeyer-Pourié et al., 2002; Daval et al., 2004). In addition, some studies suggest an apoptotic-necrotic continuum in the consequences of acute cerebral ischemia (Nakajima et al., 2000; Benchoua et al., 2001). Since the developing brain displays specific properties and sensitivity to oxygen supply (Grafe, 1994), hallmarks of cell injury may thus vary according to brain maturity, and the present study was designed to gain a better understanding of the cellular mechanisms triggered by a severe hypoxic insult at various ages of the perinatal period. For this purpose, we compared hypoxia-associated features of cell death in three different developmental models of oxygen deprivation capable of inducing brain apoptosis, i.e. in cultured neurons from the embryonic rat forebrain exposed to 95% N2/5% CO2 (Bossenmeyer et al., 1998), in the rat neonate exposed to 100% N2, a model corresponding to birth asphyxia (Grojean et al., 2003), and in the 7-day-old rat pup subjected to transient unilateral focal ischemia followed by reperfusion (Renolleau et al., 1998). In contrast, exposure to 100% N2 in 7 day-old rat induced mainly necrosis and did not enter this study (JLD, unpublished results).

Hallmarks of Apoptotic-Like Cell Death in Response to Hypoxic Injury…

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EXPERIMENTAL PROCEDURES Animals Experiments were conducted in respect to the French (Statement N° 04223) and European Community guidelines for the care and use of experimental animals. SpragueDawley female rats (R. Janvier, Le Genest-St-Isle, France) in the proestrus period, as shown by daily vaginal smears, were housed together with males for 24 h, and then maintained during gestation in separate cages under standard laboratory conditions on a 12:12 h light/dark cycle (lights on at 6:00 a.m.) with food and water available ad libitum.

Hypoxia in Neuronal Cell Cultures Primary cultured neurons were obtained from the embryonic rat brain as previously described (Bossenmeyer et al., 1998; Chihab et al., 1998; Bossenmeyer-Pourié et al., 1999a; Grojean et al., 2000). Forebrains of 14-day-old embryos were carefully collected, dissected free of meninges and dispersed in culture medium consisting of a mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium (50:50, ICN Pharmaceuticals, Costa Mesa, CA) supplemented with 5% inactivated fetal calf serum (Valbiotech, Paris, France). After centrifugation at 700 g for 10 min, the pellet was dispersed in the same medium and passed through a 46 µm-pore size nylon mesh. Aliquots of the cell suspension were transferred into 35 mm Petri dishes (Falcon, Becton Dickinson, Le Pont-de-Claix, France) precoated with poly-L-lysine in order to obtain a final density of 106 cells/dish. Cultures were then placed at 37°C in a humidified atmosphere of 95% air/5% CO2. The following day, the culture medium was replaced with a fresh hormonally defined serum-free medium corresponding to the DMEM/Ham's F12 mixture enriched with human transferrin (1 mM), bovine insulin (1 mM), putrescine (0.1 mM), progesterone (10 nM), estradiol (1 pM), Na selenite (30 nM), and also containing fibroblast growth factor (2 ng/ml) and epidermal growth factor (10 ng/ml) (Sigma Chemicals, St Louis, MO). Two days later, the culture medium was renewed with serum-free medium in the absence of growth factors. After 6 days in vitro, neuronal cells were exposed to hypoxia for 6 h by transferring culture dishes to a humidified incubation chamber thermoregulated at 37°C and filled with 95% N2/5% CO2. Cultures were then returned to normoxic atmosphere, at 37°C, whereas control cells were constantly maintained under standard conditions. When used, 1 µM cycloheximide (CHX, Sigma Chemicals) was added to the culture medium prior to hypoxia and removed by changing the medium as soon as reoxygenation began. Cells were studied as a function of time until 96 h post-reoxygenation to assess the effects of hypoxia.

In Vivo Birth Hypoxia Between 8 to 24 h after delivery, the litter size was reduced to 10 pups for homogeneity, and 5 neonates were placed for 20 min in a thermostated plexiglas chamber flushed with 100% N2, whereas the remaining 5 pups were taken as matched controls and exposed to 21% O2/79% N2 for the same time. The rate of gas delivery inside the box was calculated to

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prevent any overpressure, and was fixed to 3 liters/min. Gas in excess was evacuated through a central lengthwise split on the top of the box. The temperature inside the chamber was adjusted to 36°C to maintain body temperature in the physiological range. Following exposure to gas, all rats were allowed to recover for 20 min in normoxic conditions, and they were then returned to their dams. In these conditions, the final rate of mortality in the hypoxia group was 4%, and surviving animals did not display significant suckling problems. Hypoxic and control rats were finally sacrificed by decapitation at various time intervals between 1 day and 13 days post-exposure. Their brains were rapidly collected and immediately frozen in methylbutane previously chilled to -30°C, and stored at -80°C in plastic bags until used. Thereafter, the brains were coated with embedding medium (4% carboxymethylcellulose in water), and cut at -20°C in a cryostat (Reichert Jung, Frigocut 2800, Les Ulis, France) to generate 5-µm coronal sections at the level of the rostral hippocampus, according to the developing rat brain atlas of Sherwood and Timiras (1970), and tissue sections were mounted onto glass slides for subsequent analyses.

In Vivo 7-Day Ischemia Ischemia was performed in 7 day-old rats (17-21 g) of both sexes, as previously described (Renolleau et al., 1998). Rat pups were anaesthetized with intraperitoneal injection of chloral hydrate (300 mg/kg). After 15 min, rats were positioned on their back and a median incision was made in the neck to expose left common carotid artery. Rats were then placed on the right side and an oblique skin incision was made between the ear and the eye. After excision of the temporal muscle, the cranial bone was removed from the frontal suture to a level below the zygomatic arch. Then, the left middle cerebral artery, exposed just after its apparition over the rhinal fissure, was coagulated at the inferior cerebral vein level. After this procedure, a clip was placed to occlude the left common carotid artery and was removed after 1 h. Carotid blood flow restoration was verified with the aid of a binocular loupe. Both neck and cranial skin incisions were then closed. During surgery, body temperature was maintained at 37-38°C. After awake, rat pups were transferred to their mother for time survival at 6, 24 and 48 h and 3 to 5 days after reperfusion.

Evaluation of Cell Damage Cell viability was measured in cultured neurons by the spectrophotometric method using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), according to Hansen et al. (1989). Neurons were incubated for 3 h at 37°C with MTT (500 µg/mL, Sigma Chemicals), washed twice with ice-cold phosphate-buffered saline (PBS), and lysed in dimethyl sulfoxide (DMSO) which solubilizes the residual formazan salt for subsequent quantification. Optical density was measured at 519 nm and data were compared to those obtained from sister control cells to which 100% viability was assigned. In vivo cell counts were performed to determine the percentage of damaged neurons in cerebral cortex. For this purpose, brain sections were fixed for 10 min in a mixture of ethanol:acetic acid (3:1), washed for 30 seconds in distilled water, air-dried, and then stained for 10 min with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI, Sigma Chemicals)

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in phosphate-buffered saline (0.5 µg/ml) (Wolvetang et al., 1994). Sections were washed twice with distilled water, air-dried, and treated with anti-fading medium (10 mg/ml pphenyldiamine in 90% glycerol, pH 9.0). The number of cell nuclei which were labeled by DAPI was scored at an excitation wavelength of 365 nm under fluorescence microscopy (Zeiss Axioscop, Strasbourg, France). Cell density was measured in the infragranular part of the parietal cortex (layers III-V) at a 40 x magnification in at least 3 separate experiments by counting cells in 3 distinct section areas delineated by an ocular grid of 1/400 mm2. For each selected field, only cells with their nuclei present in the focal plane were counted. Numbers of cells were calculated per mm2 and finally reported as percentages of change from matched controls.

Monitoring of Apoptosis and Necrosis Morphological hallmarks of apoptosis and necrosis were analyzed both in cultured neurons and tissue sections after nuclear labeling by DAPI, as previously documented (Bossenmeyer et al., 1998; Chihab et al., 1998; Bossenmeyer-Pourié et al., 1999b; Grojean et al., 2000; Park et al., 1997). Indeed, it has been demonstrated that healthy cells exhibit intact round-shaped nuclei with diffuse fluorescence, indicative of homogeneous chromatin. Necrotic cells are characterized by highly refringent smaller nuclei with uniformly dispersed chromatin, while condensation and fragmentation of chromatin lead to typically shrunken nuclei in apoptotic cells, along with apoptotic bodies. Characteristic nuclei were scored under fluorescence microscopy (Zeiss Axioscop, Strasbourg, France) at an excitation wavelength of 365 nm in at least 3 separate experiments by counting concerned cells in at least 3 distinct areas of 100 cells.

Electrophoretic Detection of DNA Fragmentation At 48 h and 96 h post-reoxygenation, hypoxic and control cultured neurons were washed twice with PBS, scraped off in 2 ml PBS, and the contents of 5 dishes were pooled to be centrifuged for 5 min at 700 g. Following in vivo birth hypoxia or ischemia, rat pups were sacrificed at various time intervals and the brains were rapidly removed, dissected on a cold plate and stored at -80° C. Cell pellets or tissue samples from separate brains were processed for DNA isolation according to Laird et al. (1991).They were gently homogenized and lysed in 0.5 ml of lysis buffer (100 mM tris-HCl at pH 8.5, 5 mM EDTA, 0.2% sodium dodecyl sulfate (SDS), and 200 mM NaCl) containing 100 µg/ml proteinase K. After 16 h incubation at 55°C, DNA was precipitated by adding one volume isopropanol with continuous agitation for 15 min at room temperature. Following centrifugation at 13000 g for 5 min the pellet was air dried and dissolved in TE buffer (several hours), treated with RNAse A (20 µg/ml) for 2 h at 37°C and DNA content determined spectrophotometrically. DNA (10 µg/lane) was electrophoresed on a 1% agarose gel in 100 mM tris borate (60 V for 4 h) in the presence of 0.3 mg/ml ethidium bromide and visualized with UV illumination.

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Detection of DNA Breaks by Nick End-Labeling Sections were processed for DNA strand breaks (TUNEL assay) using the in situ Cell Death Detection Kit, Fluorescein (Roche, Meylan, France) according to the manufacturer’s instructions. TUNEL assay reveals apoptosis and necrosis as previously reported (CharriautMarlangue and Ben-Ari, 1995).

Bax and Bcl-2 Immunohistochemistry The expression of the two prototypic apoptosis-related proteins Bax and Bcl-2 was analyzed in cultured neurons and rat brain coronal sections. In vitro neuronal cells were rinsed twice with PBS, then fixed for 10 min in methanol at -10°C, and rinsed again with PBS. Non-specific binding sites for IgG were blocked by incubating the cells for 20 min with 10% horse serum (Gibco-BRL, Inchinnan, U.K.) in PBS. Thereafter, cultures were incubated for 60 min at room temperature in buffer containing the primary antibody., i.e. a rabbit polyclonal antibody against Bax (N-20, Santa Cruz, Tebu, France) diluted at 1/20 or a goat polyclonal antibody against Bcl-2 (N-19, Santa Cruz) diluted at 1/40. Following two washing steps to remove unfixed antibody, the cells were incubated for 120 min in the presence of a second-step antibody corresponding to either an anti-rabbit IgG conjugated to indocarbocyanine (Cy3, dilution 1/80) or an anti-goat IgG conjugated to rhodamine (TRITC, dilution1/100), both from Jackson ImmunoResearch Laboratories (West Grove, PA). Cultured cells were finally washed 3 times with PBS, coverslipped using mounting medium (Aquapolymount), and kept in the dark until fluorescence analysis by means of a Zeiss Axioscop microscope. For quantitative analysis, cell fluorescence activity was computerized from microphotographs, and mean intensity was calculated by using Adobe Photoshop® software and expressed as arbitrary units of mean emission per 1 000 pixels (Bossenmeyer-Pourié et al., 2002; Bossenmeyer-Pourié and Daval, 1998). The results were finally reported as Bax/Bcl-2 protein ratios as a function of time after reoxygenation as well as in normoxic control cultures processed in parallel. Both proteins were also analyzed in rat brain coronal sections of 7 µm-thickness previously fixed by incubating the slides for 10 min at 4°C in acetic acid:ethanol (1:3) in the presence of 30% hydrogen peroxide. In these conditions, brain sections were incubated at 4°C for 48 h with the primary antibody against Bax (dilution 1/20) or Bcl-2 (dilution 1/30), and secondary antibodies were used at 1/50 and 1/100, respectively, for subsequent measurements as described above. In the case of coronal sections from 7 day-old ischemic pups, Bax and Bcl-2 immunoreactivity was visualized by the avidin-biotin peroxidase (Elite ABC kit, Vecstastain, Vector, AbCys, France). The peroxidase activity was evidenced with the use of 3,3'-diaminobenzidin (DAB) and 0.02 % hydrogen peroxide. The counting of Bax- and Bcl-2 positive cells within the core and penumbra was performed in 3-5 sections (at the level of the anterior commissure) using a x40 objective, and results were reported as indicated above.

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RESULTS Hypoxia in Neuronal Cell Cultures As illustrated in Table 1, a hypoxic episode in cultured neurons from the embryonic rat brain induced delayed cell death inasmuch as cell viability was not yet different from controls at 48 h post-reoxygenation. Thereafter, the number of living cells gradually decreased to reach 64% from controls at 96 h. At this experimental time point, a significant number of cell nuclei stained by the fluorescent dye DAPI exhibited characteristic apoptosis-related morphological features, such as condensed chromatin, whereas the presence of apoptotic bodies could be seen. Necrotic cells were also depicted, but the percentage of apoptotic nuclei increased more markedly, as revealed by cell counts. Moreover, a concomitant treatment with cycloheximide, a potent inhibitor of protein synthesis, had beneficial effects. In the presence of CHX, cell viability was not significantly affected and the number of apoptotic nuclei remained within control values (Table 1), suggesting that hypoxia triggers a programmed death process in cultured neurons. When DNA fragmentation was monitored on agarose gel, no significant degradation was observed at 48 h, in good agreement with viability and morphology data, whereas DNA fragmentation was shown at 96 h after the hypoxic insult (Figure 1). However, DNA alteration was only reflected by a smear, without visualization of a ‘ladder pattern’. Finally, immunohistochemical studies revealed detectable baseline values of the prototypic apoptosis-related proteins Bcl-2 and Bax. Following exposure to hypoxia, expression of the survival Bcl-2 protein transiently increased above control values at 48 h post-insult without apparent change in Bax expression, leading to a significantly reduced Bax/Bcl-2 ratio (Figure 2). At 96 h, Bcl-2 levels abruptly declined, while the expression of Bax was markedly stimulated, resulting in a robust increase of the final Bax/Bcl-2 ratio (Figure 2). Table 1. Effects of a 6-h exposure to hypoxia of cultured rat forebrain neurons on cell viability and nuclear hallmarks of necrosis and apoptosis at 48 h and 96 h postreoxygenation. Influence of cycloheximide.

Controls 48 h + 1 µM CHX Hypoxia 48 h + 1 µM CHX Controls 96 h + 1 µM CHX Hypoxia 96 h + 1 µM CHX

Cell viability (% from controls) 100.0 ± 4.6 90.6 ± 5.3 96.6 ± 4.3 92.0 ± 6.1 100.0 ± 5.8 86.4 ± 6.0* 63.6 ± 6.2** 88.5 ± 5.1*°°

Necrosis (% of total neurons) 4.9 ± 1.2 9.1 ± 2.3* 5.2 ± 1.1 11.0 ± 2.6*°° 5.6 ± 1.5 13.4 ± 2.6** 10.8 ± 2.8** 16.1 ± 3.0**°

Apoptosis (% of total neurons) 1.6 ± 0.6 1.2 ± 0.9 1.9 ± 0.9 1.3 ± 0.8 2.7 ± 0.8 2.8 ± 1.0 21.2 ± 5.7** 3.0 ± 1.2°°

Cell viability as well as rates of necrosis and apoptosis were analyzed as described in the method section. Data are reported as means ± S.D. obtained from 3 separate experiments. Statistically significant differences from controls: *p 90%), the resulting overall NF transport appears slow. NFs seem to use the conventional kinesin and dynein motor system (Shah et al., 2000; Yabe et al., 2000), and appear to dissociate from these motor systems after phosphorylation (Yabe et al., 1999). NFs are also translocated in dendrites of specific types of neurons, and seem required for the proper dendritic arborization of large motor neurons (Kong et al., 1998; Zhang et al., 2002). Two major modifications are added post-translationally on NFs: phosphorylation and glycosylation. These modifications are dynamic and thought to regulate assembly, transport, structure and functions of NFs. Various phosphorylation sites have been identified in the head (N-terminal) and tail (Cterminal) regions of NFs. The head region of NFL and NFM can be phosphorylated at different positions (Figure 1) by protein kinases A, C and N (Sihag and Nixon, 1989; Sihag and Nixon, 1991; Hisanaga et al., 1994; Mukai et al., 1996; Cleverley et al., 1998; Nakamura et al., 2000). The phosphorylation of the NFL and NFM head region occurs rapidly after protein synthesis in the neuronal cell body, and inhibits the NF filament assembly in perikaria (Gibb et al., 1996; Gibb et al., 1998; Ching and Liem, 1999). This phosphorylation is transient, and the dephosphorylation of the NFL and NFM head region is a prerequisite for the axonal NF assembly in filaments (Gibb et al., 1998). Moreover, the transient phosphorylation of the head region of NFM also inhibits the phosphorylation of its C-terminal tail region (Zheng et al., 2003). Thus, before NF translocation in the axons, the phosphorylation of the head region of NFL and NFM protects neurons from a pathological accumulation of NF aggregates in their cell bodies . Upon entry of NFs into the axon, the C-terminal side-arm domain of NFM and NFH, as well as the short C-terminal region of NFL, become phosphorylated. In particular, NFM and NFH are phosphorylated on Lys-Ser-Pro (KSP) repeat domains (Figure 1). In humans, NFM has 13 KSP repeats, while NFH exists with two polymorphic forms of either 44 or 45 KSP repeats (Figlewicz et al., 1993). Most of the serine residues of the KSP repeats can be phosphorylated, meaning that each mole of NFM and NFH can contain about 10 and 50 moles of phosphate, respectively (Julien and Mushynski, 1982; Grant and Pant, 2000). In axons, more than 99% of assembled NFM and NFH proteins are phosphorylated on their KSP repeats, in particular in myelinated internodal regions, while this proportion is much weaker in cell bodies, dendrites and nodes of Ranvier (de Waegh et al., 1992; Hsieh et al., 1994). Unphosphorylated NFs represent only ~1% of total NFs in the neurons. In the axon, NFs are

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phosphorylated in a proximal to distal gradient (Sternberger and Sternberger, 1983; Pant and Veeranna, 1995). The C-terminal region of NFL is phosphorylated by caseine kinase II (Nakamura et al., 1999), while the kinases that phosphorylate NFM and NFH KSP repeats in their C-terminal tail domains include GSK-3α/β, cdk5/p35, ERK1/2 and JNK1/3 (Guan et al., 1991; Giasson and Mushynski, 1996; Sun et al., 1996; Li et al., 2001). In axons, the phosphorylation of multiple KSP repeats increases the negative charge of NFM and NFH, resulting in side-arm formation of their C-terminal tail and increased interneurofilament spacing (Nixon et al., 1994). This allows the radial axonal growth (i.e. regulation of axonal caliber), which increases axonal conduction velocity (de Waegh et al., 1992; Yin et al., 1998). The C-terminal phosphorylation of NFs also slows down their transport rate in axons, and mediate interactions with other cytoskeleton proteins, in particular microtubules (Hisanaga et al., 1991; Yabe et al., 2001; Shea et al., 2003). The phosphorylation of NFM seems preferentially responsible for the radial axonal growth, while the phosphorylation of NFH acts on the NF transport rate and their interactions with other proteins (Lewis and Nixon, 1988; Rao et al., 1998; Rao et al., 2003). The myelination of axons, both by Schwann cells in peripheral nerves and by oligodendrocytes in CNS, promotes the phosphorylation of NFM and NFH C-terminal tail, thus promoting the radial growth of myelinated axons and increasing their conduction velocity (de Waegh et al., 1992; Sanchez et al., 1996; Yin et al., 1998; Sanchez et al., 2000). NFL, NFM and NFH are also post-translationally glycosylated by addtion of O-linked Nacetylglucosamine moieties on serine and threonine residues located in their head regions (NFL, NFM and NFH) as well as in their KSP repeat carboxyterminal region (NFM and NFH) (Figure 1) (Dong et al., 1993; Dong et al., 1996). The proximity of the OGlcNAcylation and phosphorylation sites in the NF head domain suggest that competition between the two modes of post-translational modifications regulates NF assembly (Gill et al., 1990; Wong and Cleveland, 1990; Chin et al., 1991; Dong et al., 1993). On the other hand, in the nodes of Ranvier where NFs are more closely packed than in the internode axonal segments, O-GlcNAcylation probably replaces phosphorylation in the carboxyterminal KSP repeat region of NFM and NFH, rendering interactions between NFs more attractive than repulsive. Therefore, phosphorylation / dephosphorylation and glycosylation / deglycosylation of NFs (by kinase / phosphatase and O-GlcNAc transferase / N-acetyl-β-D-glucosaminidase respectively) contributes to the assembly, structure and functions of NFs (Dong et al., 1993; Nixon, 1993; Xu et al., 1994; Dong et al., 1996). Many neurons extend very long axons, up to 1 m in humans. To maintain the integrity and functions of these axons, some of their structural proteins, including those of the axonal cytoskeleton, have long lifetimes. For NFs in the human sciatic nerve, this average lifetime was estimated to 1 to 2 years (Lee and Cleveland, 1996). This very high stability of NFs is thought to be due, at least in part, to their phosphorylation which protects them from protease degradation (Goldstein et al., 1987; Pant, 1988). In physiological conditions, NF degradation only occurs in the axon terminus (presynaptic compartment), where NFs are dephosphorylated by protein phosphatase 2A (PP2A) (Gong et al., 2003), and then digested by calmodulin, a Ca++-dependent protease (Maxwell et al., 1997).

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Apart from their major role in regulating axonal caliber in function of their state of phosphorylation, NFs have been demonstrated or are postulated to have other functions in the axon. While gene knockout experiments demonstrated that NFs are not essential for axonal elongation, they nervertheless might facilitate it by stabilization of cytoskeletal elements and inhibition of axonal retraction (Zhu et al., 1997; Elder et al., 1998a; Elder et al., 1998b; Elder et al., 1999a). NFs participate, together with microtubules and microfilaments, to the axonal structural integrity, to the neuronal shape as well as to the axonal mechanisms of transport. They do so by direct or indirect interactions with microtubules (Hisanaga et al., 1991) or motor proteins like dynein, kinesin and myosin Va (Yabe et al., 1999; Shah et al., 2000; Yabe et al., 2000; Rao et al., 2002a), or with other crosslinking proteins like dystonin (Yang et al., 1999; Chen et al., 2000). NFM has been shown to interact with the D(1) dopamine receptor in subsets of neurons (Kim et al., 2002). Finally, of peculiar importance for the neuronal and axonal long term stability, NFs seem to protect axons from toxic components, by sequestrating for example Cdk5/p25 complexes which induce apoptosis (Nguyen et al., 2001), or by coupling of carbonyl groups issued of the oxidative stress on the lysine residues of KSP repeats (Wataya et al., 2002).

NEUROFILAMENT PROTEINS IN BRAIN DISEASES As discussed above, the tight regulation of NF subunits expression, post-translational modifications, stoichiometry between NFL, NFM and NFH, and NF axonal transport, allows the correct assembly of NF filaments. This in turn contributes to the normal axonal growth, maturation, and stability along time. Any dysregulation of these precise mechanisms of NF regulations is susceptible to induce severe pathological consequences on neurons. In particular, the hallmark of numerous human neurological diseases is the abnormal accumulation of NFs in neuronal perikarya (for recent reviews, see Al-Chalabi and Miller, 2003; Liu et al., 2004; Lariviere and Julien, 2004; Petzold, 2005), which alters axonal growth, mechanisms of particles and organelles transportation, stability, and dynamic of interactions between NFs and other axonal proteins (Herrmann and Griffin, 2002). For a long time, it was admitted that NF abnormalities in human neurological disorders were secondary to neuronal dysfunctions. Recent studies demonstrate however that dysregulations of NFs themselves can be the cause of these pathologies. The second part of this review will focus on NF dysregulations in neurodegenerative, neurodevelopmental and metabolic diseases of central and peripheral nervous systems, as well as on the use of NFs as markers of specific diseases.

NFS IN NEURODEGENERATIVE DISEASES Amyotrophic Lateral Sclerosis (ALS) ALS is a progressive neurodegenerative disease affecting motor neurons in the brain and spinal cord, with a typical onset between 40 and 60 years of age. ALS patients usually die wihin 5 years after ALS diagnosis, due to motor neurons death and loss of function of the

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relative innervated muscles, and progressive partial or total paralysis. Most of the cognitive functions in ALS patients remain preserved. ALS is a heterogeneous syndrom, in which the neuropathological hallmark is an abnormal aggregation of NFs in the degenerating motor neurons (Manetto et al., 1988; Munoz et al., 1988). 5-10% of ALS cases are familial (autosomal dominant), while all the remaining cases are sporadic. 1-2% of all ALS cases (2025% of familial ALS cases) are due to mutations in the Cu/Zn superoxide dismutase (SOD1) gene (Andersen, 2006), while the basis of the remaining ALS cases is still not known with precision. Mutations in SOD1 are thought to be linked to abnormal accumulation of NFs in ALS (Rouleau et al., 1996). Due to the abnormal accumulation and aggregation of hyperphosphorylated NFs in the ALS degenerating neurons, mutations in the NF genes have also been sought for a long time as good causative candidates for ALS. Indeed, different mutations have been found in NFs, in association with ALS (Figures 2,3,4). Codon deletions and insertions have been identified in the KSP regions of NFH in association with few sporadic cases of ALS (Figure 4) (Figlewicz et al., 1994; Tomkins et al., 1998; Al-Chalabi et al., 1999). More recently, missense mutations have also been found in the head and rod domains of NFH in other ALS cases (Garcia et al., 2006) (Figure 4). In association with ALS, the same group also identified recently a deletion in the tail domain of NFL (Figure 2), as well as missense mutations in the head, rod and tail domains of NFM (Figure 3) (Garcia et al., 2006). However, none of the mutations found in NF genes have been clearly identified as causative agent of ALS, nor linked to the familial dominantly inherited ALS (Al-Chalabi and Miller, 2003; Garcia et al., 2006), and it is thought now that these mutations in NF genes have to be considered as risk factors for sporadic ALS. However, the alteration of NF homeostasis seems to be an important part of the pathogenesis of ALS (Figures 2,3,4). As shown with mutant SOD1 transgenic models of ALS (Nguyen et al., 2001), the deregulation of specific NF kinase pathways (e.g: cdk5/p35) might cause the aberrant hyperphosphorylation of NFH and NFM side arms. This in turn might slow the axonal transport of NFs, which accumulate in neuronal perikarya (Williamson and Cleveland, 1999). The abnormal accumulation of NFs in the ALS degenerating neurons has also been associated with a significative decrease of NFL mRNA, which could increase the imbalance between NF subunits and precipitate further the neuronal degeneration (Bergeron et al., 1994; Wong et al., 2000). This decrease in NFL mRNA seems due to the direct binding of mutant SOD1 to NFL mRNA, which destabilizes it (Ge et al., 2005). Interestingly, the two main posttranslational modifications of NFs, i.e. phosphorylation and glycosylation, might be conversely deregulated in ALS, as Oglycosylation of the C-terminal tail domain of NFM is decreased, while its phosphorylation is increased, in a transgenic rat model of ALS (Ludemann et al., 2005).

Charcot-Marie-Tooth Disease (CMT) CMT is the most common inherited neurological disorder of the peripheral nervous system, affecting 1-4:10’000 individuals. CMT clinical phenotype is characterized by the progressive degeneration of motor and sensory neurons in the distal part of the limbs, leading to the slow loss of normal use of feet, legs, arms and hands (Skre, 1974; Reilly, 2000). CMT neuropathies are heterogeneous in the genes involved and, based on electrophysiological criteria, are classified in CMT1, a primary demyelinating form with reduced nerve conduction

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velocities, and CMT2, a primary axonal loss form. Some forms of CMT with overlapping characteristics between CMT1 and CMT2 have been classified as intermediate CMT. CMT is generally inherited with an autosomal dominant pattern. Recently, different missense mutations and one amino acid deletion have been identified in the NEFL gene (coding NFL) in several families in association with CMT (Figure 2) (Mersiyanova et al., 2000; De et al., 2001; Georgiou et al., 2002; Yoshihara et al., 2002; Jordanova et al., 2003; Choi et al., 2004; Zuchner et al., 2004). All these mutations are associated with the primary axonal loss form CMT2, with the exception of Glu397Lys being associated with the demyelinating form CMT1. These mutations in NFL are thought to disrupt NF assembly and axonal transport, as well as to alter NFL post-translational modifications. Other forms of CMT (CMT1) are caused by mutations in genes primarily expressed in Schwann cells and involved in myelin formation. These mutations lead to alterations in myelination, which in turn alter NFL, NFM and NFH phosphorylation states (Watson et al., 1994). The disruption of NF assembly and the alteration of NF phosphorylation states are thought to contribute, at least in part, to the CMT disease mechanisms leading to axonal degeneration.

Figure 2. Schematic representation of NFL alterations in various brain diseases. Mutations identified in association with diseases are indicated above the NFL scheme, while the disease effects on NFL are indicated below the NFL scheme. AD: Alzheimer’s disease; ALS: amyotrophic lateral sclerosis; CMT1, CMT2: Charcot-Marie-Tooth disease; PD: Parkinson’s disease; Δ: deletion.

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Figure 3. Schematic representation of NFM alterations in various brain diseases. Mutations identified in association with diseases are indicated above the NFM scheme, while the disease effects on NFM are indicated below the NFM scheme. AD: Alzheimer’s disease; ALS: amyotrophic lateral sclerosis; CMT1, CMT2: Charcot-Marie-Tooth disease; MMA: methylmalonic aciduria; NH4: hyperammonemia; PA: propionic aciduria; PD: Parkinson’s disease.

Figure 4: Schematic representation of NFH alterations in various brain diseases. Mutations identified in association with diseases are indicated above the NFH scheme, while the disease effects on NFH are indicated below the NFH scheme. AD: Alzheimer’s disease; ALS: amyotrophic lateral sclerosis; CMT1, CMT2: Charcot-Marie-Tooth disease; i: insertion; MMA: methylmalonic aciduria; PA: propionic aciduria; PD: Parkinson’s disease; Δ: deletion.

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Parkinson Disease (PD) PD is a progressive neurodegenerative CNS disorder affecting dopaminergic neurons of substantia nigra and leading to decreased dopamine availability. The principal pathological modifications in PD affected neurons are the so-called Lewy bodies, which are inclusions of accumulated proteins in neuronal perikariya and are made of numerous proteins, including NFL, NFM and NFH, α-synuclein, ubiquitin and subunits of the proteasome (Galloway et al., 1992; Trimmer et al., 2004). In particular, abnormally phosphorylated NFs have been identified in PD associated Lewy bodies (Hill et al., 1991; Trojanowski et al., 1993), but the reasons for this alteration of NF phosphorylation have not been precisely identified so far (Figures 2, 3, 4). Familial forms of PD have also been identified, in which the principal mutations found are located in the parkin, α-synuclein and ubiquitin C-terminal hydrolase L1, all three related to cellular ubiquitin proteasomal system (Lim et al., 2003). A significative decrease of NF mRNAs and proteins has also been observed in the PD affected neurons of substantia nigra (Hill et al., 1993; Basso et al., 2004) (Figures 2, 3, 4). Recently, a point mutation in the NEFM gene, located in the rod domain 2b of NFM and changing Gly to Ser (Gly336Ser) (Figure 3), has been identified in a patient that developed PD very early, at the age of 16 (Lavedan et al., 2002). Due to the position of this mutation in the very highly conserved region of IFs (rod, α-helical coils) involved in their assembly mechanism, it was speculated that this mutation could alter NFM assembly into NF filaments (Lavedan et al., 2002). As this mutation has been found in only one PD patient which moreover had three unaffected siblings (Lavedan et al., 2002; Han et al., 2005), it is not sure so far that this mutation is really causative of PD. If yes however, the NFM G336S mutation does not seem to interfere with either assembly nor cellular distribution of NFs (Perez-Olle et al., 2004), but could rather alter interactions of NFM with other PD susceptibility proteins (Al-Chalabi and Miller, 2003).

Alzheimer’s Disease (AD) Among neurodegenerative diseases, AD is the leading cause of dementia, with risks over 65 years of age varying from 6-10% for men to 12-19% for women (Seshadri et al., 1997). CNS regions involved in memory and thinking skills are the first affected, followed by neuronal death in other brain regions as disease progresses, which eventually causes the death of the patient. Despite intensive work on AD, its precise cause is still unknown. One of the important secondary features of AD is the neuronal cytoskeleton disruption, due to the inappropriate hyperphosphorylation of cytoskeletal proteins such as tau or NFs (Sternberger et al., 1985; Gong et al., 2000) (Figures 3, 4). In particular, hyperphosphorylated NFH accumulates in neuronal perikaryon and proximal axon (Sternberger et al., 1985), due most probably to an imbalance between kinase and phosphatase activities (Trojanowski et al., 1993; Maccioni et al., 2001; Veeranna et al., 2004). After accumulation in neuronal perikarya, these cytoskeletal proteins aggregate in abnormally modified filaments, and progressively form the neurofibrillary tangles and AD senile plaques, which are the hallmarks of AD. Recently, hyperphosphorylated NFM has also been identified in AD amyloid plaques (Liao et

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al., 2004). NFL mRNA is also significatively decreased in AD degenerating neurons (McLachlan et al., 1988) (Figure 2).

NFs in Other Neurodegenerative Diseases The expression and post-translational modifications of NFs have been found altered in a number of other neurodegenerative conditions (summarized in figures 2, 3). Giant axonal neuropathy (GAN) is a rare autosomal recessive neurodegenerative disorder progressively affecting both peripheral and central nervous system. GAN is due to mutations of the gene encoding gigaxonin, a protein suggested to be associated to IFs (Bomont et al., 2000; Herrmann and Griffin, 2002). GAN, due to the gigaxonin disruption, is thus characterized by the presence of giant axons filled with massive segmental accumulations of disorganized NFs (Asbury et al., 1972; Herguner et al., 2005). A recent work has shown that leprous nerve atrophy, characterized by a diminution of axonal calibre and paranodal demyelination, might be due to dephosphorylation of NFM and NFH (Save et al., 2004). NFH have been shown to be dephosphorylated in an experimental model of glaucoma, a neurodegenerative condition affecting the optic nerve in association with high intraoccular pressure (Kashiwagi et al., 2003). Glutamate excitotoxicity induces a rapid degradation of the neuronal cytoskeleton. It was shown recently that glutamate toxicity, primarily mediated by NMDA receptor, initiates a rapid loss of NFs in the affected axons, while other axonal markers remain intact for a longer period (Chung et al., 2005). Distal hereditary motor neuronopathies (dHMNs) are a heterogeneous group of disorders in which motor neurons selectively undergo age-dependent degeneration. Mutations in the small heat-shock protein HSPB1 (also called HSP27) are responsible for one form of dHMN. The mutant forms of HSPB1 seem to disrupt NF assembly, to alter axonal transport system, and lead to the accumulation and aggregation, in neuronal perikarya, of cellular components, including NFM (Ackerley et al., 2006). Huntington's disease (HD) is caused by a polyglutamine repeat expansion in the Nterminal domain of the huntingtin protein. Huntingtin is localized in the cytoplasm where it may interact with cytoskeletal and synaptic proteins. The mechanism of HD pathogenesis remains unknown but recent investigations suggest that the mutant huntingtin found in HD might interact aberrantly with cytoskeletal proteins, including NFs, and thus affect the axonal cytoskeletal integrity (DiProspero et al., 2004). Neuronal intermediate filament inclusion disease (NIFID) is a recently described novel neurological disease of early onset, presenting considerable variability in clinical phenotypes, including frontotemporal dementia, as well as pyramidal and extrapyramidal signs. The pathological hallmark of NIFID is the presence of abnormal aggregates of α-internexin, NFL, NFM and NFH in the affected neurons (Cairns et al., 2004). α-internexin, a class IV IF protein, has not been identified in any pathological protein aggregates of any other neurodegenerative disease.

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NFS IN NEURODEVELOPMENTAL AND METABOLIC DISEASES Diabetes Neuropathy Diabetes is associated with a symmetrical distal axonal neuropathy predominantly affecting sensory nerves and neurons of dorsal root ganglia. Diabetic neuropathy is characterized by a reduced conduction velocity, and axonal atrophy. Both in human diabetic patients and in streptozotocin-induced diabetic rats, abnormal aggregations of NFs and other cytoskeletal proteins have been observed in the affected neurons, together with an abnormal increase of NFM and NFH phosphorylation (Figures 3, 4) (Schmidt et al., 1997; Fernyhough et al., 1999). These alterations of NF phosphorylation seem to occur through the activation of the NF kinase c-Jun N-terminal kinase (JNK) (Fernyhough et al., 1999; Middlemas et al., 2006). NFs mRNAs are reduced. The affected neurons present defects of axonal transport mechanisms, a reduction in axon calibre, and a diminished capacity of nerve regeneration, all characteristics relying on the integrity of axonal cytoskeleton. It appears thus that NF abnormalities seem to be a primary cause of diabetic neuropathy, and not only a marker of the pathology (McLean, 1997). A recent work has shown that diabetic neuropathy in an experimental model, the insulin KO mouse, does not alter only peripheral axons, but also affects central neurons, where hyperphosphorylation of NFs together with alteration of different NF kinases activities have been demonstrated (Schechter et al., 2005).

Hyperammonemia during CNS Development Poorly understood irreversible damages to CNS development occur in neonates and infants with hepatic deficiency or inherited defects of ammonium (NH4+) metabolism, manifesting on the long term as mental retardation (Bachmann, 2002; Bachmann, 2003). We have shown, in brain cell 3D primary cultures exposed to NH4+ as experimental model of hyperammonemia during CNS development, that NH4+ impairs axonal growth (Braissant et al., 1999; Braissant et al., 2002). NFs appear to be affected in this process, as both NFM expression and phosphorylation are decrease by NH4+ exposure (Figure 2) (Braissant et al., 2002). The correct expression and phosphorylation of NFM seem to depend on levels of creatine (Braissant et al., 2002), which can be synthesized by brain cells including during development (Braissant et al., 2001; Braissant et al., 2005). Axonal growth, as well as NFM expression and phosphorylation, are protected under NH4+ exposure by co-treatment with creatine in a glial cell dependent manner (Braissant et al., 2002). Our results are consistent with clinical findings in hyperammonemic neonates or infants presenting irreversible brain lesions compatible with neuronal fiber loss or defects of neurite outgrowth. The alteration of NF phosphorylation under NH4+ exposure might occur through the dysregulation of MAPK, which are NF kinases and present altered levels of phosphorylation and activity in brain cells exposed to NH4+ (Schliess et al., 2002; Jayakumar et al., 2006; Cagnon et al., 2006).

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Methylmalonic (MMA) and Propionic (PA) Acidemias Among the most frequent organic acidemias, PA and MMA are due to deficiencies in propionyl-CoA carboxylase and L-methylmalonyl-CoA mutase, respectively, and lead to the increase of free propionic acid in blood and its accumulation in tissues (PA), and to the tissular accumulation of L-methylmalonic acid and secondarily of propionic acid (MMA). The levels of these metabolites in blood and cerebrospinal fluid can rise as high as 5 mM and may be even higher in neuronal cells. PA and MMA lead to chronic neurologic disabilities, seizures and developmental delay. Damages to basal ganglia, a general hypomyelination, cerebral atrophy and white matter edema are frequently encountered. So far, the exact underlying mechanisms of brain damage in PA and MMA remain to be elucidated. However, NFs might be implicated in the neuropathological aspects of MMA and PA (Figures 2, 3 4). Indeed, MMA and PA experimental models have provided evidence that neuronal NFL and NFM expression and phosphorylation are reduced under L-methylmalonic acid and propionic acid exposures (de Mattos-Dutra et al., 1997a; de Mattos-Dutra et al., 1997b; de Mattos-Dutra et al., 1998), while they are increased for NFH (Vivian et al., 2002).

NFs in other Neurodevelopomental and Metabolic Diseases Phenylketonuria (PKU) is one of the most frequent inborn errors of metabolism, is due to the deficiency of the hepatic enzyme phenylalanine hydroxylase and results in hyperphenylalaninemia. Among other pathological characteristics, untreated PKU leads to mental retardation. Untreated PKU patients show a severe hypomyelination of their CNS. Experimental evidence has been shown that hyperphenylalaninemia delays axonal maturation and myelination during critical period of CNS development, probably through a deficit of NFH as well as myelin basic protein expression (Reynolds et al., 1993). Progressive encephalopathy syndrome with edema, hypsarrhythmia and optic atrophy (PEHO syndrome) is a form of infantile progressive encephalopathy showing severe hypotonia, convulsions, profound mental retardation, hyperreflexia, optic atrophy and brain atrophy, in particular in cerebellum and brainstem. PEHO seems to occur in the postnatal period, without exclusion of potential prenatal onset. Interestingly, PEHO patients presented an aberrant expression of NFH in the perikarya of their cerebellar Purkinje cells, demonstrating an important disorganization of their cytoskeleton (Haltia and Somer, 1993).

NFS AS MARKERS OF DISEASES NFs, as the principal components of the axonal cytoskeleton, are released in the interstitial fluid after axonal injury or degeneration, and diffuse into cerebrospinal fluid (CSF), where they can be quantified to monitor axonal degeneration, as well as disease activity and progression. Increasing studies are published making use of NFs as markers of neuronal injury. A lot of work has been done on the measure of NFL and NFH released in CSF, as markers of axonal degeneration, to help the prediction and monitoring of the

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neurological decline in people with multiple sclerosis (MS). Different studies have shown that NFL CSF concentration is higher in patients with MS than in controls, making of NFL a promising marker to discriminate MS patients from patients with other neurological diseases. On the other hand, CSF NFH seems interesting for the follow up of the progression of the disease in MS patients, as it is increased during the progressive phase of MS. For more specific informations on the use of NFs as markers of MS, the reader is invited to read two detailed and recent reviews (Petzold, 2005; Teunissen et al., 2005). As new but non-exhaustive examples, the use of NFs as markers of three other neuropathological conditions will be briefly discussed here: ALS, subarachnoid hemorrhage (SAH), and brain damages as consequence of cardiac arrest. As discussed in a previous chapter, ALS is the most common form of motor neuron disease, presenting as neuropathological hallmark an abnormal aggregation of NFs in the degenerating motor neurons. A recent work proposes that phosphorylated NFH might be a valuable marker of axonal damage in ALS, discriminate between different categories of ALS, and be used as marker for therapeutic trials (Brettschneider et al., 2006). Axonal degeneration is thought to be an underestimated complication of SAH, which can continue for days after the primary injury, and extend into the period of delayed cerebral ischemia. A recent study shows that phosphorylated NFH, measured daily in CSF during 14 days after the SAH episode, is significatively increased in SAH patients with bad outcome (measured at 3 months) (Petzold et al., 2005). This work demonstrates the secondary axonal degeneration following SAH, and show that the levels of phosphorylated NFH in CSF are highly predictive of a bad outcome for SAH patients. The majority of patients surviving resuscitation after an out of hospital cardiac arrest present neurological complications due to global anoxia. Outcome prediction for these patients mainly rely on clinical observations, and on the recent measure of biochemical markers of brain damage in serum, such as brain specific proteins S-100 or NSE (Rosen et al., 2001). A recent study has shown that the levels of NFL in CSF give a reliable measure of brain damage, and are highly predictive of poor outcome for these patients (Rosen et al., 2004).

CONCLUSION NFs are essential cytoskeletal proteins of the neuron, which participate in axonal rigidity, tensile strength, stability along time, regulation of calibre, and transport guidance of organelles and particles. NFs alterations have been identified in many different brain pathologies, ranging from neurodegenerative, neurodevelopmental to metabolic diseases. This list of diseases showing abnormalities in NFs will certainly increase in the near future. The identified NFs alterations range from genetic mutations, to abnormal expression, posttranslational modifications and aberrant localization or accumulation in neuronal perikaryion. From this diversity of NF dysregulation in so many brain diseases, the future experimental work on NFs may unravel common mechanisms of IF accumulation and aggregation, and hopefully allow the design of better treatments for the patients suffering of these neurodegenerative diseases.

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ACKNOWLEDGMENTS Our work is supported by the Swiss National Science Foundation, grants n° 31-63892.00 and 3100A0-100778.

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In: Brain Mapping and Diseases Ed: Diane E. Spinelle

ISBN: 978-1-61122-065-0 © 2011 Nova Science Publishers, Inc.

Chapter 9

SEGMENTATION PROPAGATION FROM DEFORMABLE ATLASES FOR BRAIN MAPPING AND ANALYSIS Marius George Linguraru∗1, Tom Vercauteren2,3, Mauricio Reyes-Aguirre4, Miguel Ángel González Ballester4 and Nicholas Ayache2 1

Diagnostic Radiology Department, Clinical Center, National Institutes of Health, Bethesda MD, USA 2 Epidaure/Asclepios Research Group, INRIA, Sophia Antipolis, France 3 Mauna Kea Technologies, Paris, France 4 MEM Research Center, Institute for Surgical Technology and Biomechanics, University of Bern, Switzerland

ABSTRACT Magnetic resonance imaging (MRI) is commonly employed for the depiction of soft tissues, most notably the human brain. Computer-aided image analysis techniques lead to image enhancement and automatic detection of anatomical structures. However, the intensity information contained in images does not often offer enough contrast to robustly obtain a good detection of all internal brain structures, not least the deep gray matter nuclei. We propose digital atlases that deform to fit the image data to be analyzed. In this application, deformable atlases are employed for the detection and segmentation of brain nuclei, to allow analysis of brain structures. Our fully automatic technique is based on a combination of rigid, affine and non-linear registration, a priori information on key anatomical landmarks and propagation of the information of the atlas. The Internet Brain Segmentation Repository (IBSR) data provide manually segmented brain data. Using prior anatomical knowledge in local brain areas from a randomly chosen brain scan (atlas), a first estimation of the deformation fields is calculated by affine registration. The image alignment is refined through a non-linear transformation to correct the segmentation of nuclei. The local segmentation results are greatly improved. They are ∗

E-mail: [email protected]

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Marius George Linguraru, Tom Vercauteren, Mauricio Reyes-Aguirre et al. robust over the patient data and in accordance with the clinical ground truth. Validation of results is assessed by comparing the automatic segmentation of deep gray nuclei by the proposed method with manual segmentation. The technique offers the accurate segmentation of difficultly identifiable brain structures in conjuncture with deformable atlases. Such automated processes allow the study of large image databases and provide consistent measurements over the data. The method has a wide range of clinical applications of high impact that span from size and intensity quantification to comprehensive (anatomical, functional, dynamic) analysis of internal brain structures.

Keywords: MRI, brain, gray matter nuclei, atlas, registration, deformation, segmentation.

1. INTRODUCTION The advent of medical imaging modalities such as X-ray, ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) has greatly improved the diagnosis of human diseases. Until recently, the most common procedure to analyze imaging data was visual inspection on printed support. In the last decade, computer-aided medical image analysis techniques have been employed to provide a better insight into the acquired image data [Duncan and Ayache 2000]. Such techniques allow for quantitative, reproducible observation of the patient condition. Furthermore, the computing power of modern machines can be used to combine information from several images of the same patient (i.e. image fusion) or add prior information from a database of images. In this chapter, we present a fully automated medical image analysis technique aimed at the detection of internal brain structures from MRI data. Such automated processes allow the study of large image databases and provide consistent measurements over the data. In our case, we employ a priori anatomical knowledge in the form of digital brain atlases. Relevant background information about MRI and brain anatomy is provided next. In Methods we describe the different components of our image processing framework, which segments and quantifies internal brain structures by propagating deformable models of internal nuclei. Finally, results are presented and the algorithm is assessed.

1.1. Magnetic Resonance Imaging MRI has become a leading technique widely used for imaging soft human tissue. Its applications are extended over all parts of the human body and it represents the most common visualization method of human brain. Images are generated by measuring the behavior of soft tissue under a magnetic field. Under such conditions, water protons enter a higher energy state when a radio-frequency pulse is applied and this energy is re-emitted when the pulse stops (a property known as resonance) [Hornak]. A coil is used to measure this energy, which is proportional to the quantity of water protons and local biochemical conditions. Thus, different tissues give different intensities in the final MR image. From the brain MRI perspective, this quality makes possible the segmentation of the three main tissue classes within the human skull: gray matter (GM), white matter (WM) and cerebrospinal fluid (CSF).

Segmentation Propagation from Deformable Atlases for Brain Mapping and Analysis 181 Their accurate segmentation and sub-classification remains a challenging task in the clinical environment. The relative contrast between brain tissues is not a constant in MR imaging. In most medical imaging applications, little can be done about the appearance of anatomically distinct areas relative to their surroundings. In MRI, the choice of the strength and timing of the radiofrequency pulses, known as the MRI sequence [Stark et al.1999], can be employed to highlight some type of tissue or image out another, according to the clinical application. However, the presence of artifacts due to magnetic field inhomogeneity (bias fields) and movement artifacts may hamper the delineation of GM versus WM and CSF and make their depiction difficult [Fennema-Notestine et al. 2006; Guillemaud et al.1997; Han et al. 2006; Sled et al.1998; Van Leemput et al. 1999]. Several MRI sequences are used in common clinical practice. T1-weighted MRI offers the highest contrast between the brain soft tissues and is arguably the most popular MR acquisition technique used for brain diagnosis. On the contrary, T2-weighted and Proton Density (PD) images exhibit very low contrast between GM and WM, but high contrast between CSF and brain parenchyma. In other MRI sequences, like the Fluid Attenuated Inversion Recovery (FLAIR) sequence, the CSF is eliminated from the image in an adapted T1 or T2 sequence. More about these specific MRI sequences and their variations can be found in [Brown and Semelka 1999]. MR images depict a 3D volume where the organ or part of the body of interest is embedded. This information can be used to build a 3D representation of the structure of interest. This applies both to 2D sequences, where images are acquired in slices, and to the recently developed 3D sequences, where the data are captured in the 3D Fourier space, rather than each slice being captured separately in the 2D Fourier space [Brown and Semelka 1999; Stark et al. 1999].

1.2. Deep Gray Matter Nuclei The neurons that build up the human brain are composed of a cellular body and an axon. The latter projects its dendritic connections to other neurons in remote cerebral regions. In essence, gray matter corresponds to the cellular bodies, whereas the axons constitute the white matter. Cerebral gray matter is mainly concentrated in the outer surface of the brain (cortex), but several internal GM structures exist, as seen in Figure 1. These are known as deep gray matter nuclei and they play a central role in the intellectual capabilities of the human brain. Additionally, deep brain gray matter nuclei are relevant to a set of clinical conditions, such as Parkinson’s and Creutzfeldt-Jakob diseases [Summerfield et al. 2005; Linguraru et al. 2006]. The size and appearance of gray nuclei can be indicators of abnormality. However, their detection in MRI data sets remains a challenging task, due to their small size, partial volume effects [González Ballester et al.2002], anatomical variability, lack of white matter-gray matter contrast in some sequences and movement artifacts. A methodology for the robust detection of deep brain gray matter nuclei in multi-sequence MRI is presented in this chapter.

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1.3. Segmentation Based on Deformable Atlases Brain atlases are images that have been segmented and thus contain information about the position and shape of each structure. Such atlases can be binary (1 for the location of a structure and 0 for “outside”) or probabilistic, in which case the values correspond to the probability of a voxel containing the structure of interest. In order to locate such structures in a given patient image, the atlas image is deformed to match the shape of the patient brain through registration. Depending on the number of degrees of freedom and the type of geometric deformation allowed, registration can be rigid, affine, or non-linear (a deformation field specifying the displacement applied to each point).

Figure 1. The map of gray matter nuclei in axial view. To the left, an annotated map of deep gray matter internal nuclei reproduced from the Talairach and Tournoux atlas [Talairach and Tournoux 1988]: the caudate (C), putamen (P), globus pallidus (G) and thalamus (T). To the right, deep gray matter internal nuclei as seen in a normal T1 weighted axial MR image with good contrast between WM, GM and CSF.

Registration to a digital atlas has become a common technique with the introduction of popular statistical algorithms for image processing, such as Statistical Parametric Mapping (SPM) [Ashburner and Friston 2000] or Expectation Maximization Segmentation (EMS) [Van Leemput et al. 2001]. A widely-used probabilistic atlas is the MNI Atlas from the Montreal Neurological Institute at McGill University [Collins et al. 1998]. It was built using over 300 MRI scans of healthy individuals to compute an average brain MR image, the MNI template, which is now the standard template of SPM and the International Consortium for Brain Mapping [Mazziotta et al. 2001]. However the averaging is performed on the entire brain and the three main tissue classes: GM, WM and CSF. More anatomical details can be found in manually segmented brain scans, and popular or new options are the Zubal Atlas from Yale University [Zubal et al. 1994], the SPL Atlas from Harvard Medical School [Kikinis et al. 1996], the basal ganglia atlas build from histological data from Pitié-Salpêtrière Hospital in Paris [Yelnik et al. 2007] and IBSR from Massachusetts General Hospital, Harvard Medical School, which is employed in this work.

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1.4. Gray Nuclei Segmentation The challenging nature of the problem of segmenting gray matter nuclei from MRI images stems from the lack of contrast, limitations of image resolution, and possible imaging artifacts. Few works have attempted to provide a fully automated algorithm for their identification and accurate delineation. [Dawant et al. 1999] propose a method for the segmentation of internal brain structures based on similarity and free-form deformations to register one segmented image. No statistical atlas information is employed for spatial normalization. [Joshi et al. 2004] propose a method for unbiased diffeomorphic atlas construction, and they show results on the segmentation of the caudate nucleus within the context of a study on autism. [Pohl et al. 2006] propose a method for joint segmentation and registration based on the Expectation-Maximization (EM) algorithm, and apply their method to the segmentation of the thalamus. We propose digital atlases that deform to fit the image data to be analyzed. Our fully automatic technique is based on a combination of rigid, affine and non-linear registration. A priori information on key anatomical landmarks is used to propagate the information from the atlas employing the computed deformation field. The technique offers the robust segmentation and quantification of difficultly identifiable brain structures in conjuncture with deformable atlases.

2. METHODS 2.1. Data For the analysis of deep gray nuclei in this chapter, we used the Internet Brain Segmentation Repository1 (IBSR) from the Center for Morphometric Analysis, Massachusetts General Hospital, Harvard Medical School. Boston, MA. The database consists of 18 highresolution T1 MR scans of normal subjects. For each scan, 43 individual brain structures, including the deep gray nuclei, are manually segmented. The MR image data are T1-weighted 3D coronal acquisitions. The image resolution is between 0.93x0.93x1.5 mm3 and 1x1x1.5 mm3. There are 4 female and 14 male datasets with ages between juvenile and 71 years, covering a large variability of brain anatomies.

Figure 2. The IBSR database. We present a case from the ISBR database: from left to right, the coronal MR T1 scan; the segmentation of WM (white), GM (yellow) and CSF (red); the map of the manually segmented 43 brain structures. 1

http://www.cma.mgh.harvard.edu/ibsr/

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A subject image from the IBSR database is shown in Figure 2. T1-weighted volumetric images from IBSR have been positionally normalized into the Talairach orientation (rotation only). This rigid transformation provides a first level of inter-subject alignment.

2.2. Spatial Normalization The large variability inherent to human anatomy and the differences in patient positioning across scans leads us to consider further spatial normalization for the identification of deep gray nuclei. This will allow localizing the areas of interest with the help of an atlas of the brain. Furthermore, it will make automatic inter-patient comparisons possible. For the construction of the statistical atlas, we chose using the data from IBSR, as it contains 18 manually segmented scans; they can provide the level of necessary information to guide the segmentation of brain structures, but also be used for the quantitative validation of the segmentation method. Given that the IBSR images are already aligned rigidly to the Talairach space, we perform a first refinement of the rigid registration using an affine transform. One random image from the database is selected as atlas. The atlas selection may introduce a bias, as the chosen atlas is not an average morphology and the segmentation does not account for intra-observer variability. The atlas T1-weigthed scan is registered to each of the other 17 T1 scans. We employ a robust block-matching algorithm to estimate the affine deformation between subjects’ scans. [Ourselin 2000, Ourselin 2001]. The block matching strategy is a two-step iterative method. The standard assumption behind the algorithm is that there is a global intensity relationship between the template or reference image, I, and the one being registered to it or floating image, J. The result is reflected by the registered image J’ = J ◦ T, with T being the registration transformation. In the first step, each block of I, BI, is locally translated over J and a correlation coefficient CC is maximized to blocks of J, BJ. We use a correlation coefficient, as the registration is performed between monomodal T1-weighted MR images. CC (B I , B J ) =

1 N2

i, j

(

)(

)

⎡ x i − μ BI y i − μ B J ⎤ ⎥ σ BI σ B J ⎢⎣ ⎥⎦ ,

∑⎢

where xi are elements of BI, yj are elements of BJ, and by μ and σ we denote the mean values and standard deviations. Thus, the transformation between the two images is computed block by block and a displacement field is generated after removing outliers. In the second step, a parametric transformation, in this case affine, is estimated by regularizing the deformation field to explain most of the block correlations. A least trimmed squared regression approximates the affine transformation by minimizing the residual error

min ∑ ri:n T

i

ri:n

2

,

2

where are the squared ordered Euclidean residual norms number of displacement vectors.

( )

ri = B I i − T B J i

and n is the

Segmentation Propagation from Deformable Atlases for Brain Mapping and Analysis 185 To improve robustness, this procedure is repeated iteratively at multiple scales. Resulting registered data are interpolated using a linear function. More details can be found in [Ourselin 2001]. The alignment of the atlas to all individual scans allows a more robust inter-subject analysis and statistical algorithms can be applied.

2.3. Refined Segmentation To be able to segment GM and WM in MRI data, a good contrast between these types of tissue in T1-weighted images is desired. Although image acquisition has radically improved over the last years, the variation in parameters and patient motion brings artifacts and variations in image appearance. Bias field inhomogeneities further contribute to the degradation of image quality. Hence, the segmentation of GM cannot be done reliably only from the patient images. An affine transformation provides a better level of inter-subject GM alignment than a rigid transformation. However, to segment small GM sub-structures a more precise registration is necessary. For the examples in this chapter, we will focus on the basal ganglia. Hence, we create a mask with the caudate, globus pallidus, thalamus and putamen, which will be referred as internal nuclei for the rest of this paper, from the atlas (Figure 3). We aim to use this mask for the segmentation of internal nuclei in the other subject images. Non-linear (free-form) registration is used to align the T1 scans of the affinely registered atlas and the corresponding T1 images of the 17 subjects. We employ a diffeomorphic nonlinear registration algorithm based on Thirion’s demons algorithm [Thirion 1998; Vercauteren et al. 2007a; Vercauteren et al. 2007b]. This algorithm has an open-source implementation [Vercauteren et al. 2007c] and is used in the free MedINRIA v 1.6.0 package2 from the Asclepios Research Group, INRIA [Toussaint et al. 2007].

Figure 3. The mask of internal nuclei. We show an axial view of the T1 image chosen as atlas and the corresponding mask of manually segmented basal ganglia, including the caudate (orange), globus pallidus (yellow), putamen (white) and thalamus (red).

It has been shown in [Pennec et al. 99] that the demons algorithm could be seen as an optimization of a global energy. The main idea is to introduce a hidden variable in the registration process: correspondences. We then consider the regularization criterion as a prior 2

http://www-sop.inria.fr/asclepios/software/MedINRIA/

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on the smoothness of the transformation T. Instead of requiring that point correspondences between image pixels (a vector field C) be exact realizations of the transformation, one allows some error at each image point. Given the template image I and the floating image J, we end up with the global energy

E (C , T ) = σ i−2 Sim( I , J o C ) + σ x−2 dist(T , C ) 2 + σ T−2 Reg(T ) ,

Sim( I , J o C ) =

1 2 I − J oC , 2

where σi accounts for the noise on the image intensity, σx for a spatial uncertainty on the correspondences, and σT controls the amount of regularization we need. We classically have dist(T,C) = ||C-T|| and Reg(T) = ||∇T||2, but the regularization can also be modified to handle fluid-like constraints [Cachier et al. 2003]. Within this framework, the demons registration can be explained as an alternate optimization over T and C. The optimization is performed within the complete space of dense non-linear transformations by taking a series of additive steps, T←T+u. The most straightforward way to adapt the demons algorithm to make it diffeomorphic is to optimize E(C,T) over a space of diffeomorphisms. This can be done as in [Malis 2004; Mahony et al. 2002] by using an intrinsic update step

T ← T o exp(u) , on the Lie group of diffeomorphisms. This approach requires an algorithm to compute the exponential for the Lie group of interest. Thanks to the scaling and squaring approach in [Arsigny et al. 2006], this exponential can efficiently be computed for diffeomorphisms with just a few compositions: Algorithm (Fast Computation of Vector Field Exponentials). · · ·

Choose N such that 2-N u is close enough to 0, e.g. maxp ||2-N u(p)|| ≤ 0.5; Perform an explicit first order integration: v(p) ← 2-N u(p) for all pixels; Do N (not 2N!) recursive squarings of v: v ← v ◦ v.

By plugging the Newton method tools for Lie groups within the alternate optimization framework of the demons, we proposed in [Vercauteren et al. 2007a] the following nonparametric diffeomorphic image registration algorithm: Algorithm (Diffeomorphic Demons Iteration). · · · ·

Compute the correspondence update field u using a regular demons step; If a fluid-like regularization is used, let u ← Kfluid * u; Let C ← T◦exp(u), where exp(u) is computed using the above fast algorithm; If a diffusion-like regularization is used, let T ← Id + Kdiff * (C-Id) (else let T ← C).

Segmentation Propagation from Deformable Atlases for Brain Mapping and Analysis 187 Having the deformation fields computed, we apply them to the mask of internal nuclei of the atlas, deforming the mask according to the position and size of the internal nuclei in each subject image. A diagram of the algorithm is shown in Figure 4. The deformed mask is used to segment the internal nuclei of the patient, namely the caudate, globus pallidus putamen, putamen and thalamus.

Figure 4. Diagram of the algorithm for segmentation and quantification of the brain deep gray matter nuclei.

In order to preserve the correct values of the segmentation labels posterior to the application of the transformation, nearest-neighbor interpolation is performed, as opposed to the case of patient image registration, which employed linear interpolation.

2.4. Quantification For each internal nucleus, we compute a segmentation overlap between the automatic and the manual segmentations as a quantifiable measure of the success of the algorithm. The metric for validation is based on the Dice Coefficient (DC)

DC =

2 S A ∩ SM S A + SM

,

where SA is the segmented region of the automatic method, and SM is the manually segmented region by an expert. The volume estimation between manual and automatic volume measurement is computed for each type of internal nucleus. To correlate the manual and automatic estimates, we use the R-squared (R2) value of the best linear fit of data correlation

R 2 (V A , VM ) =

cov(V A , VM )

σV σV A

,

B

where cov represents the covariance between the manual (VM) and automatic (VA) estimates of nucleus volume and σ the standard deviation.

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3. RESULTS You will note that we present our results alternating between coronal and axial views. Images in the IBSR database are acquired in a coronal view, but for visualization we show them in radiological convention view as well. We compared registration results at three levels of deformation: rigid, affine and nonlinear. Figure 5 presents one subject scan and the atlas being deformed to best match the subject. The rigid registration on the Thalairach space provides a good alignment, but does not handle any anatomical differences. The affine transformation is better suited for the registration, but still insufficient to align small structures, such as the internal nuclei. Finally, the non-linear refinement provides the best fit between the two 3D images. Note the adaptation of size and shape of the ventricles and thalamus. Checkerboard comparative images for the three levels of registration are presented in Figure 6 for a better visual assessment. Note the better correspondence between brain structures after non-linear registration. Hence, we save the non-linear transformation field presented in Figure 7 and apply it to the mask of internal nuclei (Figure 3).

Figure 5. Inter-patient registration: (a) the target image; (b) the source image after rigid registration; (c) the source image after affine registration; (d) the deformed source using non-linear registration.

Figure 6. Comparative registration results using checkerboards: (a) after rigid registration (b) after affine registration; and (c) using non-linear transformations;

Segmentation results for the group of deep gray matte nuclei (caudate, globus pallidus, putamen and thalamus) are illustrated in Figure 8. For visual assessment of the impact of the registration on segmentation, the results are shown after rigid, affine and non-linear registration and compared to the manual segmentation of nuclei. Segmented nuclei mask are overlaid on the T1 scans of the subject. Once more, we observe the superior segmentation

Segmentation Propagation from Deformable Atlases for Brain Mapping and Analysis 189 provided after non-linear registration. In Figure 9 we present difference images between nuclei mask using automatic and manual segmentations. The error in volume estimation using non-linear registration is significantly smaller than using transformations with fewer degrees of freedom.

Figure 7. Deformation fields. We present the source image (atlas) used in the registration and the deformation filed resulting from the non-linear registration to the target image.

Figure 8. Segmentation of gray nuclei: (a) the manual segmentation; (b) after rigid registration; (c) using affine registration; and (d) using the non-linear transformation fields.

Figure 9. Segmentation overlap with the manual segmentation: (a) the difference image after rigid registration; (b) after affine registration; and (c) using non-linear registration.

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Marius George Linguraru, Tom Vercauteren, Mauricio Reyes-Aguirre et al. More segmentation results using non-linear registration are shown in Figure 10 and 11.

Figure 10. Segmentation of gray nuclei in radiological convention. We present segmentation results in axial (a), sagittal (b) and coronal (c) views. The bottom row shows the MR image for the visual evaluation of the automatic segmentation results.

Figure 10 shows typical segmentation results in a subject scan. We separate the nuclei using a color code: orange for caudate, yellow for globus pallidus, white for putamen and red for thalamus. Axial, sagittal and coronal views are presented for 3D assessment. In Figure 11 we browse through the 3D coronal space of the subject and compare the manual and automatic segmentation of the four internal nuclei. Finally, a 3D map of the segmented nuclei is illustrated in Figure 12 using 3D rendering. To quantify the quality of the segmentation for the 17 subject data, the overlap ratios and errors in volume estimation between the manual and automatic segmentations were computed. Values were calculated for each type of nuclei (caudate, globus pallidus, putamen and thalamus) and for all nuclei together, as denominated by gray nuclei. Numerical figures are presented in Table 1. As expected, numbers look better for the larger nuclei, as they are correlated with the structure size. The charts of the overlap ratio and error of volume estimation are seen in Figure 13 and Figure 14 respectively. The correlations between manual and automatic segmentation is presented in Figures 15 and 16. Figure 15 shows the best linear fit of the correlated data and the R-squared (R2) value for each category of nuclei (caudate, globus pallidus, putamen and thalamus). In Figure 16 we present the correlation for all internal nuclei together.

Segmentation Propagation from Deformable Atlases for Brain Mapping and Analysis 191

Figure 11. 3D segmentation of nuclei. We present comparative results between the manual and automatic segmentations of deep gray nuclei at six coronal locations along the 3D volume of the brain.

Figure 12. A 3D map of the segmented gray nuclei.

Table 1. Segmentation error. The rows present the overlap ratio and volume estimation error for four categories of gray nuclei (caudate, globus pallidus, putamen and thalamus) and the total volume of the nuclei (gray nuclei)

Overlap ratio Volume error (%)

Caudate 0.824±0.038 9.59±4.268

Globus Pallidus 0.788±0.045 11.112±7.02

Putamen 0.855±0.023 4.387±2.348

Thalamus 0.883±0.033 3.299±2.078

Gray Nuclei 0.855±0.018 2.613±2.058

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Figure 13. The computed overlap between manual and automatic segmentation of deep nuclei of the brain. The bar corresponding to “gray nuclei” refers to the combined volume of caudate, globus pallidus, putamen and thalamus.

Figure 14. The computed error in volume estimation between manual and automatic segmentation of deep nuclei of the brain.

It has been shown that the error induced by MRI partial volume effects in small structures can be in the range 20-60 % of the volume [González Ballester et al. 2000]. Taking into account the size of grey matter nuclei and the good correlation with manual segmentations, our results show the suitability of our approach for neuroanatomical studies.

Segmentation Propagation from Deformable Atlases for Brain Mapping and Analysis 193

Figure 15. The best linear fits and R-squared values for correlated volume estimations of the four categories of gray nuclei. The horizontal axes correspond to the manual segmentation, and the automatic segmentation estimates are shown on the vertical axes.

Figure 16. The best linear fit and R-squared value for correlated volume estimations of the total volume of the segmented gray nuclei: caudate, globus pallidus, putamen and thalamus.

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Given the high resolution and good contrast in the IBSR images, the inter-subject registration of T1-weighted MR images is sufficiently robust to govern the segmentation of small internal nuclei. However, in clinical practice data quality is variable and the intensity information from MRI may be inadequate to find an accurate alignment between scans. In these situations, it is desirable to use anatomical landmarks for the definition of more precise transformations. We proposed to employ easily identifiable anatomical structures in the brain, such as the lateral ventricles and cortex boundary. For more detail please refer to [Linguraru et al. 2006; Linguraru et al. 2007].

CONCLUSION We proposed digital atlases that deform to fit the image data to be analyzed. Our fully automatic technique is based on a combination of rigid, affine and non-linear registration. A priori information on anatomical landmarks was used to propagate the information from the atlas employing the computed deformation field. The technique offers the robust segmentation and quantification of difficultly identifiable brain structures in conjuncture with deformable atlases. In this chapter, we focused on the segmentation of the basal ganglia to present our algorithm for the segmentation of deep gray matter nuclei. An identical approach can be used for other inner brain structures to accurately segment them in patient images.

REFERENCES Ashburner J, Friston KJ. Voxel-based morphometry--the methods. Neuroimage. 2000 Jun;11(6 Pt 1):805-21. Brown MA, Semelka RC. MR imaging abbreviations, definitions, and descriptions: a review. Radiology. 1999 Dec;213(3):647-62. Cachier P, Bardinet E, Dormont D, Pennec P, Ayache N. Iconic feature based nonrigid registration: The PASHA algorithm. Computer vision and image understanding. 2003 Feb; 89(2-3):272-298. Collins DL, Zijdenbos AP, Kollokian V, Sled JG, Kabani NJ, Holmes CJ, Evans AC. Design and construction of a realistic digital brain phantom. IEEE Trans Med Imaging. 1998 Jun;17(3):463-8. Dawant BM, Hartmann SL, Thirion JP, Maes F, Vandermeulen D, Demaerel P. Automatic 3D segmentation of internal structures of the head in MR images using a combination of similarity and free-form transformations: Part I, Methodology and validation on normal subjects. IEEE Trans Med Imaging. 1999 Oct;18(10):909-16. Duncan J, Ayache N. Medical Image Analysis: Progress over Two Decades and the Challenges Ahead. IEEE Transactions on Pattern Analysis and Machine Intelligence 2000; 22(1):85-106. Fennema-Notestine C, Ozyurt IB, Clark CP, Morris S, Bischoff-Grethe A, Bondi MW, Jernigan TL, Fischl B, Segonne F, Shattuck DW, Leahy RM, Rex DE, Toga AW, Zou KH, Brown GG. Quantitative evaluation of automated skull-stripping methods applied to

Segmentation Propagation from Deformable Atlases for Brain Mapping and Analysis 195 contemporary and legacy images: effects of diagnosis, bias correction, and slice location. Hum Brain Mapp. 2006 Feb; 27(2):99-113. González Ballester MA, Zisserman AP, Brady M. Segmentation and measurement of brain structures in MRI including confidence bounds. Med. Image Anal. 2000; 4(3):189-200. González Ballester MA, Zisserman AP, Brady M. Estimation of the partial volume effect in MRI. Med. Image Anal. 2002 Dec;6(4):389-405. Guillemaud R, Brady M. Estimating the bias field of MR images. IEEE Trans Med. Imaging. 1997 Jun;16(3):238-51. Han X, Jovicich J, Salat D, van der Kouwe A, Quinn B, Czanner S, Busa E, Pacheco J, Albert M, Killiany R, Maguire P, Rosas D, Makris N, Dale A, Dickerson B, Fischl B. Reliability of MRI-derived measurements of human cerebral cortical thickness: the effects of field strength, scanner upgrade and manufacturer. Neuroimage. 2006 Aug 1;32(1):180-94. Hornak JP. The Basics of MRI, http://www.cis.rit.edu/htbooks/mri/ Joshi S, Davis B, Jomier M, Gerig G. Unbiased diffeomorphic atlas construction for computational anatomy. Neuroimage. 2004;23 Suppl 1:S151-60. Kikinis R, Shenton ME, Iosifescu DV, McCarley RW, Saiviroonporn P, Hokama HH, Robatino A, Metcalf D, Wible CG, Portas CM, Donnino RM, Jolesz FA. A digital brain atlas for surgical planning, model-drivensegmentation, and teaching. IEEE Transactions on Visualization and Computer Graphics 1996; 2(3):232-41. Linguraru MG, Ayache N, Bardinet E, Ballester MA, Galanaud D, Haïk S, Faucheux B, Hauw JJ, Cozzone P, Dormont D, Brandel JP. Differentiation of sCJD and vCJD forms by automated analysis of basal ganglia intensity distribution in multisequence MRI of the brain--definition and evaluation of new MRI-based ratios. IEEE Trans Med Imaging. 2006 Aug;25(8):1052-67. Linguraru MG, Gonzalez Ballester MA, Ayache N. Deformable Atlases for the Segmentation of Internal Brain Nuclei in Magnetic Resonance Imaging. International Journal of Computers, Communication and Control 2007;2(1):26-36. Mahony R, Manton J.H.. The geometry of the Newton method on non-compact Lie-groups. Journal of Global Optimization. 2002 Aug; 23(3):309-27. Malis E. Improving vision-based control using efficient second-order minimization techniques. IEEE Int. Conf. Robot Automat. 2004. Mazziotta J, Toga A, Evans A, Fox P, Lancaster J, Zilles K, Woods R, Paus T, Simpson G, Pike B, Holmes C, Collins L, Thompson P, MacDonald D, Iacoboni M, Schormann T, Amunts K, Palomero-Gallagher N, Geyer S, Parsons L, Narr K, Kabani N, Le Goualher G, Boomsma D, Cannon T, Kawashima R, Mazoyer B. A probabilistic atlas and reference system for the human brain: International Consortium for Brain Mapping (ICBM). Philos. Trans R Soc. Lond B Biol. Sci. 2001 Aug 29;356(1412):1293-322. Ourselin S, Roche A, Prima S, Ayache N. Block matching: a general framework to improve robustness of rigid registration of medical images. In: DiGioia AM, Delp S editors. Medical Robotics, Imaging and Computer Assisted Surgery (MICCAI 2000). Lectures Notes in Computer Science 1935, Berlin Heidelberg: Springer 2000; 557-566. Ourselin S, Roche A, Subsol G, Pennec X, Ayache N. Reconstructing a 3D structure from serial histological sections. Image and Vision Computing 2001;19(1-2):25-31. Pennec X, Cachier P, Ayache N. Understanding the demon’s algorithm: 3D non-rigid registration by gradient descent. . Med Image Comput Comput Assist Interv Int Conf Med Image Comput Comput Assist Interv. 1999; 597-605.

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Pohl KM, Fisher J, Grimson WE, Kikinis R, Wells WM. A Bayesian model for joint segmentation and registration. Neuroimage. 2006 May 15;31(1):228-39. Sled JG, Zijdenbos AP, Evans AC. A nonparametric method for automatic correction of intensity nonuniformity in MRI data. IEEE Trans Med Imaging. 1998 Feb;17(1):87-97. Summerfield C, Junqué C, Tolosa E, Salgado-Pineda P, Gómez-Ansón B, Martí MJ, Pastor P, Ramírez-Ruíz B, Mercader J. Structural brain changes in Parkinson disease with dementia: a voxel-based morphometry study. Arch. Neurol. 2005 Feb;62(2):281-5. Stark DD, Bradley WG, Bradley JR. WG. Magnetic Resonance Imaging. St Louis, Mosby 1999. Talairach J, Tournoux P. Co-Planar Stereotaxic Atlas of the Human Brain. New York, Thieme Medical Publishers 1988. Thirion JP. Image matching as a diffusion process: an analogy with Maxwell's demons. Med Image Anal. 1998 Sep;2(3):243-60. Toussaint N, Souplet J-C, Fillard P. MedINRIA : Medical image navigation and research tool by INRIA. MICCAI’07 Workshop on Interaction in Medical Image Analysis and Visualization. 2007. Van Leemput K, Maes F, Vandermeulen D, Colchester A, Suetens P. Automated segmentation of multiple sclerosis lesions by model outlier detection. IEEE Trans Med. Imaging. 2001 Aug;20(8):677-88. Van Leemput K, Maes F, Vandermeulen D, Suetens P. Automated model-based bias field correction of MR images of the brain. IEEE Trans Med Imaging. 1999 Oct;18(10):88596. Vercauteren T, Pennec X, Malis E, Perchant A, Ayache N. Insight into efficient image registration techniques and the demons algorithm. Inf. Process Med. Imaging. 2007a;20:495-506. Vercauteren T, Pennec X, Perchant A, Ayache N. Non-parametric diffeomorphic image registration with the demons algorithm. Med. Image Comput Comput Assist Interv Int. Conf Med. Image Comput. Comput Assist Interv. 2007b;10(Pt 2):319-26 Vercauteren T, Pennec X, Perchant A, Ayache N. Diffeomorphic Demons Using ITK's Finite Difference Solver Hierarchy. Insight Journal, ISC/NA-MIC Workshop on Open Science at MICCAI 2007. 2007c. Yelnik J, Bardinet E, Dormont D, Malandain G, Ourselin S, Tandé D, Karachi C, Ayache N, Cornu P, Agid Y. A three-dimensional, histological and deformable atlas of the human basal ganglia. I. Atlas construction based on immunohistochemical and MRI data. Neuroimage. 2007 Jan 15;34(2):618-38. Zubal IG, Harrell CR, Smith EO, Rattner Z, Gindi G, Hoffer PB. Computerized threedimensional segmented human anatomy. Med. Phys. 1994 Feb;21(2):299-302.

In: Brain Mapping and Diseases Ed: Diane E. Spinelle

ISBN: 978-1-61122-065-0 © 2011 Nova Science Publishers, Inc.

Chapter 10

BRAIN MAPPING ALTERATIONS IN STRABISMUS Martín Gallegos-Duarte1, Héctor F. Rubio-Chevannier2 and Jorge Mendiola-Santibañez3 1

Instituto de Enfermedades Congénitas. Querétaro, México 2 Unidad Neurológica Satélite, México, D.F. 3 Universidad Autónoma de Querétaro, México

ABSTRACT Congenital strabismus affects 3% of world population. Millions of persons suffer this condition, but still its origin or the reasons why not all patients respond to the traditional treatment are unknown. Until very recently, it was believed that congenital strabismus had no relation to cortical alterations; therefore, neuroimaging studies were only required when strabismus was present in premature infants or when brain damage was suspected. A preliminary study on strabismal patients in 1968 provided some insight into the incidence of the different presentations of strabismus in our institution, as well as the correlation among the various clinical signs. Based on this experience we decided to enlarge our sample. Using conventional EEG and digitized brain mapping (DBM) methods, we analyzed 195 young patients with clinical diagnosis of congenital strabismus –111 females (56.92%) and 84 males (43.08%); the age range was from 2 to 14 years. The DBM approach was done in real time. Given its low cost, security and availability, DBM turned to be a useful tool to evince some alterations in cerebral cortex related to congenital strabismus, especially dissociated strabismus. We also employed complementary neuroimaging methods for research purposes. From 195 DBM images, 56.4% exhibited various neuroelectric alterations, whereas 43.6% were considered normal. Abnormal DBM were more frequent in the dissociated strabismus group (64.95%) than in non-dissociated strabismus patients (42.6%); the rate of altered DBM images was higher in horizontal dissociated deviation cases (73.3%). Based on these findings, we recommend the use of DBM in patients with dissociated strabismus, and in some cases the treatment must go beyond surgery and glasses. Some of our patients were subjected to different neuroimaging methods, such as single Photon emission tomography (SPECT), magnetic resonance imaging (MRI),

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Martín Gallegos-Duarte, Héctor F. Rubio-Chevannier et al. granulometry, and proton nuclear magnetic resonance spectroscopy (1H NMRS) with the aim of correlating this data and gain further understanding on the origin of congenital strabismus, particularly dissociated strabismus cases. This chapter addresses aspects of congenital strabismus, as well as some of its cortical implications –neuroelectric, neurometabolic and morphometric. The illustrations are meant to make this interesting and scarcely-explored topic more accessible.

1. STRABISMUS Congenital strabismus affects 3% of world population [1]. This condition refers to the pathological ocular deviation [Photo 1], [1], that is, while the dominant eye controls the visual sense and direction, the deviant eye exhibits a different alignment to the purpose and direction of the dominant eye [2]. Since no neurological damage is evident in this condition, the question that prompts is: What is congenital strabismus and where is its origin? [3] [Hoyt and Good]. To gain more insight into this problem, we employed clinical neurological studies and neuroimaging to get some answers. There are always small differences in visual perception and in the movement of both eyes. In normal conditions, these variations are harmonized and integrated in the cerebral cortex, so that perceived images from each eye are fused into one. In strabismus these differences are large and anomalous relations are established between both eyes originating a functional competence between both visual fields. This in turn produces neurosensory variations such as amblyopia and suppression [4]. Congenital strabismus appears before infants are one year old and its clinical manifestations are varied [1,2,3,5]. From these manifestations the distinct sensory-motor descriptions of this condition have been integrated. However, more important than the time of appearance of this condition, is the fact that congenital strabismus is accompanied or not of dissociated movements [4]. Primary dissociated movements by nature are always congenital [4]; they can be detected when one of the eyes is fixed on a determined object, then the other manifests smooth and intermittent movements with variable angles that are regulated independently of the supranuclear control and Hering’s law [Photo 2a, 2b] [6,7,8]. Dissociated movements are identified through their clinical manifestations as: dissociated vertical deviation (DVD) when the predominant dissociated movement is upward [Photos 2a,2b] and dissociated horizontal deviation (DHD) when the movement is variable, asymmetric and directed outward [Photos 3a, 3b, 3c, 3d, 4a, 4b, 4c] [6,7].

A in Strabismus Brrain Mapping Alterations

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Phhotograph 1. In nfant with congeenital esotropia,, variety Ciancia. The left eye fixes f the gaze while w the rigght eye deviates inward.

In addition n, when the disssociated preddominant movvement is variaable and inwaard and it is acccompanied by DBM allterations, laatent nystagm mus, DVD, suppression, horizontal unncommittancee, hyperopia astigmatysmus a s, amblyopia and lack of toorticollis encoompasses a syyndrome know wn in Mexico as strabismic syndrome of angular variabbility (SSAV)) [8] [Photo 5aa, 5b, 5c, 5d].. DHD and SS SAV will be discussed d in more m detail in this chapter, since these foorms exhibit more m cortical alterations thhan the rest annd therefore have h been stuudied more thhoroughly.

Phhotographs 2a and a 2 b. In the left Photograph the left eye preesents a slight uppward deviationn due to a diissociated verticcal deviation in low degree thaat can be enhancced by diminishhing the entrancce of light to thhe eye or the quality of the imaage with a transllucent occlusor. These movem ments are independent or not asssociated with Hering’s H Law.

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Phhotographs 3a, 3b, 3c and 3d. Photographic P seequence taken in partial darkneess using the nigght shoot m mode. 6-year old d boy with DHD D, exhibits an inntermittent and variable v exodevviation when the right eye is fixed (the pupiil shows a light background refflection or Brokker's reflex withh a luminous spot at the e is deviated outward o and upward; the samee thing happens when the left eye is fixed, ceenter), the left eye thhe right eye then n gets deviated. Notice that devviation measurees are different for each eye. Thhis assymmetry and variability v in thee presentation angle a accompannied by other altterations such as a suuppression and amblyopia are not n a good signn in accordance with the normaativity of deviatiions reeported in neuro ophysiological studies s carried out o in our patiennts.

Phhotographs 4a, 4b and 4c. Patieent with DHD. Top: Exodeviattion of the rightt eye can be apppreciated beehind the translu ucent occlusor, while the left eye e is fixed. Bottom: The samee patient looking upward (ssupraduction). Notice N the asym mmetry betweenn the deviations of either eye; thhis clinically obbserved assymmetry is chaaracteristic of dissociated d strabbismus. Studies using digitizedd brain mappingg and SP PECT have sho own irregularitiees in the power,, symmetries annd coherences of o the different band b frrequencies that suggest s alteratioons in the neuroonal tracts –intrra-hemispheric and inter-hemisspheric.

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Photographs 5a, 5b, 5c and 5d. Six-year old, female patient with a variety of dissociated strabismus called “strabismic syndrome of angular variability” (SSAV). Upper Photos: Patient turns to her right, to the front and to her left during a stable phase (non-variable). Lower Photos: Patient tries to turn to her left during the variable phase. The left eye can not shift itself possibly due to the isometric contraction of the medial straight muscle that is not able to relax while the right eye gradually increases its deviation angle. This condition might be propitiated by the active cortical inhibition following Hering’s law possibly hindered by cortical disturbances evinced in Digitized Brain Mapping.

In cases of congenital strabismus, in which dissociated movements are rather evident, ophthalmologists usually call it “dissociated strabismus”. This condition entails a

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combination of movements in the horizontal, vertical and torsional directions, but the direction of the predominant movement is the one that gives name to the particular type. These dissociated movements can manifest themselves spontaneously, especially when the child in under physical or emotional stress: angry, tired, irritated, dehydrated or ill. There are some clinical manipulations that can induce this type of movements such as the cover test and cover-uncover test [9, 10] [Photos 2a, 2b]. Dissociated strabismus is usually related to neuroelectric, granulometric, sensory and perceptual alterations that will be addressed in this chapter. With regard to the origin of congenital strabismus, its multifactor character with certain family predisposition is generally accepted [5]. Electro-occulography studies (EOG) in first degree relatives considered healthy show 63% alterations in eye pursuit movements when they have a strabismic relative [11]. Although congenital strabismus does not follow a Mendelian inheritance pattern, in some occasions it behaves as a dominant autosomic disease of incomplete penetration and in other occasions as a multifactor disease [12]. In our sample, 27% of our patients [10] admit having at least other relative with strabismus. Among the many factors originating this disease, prematurity in newborns is an important risk factor to presenting strabismus, retinal alterations, refractive errors and neuronal immaturity [12]. In addition, newborns with low weight and respiratory distress [13,14,15] are especially susceptible to presenting periventricular, intraventricular or parenchymal hemorrhages. In premature newborns periventricular circulatory self-regulation is believed to be passive and dependent on perfusion pressure; meaning that fluctuations in arterial or central venous pressure, such as those occurring in traumatic births, due to respiratory alterations, the use of positive pressure and other maneuvers, can produce lesions in the vessels of the stem matrix [16,17]. Biochemical changes at this level [14] can also induce damage to neuronal interconnection pathways. This in turn produces various clinical manifestations, including strabismus. When early onset of strabismus is accompanied by evident neuronal damage, for example, infant cerebral palsy or psychomotor retardation, neuroimaging exams such as intracranial ultrasound, EEG and magnetic resonance imaging (MRI) are carried out as soon as possible to determine the type and extension of the brain damage. It is infrequent that a physician requests a BDM study to gain further insight beyond that attained with an EEG, though the former method is useful in congenital strabismus for providing information on cortical activity. Thus, patients with congenital strabismus are regularly prescribed lenses, visual therapy, orthoptic eye patches and surgery, but they could be presenting cortical malfunctioning, not clinically detectable. [8] Predisposition of patients to DBM alterations are those that are less than 7 years old and simultaneously present suppression, amblyopia, latent nystagmus and dissociated vertical deviation [Table 1].

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Table 1. In a group of 68 patients with congenital strabismus the clinical signs (Pearson’s correlations) were analyzed to establish correlations. Four signs with high correlations –latent nystagmus, dissociated vertical deviation, amblyopia and suppression-- were identified in dissociated strabismus. These four elements were also correlated with altered brain mappings. Pearson´s Correlations

Suppression

Amblyopia

Latent Nystagmus

Dissociated Vertical Deviation

Latent Nystagmus

Dissociated Vertical Deviation

Suppression 1

Amblyopia

.

.001

.000

.000

68

68

68

68

.396**

.396**

.442**

.432**

1

.194

.217

.001

.

.112

.075

68

68

68

68

.803**

.194

1

.000

.112

.

.000

68

68

68

68

.803**

1

.442**

.432**

.217

.000

.075

.000

.

68

68

68

68

**

Correlation is significant at the 0.01 level (2-tailed). Analysis.

2. PERCEPTUAL ASSESSMENT AND STRABISMUS Although CS does not affect what Piaget and Vygotski [18,19] called “superior functions”, there is evidence indicating that various association areas such as Broca’s, Wernicke’s, angular gyrus, and parieto-occipital regions, as well as motor areas might be involved; the results of perceptual assessments [20] and neuroelectric alterations in DBM [21] support this hypothesis. The former can be understood when the cerebral cortex is visualized as a vast interconnection network. The visual cortex not only has feedback circuitry but also conveys information to other extra-striatum cortical zones. For example, the visual system is involved in the process of turning ideas into semantic and reading-writing messages [22], but impairments such as strabismus and amblyopia can deteriorate these abilities. It has been suggested that letter and word perception and reading comprehension are affected by visual, semantic, and environmental aspects where such reading occurs [22,23]. Moreover, lexicological and semantic levels affect the perceptual process in the phonological and logographical aspects, respectively. It is also known that patients with CS exhibit difficulty in second and third degree stereopsis; this deficit indicates impairment in cortical integration. A subjective manner to assess the performance of the visual system is through perceptual evaluations. These tests are designed to assess the capacity of the individual to give meaning

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to the information provided by the visual system, cerebral cortex and association areas [23]. Through these tests it is possible to establish whether the responses are adequate for certain visual stimuli. Patients with strabismus present low spatial coordination, impairment in locomotor activity and a low profile in the results of perceptual tests. In a prospective study in which a screening perceptual assessment was carried out in a homogeneous group of 7-year old children with dissociated strabismus( n= 10), we found that all presented a low profile in visual perception, especially stereopsis, forms, sizes and spatial localization [21]. The mean values of these findings are given in Table 2. Table 2. Perceptual assessment. An average of visual abilities was evaluated in 10 children (7 year-old) with dissociated strabismus. The outcome of each test was always deficient (less than 100%) especially in stereopsis, velocity of perception, perceptions of forms and sizes, and peripheral vision. Strabismus Dissociated and Perceptual Alterations n = 10 Perception of depth (estereopsia) Visual memory Speed of perception Space perception Saccadic movements Peripheral vision Perception of fundamental elements Perception of forms and sizes Movements of pursuit

0% 77.50% 64.25% 56.25% 71.25% 41.75% 87.75% 41.75% 81.25%

All patients had average school performance and adequate socialization within parameters considered as normal by parents, pediatricians and teachers. However, when some parents were interviewed they recognize that their sons tended to mix–up letters when reading or writing, were slow at reading and doing their homework, got easily distracted, placed their faces at a very short distance from their notebook or had a bad quality in handwriting. Although these children presented oculomotor alterations without overt neurologic manifestations, they revealed visual perceptual deficits, possibly related to impairment in neuronal interconnection pathways between striatum cortex and cortico-cortical and interhemispheric areas participating in image processing –recognition, meaning, spatial localization, depth and visual memory, among others.

3. NEUROFUNCTIONAL STUDIES AND STRABISMUS In the study of strabismus and amblyopia different neurofunctional and morphometric methods have been utilized. Thouvenin [25] in France uses brain electrical activity mapping (BEAM) as a screening method to verify visual reactivity at cortical level in small children with strabismic amblyopia. Horton [26, 27] in the USA analyzes brain cortex in primates with cytochemical techniques; he has concluded that amblyopia is a cortical malfunction with

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ophthalmologic manifestations. In the same country, Mendola [28] using Voxel analysis, reports diminution in gray matter in areas including the calcarine sulcus, parieto-temporal regions, and ventral temporal areas. Suk et al [29] in Hong Kong have used this same technique to analyze the redistribution of gray matter, which they think is due to brain plasticity, in adult Chinese patients with exotropia employing Voxel-based morphometry (VBM). Morphometric findings reported by Mendola [28] and Chan [29] are in agreement with the neurofunctional results by Gallegos et al. [8,9,21,30] [Photo 6] showing that areas of the extra-striate cortex are affected in dissociated strabismus. Gallegos-Duarte et al. [31] and Moguel-Ancheita et al. [32] using SPECT [Photo 7, 8] and DBM [9a, 9b]demonstrate the existence of improved cortical metabolic changes after strabismus treatment [30,31,32]. Gastaut [30] in 1982 and Panayiotopoulos [33, 34] in 1989, using EEG, described the paroxysmal symptomatology in occipital lobes as a different form of idiopathic partial epilepsy. This condition refers to occipital lobe epilepsy in children with its vast symptomatology, but in which ocular deviation is exceptional [35]. On the other hand, Gallegos [36] reports two DHD patients with a paradoxical cortical response to light, to whom intermittent Photo stimulation induces regularization in paroxysmal brain activity. [Photos 10a, 10b and 10c] Gallegos and Moguel [30,31,32] using SPECT, DBM and EOG demonstrate neuroadaptive changes taking place after the application of the botulism toxin to a girl with SSAV, which is attributable to an epileptogenic focus in ictal phase in the temporal lobe [Photo 7]. In addition, Gallegos et al. [21], using DBM [Photo 9a and 9b] and 1H-NMRS show the existence of active neuronal distress in cerebral cortex in patients with DHD [Photo 11a ] and SSAV [Photo 11b] manifested as the diminution in aspartate levels, lactate enhancement and loss of the relationship between creatine and choline in cases of dissociated strabismus.

Photograph 6. Photograph of an eight year-old boy with dissociated strabismus (DHD), attention deficit disorder, and low school performance. He exhibits irritative discharges during sleep in left frontotemporal regions (electrical activity was interpolated to brain mapping). EEG traces during wakefulness (not represented here) are anomalous, consisting of the slowing-down of the left temporal region and parieto-temporal asymmetry.

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Photograph 7. Scintillography from tomographic images taken with SPECT scan analyzed 45 minutes after the iv administration of tecnecium 99 ethyl cysteinate dimer to be detected under spectrometric observation in the red-violet scale performed in a 5 year-old girl with SSAV. In zones 3 and 4 corresponding to right temporal area (left side of the image) an epileptogenic area can be appreciated. Zones 7, 8, and 9 –corresponding to left fronto-temporal area (right side of the image)—exhibit low expenditure of a glucose analog; these areas presented the lowest voltages. EEG showed slow and paroxysmal activity with hyperactivity in right fronto-temporal regions and higher power in right temporal region.

Photograph 8. A second brain SPECT was taken three months later, pharmacological treatment. The right temporal region has no signs of hyperactivity; a low to moderate hypoperfusion zone can be appreciated in the left fronto-parietal region, patent in transaxial and coronal sections. An increase in metabolic activity in occipital lobes is encountered when compared to the previous study.

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Phhotograph 9a. Digitized D Brain Mapping of thee same case of Photo P 7. The stuudy shows increeased delta acctivity in both frontal fr lobes, spreading in lesseer degree to botth paracentral heead regions, andd diiminished anterro-posterior alphha gradient.

Phhotograph 9b. Digitized D Brain Mapping (DBM M) of the same case of Photo 8. 8 A second braiin mapping w taken three months was m later pharmacological treatment. t The study s revealed a discreet improovement in thhe distribution of o the power, a diminution d of dysynchrony d andd a discreet delaay in the cerebrral ellectrogénesis. A year later anotther DBM was taken t that was reported r like noormal.

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Martín Gallegos-Duarte, Héctor F. Rubbio-Chevannieer et al.

Phhotograph 10a. EEG Six-year old, o female withh DHD diagnossis. The EEG shhows unusually increased slow activity locaalized mostly both posterior heead regions shortly after the hyyperventilation began. b M Mixed with musccle and movemeent artifacts.

Phhotograph 10b. EEG of seem patient, p awake and a with good driving d responsee with the interm mittent Phhoto stimulation n with stroboscopic light (flashhing of 8 Hz/s).

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Phhotograph 10c. The EEG of thee same patient shows s a focal sppike and show wave w dischargee emerging onn the right temp poral and parietaal region with secondary s spreaad to left homologous head reggions, event innterpolated a braain mapping.

These auth hors suggest thhat the dissociated movemeents are the oculomotor maanifestation off the epileptog genic disease [21, 30]. Bessides, Gallegoos et al [37] fiind, using graanulometry, thhat DHD and SSAV S have diifferent degreees of neuronall maturity that were determiined by this teechnique. mpleting the piccture of corticcal alterations underlying The above mentioned stuudies are com sttrabismus, and d it has becom me clear thatt the striate cortex and thee extra-striate cortex are innvolved in con ngenital dissocciated strabism mus. Moreoveer, neuroelectric studies of thhe cerebral coortex have sheed a considerab able amount off light on manyy aspects of thhis disease.

4.. DIAGNOSSTIC NEURO OPHYSIOLOGICAL METHODS APPPLIED IN STRABISMIC C STUDIES S S made the first reeport showingg that 30% off strabismic In 1950 Leevinson and Stillerman paatients presen nted electroenncephalographhic malfunctiooning. Stillerm man thought that t ocular allterations inclluding strabismus induced irritation in the occipital cortex, that is, for this auuthor strabism mus caused coortical changees, whereas Smith S and Keellaway propoounded that occcipital alterattions found inn electrical reccordings of some strabismicc patients were projected frrom thalamic nuclei and thhat the physioopathology off strabismus should be diffferent from eppileptogenic leesions [38]. Given the difficulties reesearchers in strabismus s hadd to confront at that time in i trying to unnderstand thiss condition byy means of EE EG studies, Sooto de la Vegaa and Romeroo-Apis [38] puublish in 197 70 the first series of 116 cases in straabismus (including some neurogenic n

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associated diseases) showing that 92% of patients presented electroencephalographic anomalies, especially the presence of slow waves. In this study there is a diagnosed case of “intermittent exotropia” along with an electroencephalographic recording exhibiting “clear spike paroxysmal activity from temporo-occipital (right and left) and occipito-occipital derivations”. In retrospect, this case highly probably corresponded to a patient with dissociated strabismus, similar to intermittent exotropia, now known as DHD. Three fundamental facts originated from those early studies: a) a high incidence of cortical anomalies in the strabismic population, b) slow brain waves, and c) the presence of paroxysms. However, a long time had to elapse before many factors associated with dissociated strabismus could be correlated, namely clinical findings, neuroelectric recordings, neuroimaging studies and physiopathogeny. Based on those studies, Gallegos and Moguel [8] prospectively study DBM information from 11 patients presenting congenital esotropia with variability and uncoordinated manifestations, but now related to anomalies in the brain cortex (Fisher test, p< 0.001). These authors conclude that this syndrome has own characteristics. It is characterized by the presence of congenital esotropia, variability, limited abduction, latent nystagmus, lateral nystagmus, dissociated vertical deviation (DVD) amblyopia, suppression, asymmetric horizontal movements and hyperopic astigmatism. [8,9,10]. Preliminary studies of the 11 patients mentioned previously showed lateralized corticosubcortical dysfunction in 4 cases, diffuse dysfunction in 2 cases, asymmetry in frequency and power, electrooculogenesis delay in 2 cases, and non-lateralized irregular bursts of probable sub-cortical or centro-encephalic origin in 3 cases [8]. The combination of neuroimaging methods with electrophysiological studies allows a better localization of the origin of some alterations such as epilepsy, since the time-spatial resolution is enhanced [39]. A given example is the analysis of both, the neuroelectric behavior with DBM and the neurometabolic performance with SPECT, to study strabismus. [30, 31]. Using this combination of techniques, in one of our series of patients [30] the presence of low activity and high-voltage paroxysmal bursts and higher power in the right temporal region were evinced with EEG-DBM. Also, the presence of an epileptogenic focus in ictal phase was identified with SPECT imaging in the right temporal region originating a small symptomatogenic area (=194) with relative metabolic hyperactivity (64.7 U) [Photo 7]. This patient showed no neurological manifestations other than strabismus; this condition was characterized by: slow, variable and intermittent movements in the right eye, sometimes vertical and upward. These movements were present several times a week, particularly when she was tired or sleepy [Photo 4a]. The correlation between neurometabolic [Photos 7, 8] and neuroelectric alterations [Photos 6, 9a and 9b] could be related to the dissociated eye movements in a similar manner to some interictal EEG abnormalities observed in epileptic disorders. After treatment with botulism toxin, visual therapy and neurological handling, the symptomatology improved remarkably. The motor aspect was stabilized; cortical, electric and metabolic responses also improved. A subsequent control study revealed a redistribution of glucose expenditure where higher quantities were present in posterior brain regions [Photo 8]. As DBM measures minute electrical pulses in brain activity, SPECT can quantify minor differences in the consumption of glucose analogs, by generating spectrometric images in two and three dimensions that can be computer analyzed in vivo [40, 41]. Given its spatial

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resolution, SPECT analysis is used to detect areas with functional deficits. [Photo 7] [31, 32, 40, 42] The positive findings encountered in the above-mentioned studies have stimulated the use of neuroimaging methods such as SPECT (Photos 7 and 8) EEG [Photos 10a,10b], DBM [Photo 10c], 1H-NMRS and MRI [Photos 11a, 11b and 11c], granulometry [Photos 12a, 12b, 12c, 12d], EOG [Photos 13, 14a, 14b, 14c], and neurometry [Figure 1, Table 3 ], in studies on cerebral cortex behavior in strabismus. For example, with the aid of neuroimaging techniques it has been possible to differentiate in an ontogenic and functional manner that congenital esotropia has two varieties: the most common was described by Dr. Alberto Ciancia and known as “Ciancia syndrome” [Photo 1], the second dissociated condition has been recently dubbed in Mexico as SSAV [Photos 5a, 5b, 5c, and 5c]. Each form has its own distinctive features [10]. Combined neuroimaging studies such as DBM and 1H-NMRS have recently contributed to the better understanding of SSAV (of recent nosologic description) and DHD (thought to be a type of intermittent exotropia until a few years ago); these ailments might be considered the oculomotor expression of a cortical malfunction [21,30]. By means of DBM it is known that DHD exhibits various malfunctions in brain electrogenesis different from those found in intermittent exotropia. [36] From 1H-NMRS information [21] it is also known that there is neuronal distress in DHD, whereas granulometry [37] has shown that this type of patients presents less granulometric density than healthy children do. [Photos 12a, 12b, 12c, 12c]

Photo 11a. Spectroscopy (1H-NMRS) of the occipital lobes of the brain of a boy of 7 years of age, with DHD diagnosis. A loss of the choline - creatine relation is observed.

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Photo 11b. Spectroscopy (1H-NMRS) performed in a 4 year-old girl with SSAV showing high lactate levels (4 units) and decreased N-acetyl-aspartate concentration (12 units). The white square in the axial and sagittal projections shows the exact location where the 1H-NMRS sample was taken from.

Photograph 11c. Normal RMI of a girl of 7 years of age with SEVA. The macroestructural morphometrics reports always are normal in these cases; nevertheless, the determined microstructural reports by means of the granulometric analysis are altered.

Photographs 12a and 12b. White substance of the occipital lobes in a healthy 7 year-old boy. 12a Abundant granulometric small forms are appraised that penetrate deeply in the gray substance and in addition are ordered in coraliform aspect. (12a).The healthy brains are gradually losing the elements of small size of the white substance during the granulometric technique (12b).

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Photographs 12c and 12d. 12c. White substance of the occipital cortex in a 7 year-old boy with DHD. Notice the heavy absence of small granulometric sized elements (12c) as well as lobulated aspect (12d) that emerge from two great pieces. During the procedure of elimination of granulometric sized elements, first the small elements disappear, later the elements of greater size are disappearing progressively. In the brain with DHD it is possible to be observed that the small elements do not exist and this is a important morphologic difference.

Figure 1. The graph shows higher left to right inter-hemispheric asymmetry values in all frequency bands from O1-O2 occipital regions. Long interconnection neurons might be responsible for this electric malfunctioning.

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Table 3. The shows the numerical values of figure 1. Positive values in bold numbers lie above established parameters. It is worth noticing that the highest inter-hemispheric asymmetry in all frequency bands is located in the occipital lobes. Monopolar Interhemispheric Symmetry in a case of DHD

Total Delta Theta Alpha Beta Comb.

Fpl-Fp2

F3-F4

C3-C4

P3-P4

O1-O2

F7-F8

T3-T4

T5- T6

-0.5 -0.58 -0.19 0.45 -0.69 -0.07

-0.18 0.19 -0.2 -0.6 -0.26 -1.5

-0.73 0 0.2 -1.98 -0.68 0.97

-1.31 -0.68 -0.67 -1.6 -1.87 0.54

3.11 2.7 3.4 2.36 2.66 1.69

0.36 0.61 0.12 0.26 0.07 -1.45

0.58 0.35 0.42 0.96 0.03 -0.25

0.27 -0.17 0.19 0.58 0.36 -1.14

5. SPECTROSCOPY (1H-NMRS) In a study on children with dissociated strabismus [21] the biochemical composition of cerebral cortex was analyzed with 1H-NMRS to determine whether biochemical alterations could be found in occipital lobes in cases of SSAV and DHD. 10 children participated in this research in which metabolic alterations were reported, such as enhanced lactate levels [Figure 2], decrease in N-acetyl-aspartate levels Figure 3] and lack of correlation between choline and creatine. [Figure 4].

Figure 2. Increase in lactate concentrations in 6 out of 10 patients with DHD. Presence of lactate reflects hypoxia and neuronal distress. Blue bars correspond with low lactate levels, whereas green bars indicate high concentrations of this metabolite.

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Figure 3. Diminution in N-Acetil Aspartate concentrations in 7 out of 10 patients with DHD. 1

H-NMRS gives information on the biochemical constitution of brain structures by measuring some metabolite concentrations. To achieve this electrons are excited in these substances by the incoming energy from magnetic resonance, this causes particles to change from a certain state α to a state β, when the electrons return to their initial state they release energy in the radiofrequency range that can be put into graphs. [43, 44] [Photo 11 a, 11 b] Among the quantifiable elements in the brain by 1H-NMRS is creatine (Cr), a metabolite associated with energy production; N-acetyl-aspartate (NAA), found in axonal projections in white matter and whose diminution indicates neuronal damage, and lactate (Lac) whose enhancement implies neuronal distress, since it is a metabolite of anaerobic glycolisis [44,45]. There is evidence showing that epileptogenic activity induces neurolectric and neurometabolic changes. Studies in rats [46] show that epileptic activity increases oxygen consumption in the affected area.

Figure 4. Creatine/ Choline relation. In normal conditions this relation must be 1:1, some patients presented changes in this proportion.

During the ictal phase [Photo 7] neurons receive an excitatory impulse that enhance oxygen consumption, which in turn reflects a subsequent depletion of energy in the affected

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area; this induces, on the one hand, electroencephalographic changes [Photo 9a] and, on the other hand, metabolic changes in the area surrounding the lesion, consisting basically in a raise in oxygen, lactate and pyruvate levels [Photo 11b, Figure 2]. It has been suggested that dissociated movements may be a form of expression of the epileptogenic condition and that the cerebral cortex actively participates in their origin [30]. In a study carried out in 7 year-old children (n=10) with dissociated strabismus and EEG paroxysms, 6 exhibited lactate enhancement [Figure 2]] and 7 showed a small diminution in N-acetyl-aspartate [18] [Figure 3]. N-acetyl-aspartate is present in high concentrations almost exclusively in brain neurons; therefore, it can be used to measure neuronal loss or diminution of gray matter [43]. A decrement in this neuronal marker has been reported in occipital cerebral cortex in children with dissociated strabismus [18]. This agrees with the findings reported by Suk [29] in the sense that a diminution in gray matter might exist in the occipital lobes of strabismic patients. The presence of lactate in occipital lobes (site where samples were taken) point to the existence of acute neuronal distress that can be related to epileptogenic activity detected with EEG and DBM methods in distant areas of visual brain cortex, connected by intrahemispheric or transcortical tracts, while N-acetyl-aspartate diminution may indicate a decrease in neuronal volume. In perspective, these findings suggest that the symptomatogenic zone is far from the visual brain cortex [21].

6. EOG AND STRABISMUS Since Dewar introduced the electrooculographic (EOG) study in humans in 1877, this technique is the most widely used to understand vestibular functioning; currently this methodology has been enriched with the use of video-oculography [47]. Initially this test was carried out by using a galvanometer and electrodes immersed in saline solution. This method has been improved not only in the technical aspects but also in its accuracy since nowadays it has incorporated EOG and video-recording to the electroencephalographic recording of children with strabismus. To obtain precise EOG records two electrodes placed in the temporal lobes and at least one nasal electrode must be employed [44]. However, we add a modification by taking the recordings from frontopolar and/or anterior temporal lobes having as reference the interorbital-supranasal electrode [Photo 13]. This renders a rather simple morphology in EOG traces [Photos 14a, 14b, 14c] appropriate for practical observations.

Photograph 13. Position of the electrodes by strabismus study.

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Phhotograph 14a. Normal electroooculographic trrace of a healthyy 7 year-old girrl. Upper trace corresponds c too left eye, lowerr trace to the rigght eye. A peak means electronnegative activityy and a valley ellectropositive acctivity. Dotted vertical v lines reepresent one-seccond intervals. The T procedure consists c in thhe patient follow wing with his/heer glance (withoout moving the head) glance a slow-moving object o diisplaced horizon ntally from left to right in a 100-s interval (slow w speed from leeft to right, VLIID, initials inn Spanish). In no ormal conditionns both eyes traces are simultanneous and keepp a 1:1 relation between b eye poositions and thee moving objectt (this relation iss called gain).

Phhotograph 14b. Electrooculogrraphic trace of a 7 year-old girll with SSAV. Inn this case the patient p must foollow with her glance g a slow moving m object frrom right to leftt (VLDI, initialss in Spanish). A decrease inn the electric potential is vieweed as a flatteningg of trace, simuultaneous to a significant delay in the puursuit movemen nt when comparred to the normal trace. Gain iss 25% higher (11:1.25), that is, the t reesponse is 200 ms m delayed withh respect to the normal one forr an object moviing horizontallyy in space.

Phhotograph 14c. The same patieent as in previouus 3 weeks afterr the applicationn of the botulinnic toxin into thhe medial straigh ht muscles. Thee procedure is too follow a slow w-moving objectt from right to left. l A Attenuation in th he trace can be appreciated a alonng with a largerr gain indicatingg a decrease in the t eclectic poower; this is clin nically called paresis, p caused in i this case by thhe chemical ageent. This sequennce carried ouut in different epochs shows ellectrooculographic variations inn the VLDI (sloow movement from f right to leeft) sequence originated by the pharmacologiccal handling of strabismus. s

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Although this technique is simple, it enables the recognition, on the one hand, of oculopalpebral potentials during the EEG recording to identify some differences in amplitude and synchrony, as well as the polarity of slow pursuit and saccadic movements in treated and untreated patients with dissociated strabismus. To obtain DBM images, the recordings are not made in the EOG recording area, since eye movements produce artifacts that can be misleading. EEG studies in primates in which strabismus has been provoked [48] indicate a difference in the velocity of each eye movement when they go from an out toward an inside position (temporal nasal direction). This movement is quicker when eyes go from the inside out; this difference in velocities can be appreciated during pursuit movements as a delay with respect to the object displaced through space. This difference is known as gain [Photo 15]. The EOG analysis in patients with CS indicates alterations in pursuit movements [49]. In addition, permanence of opto-kinetic nystagmus (OKN) has been described in this type of patients at older ages.

Photograph 15. An object (white shade) uniformly moves of left to right at a constant speed of 10º /s and a distance of 50 cm in scotopic conditions (in the dark). The patient follows the object with this left eye while we observed the position of the eye with respect to the object. A lag in the position of the eye with respect to the object is observed. This misalignment is the "gain". The presence of "gain" means that there is motor instability and angular variability, which increases the risk of which the patients show a dissociated strabismus and greater incidence of alterations in the Digitized Brain Mapping registries.

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7. NEUROPHYSIOLOGICAL STUDIES ON STRABISMUS: ELECTROENCEPHALOGRAM AND DIGITIZED BRAIN MAPPING In the study of strabismus, neurophysiological studies are ahead of other neurodiagnostic methods since they are endowed with a high temporal resolution (quasi-real). Besides, the obtained values are completely objective; hindrances being lower spatial resolution and its limitation to a bi-axial plane [50]. For these reasons data must be analyzed under strict neurofunctional parameters. The percentage of anomalous neurophysiological studies [Photo 16 and 17] in this series was considerably high [Figure 5]; therefore, we suggest that DBM must be performed in all patients with congenital and neurogenic strabismus, where there is evidence of dissociated movements, suspected hypoxia, low weight, neonatal trauma or some other gestational event. One must bear in mind that a single DBM considered as “normal” does not preclude previous or future abnormal mappings.

Photograph 16. EEG and Digitized Brain Mapping of a six year-old boy with SSAV (a form of dissociated strabismus). The study reveals bilateral irregularity; there is no antero-posterior gradient as should correspond to a patient of this age. Asymmetry due to lower voltage on right temporal lobe and bilateral slowing with irritative discharge in left frontal region interpolated to brain mapping.

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Phhotograph 17. Digitized D Brain Mapping bi-dim mensional imagge of the electricc activity of a 6 year-old giirl with DHD (o other form of diissociated strabiismus). There iss an asymmetryy in posterior regions due too the decreased beta activity in both right parieeto-occipital annd temporal regiions; this contraasts with the allpha- and theta- wave predominnance in the sam me regions ipsillateral to the noon-dominant eyee. Conventional EE EG exhibited asyynchrony in thee alpha rhythm.

Fiigure 5. We anaalyzed 195 filess with essential strabismus (nott neurogenic strrabismus). Mostt of the paatients showed neurologic n alterrations: 85 casees (56, 41%) weere reported likee normal whereas 110 paatients (63, 59% %) showed neuroelectrics alteraations.

DBM studiies are obtaineed of EEG datta, which in tuurn represent the power and the spatioteemporal relatio on of the diffferent frequenncy bands andd the electricaal conductivityy of neural grroups, especiaally of the apiccal dendrites of o the pyramiddal cells in cerrebral cortex. These T cells arre influenced by b the electriccal activity of subcortical neeurons [50]. DBM reveaals the dynam mic and functioonal character of the corticaal function in real r time. It caan be readily applied to all strabismic children. c It is employed reggularly to dettermine the im mplications an nd the possibble origin of some clinicaal manifestations of the disease. d An

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example of the practicality of this technique is the verification of positive neuroadaptive changes posterior to medical treatment or surgery [30, 31, 32, and 51]. DBM data are useful to demonstrate an improvement in the gain parameter during horizontal version movements posterior to medical treatment or surgery [30]. This confirms the hypothesis of neuroelectric [30, 31] and neurometabolic [30,32] improvements in comparison to the values of these parameters previous to pharmacological handling with botulism toxin and/or surgery [Photos 7, 8, 9a and 9b]. The neuroadaptive capacity encountered in patients having undergone surgery for strabismus is certainly due to the plasticity of the human brain; this characteristic is defined as the capacity “to minimize the effects of lesions through structural and functional changes [51] [Pascual-Castroviejo]. The best way to assess this plasticity is by analyzing “the clinical situation with respect to the congenital anomaly or to the pre- and post-treatment stages in the acquired “processes” [51], in other words, to determine a “before” and “after” with the utmost objectivity [30, 31, 32] [Photos 7, 8 9a and 9b]. Among the benefits obtained from neurophysiological and neuroimaging studies one can mention the possibility of obtaining positive changes by medical and surgical handling of strabismus [30,31,32], such as improvement in electric [30,31] and metabolic [30,31] interhemispheric coherence, power redistribution [9,21,30], input improvement to occipital cortical regions [21], and a diminution of paroxysms [9,21]. It is important to emphasize that strabismus is not only a cosmetic matter; rather it has become a neurological question, which is the true issue. In order to verify whether the medical and surgical handling of strabismus modifies in a positive manner the neuroelectric signal (using DBM technique), a comparison was made between the incidence of pathological reports of volunteers who were subjected to this technique before and 3 months after surgery; their electroencephalographic recordings were compared, the preliminary study included 68 patients. The DBM previous to surgery revealed anomalies in 37 (54.4%) of 68 subjects [Table 4]. Table 4. Sixty-eight patients with diverse types of strabismus were analyzed. A Digitized Brain Mapping was carried out prior and after medical and surgical handling. Data show 54.4% of abnormal maps previous to treatment. First Brain Mapping n = 68 Frequency Percent Valid Percent

Cumulative Percent

indeterminate

1

1.5

1.9

1.9

normal

15

22.1

28.3

30.2

abnormal

37

54.4%

69.8

100.0

Subtotal

53

77.9

100.0

Missing System

15

22.1

Total

68

100.0

In 29 volunteers whit DBM abnormal already treated, a second DBM was performed 3 months after the surgery; from these 18 (26.5%) presented abnormalities in the DBM [Table 5]. Since participants in this study were volunteers, many refused to have a second DBM;

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therefore, only a decreasing trend in anomalous DBM obtained 3 months after treatment could be shown. Table 5. After 3 months of medical and surgical handling, the incidence of abnormal Digitized Brain Mappings in a voluntary group (n = 29 patients) showed decreased to 26.5% of abnormal DBM. Second Brain Mapping n = 29 Frequency Percent Valid Percent

Cumulative Percent

normal

11

16.2

37.9

37.9

abnormal

18

26.5%

62.1

100.0

Subtotal

29

42.6

100.0

Missing System

39

57.4

Total

68

100.0

In other preliminary study the rates among the various kinds of congenital strabismus were analyzed, as well as the rates of neuroelectric alterations in DBM. A high incidence (38.24%) of dissociated strabismus (DHD, SSAV, variable esotropia) was found [Figure 6]; the more frequent neuroelectric alterations were hyperactivity and slowing-down of the activity [Figure 7].

Figure 6. Graph showing the incidence of various kinds of strabismus found in a 68-patient case study. A high incidence (38.24%) of dissociated strabismus was detected (14.71% with DHD and 23.53% with SSAV); the remaining subjects (61.76%) presented non-dissociated strabismus.

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Figure 7. General trend of neuroelectric behavior in 68 patients with strabismus exhibiting positive alterations in the Digitized Brain Mapping previous to surgery.

In 49 patients (54.4%) of this preliminary series some alterations (slowing-down of the activity and paroxystic activity) were identified in the different areas of the cerebral cortex, though the majority of these alterations were predominant in occipital and frontal areas. [Table 6]. Table 6. In 49 out of 68 Digitized Brain Mappings it was possible to identify the predominant location of electrical disturbances. The majority of alterations consisted of slowing of the activity in occipital and anterior regions of the brain. Predominant location of the electrical alterations N = 49 Anterior

Temporal

Parietal

Occipital

Central

Mixto

Hemispheric

Slowing

4

2

1

9

2

9

4

Paroxystic

4

2

1

4

2

3

2

Total

8

4

2

13

4

12

6

Based on these findings, we decided to analyze 195 cases to determine whether alterations found in DBM rendered a significant difference between dissociated and nondissociated strabismus. From the sample, 111 (56.92%) were females and 84 (43.08%) males, ages ranging from 1 to 12 years old. [Figure 8]

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60

50

Percent

40

30

20

56.92 43.08

10

0 Male

Female Sex

Figure 8. Digitized Brain Mapping analysis of 195 clinical cases; 111 (56.92%) were females and 84 (43.08%) males.

The majority of strabismic patients present neuroelectric alterations. From 195 patients, only 85 exhibited normal brain behavior, whereas 110 (56.4%) exhibited anomalies [Figure 9] Fourteen cases presented paroxysms, in 13 patients irritable zones were identified, slow waves were patent in 40 cases, a combination of slow waves and paroxysms was observed in 16 patients, and 27 patients exhibited other alterations as asynchrony, focalization, irritability, etc. [Table 7].

Figure 9. Trends in electrical behavior of 110 patients with altered Digitized Brain Mapping classified into groups: exotropia, 7 cases; esotropia, 39 cases; DHD, 22 cases; and other types of strabismus, 12. Notice the preeminence of esodeviations formed by esotropy and SSAV groups. These two add up to 69 cases or 62.7% of the whole sample (195 patients).

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Table 7. Trend in the electric behavior of cerebral cortex in 195 cases with dissociated strabismus. From these, 110 cases exhibited alterations in brain mapping divided into: paroxysms, 14 cases (7.2%); slowing-down of brain waves (this was the most significant finding), 40 cases (20.5%); irritative area, 13 cases (6.7%); combined alterations, 16 cases (8.2%), and other types of malfunctions such as asynchrony and asymmetries, among others. Trend in the electrical behavior in Strabismus. n = 195

Valid

Total

Normal Paroxism Slowness Irritative zone Combination Others

Frecuency 85 14 40 13 16 27 195

Valid Percent 43.6 7.2 20.5 6.7 8.2 13.8 100

In altered DBM, dissociated strabismus was more frequent than non-dissociated ones. The rate of altered DBM was still higher in dissociated horizontal deviations, which is the variety with more neuroelectrics alterations followed by the SSAV group. In conclusion this study shows that some neuroelectrics alterations in the cerebral cortex are related to strabismus, especially dissociated strabismus [Table 7]. A clear systematization of strabismus can be established by studying the clinical behavior of congenital strabismus. However, neuroelectrics findings in DBM do not reveal a characteristic pattern for identifying each type of this condition. In addition, a clear cause/effect correlation has not been established. Certainly, strabismus in most cases leaves its mark in the electroencephalographic recording, though it is not always revealed in the first attempt, given the existence of “false negatives”. The former could be due to the multifactor origin of the disease [3,4,5,10,11] or to the ontogenic nature of the brain [52]. There are always irregularities in the neuroelectric oscillations, a changing background that can be modified by internal and external factors [50,53]. But this situation must not hinder the research of strabismus using DBM. The finding of electric alterations in DBM not only has drawn some light into the origin of congenital strabismus and the participation of the cerebral cortex, but also this knowledge can guide us to find the best therapeutic alternatives for these patients. For example, in the case of dissociated cases with altered DBM, it is necessary to give the patient additional visual therapy, together with a visual perceptual assessment and, if necessary, refer them to visual therapy, language therapy, child psychology or neurology, as the case requires. Though we can not assume a pathognomonic electric behavior in strabismus, we can correlate with caution every neurofunctional alteration with the clinical implications of the disease. For example, when strabismus is accompanied of dissociated movements, suppression, latent nystagmus and amblyopia, then cortical alterations are more evident [5,8,21]. The proportion of neuroelectric alterations that each of these eventualities

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contributes in itself is not known, because they are intricately intermingled. A correlation of these clinical signs is shown in Table 1. Besides, it is known that these signs underlie dissociated strabismus, and this condition in turn presents more than 70% of the alterations found in DBM. The use of digitized brain mapping (DBM) as the chosen instrument to measure neurofunction in strabismus does not demerit the benefit that other methods with higher spatial resolution can offer and that may complement or be combined with the first technique, as the case requires, in a specific research problem within this field. In this sense, we have employed diverse complementary neuroimaging methods in vivo such as DBM, conventional EEG, neurometry, SPECT, 1H-NMRS, EOG, MRI, and granulometry. These techniques enable the analysis of brain electric behavior, energetic metabolism, biochemical composition, or morphometry. The information drawn from these approaches has provided insights into brain structure and function of children with strabismus. The use of one or more of these tools has provided information on the cause-effect relationship [21,30], structural maturity concept [37], evinced neuro-adaptive changes [30,31,32] posterior to medical and surgical treatments, as well as shed some understanding into the origin of the disease [21]. The combination of DBM, SPECT and 1H-NMRS has rendered solid evidence supporting the correlation between dissociated movements and epileptogenic disease [21,30]. Indeed, it is possible that when intermittent and variable involuntary oculomotor movements appearing several times a week are accompanied by positive paraclinic studies such as altered DBM are, in reality, and in agreement with the International League Against Epilepsy (ILAE) [54,55,56], a manifestation of epilepsy that has been evidently sub-diagnosed [21,30]. In other words, the presence of different irregularities of brain waves determined with EEG and DBM can be present in diverse neurological malfunctions such as it occurs in children with attention deficit disorder. In the specific case of children with dissociated strabismus, additional biochemical unbalances in cerebral cortex are detected with 1H-NMRS, as well as intermittent involuntary eye movements. By correlating these findings, there is evidence in dissociated CS not only for active neuronal distress, but also for the fact that these alterations in the cerebral cortex are more the cause than the effect of strabismus [21]. An example of the above is the marked presence of slow activity in brain electric activity. It is known that continuous slow activity may be associated with a diminution in cholinergic cortical afferences in anterior brain structures and that paroxysmal activity is linked to neurotransmission unbalances [50]. Both situations have been found in many of our patients [Figure 7 and Table 6], especially those with dissociated strabismus such as SSAV and DHD. We analyzed 27 clinical files of patients with DHD to determine the ratio between “slowing-down” and activity enhancement (paroxysms and irritative discharges. Thirteen cases (48.1%) showed positive slowing-down, whereas 6 of the 27 patients exhibited irritative discharges and 10 paroxysms. [Table 8] Age range of these patients was from 2 to 14 years old; mean age= 6.7 years and mode = 6 years. We found that brain alterations were scattered in the following manner: 25.9% temporal, 25.9% frontal, 22.2% occipital and 22% parietal as such or in combination with other regions. These findings bring to our minds those obtained with DBM approach in children suffering from attention deficit disorder (ADD). Similar to them, some patients with dissociated strabismus present a profile known as “maturity delay”, that is, enhancement of slow activity and deficit in fast activity.

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Other point in common with ADD children is a higher epileptiform activity with respect to normal population of this age, as well as a decrease in inter-hemispheric coherence and an increase in the power of theta activity; this is interpreted by some authors as signs of immaturity and by others as developmental deviation [57]. Coherence alterations have been identified with the genesis of epilepsy; coherence studies are used to assess interconnections among different brain regions. [39, 57] In this sense, we have found that some cases of congenital strabismus, hipo-coherence of the wave delta in T5-T6. [Figure 10 and Table 10]. Table 8. Brain electric activity in 27 patients with DHD. The main finding is that 48.1% showed slowing-down of brain waves. Trends in the electrical behavior in Dissociated Horizontal Deviation (DHD) n = 27 Irritative discharge Paroxistic Activity Lentification Count Count Count Indeterminate

21

17

14

Positive

6

10

13

Epilepsy is more evident than strabismus in the neuroelectric recording. This is explained by the fact that paroxysms and power changes are readily identified by the visual inspection of the electroencephalographic recording [54,55,56]. The electric behavior of the cerebral cortex comprised in the EEG recording not only shows power alterations and evident signs of focalized malfunctions, but also relevant information can be gained by analyzing intra- and inter-hemispheric functional relations. For example, in the 70’s research on the behavior of the electric coherence of frequency bands was initiated [39]. Among the reported findings were the differences between young brains and old ones [55], and between healthy ones and those suffering from Alzheimer’s [58]. Schizophrenia, Alzheimer’s, and ADD were among the diseases studied with new recording methods such as QEEG [58, 59]. The initial aim of improving the recording techniques was made by Adrian and Matthews, who in 1934, replicate the studies carried out by Berger and state that even though the EEG technique can be employed as a biological marker, it is not possible with this sole methodology to understand the functioning of the central control regulating eye movements and their complex cerebral interconnections to comprehend strabismus [60].

8. NEUROMETRY The advent of digital systems to enable the conversion of an analog signal into a digital one --with more accuracy and minimization of sign deformation as time goes on--, as well as the extraordinary development of computational technology --that allows the instantaneous numerical analysis of the signal (QEEG) using algorithms-- are two breakthroughs that in combination with statistical procedures –univariate and multivariate— have provided more precise and sensitive recordings. This reliable information has enabled further insight into

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various diseases, since it is now possible the detection of discreet changes with respect to normal “Z” values [Table 9]. Once the signal is improved, a computerized analysis of significant elements in the bioelectric activity --absolute and relative power, inter- and intra-hemispheric symmetries and coherences in different frequency ranges— is performed and compared with normal “Z” values to determine whether in congenital strabismus the identified changes occurring in the cerebral cortex are significant and thus might implicate the participation of the cerebral cortex in the physiopathogeny of this disease. In this sense, neurometry or the neurometric method is a highly valuable instrument [Table 9]. As digital computer technology developed in the 60’s and 70’s, it became feasible to assess and quantify with precision more parameters than was possible through human visual inspection of raw EEG waveforms. With these developments the field of quantitative computerized analysis (QEEG) came into existence. Bickford and his colleagues were among the earliest to introduced the compressed spectral array (Bickford, Fleming and Billinger, 1971) [61], which increased visualization. However, they did not provide a quantified evaluation regarding deviation from normal. Duffy and his associates [62] were among the first to exact meaningful information the volumes of data generated by the quantitative EEG (QEEG) techniques, by breaking continuous background activity into its spectral components, numerical values for the various components were thus obtained. By referencing these values to normal ones, a probability of normalcy can be established. Once a scaled value is established for a spectral component at each electrode site, a method for interpolating these values spatially between these sites is used to display a digitized brain map (DBM). These values are assigned hues of gray or colors within a scale which reflect changes over the scalp to illustrate the EEG findings. It is important to note that different QEEG techniques do not all share the same algorithms for determining normal deviations. John et al. [63] developed the neurometric Z score maps using transformation functions such as the log transform. These authors adjusted values for the subtle effects of aging by fitting these values to age-dependent parameters, as well as mathematical correct regression equations to reflect the influence of the electrical activity in one area of the brain over another area. Neurometric values calculated with Mahalanobis distance multivariate equations can correct the deviation activity in one hemisphere from deviations in the other hemisphere. Similarly, activity in the anterior regions can be calculated from activity in the posterior regions [62]. These calculations are conducted for both monopolar and bipolar derivations. In addition, since age correction regression methods can be used, the ability to fit an individual’s data can be tested to fit the regression curves at earlier ages, thereby assessing maturational lag of cortical development. In 1998, the Neurometric Analysis System was released for used by qualified medical professionals to perform post-hoc statistical analysis of EEG recordings [61]. This technique was certified to be Year 2000 Compliant in accordance with the guidelines for use by the FDA. Moreover, the American Academy of Neurology approved the neurometric method as a research tool over other QEEG methods.

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Table 9. Intra- and inter-hemispheric values, fronto-temporal and fronto-occipital, surpass normal “Z” values. There is evidence of a diminution in both inter-hemispheric coherence in frontal lobes and intra-hemispheric gradient, as well as a decrease in centro-parietal synchrony in this Dissociated Strabismus case. Altered neurometric parameters in dissociated strabismus 1. Monopolar interhemispheric coherence (Z) Fp1-Fp2

F3-F4

C3-C4

P3-P4

O1-O2

F7-F8

T3-T4

T5-T6

Total

-2.68

-2.14

-1.3

-1.17

0.26

-1

-0.34

-0.01

Delta

-2.24

-2.11

-0.96

-2.02

-0.7

0.07

-0.51

-3.81

Theta

-3.04

-2.44

-1.55

-1.35

0.09

-1.34

0.51

0.57

Alpha

-1.88

-1.26

-1.41

-0.33

0.95

-1.81

0.62

1.35

Beta

-1.17

-1.18

-1.11

-0.16

-0.44

-0.17

0.16

0.73

Comb.

1.29

1.08

0.15

0.94

-0.01

0.57

-1.33

2.6

F8- T6

F3-Ol

F4-O2

Ol-F7

O2-F8

2. Monopolar intrahemispheric gradient (Z) F3-T5

F4-T6

F7- T5

Total

4.34

3.12

3.34

2.53

2.39

2.53

-1.83

-2.08

Delta

4.15

2.8

2.6

2.05

1.84

1.64

-1

-1.16

Theta

4.2

2.97

3.41

2.67

2.03

1.87

-1.59

-1.61

Alpha

4.09

3.9

3.42

3

2.84

3.46

-2.39

-2.7

Beta

2.78

0.51

2.6

0.8

-0.25

0.66

-0.1

-0.88

Comb.

2.51

2.36

1.88

1.64

1.77

1.76

1.19

1.2

3. Monopolar intrahemispheric synchrony (Z)

Total

Fp1-F3

F2-F4

T3-T5

T4-T6

C3-P3

C4-P4

F3-01

F4-02

0.38

-0.21

-0.38

-0.68

-2.37

-1.2

-0.25

-0.69

Delta

0.65

-0.12

-0.55

-0.61

-3.19

-1.03

-0.08

-0.95

Theta

0.28

-0.47

0

-0.02

-2.41

-1.21

-1.21

-1.09

Alpha

0.59

-0.17

-0.43

-0.84

-2.37

-1.61

0.71

-0.72

Beta

0.47

0.36

0

-2.88

-1.39

-1.76

-0.55

-2.13

Comb.

-1.16

-0.9

-0.91

1.78

1.71

0.44

-0.42

0.95

Prior to addressing the topic of neurometry it is important to remember that congenital strabismus is especially studied in children and that these patients exhibit some normal variations in brain electric behavior. It must also be considered that strabismus entails very subtle changes. Its specificity increases after the age of 6 given that the results can be compared with normal populations (normal “Z” values). Neuroelectric integration in 6-year olds is still unstable and has a larger variability range than in adults. These changes, together with the scarcity of parameters in healthy children to establish normal values, hinder the possibility of elaborating fine neurometric analyses in children younger than this age. For this reason, our neurometric analyses always refer to children older than 6 years [60, 63].

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Normal values in our neurometric study have been validated in the asymptomatic population starting from 6 years of age. In the meantime, spectrographic analyses and their interpolation to brain mapping can be performed by means of various programs such as Persyst version 4.0. To carry out this type of studies, the American Clinical Neurophysiology Society (ACNS) has issued guidelines that recommend a minimum of 2-min conventional EEG recordings of representative epochs, free or artifacts, to produce a validated and certified neurophysiological interpretation. Neurometry or neurometric analysis is derived from quantitative electroencephalogram (QEEG) studies. Since its certification in 2000 by the FDA, its acceptance has been progressive. The major benefit of using computerized discriminatory mathematical equations means that obtained values can almost be simultaneously compared with normal “Z” values obtained from the healthy population of the same age [Figures 10, 11, 12, Photos 18, 19]. The neurometric analysis is, given its nature, eminently objective and descriptive. These properties allow the assessment of the topographic behavior without compromising the high temporal resolution of the EGG technique. Neurometry allows the correlation of the various EEG components and the delineation of its different characteristics along the various frequency ranges for each derivation. As a result very accurate information coming from multiple brain regions can be simultaneously obtained through the spectral analysis; thus our knowledge between structure and function is widened. A close relationship between electric activity and structure is established, as forming a unit. For example, coherence is thought to be mediated by the association of long and short cortico-cortical fibers, as well as by the association of cortico-subcortical fibers [58]. Thus, by coherence studies it is possible to classify different types of pathologies, such as in brain dementias, and differentiate them from healthy subjects, given that dementia patients present, among other deficiencies, a difficulty for processing and associating information [58, 64]. The incorporation of discriminatory mathematical analysis enables the acquisition of accurate information regarding various QEEG parameters and compares them with data bases to quantify deviations from normal values. Neurometry is not useful for identifying pathologies; rather it allows the perception of subtle changes that together with EEG data enable the statistical possibility of determining whether a certain value is out of the range of normal values and how this value is related to the rest of the electric activity in the brain at a certain moment [50]. This methodology makes quantitative analysis possible as well as allows the correlation of values that can be describing absolute and relative power (expressed in picowatts) from monopolar or bipolar electrodes. These results are defined, compared and combined in each frequency band. The same procedure is valid for assessing inter- and intra-hemispheric symmetry [Figure 1, Photo 18] and coherence [Photo 19, Figures 10 and 11, Tables 10 and 11].

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Photograph 18. Digitized Brain Mapping of a six year-old boy with DHD. Schematic representation of the neurometric study shows to intrahemispheric asymmetry for the bands delta and beta, with predominance in right parietals regions. The conventional EEG showed paroxystic activity in right parietal region in addition to diffuse lentification.

Photograph 19. Schematic representation showing coherence of EEG spectral bands. Color scale on the right side of the image marks the level of inter-hemispheric coherence (in percentage) from low (blue color) to high (red color) values. A diminution in coherence can be appreciated in temporal and parietooccipital regions in a 6 year-old female patient with SSAV.

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Figure 10. Distribution of inter-hemispheric coherence in a 6 year-old boy with SSAV. A decrease in delta waves (pink line) with value = -3.94 and an increment in alpha waves (green line) with value = 2.23 are present in left and right temporal regions.

Table 10. The Table 10 shows the numerical values of figure 10. Observe the great hipocoherence that is in the wave delta at level T5 and T6 (emphasized in yellow color). Alterations in the coherence of the wave delta at temporal level are as a relatively frequent finding in cases of congenital strabismus. These alterations suggest it extraestriated cortex participates in the origin of the congenital strabismus.

Total Delta Theta Alpha Beta

Monopolar interhemispheric coherence in a case of SSAV Fpl-Fp2 F3-F4 C3-C4 P3-P4 O1-O2 F7-F8 T3-T4 -1.22 -0.58 0.37 0.38 0.44 -0.5 2.14 -1.28 -0.64 0.4 0.27 -0.5 -1.34 -0.72 -1.7 -0.3 0.94 1.1 -0.52 -1.74 0.8 0.13 -0.32 0.72 0.73 0.99 0.55 2.23 -2.06 -0.41 0.19 0.47 -0.03 -2.11 1.54

T5- T6 -0.59 -3.94 0.27 0.2 0.39

Comb.

1.27

2.53

-1.78

-0.53

-0.39

-0.38

1.6

0.98

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Figure 11. Neurometry study in a 6-year old boy with DHD. Alterations in coherence are evident especially occipital hypercoherence in theta and beta bands, as well as marked hypocoherence in the alpha rhythm in frontal brain regions.

Neurometric analysis of dissociated strabismus has shown, among other things, a negative increment in the intra-hemispheric monopolar gradient in the hemisphere corresponding to the non-dominant eye. As an example a case of a 6 year-old boy with SSAV is presented; his dominant eye is the right one and the left is suppressed [Photo 10, Table 10]. Table 11. The table sows the neurometric parameters of same case of figure 11. The clinical signs of the patients showed right visual preference, isovision, alternated suppression, and asymmetric exotropy with higher horizontal and vertical deviations in the left eye. Monopolar Interhemispheric coherence in DHD Fpl-Fp2 F3-F4 C3-C4 P3-P4 O1-O2 F7-F8

T3-T4

T5- T6

Total

-1.22

-1.09

-0.74

-1.05

2.15

-1.24

1.6

-2.59

Delta

-1.0 1

-1.26

-0.73

-0.79

1.35

-0.98

0.11

-1.19

Theta

-1.68

-0.91

0.15

0.03

2.16

-0.75

1.3

-0.63

Alpha

-1.58

-1.25

-0 .25

-1.71

1.87

-2.96

1.57

0.97

Beta

-0.3

-0.08

-0.03

-0.64

2.07

-0.38

1.59

0.5

Comb.

0.38

0.17

0.49

0.88

0.97

1.54

0.44

0

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Phhotograph 20. Digitized D Brain Mapping of a 6 year-old boy with w DHD. Thiss patient displayyed a grreater vertical deviation d in the left eye and left ft eye suppressioon. He underweent surgery on thhree occcasions for "in ntermittent exotrropy" (a diagnoosis mistaken) with w a relapse inn motor symptom matology. D DBM reveals disscreet inverse assymmetry in poosterior regions,, with enhancedd slow-wave acttivity of deelta and theta baands in the left hemisphere; higgher alpha activvity is present inn right hemisphhere, as well ass fronto-polar sllowing-down duue to higher dellta activity.

One year prior to surggery, the DBM M showed geeneralized parroxysms of intermittent i cllustered spikees of slow wavves during sleeep phase II recordings. r [P Photo 16] Onee year after suurgery the co omputerized trraditional studdy showed no n paroxysmal activity, thoough some vaariations weree evident in innter-hemispherric synchrony in posterior parieto-tempor p ral regions, ass well as in occcipital areas; data showed no persistent lateralizationn. In addition, changes in innter-hemispherric coherence were detectedd in parieto-occcipital regionss. As followss an example of o a DHD casee in which cohherence alteraation is manifeest, but this tim me on anterior brain regions [Figure 11, Table T 11, Photo 6]

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Figure 12. A 6 year-old patient with DHD. The graphic show an increase in intra-hemispheric monopolar intrahemispheric gradient.

Table 12. The Table shows the numerical values of figure 12. The neurometric study exhibits an increase (in grey color) in intra-hemispheric statistical values in frontal regions of a 6 year-old patient with DHD. The yellow color indicates a decreed of the values in occipital-frontal regions. Monopolar Intrahemispheric Gradient in a case of DHD

Total Delta Theta Alpha Beta Combo

F3-T5

F4-T6

F7- T5

F8- T6

F3-Ol

F4-O2

Ol-F7

O2-F8

4.34 4.15 4.2 4.09 2.78 2.51

3.12 2.8 2.97 3.9 0.51 2.36

3.34 2.6 3.41 3.42 2.6 1.88

2.53 2.05 2.67 3 0.8 1.64

2.39 1.84 2.03 2.84 -0.25 1.77

2.53 1.64 1.87 3.46 0.66 1.76

-1.83 -1 -1.59 -2.39 -0.1 1.19

-2.08 -1.16 -1.61 -2.7 -0.88 1.2

Most of patient with dissociated strabismus present cortical alterations. We have studied a group of 10 patients with DHD and have found a great amount of alterations in the DBM [Photo 10c and 20], the neurometry [Figure 11] the spectroscopy [Photo 11b ] and the granulometry [Photos 12c and 12d] in all of them. From all image studies, neurofunctional ones are the most employed in the study of strabismus with regard to the cerebral cortex. However, in order to correlate structure with function, we have undertaken morphometric analyses in some patients, using magnetic resonance imaging to establish the absence of macro-structural alterations. However, when

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analyzing topographical sections obtained by resonance in a granulometry study, we found relevant differences between the brains of healthy children and those with dissociated strabismus.

9. GRANULOMETRY Since the discovery of X-rays by Roentgen in 1895, a great advancement has been made to visualize the structures of the human body. Morphometric image studies, such as Computerized Axial Tomography scan or MRI, provide highly precise information of relatively large structures of the human body. However, given the complexity of the brain, there are structural aspects that are not liable to evaluation with conventional methods, for they can not be perceived at plain sight [64]. Granulometry is a concept used in image processing. This concept, originally introduced by Matheron [65-68] is useful and versatile for the morphological analysis of images. Its application includes a wide range of tasks, such as size estimation of the components in an image and image segmentation, just to mention a few. Granulometry is the study of the distribution by sizes of the particles comprising an aggregate. This method is employed in diverse areas to describe the qualities of size and shape of the granules in a product. For example, in a soil study, granulometry provides information on whether the soil is sandy, clayey, etc.; when dealing with cement, granulometric studies may define its application and final performance. Granulometric studies are based on a sieving process which is carried out with the help of a sieve, a measuring instrument that enables the classification of a material according to size. The main element in the sieve is the mesh. This technique entails the possibility of analyzing images in a numerical language, whereas MCD and neurometry provide objective neurofuctional information on the brain anatomical substrate. Both techniques are rather precise and a correlation is expected to give some insight into the origin of strabismus. By using granulometry, a micro-structural study of the brain and cerebral cortex may be carried out in vivo using mathematical and granulometric analyses of Pixel (picture elements) and Voxel (volume elements) acquired from positive three dimensional magnetic resonance images (3-D RMI T1). By means of Voxel based analysis, Mendola [28] analyzed three major brain areas and found a diminution in total volume distribution of gray matter in visual cortex -–parieto-occipital and temporal-ventral regions—of patients with amblyopia. In other study, a redistribution of gray matter was reported by Suk-tak et al. [64], using Voxel morphometric analysis from MRI in adults with exotropia. These patients showed a redistribution of gray matter on the brain surface attributed to neuroadaptive changes [29]. Through Voxel and Pixel analyses, Gallegos-Duarte [37] reported that the granulometric profile shows differences between two forms of congenital dissociated strabismus (strabismus syndrome of angular variation, SSAV, and dissociated horizontal deviation, DHD) attributable to the presence of varying degrees of maturity in the brain [37, 69]. From the above mentioned evidence we decided to carry out a granulometric analysis to study the cortical brain structure from MRI to correlate structure and function in subjects with dissociated strabismus. This method was not intended to obtain the cartography of the brain cortex, but rather the volume, order and distribution of cellular elements obtained from Voxel and Pixel analyses. In addition, the role that the brain cortex plays in congenital strabismus is

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to be studied, as well as the relation of neuroelectric manifestations and the underlying brain structures. In the present communication a morphometric study is carried out. It consists in the determination of the characteristic structures of white and gray matter found in the occipital lobes of children with dissociated strabismus, and the correlation of these findings with brain tissue of normal children and that of children showing leukomalacia (a radiological sign of cerebral immaturity). Two healthy children are the control group (CG); the experimental group consists of four children with dissociated strabismus --two with DHD and two with SSAV; granulometric averages are carried out. A brain exhibiting leukomalacia is analyzed and compared with the other two groups as a reference of structural immaturity. In summary, two groups of patients are analyzed: one consisting of four patients with strabismus (two exhibiting SSAV and two DHD) and a control group. Granulometric curves are compared among strabismic and control subjects. Figure 13 shows the granulometric standard of white and gray matter in control subjects.

Figure 13. Granulometric patterns obtained from two control subjects.

These graphs were obtained by averaging the analyzed sections of healthy subjects. These patterns are useful when compared with patterns of subjects with SSAV and DHD. Patterns of subjects with strabismus are presented in Figure 14. In two control parameters (mature brains and immature brains), a comparison is made between granulometric values per group. The results are shown in Figure 15.

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Figure 14. Granulometric patterns from four subjects with strabismus.

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Figure 15. The control group (CG) shows a larger differential in the white matter (white columns) to gray matter (gray columns) ratio; the opposite occurs with the immature brain. Strabismus cases occupy an intermediate situation, in which the difference between these brain regions is smaller than in CG. Ratios of white to gray matter are the following: CG, 1.74; DHD (1) 2.15, DHD (2) 1.6, SSAV (1) 1.31, SSAV (2) 1.21, Leukomalacia 1.29.

In particular, three main groups of clear and dark structures are considered. These groups take into account the size of a square window, whose origin is in its center. The size of the window µ is computed as [2 µ+1] [2 µ+1], that is, the size µ=1 means a window [2(1)+1] [2(1)+1]=9 pixels, whereas µ=4, for example, means a square with [2(4)+1] [2(4)+1] =81 pixels. The groups are the following: Group 1. All the small structures within the sizes 3 to 5 are comprised. Notice that structures with sizes 1 to 2 are not considered, since noisy components are detected at these small sizes of the structuring element. Group 2. This group contains medium-sized structures located in the interval 6 to 9. Group 3. Finally, this group comprises large-sized structures, in the interval 10 to 15. In our case, MRI sections of occipital lobes of controls and patients with SSAV, DHD, and leukomalacia are analyzed [Figure 15]. The granulometric analysis consists in sieving the brain image prior to the calculation of a graph similar to that of granulometric density. This process is illustrated in Figure 16.

Figure 16. Image illustrating the sieving process.

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The granulometric profile of the four analyzed groups showed important differences. Specifically, differences in this profile were encountered between SSAV and DHD groups [Figure 15]. [69] This study allowed the correlation between structure and function in some cases of dissociated strabismus. In the occipital lobes, changes in granulometric density of brain cortex were found. These changes could be differentiated and compared to determine the maturity of certain structures. This in turn could be related to the presence of slow waves in MCD of dissociated strabismus. Our results suggest that dissociated strabismus shows immaturity signs in neuronal granulometry: the immaturity level is more evident in children with DHD rather than in those with SSAV. The classification of strabismus is currently controversial. Therefore, our interest is to determine, based upon physiopathogeny, whether clinical manifestations correspond to different conditions, or whether they are different expressions of the same disease with a common origin. These structural differences [Photos 12a,12b, 12c and 12d] might be related to neuroelectrical behavior, for example. Patterns in neuronal distribution are capable of modifying the electric signal in EEG; in consequence, they are potentially quantifiable. This circumstance has been presumed in attention deficit subjects. In this respect, prevalence of atopic cellular groups is present in patients with Zinder dyslexia. Although the size of the sample was not enough to establish a correlation between electroencephalographic findings and structure, our findings open a field in which the correlation of these parameters can be made with great accuracy. Granulometric studies provided information concerning structural volumetric quantities detected in white and gray matter. In both cases, DHA and SSAV, changes were evident in white and gray matter when compared to volumetric quantities detected in control subjects. Furthermore, strabismic subjects with DHD exhibited less variation in GM than strabismic subjects with SSAV. With respect to WM, changes were encountered in large structures for both DHD and SSAV subjects, when compared to controls. These findings indicate that dissociated strabismus, specifically DHD and SSAV, may be originated mainly by alterations in gray matter and in large components of white matter.

10. METHODOLOGY a) Clinical Study All patients had their strabological clinical record made; this included: cycloplegic refraction with cyclopentolate, motoricity (ductions and versions, horizontal version trajectories, pursuit movements, monitored with night shoot video-recording in ectopic and mesopic conditions), sensory perception (visual acuteness, stereopsis tests, suppression diagnosis and amblyopia).

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b) Electrooculography Ocular movements during the EOG recording were obtained with night shoot videorecording. The patient was seated facing forward. Subsequently, a stimulus to assess slow movements followed a horizontal trajectory at 10º/s. To check saccadic movements, patient was asked to look at a dim light located to his right or left, and he was indicated in which direction he had to look at. Studies were carried out in a room with neutral background, free of noises and distractions. The same parameters were used each time and were taken by the same health professional as required by the International Convention of Geneva to perform anthropometric measures in living subjects. For the simultaneous EOG and EEG recordings an additional nasal electrode was employed.

c) Perceptual Test A computerized perceptual screening was performed in an awake and cooperative patient, with optical correction, in a neutral environment free of external stimuli. A high resolution flat LCD monitor was ergonomically placed at a 60-cm distance from the patient at eye level to determine the perception of speed, space, shapes, sizes and primordial elements. Visual memory was evaluated, as well as reading and writing speed, slow pursuit and saccadic movements, peripheral vision, visual preference and manual preference. These assessments were all performed by the same personnel und following the same exploratory routine.

d) Brain Mapping A DBM was performed to all patients using the 10-20 International System with 21 channels and a 32-channel recording option with established parameters for each epoch, in printed form as well as pulse monitored. The cortical response to various stimuli was recorded and evaluated, using digital electroencephalography. Stimuli were light, hyperventilation, opening and closing of eyelids, sleep, and wakefulness. Simultaneous electrooculography (EOG) using specific electrodes and channels were employed to assess horizontal version movements both slow and saccadic.

e) Neurometry Is a method of quantitative electroencephalographic analysis (QEEG) that uses objective computer algorithms to extract a large number of quantitative from an EEG recording for comparison against a reference database. Each extracted feature is subjected to a statistical evaluation, and compared to the distribution of values of same features observed in a normative reference ("Z") database using multivariate statistical procedures

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f) Magnetic Resonance Analysis of brain sections, 1 mm thick, was made using 3–D RMI T1 approach, without contrast and medication free. Multiplanar brain images were acquired in simple phase, spine eco routine power sequences employing the FLAIR technique.

g) Spectroscopy (1H-NMRS) Proton nuclear magnetic resonance spectroscopy, 1H-NMRS, was performed in both occipital lobes. A piece, 8 cm3, of brain cortex from each occipital lobe was selected and analyzed. The equipment and protocol were the following: Intera Pilips 1.0T, version 10.6, Fast Fell Echo sequence (FFE), T1 weighted in 3-D, field of vision (FOV) 230 mm, RFOV 80%, slice thickness 1 mm, without space between slices, eco time (ET) 6.9 ms, repetition time (RT) 25 ms, deviation angle (DA) 30 degrees, number of excitations (NAS) 1, number of slices 120.

h) Brain SPECT Brain SPECT was carried out using scintillography and analyzed every 45 minutes after the intravenous administration of tecnecium 99 ethyl-cysteinate dimer to enable the spectrometric observation in the red-violet scale.

CONCLUSIONS The human brain cortex is unique. It consists of an ontogenic design with a wide integration network at all levels. The visual system consumes a large part of these neurointegrative resources, so a motor sensory dysfunction such as strabismus not only leaves a frequent mark in DBM, but also the neurological findings available with neuroimaging techniques suggest cerebral cortex participation as the origin of dissociated movements underlying congenital strabismus. It is difficult to establish whether strabismus is the cause of DBM alterations or, conversely, brain alterations in the cerebral cortex originate ocular deviations. There is evidence showing that observed neuroelectric alterations in extra-striate cortex could be directly related to the presence of dissociated movements. It remains to be elucidated whether motor alterations or, more accurately, sensory alterations are the cause of the changes in the inter- and intra-hemispheric coherences encountered in neurometric reports. One of the clinical characteristics of dissociated strabismus is the sensory-motor asymmetry. In addition to this finding, in this study, we have encountered alterations in coherence, frequency and power asymmetries possibly related to alterations in long-axon neurons of inter-hemispheric interconnection.

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When going from a major degree of ontogenic complexity to a minor one in a descending direction --from cortex to nuclei, from nuclei to effector organ-- it can be appreciated that the cerebral cortex plays a preponderant role that must not be ignored. Likewise, we can not disregard the evidence that the human being is especially prone to suffer from strabismus, an extremely rare disease in other species. Given that the human cerebral cortex is distinctively complex with respect to other species, we believe that in this structure lies the answer to some of our questions with regards to the cause of congenital strabismus and its origin. Evidence from current studies suggests that some cortical alterations related to dissociated congenital strabismus may induce motor dysfunctions as it occurs in epilepsy. In fact, there is no evidence pointing to motor alterations as the cause of paroxysms, slowing down of brain activity, cortical hyperexcitability, nor something indicating a possibility in the inverse direction; hence, our interest in trying to understand the participation of electric disturbances in association with the manifestations in the oculomotor system. The mechanisms underlying cortical disturbances in extra-striate areas apparently distant form each other may induce changes in ocular motility; though these mechanisms have not yet been elucidated. The presence of paroxysms possibly generates symptomatogenic zones capable of modifying the supranuclear control, as well as inhibiting Hering’s law. Once the latter happens, movements may become incoherent. Invariably, the eye pertaining to the hemisphere where the main cortical alterations are present corresponds to the eye presenting higher variability with regard to movement, suppression, amblyopia, dissociated deviation, and a more evident latent nystagmus. In summary, the eye with more sensory and motor alterations corresponds in general to the hemisphere exhibiting major alterations. We are aware that one study is not enough to establish accurately a cause-effect relationship between the activity of the cerebral cortex and strabismus, but by correlating various studies in this field we have been able to infer a physiopathologic basis for the origin of some alterations underlying congenital strabismus. In gaining a better understanding into the origin of congenital strabismus and into the neurological differences among the various types of this disease, better medical and surgical treatments will be available. Moreover, the opportunity of having a multi-disciplinary handling of the strabismic patient –neurodiagnostics, neurology, therapy and psychology, among others-- will be possible. Some of the bad outcomes frequently found after the surgical handling of strabismus can be associated with a bad pre-operative diagnosis [70], for example, to underestimate the cortical participation in the sensory and motor behaviors in this disease. It is currently within our reach in reference to dissociated strabismus the possibility of more precise surgeries, the emphasis on visual pre-and post-operative rehabilitation, as well as a better counseling to the family with regard to the neurological implications. In summary, a better knowledge on the origin of the problem and its implications enables a better treatment for the patient.

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ACKNOWLEDGMENTS The authors wish to thank to the foundation Mario Moreno Reyes for the financial support and M. Sanchez Alvarez for proof-reading this manuscript.

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[34] Caraballo RH, Cersosimo RO, Medina CS, Tenembaum S, Fejerman N. Idiopathic partial epilepsy with occipital paroxysms. Rev. Neurol. 1997; 25: 1052-8. [35] Gallego-Duarte, M.; “Respuesta cortical paradójica durante la fotoestimulación intermitente en el estrabismo disociado”. Cir Ciruj 2005; 73 (3): 163-167. [36] Gallegos-Duarte M, Mendiola-Santivañez JD, Ortiz-Retama JJ.: Estrabismo disociado y maduración. Boletin del Consejo Latinoamericano de Estrabismo (CLADE) 2007, 21; 2. (In press). [37] Soto-de la Vega M, Romero-Apis D. Alteraciones electroencefalográficas en el estrabismo. An. Soc. Mex. Oftal. 1970 (1): 9-18. [38] Céspedes-Gacía Y, González-Hernández J.A.García-Fidalgo J., Beguería-Santos R.A., Figueredo-Rodríguez P. Coherencia cerebral interictal en pacientes con epilepsia del lóbulo temporal. Rev. Neurol. 27 (12): 1107-1111. 2003. [39] Morales-Chacon L, Sánchez-Catasus C, Águila A, Bender J, García I, García ME, Lorigados. Contribución del SPECT cerebral en la evaluación de la epilepsia del lóbulo temporal farmacoresistente. Experiencia del CIREN. Rev. Mex. Neuroci. 2005; 6 (3) 250-256. [40] Riikonen et al.: SPECT and MRI in foetal alcohol syndrome. Developmental medicine and child neurology 1999, 41: 652-659. [41] Piovensan EJ, Cristiano Lange M, Kowacs PE, FemelliH,Werneck LC, Yamana A, et Al. Structural and functional analyses of the occipital cortex in visual impaired patients with visual loss before 14 years old. Arq Neuropsiquitr 2002; 60 (4): 949-953. [42] Onofre-Castillo JJ, Martínez HR, Arteaga M, Gómez A, Olivas-Mauregui S.; Espectroscopia por resonancia magnética en enfermedades neurológicas. Rev. Mex. Neurol. 2002; 3 (4) 213-217. [43] Warren K E.; NMR Spectroscopy and pediatric brain tumors. The oncologist 2004; (9): 312-318. [44] Poca MA, Sauquillo J, Mena MP, Vilalta A, Rivero M.; Actualizaciones en los métodos de monitorización cerebral regional en los pacientes neurocríticos: presión tisular de oxigeno, microdiálisis y técnicas de espectroscopia por infrarrojos. Neurocirgía 2005; (16): 385-410. [45] Darbina O, Rissob JJ, Carreb, Lonjonc EM, Naritoku DK. Metabolic changes in rat striatum following convulsive seizures. Brain Research 1050 (1): 124-129. 2005. [46] Doménech-Campos E, Armengol-Caroeller M, Barona de Guzmán R. Electroculografía: aportación al diagnóstico del paciente con alteraciones del equilibrio. Act Otorrino Esp (56):12-16. 2005. [47] Tychsen L; Wong AM; Burkhalter A.: Paucity of horizontal connections for binocular vision in V1 of naturally strabismic macaques: Cytochrome oxidase compartment specificity. J. Com. Neurol. 2004, 474 (2): 261-75. [48] Melek NB, Garcìa H, Ciancia AO. La elecroculografía (EOG) de seguimiento de persecución en las esotropias con limitación bilateral de la abducción (LBA). Arch. Oftalmol B Aires (54): 271-276. [49] Cabanyes-Truffino J, Cartografía cerebral: metodología y aplicaciones en la clínica neurológica. Rev. Neurol. 28 (11): 1090-1098. 1999. [50] Pascual-Castroviejo. Plasticidad cerebral. Rev. Neurol. (Barc) 1996; 24 (135): 13611366.

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[51] Hans-Rudolf Shc. Some findings concerning the relationship between ontogesesis and cytoarchitecture of the primate brain. J. Comp. Neurol. 2004; 133 (4): 411-427. [52] Herrmann CS, Demiralp T. Human EEG gamma oscillations in neuropsychiatric disorders. Clinical Neurophysiolology 116: 2719-2733. 2005. [53] Rubio D F. Epidemiología de la epilepsia, En: Manual clínico de la epilepsia, JGH Editores, México 1997: 15-20. [54] Pozo-Lauzan D, Pozo-Alonso AJ.: Nuevo enfoque conceptual de la epilepsia. Rev. Cubana Pediatr. 2001; 73 (4): 224-9. [55] Engel J Jr. ILAE commission report. A proposed diagnostic scheme for people with epileptic seizures and with epilepsy: report of the ILAE task force on classification and terminology. Epilepsia 2001; 42: 1-8. [56] Mauritis N M, Scheeringa R, Van der Hoeven J H, de Jong R. EEG coherence obtained from an auditory oddball task increases with age. Journal of Clinical Neurophysiology. (23): 395-403. 2006. [57] Calderón-González P.L, Parra-Rodriguez M.A., Libre-Rodríguez J.J., Gutiérrez J.V. Análisis espectral de la coherencia cerebral en la enfermedad de Alzheimer. Rev. Neurol. 2004; 38 (5): 422-427. [58] Snyder S M., Hall J R. A Meta-analysis of Quantitative EEG power associated with attention-Deficit hyperactivity disorder. Journal of Clinical Neurophysiology 23 (5): 440-452. (2006). [59] Adrian E.D., Matthews B.H.C. The interpretation of potential waves in the cortex. J. Physiol. (81): 440-471. 1934. on line http://jp.physoc.org/cgi/reprint/81/4/440. [60] Fleming, N.L.and Ballinger, T.W. (1971).Compression of EEG data by isometric power spectral plots. EEG Clin.Neurophysiol., 1971 (31): 632. [61] Duffy F.H. Bartels, P.H., Burtfiel, J.L. Significance probability mapping. An aid in the topographic analysis of brain electrical activity. EEG Cil. Neurophysiol. 1981 (51): 455-462. [62] John, E.R., Prichep, L., Fridman, J., Easton, P. Neurometrics: computer-assisted differential diagnosis of brain dysfunctions. Science 1988; 293 (4836): 162-169. [63] Snyder S M., Hall J R. A Meta-analysis of Quantitative EEG power associated with attention-Deficit hyperactivity disorder. Journal of Clinical Neurophysiology 23 (5): 440-452. (2006). [64] Suk-tak Ch; Kwok-win T; Kwok-cheung L; Lap-kong Ch; Mendola JD; Kwong KK. Neuroanatomy and adult strabismus: a voxel-based morphometric analisys of magnetic resonance structural scans. Neuroimage (22) 986-994, 2004. [65] Matheron G.: El´ements pour une th´eorie des milieux poreoux. Mason,Paris , 1967. [66] Vincent L.: Granulometries and Opening Trees. Fundamenta Informaticae, 41(12), 57– 90, 2000. [67] Vincent L., Dougherty E. R.:Morphological segmentation for textures and particles. In Digital Image Processing Methods E.R. Dougherty, editor,. Marcel Dekker, New York, (1994) 43–102. L. [68] Dougherty E. R.: Granulometric Size Density for Segmented Random-Disk Models. Mathematical Imaging and Vision, 17(3) (2002) 267–276.

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In: Brain Mapping and Diseases Ed: Diane E. Spinelle

ISBN: 978-1-61122-065-0 © 2011 Nova Science Publishers, Inc.

Chapter 11

ENDOVASCULAR BRAIN MAPPING: A STRATEGY FOR INTRAOPERATIVE VISUALIZATION OF BRAIN PARENCHYMA FUNCTIONALITY H. Charles Manning1,2,3 , Sheila D. Shay1, Erich O. Richter4, Swadeshmukul Santra5 and Robert A. Mericle1,3 1

Vanderbilt University Department of Neurological Surgery 2 The Vanderbilt University Institute of Imaging Science 3 Vanderbilt University Department of Radiology and Radiological Sciences 4 Louisiana State University HSC, Department of Neurological Surgery 5 University of Central Florida, Nanoscience Technology Center, Department of Chemistry and Biomedical Science Center

ABSTRACT Within the field of cognitive neuroscience, brain mapping strategies aim to localize neurological function within specific regions of the human brain. The burgeoning fields of functional magnetic resonance imaging (fMRI) and functional electrophysiology seek to map the human brain with ever-improving resolution. However, these functional strategies do not enable real-time, intraoperative discrimination of functional and nonfunctional brain parenchyma with precise, well-defined margins, as are necessary for surgical guidance and resection. To address the need for an intraoperative brain mapping strategy aimed specifically at neurosurgical guidance at resection, we have developed a novel brain mapping technique that we term preoperative endovascular brain mapping (PEBM). PEBM combines a super-selective, intraarterial approach with the delivery of visually detectable contrast agents to identify specific regions of functional and nonfunctional brain before and during craniotomy for brain resections. Our novel approach aims to avoid additional postoperative neurological deficits which would occur if functional brain parenchyma is inadvertently injured during an aggressive resection. Endovascular brain mapping aims to preserve brain function by providing a means of direct volumetric surgical guidance in real-time, whereby non-functional tissues are delineated by sharp, visible margins and can therefore be safely resected. The successful

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H. Charles Manning, Sheila D. Shay, Erich O. Richter et al. implementation of PEBM is highly dependent upon the proper selection and use of imaging probes, and we have developed a number of novel multimodal chemistries specifically aimed at PEBM. In this chapter, we will describe the PEBM technique in detail by highlighting its use in various small animal models, as well as our ongoing development of novel imaging probes suitable for PEBM.

Brain mapping is the attempt to specify the localization of function in the human brain in as much detail as possible [56]. Many brain mapping techniques exist, which have various strengths and weaknesses, and are therefore useful for specific aspects of research in cognitive neuroscience. The determination of the presence or absence of function in a potential neurosurgical resection site has critical importance in clinical neurosurgery and therefore this is the focus of the neurosurgical brain mapping techniques. Standard brain mapping techniques do not allow direct, real-time surgical visualization of nonfunctional brain parenchyma with sharp, precise, visualized margins that can be safely resected [12,14,15,33,39-41,46,56-58,61]. However, PEBM has the potential to achieve these goals, through further research efforts.

REVIEW OF EXISTING SURGICAL GUIDANCE BRAIN MAPPING TECHNIQUES The origins of brain mapping developed after physicians observed functional deficits in patients with a known lesion in a specific brain location [8, 56]. These early observations advanced our initial knowledge of brain functional localization. Lesion studies, however, can cause inaccurate functional data because gray matter subserving one function often overlies white matter carrying fibers subserving an unrelated function. Brain mapping for neurosurgical resections started to develop in the 1950’s with Penfield and Jasper [42]. They acquired functional data with intraoperative brain stimulation during craniotomy in awake patients. This technique is most useful for mapping the motor or language cortex during brain surgery. Some of the disadvantages of this approach include: 1) the increased operative time and associated risk of infection, 2) the increased cortical exposure required, 3) the pain and inconvenience of an awake patient during open craniotomy, and 4) the surgeon’s limited ability to perform detailed neurological and cognitive testing with the patient on the operating room table and in a state of conscious sedation. An adaptation of this technique is implantation of subdural cortical electrodes and depth electrodes, which can be stimulated postoperatively, to collect functional data in specialized epilepsy monitoring unit (EMU) beds. This technique is used today in patients who need epilepsy surgery. After a period of monitoring during stimulation of the various implanted electrodes, functional brain tissues are identified and mapped. A subsequent second craniotomy is then performed for removal of the electrodes and possible definitive resection, after the functional mapping is completed. Similarly, many neurosurgeons use microelectrode recording and stimulation to precisely localize deep nuclei [19]. The obvious disadvantages of these approaches are the necessity of performing two separate craniotomies and the associated increased risks and expense. The precise localization of function is also limited by the size of

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the electrodes, the distance between the electrodes, and the inability after removal of the electrodes to resect the exact tested neuron cells/tissue that were tested. Less invasive cortical brain mapping can be achieved to some extent with transcranial electrical stimulation (TES), and transcranial magnetic stimulation (TMS) [12,20,3941,61,70]. These brain mapping techniques use electrical or electromagnetic fields to induce changes within the cortex to stimulate the tissue or that behave as a temporary lesion. However, TES and TMS are of limited use in clinical neurosurgery because of patient discomfort, unacceptably poor spatial resolution leading to imprecise localization, and the possibility of inducing seizures with repetitive stimulation [12,56,70]. Passive transcranial modalities, such as electroencephalography (EEG), [13,31,63] and its magnetic counterpart, magnetoencephalography (MEG), [32] also have limitations in clinical intraoperative brain mapping because of their unacceptably poor spatial resolution. Furthermore, these techniques are very sensitive to external magnetic fields, thereby making their setup in typical operating room environments challenging. Functional MRI (fMRI), [14,15,46,57,58] positron emission tomography (PET) scanning, [3,26,44,56,59] or optical imaging [19,44,59] have limited usefulness in clinical intraoperative neurosurgical mapping because the modest spatial resolution of these maps prevent correlation of the imaging signal to the exact cells/tissue to be potentially resected during subsequent craniotomy. fMRI can accurately identify the tissue that is active during any given task, but it is difficult to determine if surrounding tissues are involved in important functional activities that were not included in the task-set at the time of imaging.

COMBINED BRAIN MAPPING AND VOLUMETRIC IMAGE GUIDANCE Some of these brain mapping techniques, especially fMRI, have been supplemented with stereotactic volumetric image-guidance, [59] but spatial resolution is a concern, and the correlation can degrade as the operation continues, because they are not real-time images. Once brain shifting occurs, brain tissue is resected, or cerebrospinal fluid (CSF) is lost, these stereotactically guided images become less useful.

The Endovascular Approach Advances in microcatheters and microwires have made preoperative embolization of AVMs and certain brain tumors safer and more effective [2,6,10,17,18,2325,37,38,47,48,50,51,60,63,67-69,71]. It is now routine to catheterize distal branches of the ACA, MCA, and PCA super-selectively during transcatheter embolization. A super-selective Wada test with microcatheter infusion of methohexital or amobarbital is sometimes performed at this distal arterial position before injection of the embolic agent to be sure that functional brain tissue is not in the pathway of the embolization [36,43,50,51]. These same techniques can be applied to a candidate for brain mapping. After the methohexital is injected into a specific location in the brain, if any functional tissue exists in that location, the patient will exhibit a temporary corresponding deficit for about 5 minutes. If the area is “nonfunctional” the patient will remain neurologically intact. When nonfunctional vascular

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territory is identified, then endovascular mapping (staining) of this nonfunctional brain tissue could be helpful during a subsequent elective craniotomy for brain resection.

Preoperative Endovascular Brain Mapping (PEBM) Approach Preoperative endovascular brain mapping (PEBM), which uses a super-selective, intraarterial endovascular approach to elucidate nonfunctional brain prior to and during craniotomy for brain resections, was recently described as a novel strategy [35]. The technique can help avoid new postoperative neurological deficits because it provides direct, real-time intraoperative volumetric image guidance of nonfunctional tissue with sharp margins that remain true throughout the resection, even with CSF loss or brain shifting. The conceptual steps of endovascular brain mapping for volumetric image guidance require, first, super-selective intraarterial catheterization of a distal target artery for delivery of testing materials to the corresponding target brain tissue. A super-selective Wada test is then performed to determine if functional activity is present at the target brain parenchyma [1,4,5,7,9,22,29,30,36,43,50,51,64-66]. If it is determined that no functional activity is present in the targeted brain, then a “mapping agent” is infused into the same microcatheter in the exact same distal arterial location where the super-selective Wada test was performed, staining corresponding target brain. The infusion is monitored with fluoroscopy and digital road-map angiography to avoid any reflux and therefore assure that the mapping agent travels to the same vascular distribution where the Wada test was performed. The mapped nonfunctional brain, with its sharp margins, can be clearly visualized at the time of subsequent elective craniotomy for resection. During the resection, no neurological deficits will occur if the resection is completely contained within the “mapped” nonfunctional (silent) tissue. At the current stage of development, we believe that this brain mapping technique is likely to be most useful for radical resection of malignant gliomas; specifically, diffuse infiltrating glioblastoma multiforme (GBM). When treating GBMs, the surgical plan could include resection of all possible nonfunctional brain tissue surrounding the infiltrating tumor, if this can be accomplished without causing new neurological deficits. In this situation, the surgical plan could match the super-selectively catheterized and stained vascular territories because only these territories would be known to be nonfunctional. The ideal endovascular brain mapping agent should possess all of the following characteristics: 1) systemically nontoxic; 2) clearly visualized in the brain at the time of subsequent craniotomy; 3) radiographically opaque for fluoroscopic and angiographic monitoring during the infusion; and 4) capable of adhering to the target tissues, either by crossing the blood-brain barrier (BBB) or by adhering to the target brain capillaries. Several dyes that are widely used in medical applications are classified as nontoxic. For example, fluorescein is a dye with minimal toxicity widely used in clinical practice, particularly in ophthalmologic applications, [11] where fluorescein angiography is an established method of retinal evaluation. Strategies to allow fluorescein passage across the BBB may involve invasive techniques (disruption of BBB) and noninvasive techniques (maintain intact BBB) [45]. Previous studies have shown that an invasive approach, such as using an osmotic agent to disrupt the BBB, can be used which allows many dyes to cross the

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BBB and stain the parenchyma [52,62]. Non-invasive methods are preferable, however, to reduce complications associated with BBB manipulation [44,62]. Most dyes used in food and medications are water-soluble and, thus, cannot penetrate the intact BBB by diffusion (passive transport). Some promising noninvasive strategies for brain mapping include: 1) the bioreversible covalent modification of a dye to improve its physicochemical properties that result in an enhancement of BBB transport ("pro-drug" approach); 2) conjugation of a mapping agent with nutrients (amino acids, glucose, etc.) actively transported into the brain; 3) linking a mapping agent to biomolecules undergoing adsorptive-mediated uptake (penetratin, pegellin, etc.); 4) receptor-mediated transcytosis (transferrin- or insulin-receptor antibodies, immunoliposomes) across the BBB; 5) linking a mapping agent to a cell-penetrating peptide (F-ANT, TAT, etc.); [53] 6) emulsification of a mapping agent (such as β carotene, organic dye, etc.) to stain brain capillary endothelial cells; or (7) grafted nanoparticle systems for brain delivery of mapping agents [27,53,54]. These noninvasive strategies require extensive development which is currently underway and planned for future studies. For the preliminary description of endovascular brain mapping for volumetric image guidance, we carried out our "proof-of-concept" study in animal models by using simple alternative techniques. These included the technically easier, but less desirable, invasive osmotic disruption of the BBB and dense staining of brain capillaries by capillary-level embolization. More recently, we have explored methods that enable PEBM without modification of the BBB as described below.

ANIMAL PREPARATION FOR PEBM STUDIES Approval for all animal studies was obtained from the University of Florida and Vanderbilt University Institutional Animal Care and Use Committees. A Blood Flow to MCA in Brain

Occipital Artery Ligation

B External Carotid Artery (ECA)

Internal Carotid Artery (ICA) Bifurcation Arterectomy

Common Carotid Artery (CCA) Ligation PE-50 tubing catheter Blood Flow to Brain

Figure 1. A. Schematic of the carotid artery in the animals illustrating point of catheterization and areas of arterial ligation. B. Cerebral angiogram in the anteroposterior view of a rabbit after occluding the ECA and occipital arteries to enhance flow through the ICA.

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Sprague-Dawley rats (Harlan, Indianapolis, IN) and New Zealand White rabbits were used for these studies. Rats were sedated with 87 mg/ml ketamine and 13 mg/ml of Xylazine (J.A. Webster, Inc., Sterling MA or Henry Schein, Denver, PA). The FDandC Green No. 3 was performed on a New Zealand White rabbit (Myrtle’s Rabbitry, Thompson Station, TN). The rabbit was sedated with 3 mg intravenously administered acepromazine before endotracheal induction of general anesthesia with 1 to 5% inhalational isoflurane (both agents from J.A. Webster, Inc.) In every animal, we used a carotid cutdown method to expose the common carotid artery. After dissecting the carotid past the bifurcation of the ICA and the ECA, the ECA was tied off along with the occipital artery, then a catheter was introduced into the lumen proximal to the ICA and a suture was used to secure the catheter within the vessel. Occluding the ECA and occipital arteries enhanced the flow through the ICA (Figure 1 above). In each of the animals, 0.2- 0.6 ml of mapping agent was infused through the right ICA directly into the right brain hemisphere with the aid of a standard infusion pump at 0.05 ml/min to 0.2 ml/minute. In each experiment, the animal was euthanized, its brain harvested, and the quality of the stain accessed on the same day by the appropriate method for each technique.

PEBM TECHNIQUES UTILIZED Administration of Fluorescein with BBB Manipulation Six minutes after the BBB disruption by arabinose using a previously described method, [49,52,62] a 10% solution of fluorescein (Sigma-Aldrich corp., St. Louis, MO) was mixed with an equal amount of iohexol contrast agent (Omnipaque; Amersham Health, Princeton, NJ). A total of 0.6 ml fluorescein was infused at 0.2 ml/minute using a standard infusion pump under fluoroscopic guidance. Modifications to the Zeiss operating microscope (Carl Zeiss Surgical, Inc., Thornwood, NY) to allow a clear visualization of fluorescence-stained brain territories have been described [28] this technique could be used intraoperatively to maximize visualization during a craniotomy for brain resection. However, for simplicity during our initial proof-of-concept studies in animals, a 365-nm hand-held ultraviolet light (UV-4B Spectroline; Spectonics Corp., Westbuty, NY) was used to visualize the fluorescein. With this method, staining was evident on the cortical surface and into the deep white matter. The fluorescence was well visualized, and the margin between the stained and the non-stained brain was relatively distinct (Figure 2). The normal brain tissue autofluoresces slightly at this wavelength, and therefore, improved visualization could be achieved with appropriate microscope filters [28]. This fluorescein approach is promising; however, it is limited by the need for fluorescent excitation and filtration; and it cannot be used routinely in humans because of the need for invasive BBB disruption.

Administration of FD and C Green No. 3 with BBB Manipulation In an attempt to avoid the extra step of fluorescent excitation, visible spectrum dyes were used to test the feasibility of endovascular brain mapping. An aqueous suspension of FD and

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C Green No. 3 (Sigma-Aldrich, St. Louis, MO) was selected because of its reported low toxicity and good visibility [21,34]. First, 0.3 ml of FDandC Green No. 3 was mixed with 0.3 ml of iohexol to add radio-opacity for visualization under fluoroscopy and DSA during infusion into the ICA. Selective BBB disruption was performed as previously described [49,52,62]. Then, 0.6 ml of the green dye and iohexol mixture was injected at 0.2 ml/min with a standard infusion pump in the right brain hemisphere. A New Zealand white rabbit was used for this experiment to determine if better angiographic images could be obtained for monitoring during the infusion, and if a more distal selective position could be attained with the microcatheter, which could possibly allow an improved delineation of the margin between mapped brain and normal brain.When FD and C Green No. 3 was infused after invasive BBB manipulation, the anterior and middle cerebral artery (MCA) distributions were clearly stained both on the cortical surface and into the deep white matter. There was a small area that remained unstained on the inferior and medial portion of the temporal lobe; this area likely corresponded to the PCA distribution that was not infused (Figure 3).

A B

Figure 2. Fluorescein mapped rat brain; (a) dorsal view and (b) coronal section after osmotic disruption of the blood brain barrier (BBB) using a hypertonic solution of arabinose.

A B

Figure 3. FDandC Green No. 3 mapped rabbit brain; (a) ventral view and (b) coronal section after osmotic disruption of the blood brain barrier (BBB) using a hypertonic solution of arabinose.

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Experiments are currently underway to confirm this. There was a relatively sharp visual margin between the stained brain and the unstained brain. The advantage of the rabbit model over the rat was the ability to visualize with fluoroscopy and digital subtraction angiography (DSA) exactly where the mapping agent was infiltrating into the brain. Unfortunately, we were not able to advance the microcatheter distally into the intracranial MCA branches. Therefore, the rat model was used for the remaining experiments. The FDandC Green No. 3 as a mapping agent is limited by the need for invasive BBB disruption. Because this successfully proved the concept, all future PEBM agents will be performed without BBB disruption.

Tantalum Particle Embolization, PEBM Contrast without BBB Manipulation Capillary-level embolization is another strategy of endovascular brain mapping that has the potential advantage of eliminating the invasive BBB manipulations. Tantalum embolization also has the potential to eliminate bleeding during the brain resection following craniotomy. To test the strategy of capillary-level embolization, we injected 0.6 ml of visible embolic agents mixed with iohexol into the right brain hemisphere through the right ICA of the rats. Tantalum was the initial agent used to test the feasibility of this approach. We obtained tantalum powder from the TruFill® n-BCA embolization kit (Cordis Neurovascular, Miami Lakes, FL); this material has FDA approval for intraarterial embolization of intracranial AVMs. We also obtained tantalum dust (Biodyne, Inc., La Mesa, CA), which consisted of much smaller particles sizes, to assess the differences in brain map quality that might result from differences in the tantalum. Because the particle size would likely be a primary determinate of the quality of the brain map, we performed electron microscopy on each sample and measured this property. Each of the two tantalum samples was then infused into the right brain hemisphere of 2 different rats. We demonstrated that the TruFill® tantalum powder became wedged in the brain arterioles, producing an inadequate result because of poor visualization and poor margins of the target brain parenchyma (Figure 4).

Figure 4. Tantalum particle mapped rat brain dorsal view. Notice the particles are too large to travel to the capillary level, therefore this produces a poor brain map.

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Results of electron microscopy studies revealed the particle sizes of the tantalum powder were too large (20-80 μm), which likely explains why they lodged in the arterioles. The smaller tantalum dust also produced an inadequate result, because the particles traveled through the capillaries and were washed out into the venous system. Electron microscopy studies revealed that all of these particles were less than 5 μm, with an average size of 3 μm, which was determined to be too small for adequate brain mapping. We concluded that in order to achieve an appropriate brain map, the tantalum particles have to be an ideal size to lodge in the capillaries, probably 5- to 10-μm particles.

Sudan Black Cocktail Embolization without BBB Disruption To overcome several difficulties encountered with tantalum, a novel mixture of agents was then tested to perform capillary-level embolization and subsequent brain mapping. To achieve the ideal penetration to the capillary level while preventing transit into the venous system, we used a mixture of 1mg Sudan Black (Sigma Aldrich, St. Louis, MO), 1 ml of NBCA (TruFill®, Cordis Neurovascular, Miami Lakes, FL), 10 ml Ethiodol™ (Savage Laboratories, Melville, NY), and 40 μl glacial acetic acid (Sigma Aldrich, St. Louis, MO) as a potential brain mapping agent. We then used a standard infusion pump to deliver 0.6 ml of the cocktail over 3 minutes at 0.2 ml/min. The use of NBCA and Ethiodol has been approved by FDA for injection into the intracranial arterial supply of AVMs for preoperative embolization. The practice of adding glacial acetic acid is sometimes used to slow the NBCA polymerization time [6,16]. The addition of Sudan Black, a fat soluble dye, allows visualization of the material at the time of subsequent craniotomy and resection. This novel combination of materials allows the mapping agent to remain liquid until reaching the capillary bed, and then it polymerizes to solid form before excessive transit to the venous system occurs. Once a target distribution of tissue has been mapped as nonfunctional and chosen for resection, the preoperative capillarylevel embolization of that territory should eliminate intraoperative bleeding. A symptomatic stroke is not likely in humans because the target vascular territory will have been identified previously as nonfunctional with a super-selective Wada test [5,9,36,43,50,51,65]. When the mixture of Sudan Black, NBCA, Ethiodol, and glacial acetic acid was used, there was excellent penetration of the mapping agent into the brain capillaries of the target brain distribution (Figure 5). This provided a dense, highly contrasted, black brain map. We were again able to achieve a relatively sharp margin between the stained brain in the MCA and ACA distribution and the unstained brain in the inferior medial temporal lobe, which was likely within the PCA distribution. This approach required manipulation of the ratios of each cocktail constituent to optimize imaging agent distribution through the capillaries into the venous structures. This injection can be further optimized by altering the injection speed, volume, and force of the infusion. Despite the gross appearance of black brain parenchyma, histological examination revealed only the capillaries were stained and the brain parenchyma cells were unaffected. The gross appearance of Sudan black-stained brain parenchyma is due to the fact that the capillaries are so numerous and dense.

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Figure 5. Sudan black cocktail mapped rat brain, ventral-lateral view. Notice the mapping agent is small enough to travel to the capillary level, therefore this produces a more useful brain map.

Administration of TAT-Conjugated Silica Nanoparticles and TATConjugated Cds: Mn/Zns Quantum Dots without BBB Manipulation More sophisticated techniques for PEBM have been recently described, such as the use of nanoparticles, including silica nanoparticles and quantum dots, as potential brain mapping agents, without the need for BBB manipulation or vascular occlusion [50,51]. It has been shown that is possible to deliver various nanoparticle-based imaging agents into cells using a TAT peptide-mediated delivery system [53-55]. TAT-conjugated silica nanoparticles and Cds:Mn/ZnS Qdots (TAT-Qdots) were prepared as described by Santra et al. [55]. The silica nanoparticles labeled brain endothelial cells, but the Qdots were small enough to also label the brain parenchyma cells, in addition to the endothelial cells. The Qdots are highly sensitive and photostable. Qdots without TAT did not label brain tissue confirming that the TAT peptide is necessary to penetrate through the BBB to the cells in the parenchyma. About 0.75 ml (10 mg/ml) of TAT-Qdot suspension in phosphate buffered saline (PBS) was loaded in a syringe and attached to a standard infusion pump and injected into a rat. The Qdots were injected over a period of 5 minutes and then PBS (pH 7.4) was injected for 15 minutes at the same rate of injection to remove residual TAT-Qdots. The ECA was then opened and collateral blood flow allowed for 3 minutes. The rat was then euthanized and the brain harvested. The whole brain was immediately placed under a hand held 366 nm multi-band UV source (Spectroline, model UV-4B) and photographed using a standard digital camera. The TAT-Qdot fluorescence is pink and the background autofluorescence in the absence of

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Qdots is blue at this 366 nm UV wavelength. With this method, the time needed to stain brain tissue is dependent on cerebral blood flow. Histological analysis was performed to show whether the TAT-Qdots penetrated the endothelial cells. It showed TAT-Qdots reached the nucleus of brain cells, supporting the fact that TAT-Qdots cross the BBB and migrated into the parenchyma of the brain to reach the cell nuclei. Endothelial cells in the blood capillaries were loaded with TAT-Qdots as expected. These grafted nanoparticle systems are limited by potential toxicity from the silica in the nanoparticles and the Cd:ZnS in the Qdots. It would therefore be difficult to receive FDA regulatory approval for either of these potential mapping agents.

Administration of Onyx®- Fluorescein Cocktail We have recently advanced our embolic approach to PEBM through the development and use of fluorescent cocktails of the commercially available embolic agent Onyx®. Onyx® is a non-adhesive liquid embolic agent that is comprised of ethylene vinyl alcohol copolymer (EVOH) dissolved in a sterile vehicle of dimethyl sulfoxide (DMSO). Since these agents are already approved by the FDA for other applications, their regulatory approval would be more likely. To provide visualization under fluoroscopy, micronized tantalum powder is also suspended in the solution matrix. Typically, the agent is delivered slowly by controlled injection through a microcatheter into the brain under fluoroscopic guidance. Chemically, upon entry into the blood stream, the DMSO vehicle dissipates rapidly into the blood and the EVOH copolymer and suspended tantalum particles precipitate in situ into a spongiform, coherent material. Onyx® is currently available in two formulations marketed as Onyx® 18 (6% EVOH) and Onyx® 34 (8% EVOH), with Onyx® 18 being considerably less viscous allowing increased penetration deeper into distant capillaries and vascular features. Capitalizing upon our ability to precisely deliver PEBM imaging agents via superselective arterial catheterization, we have explored the use of Onyx®-based PEBM emulsions that could be visualized by optical imaging. To do this, we added a small quantity of fluorescein to the standard Onyx® 18 preparation. Upon addition, we found that fluorescein was highly soluble in the DMSO/EVOH mixture and would co-precipitate with EVOH in aqueous solution forming a highly fluorescent spongiform solid that appeared suitable for imaging. We hypothesized that the improved sensitivity of fluorescence imaging (compared to gross visualization or x-ray angiography) would enable enhanced visualization of the embolic material following precipitation and possibly improve the sensitivity of our PEBM approach. An added benefit of using fluorescein as the optical probe was that fluorescence visualization could be achieved in the visible portion of the electromagnetic spectrum, and excitation of the agent could be achieved using a simple hand-held ‘black light’ or Wood’s lamp, which is operating room compatible. In Figure 6, we illustrate a typical brain map in a rat using the Onyx®-based optical imaging approach. Immediately following catheterization as described above and agent preparation, the Onyx®-fluorescein emulsion was slowly injected using an infusion pump (5mg/mL agent @ 1mL/min). After approximately 10 minutes, the animal was sacrificed and a craniotomy was performed. Gross whitelight visualization of the intact brain illustrated

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enhanced probe accumulation and staining primarily on the left hemisphere, as shown by the slight yellow tinting of the brain tissue from the dye (Figure 6 A and C). Additionally, some of the larger tantalum particles can be seen in the middle cerebral artery.

A

C

B

D

Figure 6. Endovascular brain mapping using the Onyx®-Fluorescein embolic approach in rat. (A) Gross whitelight visualization of rat brain following agent infusion and craniotomy. Under whitelight illumination, left hemisphere of brain appears slightly yellow-tinted from agent accumulation. Larger tantalum particles can be seen in the arterials because of their large size. (B) Fluorescence visualization illustrates enhanced visualization of mapping agent accumulation. Good differentiation between left and right brain hemispheres is observed, with some accumulation of fluorescence in the deep veins. (C/D) Whitelight and fluorescence images, respectively, of resected brain.

Agent localization primarily to the left hemisphere can be further appreciated by fluorescence visualization following illumination with a hand-held Wood’s lamp. In addition to agent localization to the left hemisphere, we also observed some accumulation in the posterior fossa, possibly indicating some agent passage from the arterial system to the venous system and settling in the dural sinus veins in the posterior fossa (Figure 6 B and D). In summary, it appears feasible to prepare fluorescent embolic materials using an Onyx®-based medium, and these materials appear to be promising agents for PEBM. As with any embolic material, a considerable limitation of these agents is the fact that they occlude the vasculature and are therefore unsuitable for imaging normal tissues. However, these materials and similar

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agents hold considerable promise for resection guidance of diseased tissue that will subsequently be removed or in applications where it is appropriate to leave embolics in place, such as treatment of AVMs. We are currently developing improved mapping agents that do not require BBB manipulation or vascular occlusion, as well as having the safest possible profile, to minimize potential toxicity. BBB disruption is not desirable in the ideal PEBM agent, because of its associated potential neurotoxicity and possible increased cerebral edema. Vascular occlusion is also not ideal, because nearby adjacent brain tissue could be affected by the ischemic edema arising from the non-functional mapped territory.

CONCLUSIONS In this chapter, we have described the concept for preoperative endovascular brain mapping (PEBM), and demonstrated the feasibility and utility of the technique with several potential mapping agents in animal models. In each case, using the techniques illustrated we have achieved good visual clarity and relatively sharp margins between the mapped brain and the non-mapped brain. Furthermore, we have illustrated simple PEBM techniques that require BBB manipulation to be effective, as well as alternative techniques that do not require BBB manipulation. Our concept of preoperative endovascular brain mapping for volumetric image guidance has the potential to produce clearly visible, safe margins of nonfunctional brain tissue for resection with excellent spatial resolution that remains true throughout the procedure, regardless of brain-shifting. If this approach is successful, it could significantly reduce or eliminate postoperative neurologic deficits. Although safety and toxicology studies must be performed on any potential mapping agent, there is a greater potential for toxicity, when considering nanoparticle systems as many of their characteristics remain unknown. Studies to determine the durability of each mapping agent to allow for a possible delay between brain mapping and surgical resection must also be performed. Even though further study is required to demonstrate safety, minimize toxicity, and improve the characteristics of potential mapping agents, PEBM is an emerging imaging technique that may prove complementary to other brain mapping strategies, as well as serving as a surgical guidance tool during the resection of diseased tissue.

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[54] Santra S, Yang H, Holloway PH, Stanley JT, Mericle RA: Synthesis of waterdispersible fluorescent, radio-opaque, and paramagnetic CdS:Mn/ZnS quantum dots: a multifunctional probe for bioimaging. J. Am. Chem. Soc. 127:1656-1657, 2005. [55] Santra S, Yang H, Stanley JT, Holloway PH, Moudgil BM, Walter G, et al: Rapid and effective labeling of brain tissue using TAT-conjugated CdS:Mn/ZnS quantum dots. Chem. Commun. (Camb):3144-3146, 2005. [56] Savoy RL: History and future directions of human brain mapping and functional neuroimaging. Acta Psychol. (Amst) 107:9-42, 2001. [57] Schlosser MJ, Aoyagi N, Fulbright RK, Gore JC, McCarthy G: Functional MRI studies of auditory comprehension. Hum. Brain Mapp. 6:1-13, 1998. [58] Schlosser MJ, McCarthy G, Fulbright RK, Gore JC, Awad IA: Cerebral vascular malformations adjacent to sensorimotor and visual cortex. Functional magnetic resonance imaging studies before and after therapeutic intervention. Stroke 28:11301137, 1997. [59] Sobottka SB, Bredow J, Beuthien-Baumann B, Reiss G, Schackert G, Steinmeier R: Comparison of functional brain PET images and intraoperative brain-mapping data using image-guided surgery. Comput. Aided Surg. 7:317-325, 2002. [60] Standard SC, Ahuja A, Livingston K, Guterman LR, Hopkins LN: Endovascular embolization and surgical excision for the treatment of cerebellar and brain stem hemangioblastomas. Surg. Neurol. 41:405-410, 1994. [61] Stewart L, Ellison A, Walsh V, Cowey A: The role of transcranial magnetic stimulation (TMS) in studies of vision, attention and cognition. Acta Psychol. (Amst) 107:275-291, 2001. [62] Suzuki M, Iwasaki Y, Yamamoto T, Konno H, Kudo H: Sequelae of the osmotic bloodbrain barrier opening in rats. J. Neurosurg. 69:421-428, 1988. [63] Taylor CL, Dutton K, Rappard G, Pride GL, Replogle R, Purdy PD, et al: Complications of preoperative embolization of cerebral arteriovenous malformations. J. Neurosurg. 100:810-812, 2004. [64] Trenerry MR, Loring DW: Intracarotid amobarbital procedure. The Wada test. Neuroimaging Clin. N Am. 5:721-728, 1995. [65] Urbach H, Klemm E, Linke DB, Behrends K, Biersack HJ, Schramm J, et al: Posterior cerebral artery Wada test: sodium amytal distribution and functional deficits. Neuroradiology 43:290-294, 2001. [66] van Emde Boas W: Juhn A. Wada and the sodium amytal test in the first (and last?) 50 years. J. Hist. Neurosci. 8:286-292, 1999. [67] Vinuela F, Dion JE, Duckwiler G, Martin NA, Lylyk P, Fox A, et al: Combined endovascular embolization and surgery in the management of cerebral arteriovenous malformations: experience with 101 cases. J. Neurosurg. 75:856-864, 1991. [68] Wakhloo AK, Juengling FD, Van Velthoven V, Schumacher M, Hennig J, Schwechheimer K: Extended preoperative polyvinyl alcohol microembolization of intracranial meningiomas: assessment of two embolization techniques. AJNR Am. J. Neuroradiol. 14:571-582, 1993. [69] Wakhloo AK, Lieber BB, Rudin S, Fronckowiak MD, Mericle RA, Hopkins LN: A novel approach to flow quantification in brain arteriovenous malformations prior to enbucrilate embolization: use of insoluble contrast (Ethiodol droplet) angiography. J. Neurosurg. 89:395-404, 1998.

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[70] Walsh V, Cowey A: Transcranial magnetic stimulation and cognitive neuroscience. Nat. Rev. Neurosci. 1:73-79, 2000. [71] Yakes WF, Krauth L, Ecklund J, Swengle R, Dreisbach JN, Seibert CE, et al: Ethanol endovascular management of brain arteriovenous malformations: initial results. Neurosurgery 40:1145-1152; discussion 1152-1144, 1997.

In: Brain Mapping and Diseases Ed: Diane E. Spinelle

ISBN: 978-1-61122-065-0 © 2011 Nova Science Publishers, Inc.

Chapter 12

THE BRAIN’S NEUROPROTECTIVE AND PROAPOPTOTIC EFFECTS OF ASPIRIN - A REVIEW Yair Lampl∗ Departments of Neurology, Edith Wolfson Medical Center, Holon and Sackler Faculty of Medicine, Tel Aviv University, Israel.

ABSTRACT The investigation of techniques for neuroprotection plays a key role in brain research which involves finding a protective method for acute or chronic destruction of brain tissue. These methods are aimed either toward the necrotic pathway or the apoptotic one. The ability of acetylsalicylic acid (aspirin) to alleviate both destructive pathways is increasingly being recognized, as well as there being indirect evidence for its effective use in the attenuation of severity of neurologic diseases. The relation between the neuroprotective effects and the dosage of aspirin are not yet in agreement. The rationale of action appears to be aspirin’s direct and indirect specific effect on the nuclear factor Kappa β (NF Kappa-). Other targets of aspirin activity are the mitogen activated protein kinase (MAPK), the nitro oxide synthase (NOS) and the adenosine triphosphate (ATP). The protective effect of aspirin was studied in hypoxic damage, cerebral infarction, degenerative brain disease and epilepsy. An aspirin-induced apoptotic phenomenon was documented in gastric colon, lung and cervical cancer. Evidence of the same mechanism was shown also in brain malignant glioblastoma cells. The antiapoptotic and antitumoral effects are mediated by the Bcl-2 and caspase-3 pathways, as well as the mitochondrial permeability transfer mechanism. The pro- and anti-apoptotic mechanisms studied in regards to brain ischemic events are still unresolved issues. However, data from direct and indirect in vitro and in vivo studies, as well as epidemiological studies, lead to the assumption that aspirin probably does have an in vivo protective effect in humans. The ∗

Correspondence concerning this article should be addressed to Dr. Yair Lampl, MD Department of Neurology , Edith Wolfson Medical Center Holon 58100, Israel. Tel: 972 – 3 – 502-8512; Fax: 972 – 3 – 502-8681; Email: [email protected].

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Yair Lampl promising data from these experimental studies bode well for an optimistic view for the possibility of aspirin’s therapeutic use as a neuroprotective agent in human diseases of the central nervous system.

INTRODUCTION Management of acute and chronic pathological progressive destruction of brain neural cells is one of the most fascinating and important focal issues in cutting edge neuroscience. The interpretation of degenerative devastating processes is the first step for research in the activation of neuromodulatory systems and neurogenesis. The relative new concept that death of neuronal cells is not only initiated by one single process, but can be activated in addition to passive necrosis also by the active system or apoptotic pathway, changed forever our understanding and opinions. The knowledge of the existence of programmed cell death (PCD) forced us to explore therapeutic agents which simultaneously affect both mechanisms. However, there is a wide discrepancy between real scientific knowledge of these current theories and the translation into therapeutical properties in order to find a clinically effective method to inhibit these pathological processes. Most of the studies that examined new neuroprotective agents failed or were found to have only limited effect. The investigation for agents with inhibitory effects on only one at a time of the pathways can be assumed to be the reason for this failure. Aspirin (acetylsalicylic acid – ASA), which is well known for a long time as a non selective COX inhibitor, seems to show the promising data. The research into its initial analgesic and anti-inflammatory effects were enlarged into additional study indicators, especially the antiaggregation, and lately neuroprotective, pro- and anti-apoptotic properties. The multi variant target effects of aspirin seem to be specific to this agent in comparison to other selective COX-1 or COX-2 inhibitors. Experimental data produced diverse effects of the necrotic and apoptotic pathways in neural cell death. Although a lot of this data is also limited, these new findings can let us be optimistic toward the eventual additional use of aspirin in the arsenal of neuroprotective agents under current research.

DISCUSSION Neuroprotection 1. Neuroprotection The concept of neuroprotection is based on the assumption that different pathways leading to acute or chronic progressive damage of neuronal tissue can be interrupted by external stimuli. It can be a pharmacological or non pharmacological method (example – hypothermia). The interruption of the propagation of the damaging cascade can be achieved by inhibition of excessive glutamate receptor activity, countering the accumulation of intracellular calcium ions, massive production of free radicals and the activation of the apoptotic cell death pathway. Target conditions for neuroprotective research are the degenerative diseases, such as Parkinson’s disease, Alzheimer’s disease and amyotrophic lateral sclerosis, and the acute,

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hypoxic, traumatic and ischemic brain conditions. Brain ischemia is one of the most studied with research showing a post ischemic cascade of irreversible electrophysiological changes thru release of excitatory neurotransmitters, permanent breakdown of the ionic hemostatic balance and depletion of energy donators [1-10].

1a. Neuroprotection and Aspirin There is a lot of evidence for a beneficial neuroprotective effect induced by aspirin. The protective mechanisms included various targets of brain neurochemistry. The type of mechanisms is dependent upon the specific form of action induced by the aspirin. Aspirin inhibits the excessive activation of the N methyl D aspartate (NMDA) ionotropic glutamate receptor [11,12], NFKB activity which regulates protein [13-15] and Kinase activity which down-regulates the proinflammatory enzymes of the nitric oxide synthase (NOS) family group [16-18]. It reduces the oxidative stress [19-24], inhibits the uncoupling of oxidative phosphorylation [25-27], regulates the amount of adenosine triphosphate (ATP) and increases the extracellular adenosine [28-29]. 2. Glutamate and N Methyl D Aspartate (NMDA) Glutamate is the most common excitatory neurotransmitter in the central nervous system. It is composed of four different types of receptors: 3 inotropic receptors – (a) N methyl D aspartate (NMDA), (b) α amino 3 hydroxy 5 methyl 4 isoxazolepropionic acid (AMPA), and (c) Kainate, and one metabotrophic receptor. The NMDA is subclassified into two types of receptors - NR1 (NR1AG) and NR2 (NR2AG). The NR1 receptor is diffusely represented in the brain and is a product of a single gene. The NR2 receptor is localized in various areas of the brain and is composed of four different genes. The AMPA receptor has the sub units GluR1and GluR4, and is more prevalent in the telencephalon, whereas Kainate has GluR5 and GluR7 subunits, as well as K1 and K2, and is more diffusely widespread in the brain. The metabotrophic receptor has three main subgroups – subgroup 1 (m GluR1 and m GluR5), 2 (m GluR2 and m GluR3) and 3 (m GluR4, 6, 7, and 8). The ionotropic glutamic receptors, along with the NMDA receptor, are permeable to Ca+2 and monovalent cations [30,31]. Its overactivation leads to cascade and progressive neuronal damage. Any permanent metabolic dysfunction and deficit in energy substance are responsible for ongoing depolarization and release of Mg+2. The resulting increase of Ca+2 influx and accumulation of intracellular Na+ and Ca+2 ions induce the neural cell death [32]. The neurotoxicity of NMDA also activates the phospholipase A2 pathway [33], neuronal nitro oxide synthase [34-36], and calpain [37]. Acute glutamate neurotoxicity is studied especially in stroke, hypoxia, trauma and epilepsy [38,39]. It has been hypothesized to occur in slow progressive neurodegenerative processes. 2a. Glutamate, NMDA Receptor and Aspirin Induction of cyclooxygenase 2 (COX2) is seen after seizures [40,41], spreading depression [40] and after focal and diffuse ischemia [42,43]. Amelioration of ischemic brain damage, associated with changes of NMDA activity, was shown after administration of indomethacin in gerbil hippocampus CA1 neurons [44] and after treatment with Ibuprofen leading to reduced volume of focal brain ischemia in spontaneous hypertensive rats [45]. Hewett et al [46] examined various COX-1 and COX-2 agents. They found an antineural death effect with administration of COX-1/COX-2 inhibitors and selective COX-2 enzymes, but not in selective COX-1. Aspirin had no effect when using it alone as a preventative

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treatment, but worked well in a combination of aspirin as a pretreatment and the COX-2 inhibitor as post treatment. In this combined administration, the positive COX-2 inhibitory effect was markedly increased. The neurotoxicity of NMDA is mostly induced through a pathway of activation of the NFKB. Grilli et al [15] showed in hippocampal and cerebellar neurons that blockade of the NFKB pathway is the main target of aspirin and sodium salicylate anttiglutamate activities. In mouse cortical cells after treatment with 3 mM of aspirin, Ko et al [47] found blocking of NMDA induced activation of C Jun terminal kinase (JNK) and NFKB. The effect was assumed to induce nuclear translocation of NFKB’s p50 and p65. On rat cerebellar cells, glutamate also induces p38, a subfamily of MAPK, another target of aspirin activity [48]. Crisanti et al [12] demonstrated the key role of protein kinase C zeta (PKC zeta) in the relationship of NMDA and NFKB in neural death. They showed that aspirin, but not salicylic acid, has a direct effect on PKC zeta, leading to decrease of NMDA activity and its nuclear translocation. The decrease of NMDA activity reduces neuronal cell death. The prevention of PKC zeta cleavage and its nuclear translocation by aspirin is hypothesized to have a role in the anti-acute neuronal damage, as well as antiapoptotic, mechanisms.

2b. Aspirin in Non Central Nervous System NMDA Receptors Cochlear system Some studies showed that the association between aspirin and aspirin-induced tinnitus is based on the activation of the cochlear NMDA receptors [49]. Platelets The NMDA receptor of platelets has some characteristics which have differentiated them from the NMDA receptor of the central nervous system [50], including having antiaggregating activity. This effect is potentiated in association with various COX inhibitors, including aspirin and indomethacin [51].

3. NFKB NFKB was first described by Sen and Baltimore in 1986 [52]. This eukaryotic transcription factor enzyme consists of various proteins and exists in most cells where it reacts on specific NFKB binding sites [12,53]. The NFKB DNA are induced and activated by various exogenic and endogenic stimuli. NFKB exists in the cytoplasm as homomer and heteromers of the structurally related protein – Rel/NFKB [54]. According to structure, function and properties, this family group is divided into two main subgroups [55]. The inactive form of NFKB has multiple contacts with IKB, an inhibitor protein related to the Rel/NFKB family group. The activation of the NFKB pathway is induced by splitting of it from the NFKB-IKB complex, activation of the enzyme IKB kinase, phosphorylation of IKB, ubiquitination of the protease, and finally, its degradation. The destruction of the IKB leads to nuclear translocation of the NFKB and activation of the target gene promoter [56,57]. For activation of NFKB transcription activity, the induction of other groups of NFKB proteins (Rel-A, Rel-B, c-Rel) is necessary [58-60]. Sizeable amounts of stimuli varieties – cytokine and chemokine (TNFα, TNFβ, ILα and ILβ) receptors on cell surfaces and adhesion molecules [61]; bacterial products, including lipopolysacharrides; various viruses, such as the herpes group, HTLV, Epstein-Barr virus and the influenza virus; and exposure to hypoxia and

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H2O2, irradiation and toxins – can lead to activation of NFKB. NFKB has a crucial role in protection of the cell, including cell survival [62,63], protection against signal induced cell death [64,65], cancer [66] and oxidative stress [67,68]. NFKB is activated in the early stages of brain damage, as well as in the apoptotic pathway.

3a. Aspirin and NFKB Aspirin has a proven inhibitory effect on NFKB. It is based on prevention of the activity of IKB kinase β. IKB kinase is responsible for the phosphorylation and degradation of IKB. IKB is the main factor in the stabilization of NFKB. Breakdown of the IKB kinase evades the IKB degradation leading to persistence of NFKB in the cytosol and preventing its nuclear translocation [69,70]. The supposition that aspirin may also have a direct effect on NFKB dependent gene is controversial [70,71]. The effect of aspirin on NFKB is related to the inhibition of the vascular cell adhesion molecule 1 (VCAM 1) [72,73] and E selectin [72], reduction of the intracellular adhesion molecule 1 (ICAM 1) expression [74] and changing the mobilization of TNFα and suppression of TNFα mRA [75,76]. The inhibitory effect of aspirin on NFKB is apparent in its blockage of viral activity. 1-2 mM of aspirin inhibits Chlamydia pneumonia by inducing NFKB activation [78]. This inhibitory effect is also shown with the cytomegalovirus [79], HIV in a dosage of 10 mM and in herpes 8 [80]. Aspirin’s NFKB blockage effects also tumor proliferation in the pancreas [81] and lung [82], colorectal cancer [83], as well as reduces angiotensin II in organ damage [84] and carotid artery plaques in humans [85]. According to the various studies, the aspirin’s effective dosage is unclear and ranges from 1mM - 20 mM. 3b. NFKB, Aspirin and the Central Nervous System Brain Hypoxia A decrease of NFKB DNA binding in brain activity was found to be characteristic of COX2 knockout mice [86]. Grilli et al [15] demonstrated and discussed aspirin’s role in neuroprotection through blockage of NFKB activity. On the oxygen and glucose deprivation rat model, Moro et al [87] showed prevention of neural damage after administration of low dose (0.1-0.5 mM) aspirin. This effect was correlated to NFKB translocation, iNOS expression and inhibition of glutamate release. Opposite to these findings, Vartiainen et al [88] in hypoxia/reoxygenation spine rat model pointed to a clear neuroprotective effect of aspirin. However, this effect was found to be secondary to inhibition of the activation of extracellular signal regulate protein (ERK), ERK-1 and ERK-2, but unrelated to NFKB. Parkinson’s Disease Aubin et al [89] demonstrated that the neurotoxic effect of MPTP in mice, an animal model used for Parkinson’s research, can be inhibited by aspirin. They hypothesized that NFKB may play a main role. On Chinese hamster ovary cells, Weingarten et al [90] found that the dopamine intracellularily activated NFKB rapidly and was resistant to aspirin, whereas dopamine extracellularily induced NFKB activity slowly and in small amounts. Only this last type of dopamine activity was inhibited by aspirin.

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Alzheimer’s Disease In human glial cell cultures, activation of NFKB by IL1β leads to release of α2 macroglobulin. This proinflammatory mechanism was assumed to be inhibited by aspirin and to act as a blocker of Aβ peptide, a key molecular chain in the development of Alzheimer’s disease [91]. On the examination of 3-30 mM of sodium salicylate in astroglial cells exposed to the Aβ peptide, the reduction of Aβ activation was up to 60% [92]. APO E Apo E and Apo J are expressed in glial cells, Apo E in microglia cells and Apo J in astrocytes. On exposure to aspirin (10 mM), this expression was blocked. It was assumed that the inhibition occurred via blocking of NFKB [93]. CNS Inflammation While exploring the actions of hepatic metabolism after infection of the central nervous system, Abdulla et al [94] found, following injection of lipopolysacharrides, down regulation of the hepatic enzyme and CYP genes and increase of NFKB activity parallel to other transcription factors. Although there was no direct investigation of the effect of aspirin, it can be hypothesized to be present through the NFKB inhibition effect. 4. Mitogen Activated Protein Kinase (MAPK) MAPK is a family of serinekinase proteins, acting as nuclear transcription factors. The MAPK protein family activity is initiated on the cell surface and acts by transferring γ phosphate of the ATP to the dual serine-threonine sites in the cell’s protein. The MAPK pathways lead to cell death through necrotic or apoptosis mechanisms, as well as to cell proliferation. Three families of MAPKs have been identified: (1) extracellular signal regulate protein (ERK); (2) C-Jun NH2 terminal kinase (JNK); and (3) p38. The ERKs play a role in the mitosis and cell definition and is activated in response to growth factors [95-99]. Of its various know enzymes, the most studied are the ERK-1 and ERK-2. The route of activity is through stimulation of cell surface receptors and activation of the Ras protein. The transcriptional activation of the C-fas is induced by way of phosphorylated ERK-translocation to the nucleus and phosphorylation of the transcription factor ELK-1 [100,101]. The JNK proteins are also known as a group of stress activated MAPK. They consist of two main enzymes – JNK-1 and JNK-2 – and some other less known types. JNK-3 is characterized by its specificity for brain and testicles [102]. All theses are activated by stress, cytokines or endotoxins [103-105]. The JNK signal pathway regulates the C-Jun transcription factor, the ELK-1 and AIF2 [105]. p38 is activated by stress and cytokines and is responsible for regulation of AIF2 and ELK-1 transcription factors. 4a. MAPK and Aspirin ERK In human neutrophils, aspirin inhibits ERK activity. This characteristic is specific for aspirin and was not found with indomethacin [106]. The effect was induced also by MEK – an activator of ERK. A relationship was found between the ERK activation and the integrin

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dependent neutrophil adhesion. An interaction between ERK, NFKB and TNFα was evident with exposure to sodium salicylate [107-110].

JNK and p38 Although activity of JNK seems to be reduced by aspirin [111], Schwenger et al [112] found that p38 and JNK were induced after treatment of cells with sodium salicylate. With the use of sodium salicylate, interaction was seen between p38 and NFKB [113], but not between p38 and JNK [114]. 4b. MAPK, Aspirin and the Central Nervous System Hypoxia The pathway mechanism of NMDA receptor inhibition by aspirin has been shown to be also partially dependent upon the activation of JNK. This activation, which leads to Ca+2 dependent manner of neuroprotection, was tested at an aspirin dosage of 3mM [115]. In hypoxic injured rat spinal cord cell cultures, Vartiainen et al [88] showed that aspirin inhibits neural death. They found an inhibition of both ERK-1 and ERK-2, which were elevated after the trauma. p38 was not altered by the hypoxia and was not affected by aspirin. The therapeutic dosage of aspirin was 1-3 mM/L. Alzheimer’s Disease In the examination of APP 751 transgenic mice (the usual rodent model for Alzheimer’s disease) after exposure to focal brain ischemia, high p38 MAPK was observed in microglia [116]. Aspirin, as well as other p38 inhibitor agents, neutralized the neural vulnerability of the mice. 5. Nitric Oxide (NO), and Nitro Oxide Synthase (NOS) NO is and essential labeled messenger molecule, having the properties of neurotransmitters. The formation of NO occurs by the transmission of L arginine into L citrulline. This process includes the reaction of the quanidino nitrogen area of the L arginine with molecular oxygen in the presence of NOS enzymes. The low concentration of NO produced by NOS inhibit the expression of adhesion molecules and block the synthesis of cytokine and the adhesion of leukocytes. High concentrations of NO produced by NOS react toxic and in a proinflammatory manner [116]. There are three know NOS isomers – two of them (NOS I and NOS III) are constitutional, and one (NOS II), inducible. NOS I (neurogenic NOS-nNOS), which is located in the neuron’s cytosole, induces rapid Ca+2 influx, binding to the calmodulin and generation of NO [117,118]. It also has a crucial effect on the NMDA receptor, acting on the post synaptic calmodulin, the calcium calmodulin complex and the guanylate cyclase in the presynaptic neuron [119]. This process is necessary for inducing the phenomenon of long-time potentiation (LTP) in the creation of memory [120,121]. NOS II (inducible NOS-iNOS) is an inducible cytoplastic enzyme. It can be expressed by overestimation of the astrocytes [123]. Under normal physiological conditions, the concentrations of NOS II are very low [124]. In the presence of lipopolysacharrides, it activates the release of proinflammatory cytokines [125,126]. The main mechanism of NOS II is at least partially dependent upon the nuclear transcription of NFKB, which is inhibited by aspirin [127]. NOS III (endothelial NOS-eNOS) has similar properties to NOS I. It regulates

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the calcium-calmodulin complex system [128] and plays a key role in the vasodilatation of cerebral vessels [129-131] and in controlling the cerebral blood flow [132]. The damaging effect of NO on the brain was investigated in Alzheimer’s disease [133-134], multiple sclerosis [135], Parkinson’s disease [136], and especially, in ischemic brain damage [137139]. The rationale for these effects can be explained by the activation of reactive nitro oxide radicals, damage to the nucleic acids and activation of the poly ADP ribose synthase (PARS).

5a. NOS and Aspirin A specific characteristic of aspirin is having a least a partial inhibitory effect on iNOS expression [140-142]. Kepka-Lenhart et al [143] found that aspirin in a concentration of 3-10 mg inhibited iNOS mRNA induction in lipopolysaccharide stimulated murine macrophage cells, but enhanced iNOS mRNA in interferon gamma stimulated cells. They assumed that different pathways are involved in the aspirin effect on NO and iNOS. Amin et al [140] showed on the same cell culture type that the inhibition of iNOS expression by aspirin is based on the modification and direct action on the mechanism of translation-post translation of the iNOS catalytic activity. The specificity of this effect by aspirin was demonstrated when compared with indomethacin. Inhibition of induction of iNOS was shown on rat cardiac fibroblast selectivity after the administration of aspirin, but not with indomethacin or acetaminophen [144], and it was also shown in rabbit infracted heart in situ after exposure to 150mg-500 mg/day of aspirin [145]. The effect of aspirin on iNOS and NO synthesis in vascular smooth muscle cells was shown to be independent [146] or partially dependent [147] from the mechanism which activated the NFKB system. 5b. NOS, Aspirin and the Central Nervous System Glial Cells In rat glial cell culture, iNOS expression is suppressed by high aspirin dosage resulting in decreased NO production. The effect was hypothesized to be caused either by aspirin’s direct effect on iNOS or through an effect of the prostaglandins [148]. Brain Degenerative Diseases In the comparison of indomethacin, ibuprofen and aspirin on NOS mRNA expression, the properties of beta amyloid protein and interferon gamma to induce NO production in murine macrophage cell lines were tested. Aspirin, contrary to the other agents, had no effect [149]. However, Asanuma et al [150] showed that using the electron spin resonance spectrometry, aspirin has a direct effect on scavenger generated nitric oxide radicals. This dosage dependent phenomenon was assumed to also have neuroprotective properties in neurodegenerative diseases, especially in Alzheimer’s disease. Brain Ischemia and Anoxia Aspirin was found to reduce various types of NOS activities in nitrogen hypoxic induced brain slices. The effect is dose dependent and is expressed only at higher dosages. Aspirin’s activity rate is lower than those of salicylate acid [151]. Contrary to these findings, Vartiainen et al [88] found no involvement of the iNOS in the reactive mechanisms of early post anoxia in rat spinal cord culture. In the comparison of aspirin to triflusal (a fluorinated derivate of

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aspirin), a reduction of nitric oxide activity, calculated as a summation of nitrates, nitration and all NOS activity levels, was demonstrated. The study, which was performed on the rat brain, was conducted under anoxic reoxygenation (180 min) conditions. The reduction was up to 25% dependent upon dosage of the study medication [152].

6. ATP, Aspirin and the Central Nervous System The total volume of adenosine 5’-triphosphat (ATP) varies according to the amount of aspirin present. On cultured cortical neurons exposed to oxygen-glucose deprivation, aspirin blocked the ATP decrease after stress or ischemia in about 40% of the cultures. These characteristics are specific for aspirin and salicylate acid, not for indomethacin. In rats during hypoxia [153], aspirin induced ATP increase and inhibits the negative effect of iNOS induction [154]. The rate of ATP was also increased in a focal cerebral ischemic-reperfusion model [155] varying up to 120%. The mechanism was assumed to be due to the inhibition glutamate recovery by restoration of the ATP level [154]. However, in the examination of the rat glioma growth characteristics in vitro studies, the synthesis of ATP was reduced up to 34% in the presence of aspirin and salicylic acid, while being associated with inhibition of malignant cell growths [156]. 7. Heat Shock Proteins (HSP) Heat shock proteins (HSP) are members of a family of proteins located in intracellular structures – cytoplasm, nucleus, mitochondria, or endoplasmic reticulum. Under normal physiological conditions, they act as protease of chaperons. Their concentrations increase three fold under ischemic and hypoxic conditions, in chemical and toxic injuries, with thermic stress and viral infections [157-159]. The HSP family is divided into six main groups according to their molecular weight – small HSP, HSP 40, HSP 60, HSP 70, HSP 90, and HSP 100. In brain ischemia and the presence of protein synthesis reduction, the HSP 70 is upregulated and the HSP 70 mRNA and protein transcription is induced. The location of increased HSP 70 is in the surrounding area of the post ischemic necrotic core (penumbra). It is hypothesized that the HSP 70 increase has a protective influence on ischemic tissue and facilitates the renaturation of protein after the onset of reperfusion [160-162]. Administration of HSP inducer demonstrated reduced infarct volume in the permanent MCA occlusion model [163]. 7a. Heat Shock Protein (HSP), Aspirin and the Central Nervous System Studies in cultured cells have shown that aspirin can induce HSP70 expression and increase its amounts. After injection of 100 mg/Kg of aspirin combined with heat treatment, Fawcett et al [164] showed on rats a three- to four-fold HSP 70 level in lungs, liver and kidney. Without the associated heat treatment, no alteration of HSP 70 was seen. However, in the examination of rat brain after induced focal cerebral ischemia, HSP 70 was found not to be different after treatment with aspirin than the vehicle control group in the cortex and striate regions. In this study, also found, was no difference in infarct volume among groups. The dosage of aspirin was 100 mg/Kg administered intraperitoneally, and the rats were sacrificed 24 hours after treatment [165].

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8. Metalloproteinases (MMP’s) The matrix metalloproteinases (MMP’s) are a group of enzymes which are responsible for the allostasis of the cell matrix composition and for the cleavage of various extracellular matrix components and extramatrix properties. The origin of these enzymes has already been detected in invertebrates. All MMP’s share a common structure. They contain a single peptide, a propeptide and a catalytic domain. They consist of two zinc ions needed for the catalytic process and at least one of calcium ion. The enzymes, which are active in a neutral milieu, are mostly synthesized as an intact latent enzyme and are activated by plasminogen activators and furin. There are more than 20 members in this enzyme group divided into subfamilies – the classical MMP’s, membrane-bound MMP’s, the ADMS (adamlysins) and the ADMATS (adamlysins and metalloproteinase with a thrombospandin motif). The MMP’s include collagenase, stromelysines, elastases and aggrecanases. A relationship has been found between some of the MMP’s and MAPK [166-168], as well as with pro TNF α [169]. MMP’s have a crucial role in the embryogenesis and normal healing [170], but also in pathological processes, such as arthritis, inflammatory and pulmonary diseases and cancer [171,172]. The effects are partially dependent upon the MMP’s role in angiogenesis. In the central nervous system, the role of MMP’s includes regulation of the blood brain barrier, neoplastic processes cerebrovascular diseases [173-177] and malignant brain tissues [178]. 8a. Metalloproteinase and Aspirin Evidence has indicated that there is an inhibitory effect of aspirin on metalloproteinase. However, the studies were performed only on a part of this protein family group and only on selective cell tissues. It was hypothesized that the effect is at least partially dependent upon the involvement of the NFKB, C JUN and ERK pathways. MMP 1 Aspirin inhibits human endothelial cells. The decrease of expression is selectively specific for aspirin and salicylate, not for other COX inhibitors and is dose dependent (1-5 mM). The effect is correlated to down regulation of oxidized low density lipoproteins and suppression of lectin-like receptors [179]. The dissociative effect between aspirin and selective COX-2 inhibitors was shown also in fibroblast-like synoviocytes [180]. MMP 2 An inhibitory effect was shown to be similar to aspirin in COX-2 inhibition of MMP 2 by its acting on fibronectin [181]. This inhibitory effect was found in lung cancer tissue [182], breast cancer [183] and other neoplastic tissues [184]. MMP 9 The inhibition of MMP 9 expression caused by aspirin was demonstrated in mice tumors [185] and in human breast tumors [183]. No studies have been performed to analyze the efficacy of aspirin on MMP in the central nervous system.

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9. Aspirin and Diseases of the Central Nervous System 9a. Stroke, Brain and Aspirin in Vivo Various studies have identified significantly better outcome of stroke after aspirin administration. Aspirin was acknowledged to be the protective agent in hypoxia as seen in vitro and in vivo studies. Riepe et al [153] showed post anoxic recovery of population spike amplitude and the ATP content after administration of 20 mg/Kg aspirin before pretreatment of white Wistar rats. The recovery rate was about 65%-87% in the population spike recovery and dependent upon time of treatment. Khayyam et al [186] found clear infarct volume reduction by administration of 15 mg/Kg aspirin 30 minutes and 2 hours prior to both common carotid and middle cerebral artery occlusion, but not in the earlier phase (8-24 hours) or after inducing stroke. In a study by De Cristobal et al [187], a reduction of volume was demonstrated in the rat after permanent focal cerebral ischemia with the administration of 30 mg/Kg, 2 hours prior to ligation. Berger et al [188] induced focal ischemia in male Wistar rats by intraperitoneal injection of aspirin. They found a decrease of more than 50% reduction after repeated injections of 40 mg/Kg, not by another lower dosage or single bolus treatment. 9b. Alzheimer’s Disease and Aspirin Studies, which analyzed the efficacy of COX-1 and COX-2 inhibitors to prevent Alzheimer’s disease, reported controversial conclusions. The Alzheimer’s Disease Corporation Study, a multi-center, randomized, placebo-controlled study with a duration of one year, found no effect in treatment with naproxen or fecoxib. Aspirin was not studied [189]. In a Swedish population based sample study, a cross sectional and longitudinal study, which included 702 participants, found an association between low prevalence of Alzheimer’s disease and high dosage use of aspirin [190]. A reduced occurrence of Alzheimer’s disease was found among aspirin users in The Cache Country Study of 201 patients [191] and in the Baltimore longitudinal study of aging with 1,686 participants. The effect was dependent upon duration of aspirin treatment (overall RR 0.4 more than two year duration and RR 0.74 less than two year duration)[192]. Contrary to this finding, in a twin cohort controlled study of 50 pairs and in a case-controlled study of 302 participants [193], only weak correlation toward efficacy was found [194]. In a cross-sectional retrospective study which analyzed the data of 2,708 patients, no effect of aspirin was found contrary to non aspirin NSAIDS [195]. A cohort Kungsholmen Project study of 1,301 patients, with a follow-up period of six years, found a relative risk (RR) of 1.8 in Alzheimer’s disease patients of the APO E α negative group using aspirin [196]. The positive results in some of these studies may be explained by the association between Alzheimer’s disease and aspirin’s neuroprotective effect which involves the amyloid beta, the most important morphological component of Alzheimer’s disease. Aspirin was shown to affect the inhibition of the amyloid beta aggregation [197], the complete inhibition of the fibrillogenesis of amyloid beta (1-42 peptide)[198] and the inhibition of the glial expression of apolipoprotein E, which plays a role in the Alzheimer’s disease metabolism [199]. 10. Parkinson’s Disease and Aspirin In an epidemiological prospective study from a cohort of 45,000 persons, the association between aspirin and parkinsonism was analyzed. Among 415 patients, there was a non significant low risk of parkinsonism with the aspirin users (RR 0.56) [200].

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Contrary to the epidemiological studies, data of in vitro and in vivo experimental studies are promising. The ability of aspirin to protect mesencephalic culture cells against 6 hydroxy dopamine and 1 methyl 4 phenylpyridium (MPP+) was shown to be selective for dopaminergic neurons [201]. The neuroprotective effect of aspirin and salycilate was shown in in vivo studies in the MPTP mice model. The dopamine depletion increased up to 38.6%, dependent upon the dosage of administered aspirin (50 to 100 mg/K)[202].

11. Amyotrophic Lateral Sclerosis (ALS) and Aspirin Barneoud et al [203] in one study examined the effect of soluble aspirin (lysine acetylsalicylate) on the animal model familial ALS mice with SOD (superoxide dismutase) mutation. A better functional outcome was demonstrated with early onset of aspirin treatment. This effect was not present with a later onset of treatment. There was no difference in the life expectancy of these groups.

Apoptosis 1. Overview of Apoptosis Apoptosis is an energy-dependent process of the active cell death, induced by the cell itself. It is characterized by specific morphological parameters which distinguish this cell pathway process from the more commonly known one of necrosis. As first described by Kerr et al [204], the parameters in apoptosis are shrinkage of the cell volume, chromatin margination of the nucleus and irregimentation of cells by persistence of the membrane integrity up to the last stage of cell disruption [205]. The apoptotic cell death pathway was first interpreted as being only a normal component of cell and tissue homeostasis [206]. However, an increase of apoptosis was observed in various pathological disorders. In the central nervous system, it was found to be present in neoplasm [207-208], epilepsy [209,210], ischemic brain damage [211,212], trauma [213] and neurodegenerative diseases [214]. Its role in neoplastic disease was described as being due to its activation after processes which are associated with tissue damage due to DNA mutation or chemotherapy [208]. The genetic regulation of apoptosis was first identified in the nomads of the genes Ced-3 and Ced-4 as a proapoptotic gene, and in Ced-9, as an inhibitor gene [215]. In the mammalian form, overexpression of Bcl-2 was found to have a significant anti-apoptotic effect by acting on the ICE and c-Myc proteins [216], both members of a proapoptotic gene group. Other family members of the Bcl gene group have properties involved in the apoptotic process, including Bax, Bad, Bak and Bcl-x5 as proapoptotic, and Bcl-x4, as inhibitor factors [217]. The hemostasis between the effects of the various members of this group is probably the reason for a stable hemostasis in healthy individuals. p53, a DNA transcription factor, is another protein involved by activation of the proapoptotic Bax gene, in the steady state of apoptosis. Apoptosis is activated in two parallel pathways – the intrinsic and the extrinsic pathway. The extrinsic pathway is advanced by the ligand of the TNF family. These components, along with TNF α1 receptor, lead to trimerization of the receptor and activation of the TNF- and Fas-associated death domains, and finally, targeting caspase-8 to recruit the apoptotic pathway. The intrinsic pathway is a mitochondrial target one which leads to release of cytochrome C and apoptosis induced factor (AIF). The AIF acts on the Apat-1, caspase-9, and finally, caspase-3, in order to enhance the apoptosis. The Bcl protein family has a mean role

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in the regulation of this pathway, including the Bcl-2 and Bcl-xl proteins which also have antiapoptotic properties that act by modulation of the VDAC channel, inhibition of the cytochrome release and translocation to the mitochondria during apoptosis. This mechanism is a caspase-dependent process. The intramembranous space’s acting Smac/Diablo protein functions as a regulator to the caspase activator. The proapoptotic Bcl family members, in the form of Bax, Bak and Bid, act by changing the configuration of the channels on the outer membrane of the mitochondria leading to release of cytochrome C and Smac proteins [218]. Apoptosis seems to have a key role in ischemic brain damage. In animal models, activation of the apoptotic process was identified 30 minutes after onset of stroke, increasing to its maximal point after 24-48 hours, with a persistence of the activity for up to four weeks [219]. In parts of the traumatic brain, apoptosis has appeared after eight hours, peaking in activity 24-48 hours, as well [220]. The increase in activity was associated with elevation of the Bax gene [220]. Evidence of the existence of apoptosis was found also in epilepsy [209-210], as well as in various neurodegenerative diseases of the central nervous system, including amyotrophic lateral sclerosis [221], Parkinson’s disease [222] and Alzheimer’s disease [214]. However, the meaning of the increase in apoptosis versus decrease of the process is not well understood in the central nervous system, and it seems that at least part of the process has a favorable effect, whereas other effects lead to transient or permanent undesirable outcomes.

2. Apoptosis and Glutamate Receptors Glutamate receptors are frequently involved in toxic mechanisms, leading to necrotic damage in the central nervous system, especially in brain ischemia, brain traumatic lesions and epilepsy. The excitatory neurotransmitter substrates are responsible for mediation of glutamate receptors which results in this irreversible damage. It is also known that, apart from the excitatory necrotic pathway, a second, late stage and non-necrotic neural cell death pathway is activated by glutamate. This traumatic process is based on activation of the calcium influx process by opening calcium channels, which are regulated by the glutamate receptors [213,223,224]. This type of neural cell death can be identified for an extended period of time (up to hours) after the induction of ischemic or anoxic brain damage. This late phase of injury induces changes compatible with apoptosis. The association between the glutamate receptor, glutamate excitatory cell toxicity and apoptosis was shown in cortical, hippocampal and cerebral neural cells. Glutamate was shown to trigger the internucleosomal DNA cleavage in cortical and hippocampal neural cells [225]. The morphological changes were characterized by internucleosomal DNA fragmentation, compatible with apoptotic changes. DNA fragmentation and chromatin margination was found also in cerebellar cells in a second phase of injury and after reactivation of damaged mitochondrial functions [226]. The type of damage (necrotic or apoptotic) was dependent upon the intensity of NMDA receptor stimulation. The less intense activation was responsible for the delayed apoptotic injury, whereas the high intense was responsible for the early necrotic damage. An association between apoptosis and other inducing factors was found also in oxidative stress-induced mediators. Adcock et al [227] identified morphological changes characteristic for apoptosis after activation of NFKB followed oxidative stress.

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3. The Proapoptotic Effect of Aspirin The proapoptotic effect of aspirin was identified in malignant colorectal, gastric and breast cancer and leukemia. Retrospective studies showed a decrease of colorectal cancer up to 50% in patients who were under permanent aspirin treatment. However, other studies, especially post hoc analysis of larger vascular studies, did not share this optimistic finding [228-231]. The specific role of aspirin in comparison to other Cox inhibitor substrates was hypothesized to be caused either by a covalent modifying action alternating the biological role of Cox-2 or the special effect of the more potent metabolite of salicylic acid, both acting through the enhanced effect of the metabolite 15 R hydroxyeicosatetraenoic acid, a tumor inhibitor [232]. These studies analyzed the mechanisms of the anti-tumoral effect of aspirin and demonstrated a proapoptotic effect on the early stage of carcinogenesis [233,234]. The exact location where aspirin affects the apoptotic pathway seems to be on its intrinsic and extrinsic pathway areas. Mitochondria play a critical role in the aspirin-induced apoptosis [235], lowering the mitochondrial permeability [236], for example. A direct effect by aspirin was shown on the actions of NFKB [237,238], interleukins [239,240], TNFα [204,241] and the caspase family members [242]. Aspirin also acts by modification of various antiapoptotic and proapoptotic protein members of the Bcl2 family group. In an analysis of the deubiquitinating enzymes, Brummelkamp, et al [243] found that the inhibition of the enzymes activates the transcription factor NFKB and leads to the antiapoptotic mechanism. An inhibition of NFKB reverses this effect by acting on the TNFα receptor and TNF receptor associated protein factor (TNF/RAF), as well as working downstream on the IKB/NFKB complex which seems to be the initiating factor. Although the aspirin-induced proapoptotic effect was mostly studied on cell culture specimens, the assumption is that this phenomenon has a very important role in controlling the growth of premalignant or malignant cells. 3a. Gastric Cancer Various reports have indicated that aspirin induced gastric mucosa cancer cell death. In an epidemiological study, Thun et al [231] showed a 40% reduction of death from gastric cancer among aspirin users. Studies performed on gastric epithelial cells showed that the antitumoral effect of aspirin is based on activation of the apoptotic pathway. Zhou et al [244] noted that the antiapoptotic induced by aspirin is brought about by the upregulation of Bax and Bak. Zhu et al [245] showed that the apoptosis is blocked by protein C activation through inhibition of c-Myc. In a study performed on the AGS line of gastric mucosa cells, Power et al [246] demonstrated early activation of caspase-8 and caspase-9, associated with activation of caspase-3, caspase-6 and caspase-7 and cleavage of the PARP protein. These findings stress the main role of the mitochondrial pathway in the aspirin induced apoptosis in gastric cells. On the same gastric cell line, this research group also demonstrated down regulation of cytosolic Bcl-2, translocation of Bax from cytosol into the mitochondria and Bid processing. The apoptosis induced by enhanced aspirin concentration was more dependent upon the caspase-9 activating pathway. This finding underscores not only the main role of the Bcl-2 protein family, but also the more important role it plays in the activation of the mitochondrial pathway. These results were also confirmed in other studies on other malignant gastric cell lines [247,248].

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3b. Colorectal Cancers The clinical role of aspirin in colorectal cancers was demonstrated by prospective and retrospective epidemiological studies and showed reduction of risk of fatal colon cancer with its use. Its preventative effect was shown to be enhanced when the subject was included in the case-controlled study of the Nottingham Faecal Occult Blood Screening Programme [249]. A randomized trial of aspirin to prevent colorectal adenoma found that 81 mg of aspirin has a preventative effect in subjects at risk for developing colorectal cancer [250]. In animal models, aspirin’s protective effect was shown in colon cancer of the rat [251], and also in the mouse min/+ model [252]. In cell culture studies, Din et al [253,254] found various specific effects on colorectal cancer cell lines compared with breast and gynecologic cancers. They found concentrator dependent IKBα degradation, NFKB nuclear translocation and inducing of apoptosis in all colorectal cell lines compared with no effect in the non-colorectal ones. These effects are dependent upon the p53 protein and DNA repair status. This finding was not in agreement with previous studies assuming that the DNA mismatched repair protein is responsible for the aspirin induced proapoptotic effect in human colorectal cancer. The protective effect of aspirin against apoptosis through activation of the phosphatidylinostal 3 Kinase/AKT/p21 pathway [255] leads to the conclusion that aspirin may have a proapoptotic, but also a preventative effect, so that, therefore, aspirin may really have a regulatory effect on apoptosis. 3c. Endometrial and Human Cervical Cancers In one study performed on three different endometrial cancers, the antiapoptotic properties of aspirin, as well as other selective Cox-2 inhibitors, were analyzed. Aspirin was shown to trigger apoptosis by release of cytochrome C, activation of caspase-9, caspase-3 and cleavage of PARP. The Bcl-2 and Bcl-x1 were downregulated and Bax and Bcl-x5 upregulated. These findings were confirmed on three different endometrial cancer cell lines. In other studies, human cervical adenocarcinoma and aspirin was analyzed mostly on the HeLa cell line, and as has been proven elsewhere, its role in the inhibition of TNFα and IL-1induced NFKB was shown. This effect was found to be dose-dependent and based upon phosphorylation and degradation of IKBα, a protein belonging to the inhibitory protein group, acting upon the NFKB transcription factor [256]. Other studies have examined aspirin in combination therapy, and its subsequent effect on apoptosis. In this type of cell line, Kim et al [257] showed there was an enhanced aspirin antitumoral effect, which was mediated by Bcl-2 and caspase-3, when combined with radiation. 3d. Leukemia Analysis of the behavior of human leukemic T cells was mostly performed on the Jurkat cell line [258]. The phenomenon of aspirin-induced apoptosis was shown, at least partially, upon inhibition of the Mcl-1 protein activity. This is an early induction gene, identified in human myeloid leukemia. Other hypotheses tested on the same cell line, which caused the apoptosis, were not confirmed. 3e. Hepatoma In cultured hepatocytes, Bradham et al showed the mediation of the mitochondrial permeability transition (MPT) event in TNFα- and Fas-mediated apoptosis [259]. In cultured heptocyte cell line testing, aspirin was shown to activate the process of MPT leading to cell

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death [260]. The intensity of death was dependent upon aspirin concentration. It was hypothesized that this mechanism is responsible for Reye’s syndrome. Oh et al [236] had determined that aspirin induced MPT pathways promoted apoptotic, as well as necrotic cell death. The apoptosis was mediated by acceleration of caspase-3 activation.

3f. Breast Cancer Whereas, Cox inhibitors were shown to prevent breast cancer progression in an animal model [261-263], cell culture examination could find similar characteristic apoptosismediating factors also for aspirin [240,264]. However, evidence for the efficacy of aspirin as an antiapoptotic agent was found also in breast cancer. In MCF7 breast cancer cell culture, aspirin prevents the upregulation of survivin. Survivin is a family member of the IAP (inhibitor of apoptosis protein) and prevents apoptosis. This effect is nonspecific for aspirin, but more effective in comparison to selective Cox-2 inhibitors [265]. 4. Aspirin-Inducing Proapoptotic or Antiapoptotic of the Central Nervous System Analysis of the behavior of cerebellar granular cells to exposure of glutamate showed that, in addition to the induction of the necrotic mechanism, there was also a prominent activation of the apoptotic pathway. These results involving aspirin on brain NFKB play a crucial role in helping to understand the association between aspirin and brain neuroprotection. Pretreatment of the cell culture with aspirin induced inhibition of P50, a member of the NFK family group, with induction of apoptosis. The relation between glutamate and apoptosis was shown also for p53, p21 and MSH2, proteins which are not related to aspirin [266]. 4a. Malignant Brain Tumor Studies which analyzed the effect of aspirin on glioma cell lines demonstrated inhibition of cell growth. Aas et al [156] showed that aspirin inhibited the proliferation of cells in vitro and in vivo in rat glial cells. The antitumoral drug effect on glioma cells was demonstrated also in other studies [267]. The correlation between aspirin, reduction of cell proliferation and apoptosis was shown by Amin et al [268]. They analyzed the antiproliferation on gliobastoma cell lines T98G. Aspirin exhibited clear proapoptotic and antiproliferative effects. This was dependent upon dosage and time, reading the maximal intensity of inhibition after 24 hours. The dosage dependency was shown also on rats, impregnated with C6 glioma cells. With the administration of low dosage (12.5-25 mg/Kg per day), the tumor size decreased; whereas, high dosage (200-400 mg/Kg per day) initiated a paradoxical effect with increase of the tumor volume, cell proliferation and mitotic index [269]. 4b. Brain Ischemia The mechanism of activation of brain apoptosis after ischemia is not clear. Lee et al [270] studied the antiapoptotic effect of antiaggregation and antitoxic drugs in induced focal ischemia in rats. After administration of aspirin, no suppression of the DNA fragmentation phenomenon was seen in the surrounding cortical penumbra tissue. A similar observation was noticed after clopidogrel and other antioxidant agents. We examined the role of Annexin V in acute stroke [unpublished data]. Annexin V, an endogenous human protein, is attached to the membrane-bound phosphatidylserine. In apoptosis, there is a translocation of the phosphatidylserine from the inner to the outer leaflet of the cell membrane [271,272].

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Annexin V radiolabeled to Tc99m can be used in vitro and in vivo to detect apoptosis. We determined that radiolabeled Annexin V had the ability to concentrate in apoptotic cell death in humans with acute cerebral stroke. From a total of 12 patients, eight had a positive finding, and in five of these, there was a high concentration of Annexin V found not only in the infarct area, but in a distant one which included the contralateral hemisphere. There was a correlation between this finding and the usage of aspirin prior to the stroke. Although preliminary, these results may raise the hypothesis that aspirin may play a proapoptotic role in stroke. Also, as verification of this assumption, a new compound – nitric oxide aspirin (NCX 4016) (a nitric oxide releasing aspirin agent) – was shown to prevent apoptosis in the mouse model [273] and reduce brain damage in focal cerebral ischemia in the rat [165].

CONCLUSION 1. Promising Data The data concerning the neuroprotective effect of aspirin on the brain is very promising. It seems to be activated by the use of various pathways and target locations. The majority of emphasized the specificity of aspirin’s proneuroprotective effect in comparison to the other COX inhibitors. Additionally, the exact dose needed for good efficacy is not yet clear. According to various studies, efficacy was achieved by low up to high concentrations. The conclusion that aspirin has a neuroprotective effect is based upon epidemiological, in vitro and in vivo animal models, but not on human in vivo studies. For confirmation of this promising conclusion and to determine its implication on therapeutic management, human clinical studies must be performed.

2. Aspirin in Apoptosis of the Central Nervous System The overall effect of aspirin in the brain proapoptotic and antiapoptotic mechanisms is not yet clear. Nevertheless, studies have demonstrated pathways of action connecting aspirin and apoptotic cell death. Furthermore, it would seem that the proapoptotic characteristics of aspirin demonstrated in other tissues are relevant also for the brain. Therefore, this may result in the assumption that aspirin plays a role in protection against brain tumors. It may be hypothesized that Aspirin may have an additional neuroprotective effect against other diseases of the central nervous system. However, as the exact behavior of aspirin in inducing proapoptotic or antiapoptotic mechanisms is not yet entirely clear, more data is necessary in order to establish more definitive conclusions.

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[243] Brummelkamp TR, Njman SM, Dirac AM, Bernards R. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB. Nature. 2003;424(6950):797-801. [244] Zhou XM, Wong BC, Fan XM, Zhang HB, Lin MC, Kung HF, et al. Non-steroidal antiinflammatory drugs induce apoptosis in gastric cancer cells through up-regulation of bax and bak. Carcinogenesis. 2001;22(9):1393-7. [245] Zhu GH, Wong BC, Eggo MC, Ching CK, Yuen ST, Chan EY, et al. Non-steroidal anti-inflammatory drug-induced apoptosis in gastric cancer cells is blocked by protein kinase C activation through inhibition of c-myc. Br J Cancer. 1999;79(3-4):393-400. [246] Power JJ, Dennis MS, Redlak MJ, Miller TA. Aspirin-induced mucosal cell death in human gastric cells: evidence supporting an apoptotic mechanism. Dig Dis Sci. 2004;49(9):1518-25. [247] Gu Q, Wang JD, Xia HH, Lin MC, He H, Zou B, et al. Activation of the caspase-8/Bid and Bax pathways in aspirin-induced apoptosis in gastric cancer. Carcinogenesis. 2005;26(3):541-6. [248] Tomisato W, Tsutsumi S, Rukutan K, Tsuchiya T, Mizushima T. NSAIDs induce both necrosis and apoptosis in guinea pig gastric mucosal cells in primary culture. Am J Physiol Gastrointest Liver Physiol. 2001;281(4):G1092-100. [249] Baron JA, Cole BF, Sandler RS, Haile RW, Ahnen D, Bresalier R, et al. A randomized trial of aspirin to prevent colorectal adenomas. N Engl J Med. 2003;348(10):891-9. [250] Little J, Logan RF, Hawtin PG, Hardcastle JD, Turner ID. Colorectal adenomas and diet: a case-control study of subjects participating in the Nottingham faecal occult blood screening programme. Br J Cancer. 1993;67(1):177-84. [251] Barnes CJ, Cameron IL, Hardman WE, Lee M. Non-steroidal anti-inflammatory drug effect on crypt cell proliferation and apoptosis during initiation of rat colon carcinogenesis. Br J Cancer. 1998;77(4):573-80. [252] Mahmoud NN, Dannenberg AJ, Mestre J, Bilinski RT, Churchill MR, Martucci C, et al. Aspirin prevents tumors in a murine model of familial adenomatous polyposis. Surgery. 1998;124(2):225-31. [253] Din FV, Dunlop MG, Stark LA. Evidence for colorectal cancer cell specificity of aspirin effects on NF kappa B signaling and apoptosis. Br J Cancer. 2004;91(2):381-8. [254] Din FV, Stark LA, Dunlop MG. Aspirin-induced nuclear translocation of NFkappaB and apoptosis in colorectal cancer is independent of p53 status and DNA mismatch repair proficiency. Br J Cancer. 2005;92(6):1137-43. [255] Ricchi P, Palma AD, Matola TD, Apicella A, Fortunato R, Zarrilli R, et al. Aspirin protects Caco-2 cells from apoptosis after serum deprivation through the activation of phosphatidylinositol 3-kinase/AKT/p21Cip/WAF1 pathway. Mol Pharmacol. 2003;64(2):407-14. [256] Kutuk O, Basaga H. Aspirin inhibits TNFalpha- and IL-1-induced NF-kappaB activation and sensitizes HeLa cells to apoptosis. Cytokine. 2004;25(5):229-37. [257] Kim KY, Soel JY, Jeon GA, Nam MJ. The combined treatment of aspirin and radiation induces apoptosis by the regulation of bcl-2 and caspase-3 in human cervical cancer cells. Cancer Lett. 2003;189(2):157-66. [258] Iglesias-Serret D, Pique M, Gil J, Pons G, Lopez JM. Transcriptional and translational control of Mcl-1 during apoptosis. Arch Biochem Biophys. 2003;417(2):141-52.

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In: Brain Mapping and Diseases Ed: Diane E. Spinelle

ISBN: 978-1-61122-065-0 © 2011 Nova Science Publishers, Inc.

Chapter 13

REGIONAL DIFFERENCES IN NEONATAL SLEEP ELECTROENCEPHALOGRAM Karel Paul1,*, Vladimír Krajča2, Zdeněk Roth3, Jan Melichar1 and Svojmil Petránek2 1

2

Institute for the Care of Mother and Child, Prague, Czech Republic Faculty Hospital Bulovka, Department of Neurology, Prague, Czech Republic 3 National Institute of Public Health, Prague, Czech Republic

ABSTRACT Background and purpose: While EEG features of the maturation level and behavioral states are visually well distinguishable in fullterm newborns, the topographic differentiation of the EEG activity is mostly unclear in this age. The aim of the study was to find out wether the applied method of automatic analysis is capable of descerning topographic particulaities of the neonatal EEG. A quantitative description of the EEG signal can contribute to objective assessment of the functional condition of a neonatal brain and to rafinement of diagnostics of cerebral dysfunctions manifesting itself as “dysrhytmia”, “dysmaturity” or “disorganization”. Subjects and methods: We examined polygraphically 21 healthy, full-term newborns during sleep. From each EEG record, two five-minute samples were subject to off-line analysis and were described by 13 variables: spectral measures and features describing shape and variability of the signal. The data from individual infants were averaged and the number of variables was reduced by factor analysis. Results: All factors identified by factor analysis were statistically significantly influenced by the location of derivation. A large number of statistically significant differences was also found when comparing the data describing the activities from different regions of the same hemisphere. The data from the posterior-medial regions differed significantly from the other studied regions: They exhibited higher values of spectral features and notably higher variability. When comparing data from homotopic regions of the opposite hemispheres, we only established significant differences between *

Karel Paul; Institute for the Care of Mother and Child; 14 710 Prague 4; Czech Republic; Tel. : +420 296511498 Fax. : +420 241432572 e-mail: [email protected]

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the activities of the anterior-medial regions: The values of spectral features were higher on the right than on the left side. The activities from other homotopic regions did not differ significantly. Conclusion: The applied method of automatic analysis is capable of discerning differences in the sleep EEG activities from the individual regions of the neonatal brain. Significance: The capability of the used method to discriminate regional differences of the neonatal EEG represents a promise for their application in clinical practice. Keywords: Full-term newborn; EEG; Regional differences; Automatic analysis.

INTRODUCTION When analyzed visually, the EEG activity of sleeping full-term newborns at the first glance appears topographically non-differentiated for the most part. The EEG atlases dealing with the earliest age either do not mention the regional differences in the EEG signal in fullterm sleeping newborns at all [1,2], or the EEG activity of these infants is being described as ‘uniformly distributed’ [3]. The reason for this is probably the fact that the human eye will not discern differences in the activities from the individual cranial regions. However, the application of computing technology has proven that the electroencephalogram of a full-term newborn is in fact topographically differentiated. The regional differences in the values of spectral energies were described [4-8]. Intrahemispheric and interhemispheric coherence of the EEG activity had been studied [8-14]. Automatic brain mapping was applied to the neonatal EEG [15,16]. Topographic interdependencies of the neonatal EEG have been examined by the means of non-linear methods [17-20]. In the present study, we have applied a multi-channel automatic method based on adaptive segmentation [21] in order to describe the EEG activity from the specific regions of the neonatal brain. This method evaluates not only spectral measures but also additional features as amplitude level, shape and variance of the signal, in which it comes close to visual analysis. The objective of the study is to verify whether the applied method is capable of discerning the differences in the EEG activities of the specific brain regions. It is possible to suppose that if the used method is able to discerne physiological regional EEG differences it will be possible to use the method in a detection and objective description of topographic deviations in patients with a cerebral pathology.

SUBJECTS We included 21 healthy full-term newborns in the study. They were born in the 39th to the 40 week of gestation, the Apgar score was >7 in the first minute and >8 in the fifth minute, and their birthweights ranged from 3010 to 3950g. The infants were examined in the 4th and 10th day of their life. Parents of the infants were informed of the methods and purposes of the examination and gave their consent. The project was approved by the institute’s ethical commission. th

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METHODS The examinations were carried out in an EEG laboratory in standardized conditions after morning feeding and lasted 90-120 minutes. The examination room was noise-protected and background noise level did not exceed 45dB. The illumination level was reduced to a degree that would enable the observer to just perceive changes in infant’s behavior. Room temperature was in the 23-25°C range. Disturbing environmental stimuli were excluded. Infants were examined in a crib, placed in supine position. The EEG activity was recorded polygraphically from eight bipolar derivations, positioned under the system 10-20 (Fp1-C3, C3-O1, Fp1-T3, T3-O1, Fp2-C4, C4-O2, Fp2-T4, T4-O2); the reference derivation, linked ear electrodes; filter setting, 0,2 and 60Hz; sensitivity, 100μV per 10mm. The respiration (PNG), ECG, EOG, and EMG of chin muscles were also recorded. Electrode impedances were not higher than 5kOhm. The recording was performed using the Brain-Quick (Micromed) digital system with sampling frequency of 128Hz and the data were stored on CDs. An observer continuously recorded any change in infant’s behavior on the polygram. Two five-minute-samples free of artifacts (segments contaminated by artifacts were eliminated by visual inspection) were selected from the EEG record of each infant. One sample was chosen from the middle part of quiet sleep, the other from the middle of the subsequent active sleep. In this study, we have defined mentioned sleep states according to the following criteria: Quiet sleep was defined as sleep with closed eyes, absence of eye movements, regular breathing, absence of body movements except for startles, and the typical EEG pattern ‘tracé alternant’. Active sleep was defined as a behavioral state in which the infant’s eyes were closed or nearly closed, eye movements were apparent, breathing was irregular, and mimic muscle movements, small movements of extremities and even large generalized movements occurred intermittently. The EEG showed the ‘activité moyenne‘ pattern [3]. Quantitative processing of EEG was performed off-line. Subject to analysis were data from the above-mentioned bipolar montage. A method based on multi-channel adaptive segmentation [21] was used. The method was selected for the following reasons: (a) The algorithm of the adaptive segmentation divides the EEG signal into quasi-stationary segments of variable length. The idea was that the feature extraction from such relatively homogeneous epochs would be substantially more effective than the feature extraction from fixed epochs. This holds especially true when analyzing the highly variable pattern as tracé alternant. (b) The division of the signal into quasi-stationary segments made it possible to evaluate length, number and proportional occurrence of these segments and thus to quantify the stability and variability of the signal. The method of applied automatic analysis was explained in detail in our previous paper [22]. Therefore it is described only briefly in this study. Using adaptive segmentation, the EEG signal from each derivation was divided into relatively homogeneous segments of variable length. The limits of the segments were in fact defined by the change in stationary character of the signal. The segments were distributed into three classes according to their maximum voltage. The segments whose amplitude didn’t exceed 50μV were placed into the 1st class, the 2nd class contained segments with voltage higher than 50μV and lower than 90μV, and the 3rd class was occupied by segments with the amplitude of 90μV and more. Examples of the application of adaptive segmentation and the distribution of segments into

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voltage classes are presented in figure 1. The activity of each segment was then described by ten features. The AV feature described the variance of the segment’s amplitude; Mm defined the value of the maximum amplitude ‘peak-to-peak’; the following five features provided information about the value of spectral amplitude in five frequency bands, δ1 in the 0.2-1.5Hz band, δ2 in the 1.6-3Hz band, θ1 in the 3.1-5Hz band, θ2 in the 5.1-8Hz band, α in the 8.115Hz band; feature D1 described the steepness of the curve; D2 described its sharpness; ØF informed about the average frequency of an activity in the segment. The data of the features describing each segment were then averaged in each class, and for each class three additional features were extracted: t% defines the time percentage of the specific class occurence; No gives the number of segments of a specific class; L provides the information about the average duration of the segments of a specific class in sec. In this manner the automatic analysis provided 312 values (8 derivations x 3 classes x 13 features) from the five-minutesample of the analyzed EEG signal. An example of the numeric output of the automatic analysis is presented in table 1.

Figure 1. An example of adaptive segmentation and distribution of segments into voltage classes. Polygraphic records of quiet sleep (QS) and active sleep (AS). Fp1-C3, C3-O1, Fp1-T3, T3-O1, Fp2-C4, C4-O2, Fp2-T4, T4-O2, eight EEG channels; PNG, respiration (movements of abdomen); ECG, electrocardiogram; EOG, electrooculogram; EMG, electromyogram of chin muscles; vertical lines, segments’ boundary; 1, 2, 3 , voltage classes.

STATISTICAL ANALYSIS The data collected from individual infants were averaged and the number of variables taken into account was reduced by means of factor analysis. Using the principal component analysis, three factors – Fc1, Fc2, Fc3 – were extracted, transformed by Varimax rotation with the Kaiser normalization and the respective factor scores were computed.

Table 1. An example of the automatic analysis output Fp1-C3 3: 2: 1:

AV 29.5 17.6 11.1

Mm 119.0 73.3 44.7

δ1 134.1 80.9 50.0

δ2 94.4 50.2 28.0

θ1 54.0 33.9 18.1

θ2 32.5 22.1 11.9

α 15.7 11.6 6.7

D1 25.3 19.7 12.4

D2 26.3 21.5 14.8

ØF 2.4 2.5 3.0

t% 21 31 48

No 27 33 37

L 2.4 2.8 3.9

Numerical data obtained by the analysis of a 5–minute-period of the EEG activity from the channel Fp1-C3 in quiet sleep. 1,2,3, voltage classes; AV,…,L, features.

Table 2. Features' representation, eigenvalues and percentage of variance of factors identified by factor analysis Factors Fc1 Fc2 Fc3

Representation of features AV,Mm,δ1,δ2,θ1,θ2,α,D1,D2, ØF No,t% L

Eigenvalues 7.38 1.49 1.31

% of variance 56.76 11.47 10.08

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Table 2 shows the list of factors identified by the factor analysis and the list of features represented by the specific factors; furthermore the table shows data about the eigenvalues of factors and the percentage of variance explained by these factors. In the first phase of the statistical analysis we tested (a) the effect of brain region, (the activity from each brain region is represented by a symbol of the individual bipolar derivation: Fp1-C3, …, T4-O2), (b) the effect of voltage class (low-, mid-, and high-voltage class), (c) the effect of sleep state (quiet and active sleep), and (d) the mutual statistical dependences of these effects on all three factors using the method of General Linear Model; the Wilks’ multivariate test (λ) evaluated by means of F-test served as criterion. Subsequently using the F-test, the effect of brain region upon each factor was tested separately, as well as the effect of voltage class, the effect of sleep state and mutual dependences of these effects. In the next phase of the statistical analysis, in order to determine the differences between the individual brain regions, we evaluated the vector of the 13 EEG features in each voltage class separately both in quiet and in active sleep. Using the General Lineal Model method, we employed the multidimensional analysis of variance, which further modifies the calculations of comparative tests with regard of mutual correlations between the 13 features, so that the final tests are not affected by these correlations. Following the initial parallel analysis of the 13 features, we compared in detail the effects of individual brain regions for each of the 13 features using the test according to Šidák. These tests for the individual features serve as an explanatory supplement to the basic multidimensional tests and they illustrate which brain areas and which features participate in the topographic differences, and the direction of these differences.

RESULTS The Effect of Brain Region By evaluating the effect of brain region we were testing the presence of topographic differentiation of EEG activity. The influence upon the factors identified by factor analysis are shown in table 3. It is apparent that both the entire set of factors – Fc1, Fc2, Fc3 – and even each individual factor are highly significantly influenced by the brain region. This means that both the factor Fc1 representing above all spectral features, and the Fc2 and Fc3 factors, which represent non-spectral features, are influenced. Table 3. The effects of brain area, voltage class and sleep stat upon the factors identified by factor analysis and the effects' interactions Effects Factors Fc1,Fc2, Fc3 Fc1 Fc2 Fc3

Brain area F p

Volt. class F p

Sleep state F p

Area x Class F p

Area x Sleep F p

4.76

< .001

317.00

< .001

298.66

< .001

6.86

< .001

2.27 = .001

3.60 5.19 6.33

< .001 < .001 < .001

610.09 258.64 412.28

< .001 < .001 < .001

46.21 436.92 452.85

< .001 < .001 < .001

2.51 13.16 4.72

= .002 = .037 < .001

3.06 = .003 1.24 = .274 2.50 = .015

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The Effect of Voltage Class and Dependence between the Effects of Brain Region and Voltage Class (Table 3) When analyzing the effect of voltage class we were testing whether the studied features of EEG signal differ significantly in individual voltage classes. We established that the effect of voltage class significantly influenced the entire set of factors as well as each factor in particular. We have also found a statistically significant dependence between the effect of brain region and the effect of voltage class for all three factors together and for each factor in particular, which points to the fact that the effect of brain region is different in each voltage class. The Effect of Sleep State and Dependence between the Effects of Brain Region and Sleep State (Table 3) The sleep state also significantly influenced all the analyzed factors together as well as each factor separately. We have also proven the presence of a significant dependence between the effect of brain region and the effect of sleep state, documenting that the brain region effect is influenced by the sleep state, for all the three studied factors as a whole and for factors Fc1 and Fc3. The Effect of Brain Region on the EEG Features The results of the comparison of measured values of the EEG features between the individual brain areas with respect to the voltage class and to the sleep state are depicted synoptically in figure 2. First we compared the data from the specific regions of the given hemisphere to one another, so that each region was compared to the other regions of the hemisphere (Fp1-C3 vs. C3-O1, …, C4-O2 vs. T4-O2; the activities from the studied regions are in this case represented by the symbols of the specific derivations). Then we compared the data from the homotopic regions of the two hemispheres to one another (Fp1-C3 vs. Fp2-C4, …, T3-O1 vs. T4-O2). In this way we have mutually compared the activity from the total of 12 pairs of regions altogether. In each pair of regions we were comparing 39 pairs of items (13 features x 3 voltage classes). In the end we have acquired 468 items (12 pairs of areas x 39 pairs of items) for each sleep state, which provide the information on the occurrence of statistically significant differences between the compared data, or lack thereof. While comparing data describing the activities from the individual regions of the same hemisphere, we have found a large number of significantly different values. (a) We have found most differences between the activities of the anterior and posterior medial regions (Fp1,2-C3,4 vs. C3,4-O1,2). It became evident that spectral features (AV, …, D2) and the feature No have significantly higher values in posterior regions. On the other hand the low voltage class values of the L and t% features were significantly higher in anterior regions. (b) The differences in activities of the anterior and posterior temporal regions (Fp1,2-T3,4 vs. T3,4 – O1,2) were distinguished by the following fact: While the majority of spectral features (AV, …θ2) of the high-voltage and mid-voltage class reached significantly higher values in the anterior regions, the values of the α, D1 and D2 features in the mid-voltage and lowvoltage classes were higher in the rear. The values of non-spectral features from both regions mostly did not differ. (c) We established sleep state dependent differences while comparing the values from the anterior-medial and anterior-temporal regions (Fp1,2-C3,4 vs. Fp1,2T3,4). In quiet sleep, the values of spectral features (δ2, …, α) were higher in the medial regions, on the contrary in active sleep spectral features presented higher values in lateral

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regions. (d) The differences between activities from the posterior-medial and posterior-lateral regions (C3,4-O1,2 vs. T3,4-O1,2) were noted for medially localized higher values of spectral features (AV, …, α). However in active sleep the α, D1, D2 features exhibited higher values laterally. The non-spectral features L and t% had in the low-voltage class significantly higher values temporally, while the values of the t% and No features in the high-voltage class were higher medially.

Figure 2. Differences of EEG features between compared brain areas. Results of Šidák’s test. QS, quiet sleep; AS, active sleep; AV, …, L, features; Fp1-C3 x C3-O1, …, T3-O1 x T4-O2, pairs of bipolar derivations representing the EEG activity of compared brain areas; 1, 2, 3, voltage classes; A, measured value is significantly higher (p