183 Advances in Anatomy Embryology and Cell Biology
Editors F. F. Beck, Melbourne · B. Christ, Freiburg F. Clascá, Madrid · D. E. Haines, Jackson H.-W. Korf, Frankfurt · W. Kummer, Giessen E. Marani, Leiden · R. Putz, München Y. Sano, Kyoto · T. H. Schiebler, Würzburg K. Zilles, Düsseldorf
K.N. Michailova · K.G. Usunoff
Serosal Membranes (Pleura, Pericardium, Peritoneum) Normal Structure, Development and Experimental Pathology
With 33 Figures
123
Krassimira N. Michailova. MD, PhD Department of Anatomy and Histology Medical University – Sofia 1. Sv. Georgi Sofiiski St. 1431 Sofia Bulgaria Kamen G. Usunoff, MD, DSc Department of Anatomy and Histology Medical University – Sofia 1. Sv. Georgi Sofiiski St. 1431 Sofia Bulgaria e-mail:
[email protected] and Institute of Physiology Bulgarian Academy of Sciences Acad. Georgi Bonchev St., Block 23 1113 Sofia Bulgaria e-mail:
[email protected] ISSN 0301-5556 ISBN-10 3-540-28044-8 Springer Berlin Heidelberg New York ISBN-13 978-3-540-28044-6 Springer Berlin Heidelberg New York
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List of Contents
1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8
Introduction and Review of the Literature . . . . . . . . . . . . . . . . . . General Remarks: Main Functions of the Serosal Membranes . . . . . Common Organization of the Pleura, Peritoneum and Pericardium. Mesothelial Cells. Surface Membrane Specializations . . . . . . . . . . Stomata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Milky Spots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transport Across the Serosal Membranes . . . . . . . . . . . . . . . . . . . Secretory Functions of the Mesothelium . . . . . . . . . . . . . . . . . . . Healing and Regeneration. Serosal Adhesions . . . . . . . . . . . . . . . Response to Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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3 5 7 9 11 13 15
2 2.1 2.1.1 2.1.2 2.2 2.2.1 2.2.2 2.2.3 2.2.4 2.2.4.1 2.2.4.2 2.2.4.3 2.2.4.4 2.2.5 2.2.6
Materials and Methods . . . . . . . . . . . . . . . . . . Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Human . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transmission and Scanning Electron Microscopy Staining with Ruthenium Red . . . . . . . . . . . . . . Experiments with Horseradish Peroxidase . . . . . Experimental Pathology . . . . . . . . . . . . . . . . . . Hemothorax . . . . . . . . . . . . . . . . . . . . . . . . . . Pneumonectomy . . . . . . . . . . . . . . . . . . . . . . . Peritonotis Caused by Pseudomonas aeruginosa . Application of Melted Paraffin . . . . . . . . . . . . . Injection with India Ink . . . . . . . . . . . . . . . . . . Data and Image Analysis . . . . . . . . . . . . . . . . .
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17 17 17 18 19 19 19 20 20 21 21 21 22 23 23
3 3.1 3.1.1 3.1.2
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Organization of the Human and Animal Serosal Membranes . . . . Surface Relief of the Pleura, Peritoneum and Pericardium . . . . . . . . . . . . Cell Organelles, Vesicular System and Intercellular Junctions of the Cubic and Flat Mesothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane Specializations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Basal Lamina, Elastic Membrane and Submesothelial Connective Tissue Layer . . . . . . . . . . . . . . . . . . . . . Stomata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Milky Spots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development of the Pleura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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43 47 52 56
3.1.3 3.1.4 3.1.5 3.1.6 3.2
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1 1
VI 3.2.1 3.2.2 3.3 3.4 3.4.1 3.4.2 3.4.3
List of Contents
3.4.4
Rat Prenatal and Postnatal Development . . . . . . . . . . . . . . . . . . . Human Prenatal Development . . . . . . . . . . . . . . . . . . . . . . . . . . Horseradish Peroxidase Transfer Across the Pleura and Peritoneum The Injured Serosal Membranes and Their Response . . . . . . . . . . Experimental Hemothorax . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Healing after Pneumonectomy . . . . . . . . . . . . . . . . . . . . . . . . . . Postinflammatory Changes of the Peritoneum with Special Reference to Stomata . . . . . . . . . . . . . . . . . . . . . . . . Pleura after Application of Melted Paraffin . . . . . . . . . . . . . . . . . .
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56 61 67 70 70 76
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78 88
4 4.1 4.1.1 4.1.2 4.1.3 4.1.4 4.1.5 4.1.6 4.1.7 4.1.8 4.2 4.3 4.3.1 4.3.2 4.4 4.4.1 4.4.2
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Normal Structures of the Serosal Membranes . . . . . . . . . . . . . . . . . . . . . General Organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cubic and Flat Mesothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Membrane Specializations and Lammelar Formations . . . . . . . . . . . . . . . Mesothelial Basal Lamina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Elastic Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Submesothelial Connective Tissue Layer . . . . . . . . . . . . . . . . . . . . . . . . Stomata in Normal Conditions and Postinflammatory Changes . . . . . . . . Human and Animal Milky Spots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prenatal and Postnatal Development of the Pleura . . . . . . . . . . . . . . . . . Transport Across the Serosal Membranes . . . . . . . . . . . . . . . . . . . . . . . . General Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transport of Horseradish Peroxidase Through the Pleura and Peritoneum Injured Serosal Membranes and Their Recovery . . . . . . . . . . . . . . . . . . . Early Alterations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Late Alterations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
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92 92 92 93 95 98 99 100 102 105 107 110 110 113 114 114 115
Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Abbreviations
(The abbreviations apply to all figures.) BL ECM ED EH EM EP ER GW HRP LB LL MS PD PNT PP RR RP SEM SM TEM VP
Basal lamina Extracellular matrix Embryonic day Experimental hemothorax Elastic membrane Experimental peritonitis Endoplascmic reticulum Gestational week Horseradish peroxidase Lamellar body Lymphatic lacuna Milky spot Postnatal day Experimental pneumonectomy Parietal pleura Ruthenium red Reaction product Scanning electron microscopy Serosal membranes Transmission electron microscopy Visceral pleura
Introduction and Review of the Literature
1
1 Introduction and Review of the Literature 1.1 General Remarks: Main Functions of the Serosal Membranes The major (coelomic) cavities of the body are bounded by serosal membranes (SM), which consist of a layer of loose connective tissue, covered by a limiting mesothelium over a basal lamina (BL). The mesothelium provides a frictionless movement of the organs in the pleural, peritoneal and pericardial cavities. This lubrication is achieved by a small amount of fluid and a special adaptation of the mesothelial apical membrane that bears a significant number of microvilli, covered by a surface film of hyaluronic acid-rich glycoprotein (Roth 1973; Wang 1998). The glycocalix represents a 28–57-nm thick layer (Schwartz 1974). On the mesothelial surface there is a high density of anionic sites, and it is negatively charged (Leak 1986; Gotloib et al. 1988; Ohtsuka and Murakami 1994). Besides free movement, there are several other significant functions of the SM that are important in the maintenance of serosal homeostasis. These include transport of fluid and particulate material across the SM, regulation of leukocyte migration in response to inflammatory mediators, synthesis of pro-inflammatory cytokines, growth factors and extracellular matrix molecules (ECM) for serosal repair, control of coagulation and fibrinolysis, and antigen presentation. The SM promote the deposition of glycosaminoglycans and lubricants, protect tissue from abrasion and infection, provide the material for bioprostheses, and also play a significant role in tumor implantation and dissemination (Whitaker et al. 1982a; 1982b; Rennard et al. 1984; Li 1992; Simionescu et al. 1993; Wang 1998; Visser et al. 1998; Mutsaers 2002). The physiologic properties of the peritoneal mesothelium are well known: it has been used for a life-saving procedure—peritoneal dialysis—as well as intraperitoneal nutrition, chemotherapy, immunotherapy and the treatment of ascites (Eggermont and Sugarbaker 1991; Khoroshaev et al. 1991; Mahedero et al. 1992; Garosi and Di Paolo 2001b; Krediet et al. 2002; Antony 2003). There are four major serosal cavities: peritoneal, two pleural and pericardial. In the male there are also two small cavities covered with tunica vaginalis testis. All cavities are hermetically closed, except for the female peritoneal cavity, which has the two tiny openings of the uterine tubes. The serosal cavities have a parietal sheet that builds the wall of the cavity, and a visceral sheet that covers the organs. The largest and the most complicated is the peritoneal cavity. The total area of the human peritoneum is approximately 1.8 m2 which is almost equal to the body surface area of the skin (Wittmann et al. 1994). The peritoneal covering of the anterior abdominal wall and of the diaphragm is relatively simple, whilst the parietal peritoneum of the posterior abdominal wall and the lesser pelvis, as well as the visceral peritoneum, have extremely complicated contours (Williams et al. 1995). The pericardial and pleural cavities are far more simple (Holt 1970; Wang 1974; Lai-Fook and Kaplowitz 1985; Sahn 1988; Lee and Olak 1994; Rienmuller et al. 2004). The SM contain a small amount of hypooncotic fluid and its turnover
2
Introduction and Review of the Literature
has been studied mostly in the pleural space (Agostoni 1972; Miserocchi and Agostoni 1980; Miserocchi 1997; Agostoni and Zocchi 1998; Zocchi 2002; Lai-Fook 2004). There is one very strange exception. The elephant is the only mammal whose pleural space is obliterated by connective tissue (reviewed in West 2002). Interestingly, the fetal elephant has a normal pleural space that is obliterated late in gestation (Eales 1929). During human development, the intraembryonic mesoderm on each side of the neural groove differentiates into paraaxial, intermediate and lateral mesoderm. The lateral mesoderm is continuous with the extraembryonic mesoderm covering the yolk sac and amnion. At the end of the gestation week (GW) 3, small spaces appear in the lateral mesoderm that fuses, dividing the mesoderm into two layers: the intraembryonic somatic (parietal) layer and the intraembryonic splanchnic (visceral) layer. The somatic mesoderm and overlying embryonic ectoderm form the embryonic body wall (somatopleura), whereas the splanchnic mesoderm and embryonic endoderm form the embryonic gut wall (splanchnopleura). A continuous mesothelial membrane lines the margin of these two layers and borders the intraembryonic coelom. Between GW5 and GW7, the coelom is subdivided by a process of septation into a future pericardial cavity, two pleural cavities and a peritoneal cavity. The most important structure dividing the intraembryonic coelomic cavity is formed by the septum transversum, but it leaves large openings—the pericardioperitoneal canals. At GW6 the mesoderm of the body wall is divided into two components: the thoracic wall and the pleuropericardial membrane. Since the diaphragm is still incomplete, the pleural cavities communicate with the abdominal cavity. At GW7 the pleuroperitoneal fold fuses with the septum transversum and the pleural cavities, as well as peritoneal cavity, are definitely formed. The pleuropericardial membranes are closed by embryonic day (ED) 41, whereas the septum transversum is closed after ED52 (Hesseldahl and Larsen 1969; Sadler 1990; O’Rahilly and Müller 1992; Moore and Persaud 1993). According to Whitaker et al. (1982a), Bichat in 1827 observed for the first time that surface of the serosal cavities was covered by flattened cells, similar to those lining lymphatic vessels, and he classified them as part of the lymphatic system. Von Recklinghausen (1863) introducing the ‘en face’ method, and using silver nitrate as a stain, demonstrated for the first time the cell boundaries as a characteristic ‘black net’ with minute dark areas at the junction between two or more cells. Tourneux (1874) observed the division of the mesothelial cells and concluded that their main function was to provide a boundary surface. The embryological study of Minot (1890) allowed him to define the lining of the coelomic cavities as ‘mesothelium’, the name being derived from its embryological origin in the mesoderm and its morphological resemblance to epithelium. Therefore, His (1895) referred to serosal coverings as an ‘epithelial-like tissue of mesodermal origin’. The present opinion (Whitaker et al. 1992) is that the mesothelial cells are of a primitive mesodermal origin, but share characteristics of both epithelial and mesenchymal cells. The earliest light microscopic investigation of Kolossow (1893) was devoted to the ‘ciliae’ of the mesothelial surface, but he could not find the intercellular holes and
Common Organization of the Pleura, Peritoneum and Pericardium. Mesothelial Cells
3
suggested that the mesothelium was a syncitium. MacCallam (1903) stressed the importance of phagocytosis and intercellular passage of particulate matter across the mesothelium. Buxton and Torrey (1906) attributed removal of carbon and bacteria to lymph nodes and discussed that the structure of the peritoneal side of the diaphragm was different. New mesothelium was formed by submesothelial fibroblasts, declared Hertzler (1919) after observations of transformations of spindle-like cells. Lewis (1923) examined pericardial mesothelium in culture and described transition from mesenchymal-like to epithelial-like cells. Cunningham’s investigations (1926) cover the fields of the mesothelial regeneration, transport and reaction to injury. Thus, these earliest studies put several main directions, as ‘eternal’ problems of the SM, investigated to this day. 1.2 Common Organization of the Pleura, Peritoneum and Pericardium. Mesothelial Cells. Surface Membrane Specializations Some studies (Sahn 1988; Wiener-Kronish and Matthay 1988; Gotloib and Shostack 1992; Simionescu et al. 1993) indicate, that the SM are simple structures consisting of a monolayer mesothelium over BL and an uniform underlying connective tissue layer. On the other hand, the already classical and some recent morphologic studies (Miller 1947; Nagaishi 1972; Wang 1974; 1998; Obata 1978; Ishihara et al. 1980; Pinchon et al. 1980; Albertine et al. 1982; 1984; Mariassy and Wheeldon 1983; Vladutiu 1986; Knudsen 1991; Michailova 1995a; 1995b; 1996b; 1997a; 2001b; Wassilew et al. 1998; Michailova et al. 1999; 2004) demonstrate the SM as complex structures. These observations describe different types of mesothelial cells and submesothelial connective tissue as a complicated structure, built from certain layers with varieties in the two serosal sheets and in different SM. This composition suggests highly specialized capabilities. Morphological studies of the mesothelium of several mammalian species, including mouse, rat, hamster, rabbit, sheep, horse, cat, dog and humans (Odor 1954; Fukata 1963; Baradi and Hope 1964; Kluge and Hovig 1967; Tyler et al. 1971; Andrews and Porter 1973; Baradi and Rao 1976; Tsilibary and Wissig 1977; Bettendorf 1978; Madison et al. 1979; Ishihara et al. 1980; Albertine et al. 1982; 1984; Whitaker et al. 1982a; 1982b; 1982c; Gaudio et al. 1988; Michailova 1994; 1995a; 1997a; Wassilew et al. 1998; Michailova et al. 1999; 2004; Mutsaers 2002; 2004) have demonstrated with minor exceptions, that mammalian mesothelium is essentially similar, irrespective of species or anatomical site. The most common mesothelial cell is flat (squamous-like), predominantly elongated, approximately 25 µm in diameter, with the cytoplasm raised over a central oval nucleus. Organelles of these cells are located mainly centrally, close to the nucleus. The flat cells contain microfilaments, few mitochondria and poorly developed rough endoplasmic reticulum (ER) and Golgi apparatus. The high (small, cubic) mesothelial cells are more rare and are characteristic especially for the mediastinal pleura, the peritoneal side of the diaphragm, the parenchymal organs (liver, spleen), and the ‘milky spots’ (Nagaishi 1972; Barberini et al. 1977; Tsilibarry and
4
Introduction and Review of the Literature
Wissig 1977; Obata 1978; Mironov et al. 1979; Pinchon et al. 1980; Mariassy and Wheeldon 1983; Michailova et al. 1989; 1999; 2004; Michailova 1994; 1995; 1995a; 1995b; 1995c; 1996b; 2001e; 2004b; Wang 1998; Michailova and Usunoff 2004). The cubic cells have abundant mitochondria and rough ER, a well-developed Golgi apparatus, microtubules and a comparatively greater number of microfilaments, suggesting a more metabolically active state. Cells that are morphologically similar to the cubic mesothelial cells can also be identified after injury or stimulation of the SM (Whitaker and Papadimitriou 1985; Fotev et al. 1987; Isoda et al. 1987; Dobbie 1989; Michailova 1996c; 2001d; 2001e; 2004a; 2004b; Mutsaers et al. 2002). In the first electron microscopic study, Odor (1954) reported the mesothelial covering of the rat oviduct. She paid special attention to the form, distribution and internal structure of the microvilli. Stoebner et al. (1970) stated that the microvilli represent the most significant criterion for the cytologic recognition and the most characteristic obligatory feature of the mesothelial cell. A well developed microvillous border is described in many mammalian species, including rat, guinea pig, ground squirrel, mouse, dog, cat, hamster, rabbit, horse and human, as well in birds (Voth and Kohlhardt 1962; Stoebner et al. 1970; Tyler et al. 1971; Suzuki et al. 1973; Lang and Liebich 1976; Pinchon et al. 1980; Michailova and Vassilev 1988a; 1988b; 1990b; Michailova et al. 1989; Michailova 1995a; 1996b; 1997a; 1997b; Wassilew et al. 1998; Oliveira et al. 2003). The mesothelial microvilli project from the cell surface and are long, slender and less densely packed than the microvilli seen on the luminal side of the intestine and of the kidney tubules (Junqueira et al. 1987). They vary in length, shape, internal structure, and density (Vogel 1957; Fukata 1963; Staubesand et al. 1963; Baradi and Hope 1964; Baradi and Rao 1976; Fentie et al. 1986). The microvilli could increase the actual peritoneal surface area by a factor of 20 (Gotloib 1982; 1986). The fact that omental mesothelial cells can transiently increase their population of microvilli up to sevenfold suggests that under physiological conditions, their concentration in any given area could reflect functional adaptation rather than static structural variation (Wang 1974; Madison et al. 1979) as well as an alteration in the surface charge (Gotloib et al. 1988; Mutsaers et al. 1996). The mesothelial microvilli are rare objects of investigations in the early stage of the embryonic development, moreover—characterizing only the animal and the yolk sac (Tiedemann 1976)—whilst data on the late period are missing, and those on the early postnatal period are few (Emery 1970; Krause and Leeson 1975; Hislop et al. 1984; Scheuermann et al. 1988). Variations in the number of microvilli have been reported on mesothelial cells of different organs, between adjacent cells and on the surface of individual cells (Andrews and Porter 1973; Barberi et al. 1977; Leeson 1977; Tsilibary and Wissig 1977; Wang 1998; Michailova et al. 1999; 2004). The present opinion (Mutsaers 2002) is that the main function of microvilli is to trap proteins from the serosal fluid to help ensure mesothelial cell integrity. The lamellar bodies (LB), as fine, densely packed membrane storage of surfactant, were described for the first time in the lung pneumocytes type II relatively
Stomata
5
late (Stratton 1975; 1984; Otto-Verberne and Ten Have-Opbroek 1987; 1988). A new fixation method (Futaesaku et al. 1972), with a mixture of glutaraldehyde and tannic acid, showed stages of the secretion and hydratation and their expansion over the surface, as ‘a geometrically regular lattice structure called tubular myelin’ (Williams 1977). Next investigations demonstrated the extrapulmonary sites of LB secreting cells, as in other ‘sliding’ tissues, including pleura, peritoneum, pericardium, articular surface, gastrointestinal mucosa, skin, Eustachian tube, glandular epithelia and other organs (Hills et al. 1982; Ueda et al. 1986; Schmitz and Müller 1991; Dobbie et al. 1994a; Michailova 1996b; 2004a; 2004b; Bourbon and Chaillet-Heu 2001). Data from last 20 years about SM, and especially the peritoneal mesothelium, as main object of continuous ambulatory peritoneal dialysis, show it as a structure that releases lamellar material in amounts similar to those produced by the lung (Dobbie and Lloyd 1989; Dobbie 1990). In vitro, the mesothelial cells synthesize phosphatidylcholine, the major component of pulmonary surfactant and LB (Dobbie et al. 1988). By means of radiolabeling (Rooney et al. 1984) and immunostaining (Dobbie 1996) and after studies in culture (Hjelle et al. 1991) the stages of development (Hallmann et al. 1984), biosynthetic pathways (Rooney 1984) of the different components, and the importance of the macrophages (Miles et al. 1988) in the recycling of the LB were defined more precisely. Numerous biochemical investigations (Hawgood and Clements 1990; Mason et al. 1998) presented surfactant as a mixture of approximately 90% lipids and 10% protein complex of four known surfactant proteins: A (SP-A), SP-B, SP-C and SP-D with different properties of formation, stabilization, functions and homeostasis of the organized surfactant system (McCormack 1998; Pryhuber 1998). Lamellar bodies exhibited an ultrastructural homogeneity and an identical bilayered periodicity in covering cells in different sites (Dobbie 1996). These data raise a question about a common biological membrane system with considerable conservation during evolution and preserved single evolutionary origin (Bourbon and Chailley-Heu 2001). This surfactant system was immunomodulatory, formed part of the defense system, was semipermeabe, ensured fluid balanceand lubrication, and carried out many other functions (Hills 1992; Johansson and Curstedt 1997; Chen and Hills 2000). 1.3 Stomata Despite the research that started in the nineteenth century, the distribution and the fine structure of the characteristic phenomenon of the SM–stomata, remained obscure. The existence of openings between the mesothelial cells and their direct contact with the diaphragmatic lymphatic system has been the subject of considerable debate since von Recklinghausen (1863) demonstrated them for the first time. Several subsequent studies (reviewed in Wassilew et al. 1998; Michailova 2001e; Michailova et al. 1999; 2004) questioned the existence of stomata but Buxton and Torrey (1906), and especially Cunningham (1926) described the peritoneal side of the diaphragm as being different from the remaining peritoneum and permit-
6
Introduction and Review of the Literature
ting rapid transport of carbon, bacteria and chicken erythrocytes to and from the abdominal cavity, stressing the importance of the intercellular route. This early period was completed by the detailed descriptions of Allen (1936) of the gaps between three mesothelial cells and of communicating channels from the peritoneal cavity to the lymphatic system of the diaphragm. The first electron microscopic studies as well as later ones investigated samples from both diaphragm and other sources and they did confirm the existence of stomata (French et al. 1960; Voth and Kohlhardt 1962; Fukata 1963; Kluge and Hovig 1967; Fedorko and Hirsch 1971; Gaudio et al. 1988; Slater et al. 1989). The majority of observations suggested that the preferred places for stomata were restricted to the diaphragmatic peritoneum (Leak and Rahil 1978; Fukuo et al. 1990; Negrini et al. 1991; Ohtani et al. 1992; 1995; Nakatani et al. 1996; 1997; Wassilew et al. 1998; Michailova 2001e; Shinohara et al. 2003, to cite only a few). The ultrastructural investigations answered some of the questions about the route of cells, larger particles, certain tracers and fluids through stomata to the lymph nodes (Oya et al. 1993; Shinohara 1997; Azzali 1999). Some authors (Casley-Smith 1964; Wang 1974; Bettendorf 1979) described very small openings (nanometers in diameter) between junctions of two or three mesothelial cells. A second group of studies (Barberini et al. 1977; Leak and Rahil 1978; Li 1993; Wang et al. 1997) showed larger (micrometers in diameter) stomata, able to absorb larger particles, or cells. Several investigations demonstrated changes in the stomata and the underlying large lymphatic vessels–lymphatic lacunae (LL) after continued peritoneal dialysis, inoculation of pathogens, invasion of tumor cells, ascitic elimination and different experimental conditions (Dobbie et al. 1981; Jonecko 1990; Eggermont and Sugarbaker 1991; Khoroshaev et al. 1991; Li 1992; Mahon and Libshitz 1992; Mahedero et al. 1992; Marco et al. 1992; Oya et al. 1993; Uriarte et al. 1993; Michailova 2001d; 2001e). Others (Poggi et al. 1991; Nakatani et al. 1997; Shao et al. 1998; Ohtani et al. 2001) examined the process of sequential formation of stomata and the morphogenesis of the lymphatic vessels during the prenatal and postnatal periods. All authors pointed out that stomata appear late in ontogeny. Nakatani et al. (1996; 1997) found for the first time stomata in newborn rats, and their number increased exponentially until postnatal day (PD) 10. Also in rats, distinct lymphatics are noted in the subpleural space of the diaphragm periphery by ED16, and by ED19 lymphatics appear in the subperitoneal space of the diaphragm. However, it is only during the early postnatal days that endothelium projects ‘many bulges that subsequently become elongated and contacted with pores among mesothelial cells’, and direct connections of the LL to the peritoneal cavity are formed after birth (Nakatani et al. 1996; Shao et al. 1998; Ohtani et al. 2001). Data from the last 20 years has widened the extra-diaphragmatic localization of stomata: on the greater omentum (Mironov et al. 1979; Shimotsuma et al. 1993; Cui et al. 2002), parietal (costal, mediastinal and diaphragmatic) pleura (Staub et al. 1982; Mariassy and Wheeldon 1983; Masada et al. 1992; Li 1993; Ohtani et al. 1993; Lee and Olak 1994; Negrini et al. 1994; Michailova 1996b; Shinohara 1997; Wang et al. 1997; 1998; Miura et al. 2000; Michailova 2001d; Li and Li 2003), tunica vaginalis
Milky Spots
7
testis (Baradi and Rao 1980; Michailova 2001b), ligamentum falciforme hepatis (Tesch et al. 1990), mesenteric duplicatures (Ettarh and Carr 1996), liver (Wassilev et al. 1998), anterior abdominal wall (Michailova et al. 1999), and both layers of the ovarian bursa (Sui and Li 2001). The data on the presence of stomata in the pelvic peritoneum are few and are somewhat contradictory. Li and Yu (1991) declared that ‘stomata were not discovered in the pelvic and anterior wall peritoneum’, but later Li et al. (1997) reported the presence of stomata over the entire pelvic peritoneum of human fetuses and mature mice. Dobroszynska et al. (1999) examined the stomata distribution of the pig broad ligament, uterus, oviduct and paraovarian sac (Dobroszynska et al. 1998). Michailova et al. (2004), at variance with Li et al. (1997), found only occasional stomata in the pelvic peritoneum in untreated rats. Several investigations stressed the importance of stomata by the interconnections of different serosal cavities There is a pathway via the diaphragm from the peritoneal to the pleural cavity (Li and Jiang 1993; Abu-Hijleh et al. 1995; Ohtani and Ohtani 1997), as well as an anastomosis between the pericardial and pleural cavities (Nakatani et al. 1988; Matsuda et al. 1990; Takada et al. 1991; Eliskova et al. 1995). 1.4 Milky Spots In 1874 Ranvier described small whitish spots over the serosa of the greater omentum and nominated them ‘taches laiteuses’ (i.e., ‘milky spots’; MS). The human MS are larger (0.5–3.5 µm2 ), while in the small laboratory animals they measure 0.1–3.0 µm2 (Hamazaki 1925; reviewed in Lieberman-Meifert and White 1983). These tiny accumulations of lymphoid tissue surrounding small epiploic branches of the right and left gastroepiploic arteries, were soon recognized to have a protective role, and Morrison (1906) called the greater omentum the ‘policeman of the abdomen’. Seifert (1920) first differentiated the MS from the lymphatic nodes, considering the MS as a part of the reticuloendothelial system. Carr (1967) reasonably named the MS ‘a lymphoreticular organ’. The microvasculature of MS consists of a classic succession of arteriole, precapillary, capillary, postcapillary, collecting venule, and venule (Borisov 1964). Kanazawa et al. (1979) described four types of capillary formations within the MS. The capillaries of the MS are fenestrated (Hodel 1970; Takemori 1979a; Cranshaw and Leak 1990; Takemori et al. 1994). Borisov (1964) demonstrated in man MS lymphatic capillaries with blind beginnings. The mesothelial covering at the MS is disrupted (Felix 1961; Carr 1967; Hodel 1970; Mironov et al. 1979; Beelen et al. 1980; Shimotsuma et al. 1989; 1991; Cui et al. 2002; Michailova and Usunoff 2004). Cellular migration is facilitated by the lack of mesothelial BL (Felix 1961; Carr 1967; Beelen et al. 1980; Cranshaw and Leak 1990; Cui et al. 2002). The cellular composition of the MS was studied intensively (Seifert 1920; 1921; Felix 1961; Carr 1967; Beelen et al. 1980; Lieberman-Meifert and White 1983;
8
Introduction and Review of the Literature
Cranshaw and Leak 1990; Beelen 1991; Wijffels et al. 1992; Takemori et al. 1994; 1995; Krist et al. 1995; Lenzi et al. 1996; Cui et al. 2002; Michailova and Usunoff 2004; Yildirim et al. 2004). The ‘active’ MS contain numerous lymphoreticular cells (Lieberman-Meifert and White 1983). Most numerous are macrophages and lymphocytes, less numerous are macrophages with dendrites and plasmocytes. There are also mastocytes and lipocytes. Close to the capillaries there are numerous periadventitial cells; sometimes undifferentiated mesenchymal cells and fibroblasts are to be found (Felix 1961; Beelen et al. 1980; Shimotsuma et al. 1989; 1993; Krist et al. 1995). In infants with healthy peritoneum, the mean number of cells per MS is 570±33, of which 47.5%±7.5% are macrophages (Shimotsuma et al. 1989; 1991; 1993). According to Krist et al. (1995) the number of macrophages in the human omental MS is even higher—67.9%±9.4%. On the surface of MS, the mesothelial cells are often replaced by macrophages (Felix 1961; Carr 1967; Shimotsuma et al. 1988; 1989; 1993; Dux 1990; Cui et al. 2002; Michailova and Usunoff 2004). The macrophages orient toward the peritoneal cavity for trapping or permitting the entrance of foreign particles (Shimotsuma et al. 1993). Beelen et al. (1980) divide the MS macrophages into three types: exudate macrophages (monocytes), exudate-resident macrophages, and resident macrophages (more than 90% in normal steady state). Daems and collaborators presented a series of studies on the resting peritoneal macrophages (Daems and de Bakker 1982; de Water et al. 1984; de Bakker et al. 1985a; 1985b). According to Wijffels et al. (1992) the macrophage precursors are centrally localized inside the MS. In normal human MS, the second major cell component represents B lymphocytes (29.1%±5.2%) and T lymphocytes (11.7%±2.4%) (Shimotsuma et al. 1991; 1993). Krist et al. (1995) reported that B lymphocytes comprise 10.1%±3.4%, and T lymphocytes 10.2%±3.7% of the cells in the MS. After intraperitoneal injection of sheep erythrocytes the MS contain surface immunoglobulin-positive B lymphocytes in the periphery and T lymphocytes are to be found centrally (Dux et al. 1986). In normal conditions mast cells are relatively few—6.1% (Shimotsuma et al. 1993). As a rule, the MS are associated with adipose tissue (Seifert 1920; 1921; Hamazaki 1925; Hodel 1970; Young et al. 1975; 1977; Beelen et al. 1980; Lenzi et al. 1996; Zareie et al. 2001; Michailova and Usunoff 2004; Yildirim et al. 2004) and the adipocytes have delicate paracrine interaction with lymphocytes (Pond 2003). Omental MS in normal animals occasionally show neutrophil myelopoiesis (Takemori 1980). Clear indication of strong extramedullary hematopoiesis was demonstrated in New Zealand Black mice (Takemori et al. 1994). Similar results were reported in the activated omental MS of normal mice by Lenzi et al. (1996). The MS are innervated. Shimotsuma et al. (1993) commented that the release of neuropeptide Y from perivascular sympathetic fibers may influence the circulation in the MS. Krist et al. (1994) described dopamine-immunoreactive fibers with perivascular location in the MS of the human greater omentum. There are also extra-omental MS. Takemori (1979b) described MS on the parietal peritoneum over the pancreas in the mouse. More interesting are the so-called splenoportal MS in the fat bands along the splenic artery (Takemori et al. 1995).
Transport Across the Serosal Membranes
9
The presence of MS on the peritoneum of the excavatio rectouterina was mentioned recently by Fujiwara et al. (2002). We (Michailova and Usunoff 2004) described MSlike formations on the broad ligamentum of the uterus in the rat. Kampmeier (1928) encountered plaques of macrophages in the mediastinal pleura behind the heart of a human baby. These were called by other authors ‘Kampmeier’s foci’ (reviewed in Pereira et al. 1994). Aharinejad et al. (1990), Inoue and Otsuki (1992), Pereira and Grande (1992), Boutin et al. (1996) and Li and Li (2003) described the pleural (thoracic, costal, mediastinal) MS. MS facing the pericardial cavity were described by Fukuo et al. (1990), Nakatani et al. (1988) and Takada et al. (1991). The characteristic vascular network of the MS is differentiated at the mid-term of the intrauterine development (Borisov 1964). During the ontogenetic development of the MS, Seifert (1920) distinguished three types: ‘primary’, ‘passive’, and ‘active’ MS. The primary MS are to be found only in fetuses and newborns (Borisov 1964). The passive MS of the human fetus appear approximately at the sixth month of uterine life where the number of mesenchymal cells in the MS diminishes and the number of lipocytes increases (Seifert 1923; Maximow 1924). It was repeatedly suggested (Maximow 1924; Carr 1967; Liebermann-Meffert and White 1983) that the lipocytes in the MS can differentiate in active macrophages. Every irritation of the peritoneal cavity leads to reorganization of the MS, and they are transformed into ‘active’ MS (Beelen et al. 1980; Garosi and Di Paolo 2001a). Human newborn babies possess completely developed pleural MS (Kampmeier 1928; Aharinejad et al. 1990). Shimotsuma et al. (1989) found that in humans the number of MS per unit area is at its greatest in infancy and gradually decreases with age. Krist et al. (1997) found that small accumulations of cells with about 50% monocytes/macrophages were present in the MS at 20 weeks’ gestation. Starting at 29 weeks, vascularized clusters of cells were seen, and true MS were present at 35 weeks. Shimotsuma et al. (1994) examined the MS in the fetal lamb omentum under normal conditions and after intraperitoneal carbon injections in utero. Takemori and Ito (1979) described that MS on the dorsal layer of the omentum during development become significantly more numerous in female mice than in male mice. 1.5 Transport Across the Serosal Membranes As early as in the nineteenth century it was demonstrated in experimental studies that hypertonic solutions of salt, sugar and glycerine initially increase in volume, while isotonic fluids are absorbed (Wegner 1877). Starling (1895) established also that movement of isotonic solution across the peritoneal capillary walls depends on transcapillary pressure, which in turn is modulated by two opposing forces: capillary hydrostatic pressure directed out of the capillaries, and osmotic (oncotic) pressure of the plasma proteins directed into the capillaries. He demonstrated also that water-soluble dyes can be absorbed from the pleural space into the bloodstream but did not distinguish between absorption across the visceral pleura (VP) and parietal pleura (PP). Agostoni (1972) stated that pleural fluid tends to
10
Introduction and Review of the Literature
enter the pleural space from the systemic capillaries supplying PP and reabsorbed via pulmonary capillaries in the VP. In this main concept Agostoni and Zocchi (1998) include information on lymphatic drainage through the PP. The pleural fluid is largely drained via parietal lymphatics and very little fluid drains through the VP (Miserocci et al. 1982; 1993; Negrini et al. 1985). The hydrostatic and oncotic pressure of the pulmonary capillaries determines the rate of fluid absorption across small segments of the VP. Kinasewitz et al. (1983) conclude that the distribution of hydrostatic and oncotic pressure across the pulmonary capillary and serosal membrane of the VP dictated the rate of pleural fluid production, while neither the pulmonary lymphatics nor the interstitium of the lung contribute to VP fluid exchange. Negrini and co-workers (1985) describe substantial regional differences in the kinetics of fluid and regional protein absorption rates. Further, no fluid could be reabsorbed into the pulmonary capillaries, as much as they provide filtration into the subpleural interstitium (Negrini et al. 1992; Miserocchi et al. 1993). According to Zocchi (2002), liquid enters the pleural space through the PP down a net filtering pressure gradient, and liquid removal is provided by an absorptive pressure gradient through the VP, by lymphatic drainage through the stomata of the PP, and by cellular mechanisms. The concept that fluid flux across the SM is determined by the action of Starling forces was confirmed repeatedly (Agostoni 1972; Kinasewitz and Fishman 1981; Kinasewitz et al. 1983; Granger et al. 1984; Miserocchi et al. 1984a; 1984b; Negrini et al. 1985). More recently, Miserocchi (1997) insisted that the pleural fluid is produced at the PP and that reabsorption is accomplished by lymphatic vessels of the PP—assured by a porous flow model and by oligolamellar surfactant. The parietal layer of the peritoneum, a small fraction of the total peritoneal surface, receives its blood supply from the vessels of the abdominal wall, while the visceral peritoneum, the larger portion of the mesothelium, is supplied primarily by branches of superior mesenteric artery, a major division of the splanchnic circulation (Wittmann et al. 1994). The presence of a high density of anionic sites along the intercellular junctions of the adjacent mesothelial cells and underlying lymphatic endothelial cells may play a role in the rapid movement of small solutes and molecules into the lymphatic lumen (Leak 1986). Numerous ultrastructural studies, some of them with tracers, confirm the role of the SM in the transport of various fluids, particles and cells. Different transport mechanisms enhance the ability of certain substances to traverse the SM freely from and to the serosal cavities (Levine 1985; Payne et al. 1988; Michailova and Wassilew 1988; Mikhaylova and Vasilev 1988; Jonecko 1990; Konig et al. 1990; Lee and Olak 1994; Ohtani et al. 1995; Michailova and Takeva 1997). Other reports discuss only passive diffusion (Kim et al. 1979; Payne et al. 1988; Konig et al. 1990), effect of body movement (Lee and Olak 1994), differences in pressure in the vessels and in the cavities (Payne at al. 1988), main pleural and peritoneal ways via stomata (Ohtani et al. 1993; Abu-Hijleh et al. 1995; Lai-Fook 2004; see also Sect. 1.3), as well as the different mechanisms of the intercellular and transcellular mesothelial transport (Jonecko 1990). Studies with tracers have shown that particles and solutes can be
Secretory Functions of the Mesothelium
11
actively transported across the mesothelium by pinocytotic vesicles (Fukata 1963; Staubesand 1963; Baradi and Hope 1964; Fedorko and Hirsch 1971; Fedorko et al. 1971; Leak and Rahil 1978; Dobbie et al. 1981; Mikhaylova and Vasilev 1988; Zocchi 2002). When the extracellular fluid pressure is elevated, the mesothelial cells increase the number of plasmalemmal vesicles (Shumko et al. 1993). The cytochemical profile of the mesothelium and the submesothelial connective tissue cells shows them as metabolically active and with high enzyme content (Ramsey et al. 1970; Chalet et al. 1976; Raftery 1976; Efrati and Nir 1976; Whitaker et al. 1980b; 1982a; 1982c; Davila and Crouch 1993; Agostoni and Zocchi 1998). The SM actively transport fluids, particles and cells across the mesothelium (through pinocytic vesicles and intercellular junctions) and stomata, directing movement to and from the serosal cavities (reviewed by Mutsaers 2002). Tracer studies indicated also that transport occurs through the intercellular junctions and stomata (CasleySmith 1967; Cotran and Majno 1967; Cotran and Karnovsky 1968; Cotran and Nicca 1968; Kluge 1969; Fedorko and Hirsch 1971; Whitaker et al. 1980a; 1980b; Mikhaylova and Vasilev 1988; Konig et al. 1990; Agostoni and Zocchi 1998; Zocchi 2002). The resting mesothelium is mainly concerned with membrane transport, whilst cubic mesothelial cells have several functional activities (Ramsey et al. 1970; Raftery 1973a; 1973b; 1973c; 1973d; 1973e; 1976; Marsan and Cayphas 1974; Efrati and Nir 1976; Clausen et al. 1979; Whitaker et al. 1980a; 1980b; 1982a; Mutsaers 2002). Negrini et al. (1994) used the pore theory to describe the fluid and solute exchange of the PP. They modeled two pore populations with radii of 83–89 Å and 156–222 Å, while Zakaria and Rippe (1993) define a three-pore model of peritoneal permselectivity including: a transcellular ultra-small pore, small pore radius 47– 48 Å and a large pore pathway. Zocchi (2002) summarized recent experimental evidence, from in vivo and in vitro studies, and stated that the mesothelium is a less permeable barrier than previously believed, being provided with permeability characteristics similar to those of the microvascular endothelium. 1.6 Secretory Functions of the Mesothelium Large series of investigations demonstrated that the mesothelium synthesizes different substances, which are shown mostly in experiments with cell cultures (LaRocca and Rheinwald 1984; Rheinwald et al. 1984; Milligan et al. 1995; to cite only a few). The mesothelial cells are metabolitically active cells and can synthesize and secrete glycosoaminoglycans and lubricant surfactant, which provide a slippery, nonadhesive and protective surface to facilitate intracoelomic movement between parietal and visceral serosal sheets (Dobbie et al. 1988; Dobbie and Lloyd 1989; Dobbie 1990; Hills 1992;). The mesothelial cells can also secrete a diverse array of mediators in response to external signals and regulating an inflammatory response, recruiting cells into the serosal cavities and presenting antigen to T cells as well as intraserosal immunoglobulin synthesis (Mutsaers 2002; 2004). Pro-, anti- and immunomodulatory mediators include prostaglandins, prostacyclin, chemokines, nitric oxide, reactive nitrogen and oxygen species, anti-oxidant
12
Introduction and Review of the Literature
enzymes, cytokines, products of the coagulation cascade and adhesion molecules (Topley and Williams 1994; Visser et al. 1998; Bellingan et al. 2002; Antony 2003; see Sect. 1.8). The secretion of cytokines especially interleukin (IL)-8 may play a key role in the initiation and maintenance of the inflammatory reaction (Topley and Williams 1994; Visser et al. 1995). The results on the expression of adhesion molecules and fibronectin of an activated mesothelium suggest that they form a defensive sheet (Liang and Susaki 2000). The mesothelial cells play an active role in the serosal repair through the release of growth factors, ECM molecules and their proteases (Harvey and Amlot 1983; Rennard et al. 1984; Laurent et al. 1988; Owens and Grimes 1993; Owens and Milligan 1994; Owens et al. 1996; Yang et al. 1999; Ha et al. 2002; Lee et al. 2004). Cultured rat mesothelial cells produce ECM glycoproteins—fibronectin, laminin, collagens I, III and IV, and elastin (Rennard et al. 1984; Rheinwald et al. 1987; Friemann et al. 1993; Owens et al. 1996; Michailova 2001c; Lee et al. 2004). The pleural mesothelial cells can organize ECM ‘connective tissue macromolecules’ beneath mesothelial cells into thick collagen fibers, amorphous elastic fibers and BL-like structures, which resemble those same components in vivo (Rennard et al. 1984). Other groups have demonstrated increased production of the different types of collagens under hypoxic conditions and following incubation of cells with various cytokines and growth factors (Owens and Grimes 1993; Owens and Milligan 1994; Yang et al. 1999). The secretion of a mesothelial cell-derived fibroblast chemoattractant (fibronectin) may play a role in the response to injury and in the pathogenesis of the fibrosis (Kuwahara et al. 1991; 1994). Eid et al. (1994) report the ability of the epicardial mesothelial cells to synthesize and release endothelin and discuss its stimulation after application of angiotensin II. Mesothelial cells in vitro are capable of synthesizing a variety of a biglycan-like chondroitin/dermatan and several heparin sulfate proteoglycans as well as interstitial and basement membrane proteoglycans (Junqueira et al. 1987; Milligan et al. 1995). Normal mesothelium and cells of mesothelioma are unique in that they are derived from the mesoderm and coexpress the mesenchymal intermediate filaments (cytoskeletal proteins) vimentin and desmin, as well as low molecular weight cytokeratins, which characterize the epithelial cells (Wu et al. 1982; La Rocca and Rheinwald 1984; Galateau-Salle 1993). The mesothelial cells have the ability to change their phenotype in epithelial-to-mesenchymal transition (Stylianou et al. 1990; Foley Comer et al. 2002). Kupryjanczyk and Kaprinska (1998) discussed that desmin expression of the mesothelial cells discriminated them from other tissues except muscle cells. Roth (1973) first demonstrated that mesothelial cell secretes surface glycosoaminoglycans (mainly hyaluronan), present within intracytoplasmic vesicles. Hyaluronan is not derived from the circulation but is produced by the mesothelial cells (Arai et al. 1975a; 1975b). Cultured mesothelial cells also produce glycosoaminoglycans (Ohashi et al. 1988; Yung et al. 2000). Honda et al. (1986) demonstrated that the rabbit pericardium actively produces a high molecular-weight hyaluronic acid that contributes to the increased viscosity of
Healing and Regeneration. Serosal Adhesions
13
pericardial fluid. Activated cubic mesothelium produces increased quantities of hyaluronan (Satoh et al. 1987; Wang and Lai-Fook 1998). This hyaluronan production is upregulated following injury (Baumann et al. 1996; Yung et al. 2000). The hyaluronan is assembled in a pericellular matrix coat (Heldin and Pertoft 1993) and probably has protective effects against infections and cytotoxic agents. The secretion of glycosaminoglycans into serosal fluid may play an important role in preventing the dissemination and growth of tumors (Jones 2001). A large group of studies presents the factors which promote both the deposition and clearance of fibrin, as fibrinolytic substances, and which participate in preventing fibrosis and adhesion (Raftery 1981; Wu et al. 1982; Haidar et al. 1990; Davila and Crouch 1993; Baumann et al. 1996; Kramer et al. 2002). The fibrinolytic activity within normal SM is in the form of plasminogen activators located in the mesothelium, which convert plasminogen in the blood or in fibrinous exudates to plasmin, as enzyme lyses fibrin adhesions (Holtz 1984; Holmdahl et al. 1997; 1998). 1.7 Healing and Regeneration. Serosal Adhesions Data on the physiological renewal of the mesothelium and the submesothelial layer shows them as kinetic objects with regenerative possibilities (Whitaker et al. 1982a; 1982b; 1982c; Whitaker and Papadimitriou 1985). Studies by means of in vitro methods (different types of cultures) help us to understand its potential for growth and replication (Stylianou et al. 1990), and the relationship of mesothelial cells to other cells with mesenchymal origin (Kuwahara et al. 1991). Under normal conditions, the mesothelium is a slowly renewing tissue with 0.16%–0.5% of cells undergoing mitosis at any one time (Ivanova and Puzyrev 1976; Fotev et al. 1987; Mutsaers et al. 2000). The capacity of serosal renewal is of great importance as SM show significance in pathology. Response to inflammatory or other irritants, immunological reaction, ‘subclinical alterations’, different type of proliferations, hyperplasia, fibrosis, lesions, plaques, metaplastic and neoplastic changes, with special reference to ones of very aggressive tumors—mesotheliomas (Hillerdal 1983; Sahn and Antony 1984; Herbert 1986; Dardick et al. 1987; Corrin and Addis 1990; Eggermont and Sugarbaker 1991; Hott et al. 1992; Galateau-Salle 1993; Pittet et al. 1993; Adamson et al.1994; Peng et al. 1994; Owens and Milligan 1994; Jones 2001; Carbone et al. 2002; Mutsaer 2004), allow Barrett (1970) to define the SM and especially the pleura as ‘an anatomical luxury and a pathological hazard’. Undoubtedly, the greatest interest was caused by the heavy alterations of the SM following exposure to asbestos (Dodson and Ford 1985; Moalli et al. 1987; Adamson et al. 1994; Rutten et al. 1994; Cugel and Kamp 2004). Numerous authors repeatedly reported significant changes in the peritoneal membrane in chronic patients with peritoneal dialysis that develop from ‘simple sclerosis’ and adhesions to sclerosing peritonitis (Dobbie et al. 1981; 1990; 1994b; Gotloib et al. 1985; Dobbie 1989; Holmdahl et al. 1997; Garosi and Di Paolo 2001c; Krediet et al. 2002).
14
Introduction and Review of the Literature
The integrity of the mesothelial border protects the underlying structures, and ensures normal fibrinolysis. Hertzler (1919) was the first to observe that small and large peritoneal wounds healed in the same amount of time. He concluded that the mesothelium could not regenerate solely by proliferation and centripetal migration of cells at the wound edge, as occurs for the healing of epithelium. There is a general agreement (reviewed by Mutsaers 2002; Herrick and Mutsaers 2004) that the healing process begins within 24 h of injury with the appearance of a population of rounded cells, predominantly neutrophils and macrophages, on the wound surface. Mesothelial cells at the wound edge undergo cell division and the epithelial sheet temporarily transforms into spindle-shaped fibroblastic cells that migrate onto the denuded wound area (Whitaker and Papadimitriou 1985; Mutsaers et al. 2000). Proliferative factors and chemotactic factors (Warn et al. 2001; Mutsauers et al. 2002) apparently play a major role in stimulating the repair process. Irrespective of the size of the damaged wound area, type of trauma or animal species, serosal healing is complete within 7–10 days of injury when the wound area is covered by cells displaying all the characteristics of mesothelial cells (Raftery 1973a; 1973b; 1973c; 1973d; 1973e; Swanwick et al. 1973; Whitaker and Papadimitriou 1985; Wittmann et al. 1994; Michailova 1996c; 2001d; 2001e; 2004a; 2004b). It is unlikely that the processes of cell division and migration alone account for these similar healing times (Herrick and Mutsaers 2004), and numerous studies proposed additional sources for the regenerating mesothelial cells: macrophage transformation, exfoliation of mature or proliferating mesothelial cells from adjacent or opposing serosal surfaces, pre-existing free-floating serosal progenitor cells that implant on the wound and differentiate into mesothelial cells, subserosal mesenchymal precursors that convert into mesothelial cells and migrate to the wound surface, and bone marrow-derived circulating precursors (Raftery 1973a; 1973b; 1973c; 1973d; 1973e; Ryan et al. 1973; Whitaker and Papadimitriou 1985; Bolen et al. 1986; Fotev et al. 1987; Davila and Crouch 1993; Foley-Comer et al. 2002; Herrick and Mutsaers 2004). Cleaver et al. (1974) observed that the healing mesothelium was retarded following postoperative peritoneal lavage, possibly due to the removal of free-floating cells. A significantly higher number of viable free-floating mesothelial cells were recovered from experimental animals 2 days after injury compared with the control uninjured animals (Whitaker and Papadimitriou 1985; Fotev et al. 1987). FoleyComer et al. (2002) reported that the free-floating mesothelial cells are able to adhere to exposed and deposited ECM. These cells undergo cell division and integrate into the mesothelial layer. Other investigations (Raftery 1973d; 1973e; Bolen et al. 1986; 1987; Dobbie 1990; Davila and Crouch 1993; Pampinella et al. 1996; Yen et al. 1996) propose that the regenerating mesothelial cells are derived from multipotential subserosal mesenchymal cells which when appropriately stimulated, begin to differentiate into mesothelial cells while migrating to the injured surface. Raftery (1973e) described the involvement of a subserosal precursor cell in the repair of mesothelium. There are new findings (reviewed by Mutsaers et al. 2000; Herrick and Mutsaers 2004) that support the view that the mesothelial cells themselves may be multipotent and have the ability to differentiate into various different cell types.
Response to Inflammation
15
The fibrinolytic activity of the mesothelial cells is a key factor in the prevention and removal of fibrin deposits that form following mechanical injury, hemothorax, and infections of the SM. If the fibrinolytic capacity is insufficient and fibrin accumulation is not resolved, fibrous adhesions form between opposing serosal surfaces (Mutsaers 2002). There is a fine balance between fibrin deposition and breakdown in the serosal cavities, which if inappropriately regulated, might cause reduced fibrin clearance and result in adhesion formation (Holmdahl et al. 1997; 1998). Fibrin that is not resorbed becomes stabilized, infiltrated by fibroblasts, and ultimately organized into permanent adhesions (Duffy and diZerega 1994). The serosal adhesions have serious pathologic consequences, such as disturbed function of the lungs and heart, intestinal obstruction, infertility, and pain (reviewed in Duffy and diZerega 1994; Wittmann et al. 1994; Jones 2001). Traditionally, the adhesions have been thought to consist of nonfunctional scar tissue. However, recently Herrick and her colleagues changed the classical belief, showing that the adhesions have feature of a dynamic structure (Herrick et al. 2000). They found out that one-third of the adhesions contain smooth muscle cells as well as adipocytes lined by collagen fibers. The presence of nerve fibers was first described by Kligman et al. (1993) in pelvic peritoneal adhesions. Sulaiman et al. (2000) examined experimental adhesions between the cecum and anterior abdominal wall, and found out that nerve fibers grow into them from both sides. Further, these authors established that all human peritoneal adhesions contain nerve fibers that immunoreact for calcitonin gene-related peptide and substance P. 1.8 Response to Inflammation By serosal inflammation, the mesothelial cells secrete various mediators: prostaglandins and prostacyclin, chemokines (IL-8, MCP-1, RANTES, GRO-alpha, IP-10, SDF-1, Eotaxin), nitric oxide and reactive nitrogen and oxygen species, anti-oxidant enzymes, cytokines (IL-1, IL-6, IL-15 CSF-G, M, GM) and growth factors (TGF-β, PDGF, FGF, HB-EGF, VEGF, ET-1, HGF, KGF, PAF), ECM molecules (reviewed above), adhesion molecules (ICAM, VCAM, E- and N-cadherin), and products of the coagulation cascade (tissue factor, tPA, uPA, PAI) (see Topley and Williams 1994; Mutsaers 2002; 2004; Antony 2003, for painstaking reviews). After direct stimulation with staphylococci, the human mesothelial cells produce IL-8 (Visser et al. 1995). This response leads to the onset of serosal infections: a massive influx of neutrophils—the most important eliminators of bacteria—from the blood vessels (Light 1990; Brauner et al. 1993; Topley et al. 1996b). Visser et al. (1996) examined whether mesothelial cells can ingest and digest bacteria. Both the peritoneal macrophages and mesothelial cells derive mediators that are directly involved in controlling inflammation. It has been widely accepted that resident peritoneal macrophages form the first line of defense against peritoneal infection (Daems and de Bakker 1982; de Water et al. 1984; de Bakker et al. 1985a; 1985b), and interaction between them and
16
Introduction and Review of the Literature
mesothelial cells is pivotal to the activation and subsequent amplification to the peritoneum’s response to infection (Topley et al. 1996a). Knudsen et al. (2002) established that also by sterile peritonitis the local macrophages are important both for the accumulation of neutrophils in the inflamed peritoneum. The serosal inflammation is activated on the surface of the mesothelial cells with subsequent release of chemokines (Light 1990; Brauner et al. 1993; Topley et al. 1996b). Thus, secreting chemokines in a polarized manner, mesothelial cells promote directed transmesothelial migration of both neutrophils and monocytes (Mutsaers 2002). Also, Visser et al. (1998) investigated the ability of mesothelial cells to produce important chemokines: huGRO-alpha (attractant for neutrophils), MCP-1 and RANTES (monocyte attractants). The movement of leukocytes from the blood vessels to the inflammation site is facilitated by the expression of integrins and adhesion molecules. The neutrophils show minimal adherence to and migration across unactivated mesothelial monolayer, despite an extensive amount of ICAM-1 on the mesothelial membrane (Zeillemaker et al. 1996). Neutrophil migration not only requires ICAM-1 expression, but also activation and release of cytokines from the mesothelial cells. Interleukin-8 appears to be the major cytokine (Mutsaers 2002). ICAM-1 and VCAM-1 are expressed on the microvilli of the mesothelial cells (Liang and Sasaki 2000; Johkura et al. 2001). According to Mutsaers (2002), since the adhesion molecules are only expressed on microvilli, the leukocytes may not ‘crawl’ on the cell surface, but to and from microvilli. Mutsaers et al. (1996) established that the greatest concentration of microvilli on mouse testicular mesothelial cells occurred at intercellular junctions 6 days after injury, when regeneration of the mesothelium was almost complete and extravasal inflammatory cells were being cleared. Most probably, the leukocyte clearance from serosal cavities is via stomata through draining lymphatic vessels contrary to the influx from the blood vessels (Bellingan et al. 1996). The resident and inflammatory macrophages are cleared at different rates (van Furth 1992). Bellingan et al. (1996) discovered that the inflammatory macrophages normally emigrate rapidly from the peritoneal cavity during the resolution of inflammation, in contrast with resident macrophages that persist in the noninflammed peritoneum for weeks. Further, Bellingan et al. (2002) demonstrated that the macrophages adhere specifically to mesothelium overlying draining lymphatics; their emigration rate is regulated by macrophage activation and is controlled through specific adhesion molecule regulation of macrophage– mesothelium interactions. The role of the mesothelial cell in regulating clearance of leukocytes during resolution of inflammation is yet to be elucidated (Mutsaers 2002). Beelen et al. (1980) demonstrated that a single intraperitoneal injection of newborn calf serum in rats resulted in an increase in the number and size of MS, and an influx of leukocytes. The intraperitoneal administration of foreign bodies or substances is followed by an acute inflammatory reaction (Beelen et al. 1980; Liebermann-Meifert and White 1983; Cranshaw and Leak 1990). Under inflammatory conditions, leukocytes dramatically increase, lipocytes are strongly reduced,
Material
17
and the mesenchymal cells are transformed into active macrophages of the MS (Kremli and Mamontov 1990; Broche and Tellado 2001; Cui et al. 2002). Weinberg et al. (1992) found new, ‘specialized’ MS that are dedicated to active plasmacytogenesis and antibody secretion after application of Shistosoma mansoni. Lenzi et al. (1996) utilized the same experimental model and discussed pronounced lymphocytosis, plasmocytogenesis, and myelomonocytosis in activated MS. According to van Vugt et al. (1992), following intraperitoneal injection of Bacillus Calmette– Guerin (BCG) in rats, the number of dendritic cells increases. van Vugt et al. (1996) established that during the first 4 months after administration of BCG, the number and size of the MS increased enormously. Doherty et al. (1995) found out that the omentum was the only abdominal organ which showed an increase of blood flow and reported that the omental MS are the major route through which leukocytes migrate into the peritoneal cavity following administration of zymosan A.
2 Materials and Methods 2.1 Material 2.1.1 Animals Experiments were carried out on 50 Wistar rats (20 young adult males and 30 female, aged between 60 and 120 days), weighing about 200 g each. A rat is a standard laboratory animal that can be used as a convenient experimental model for investigating the mammalian SM to answer basic morphological questions. It was chosen as representative of mammals mainly because: (a) the lungs represent a typical example of the thin type of pleura; (b) the lack of detailed information about the pleura and pericardium as compared with the relatively complete characteristic of the peritoneal covering; (c) in contrast to the peritoneum, observations about the pleura may be interpreted as a more suitable model as the pleural cavity has a simple relief with only one visceral covering over the lung; (d) morphologic changes have a diffuse character over organs of the entire thoracic half, which is close to the clinic practice; (e) clear definition and easy accessibility for experimental studies. For comparison observation between the pleural visceral and parietal sheets the rat and those of different mammalian species in separate experiments were used: 20 young adult mongrel domestic cats (Felis catus) of various ages, of both sexes, random breed, 1.5–3 kg body weight; 12 guinea pigs—Cavia cobaya, aged 2–24 months, weighing 250–400 g; 10 Chinchilla rabbits; 15 ground squirrels—Citellus citellus L. aged 2–24 months, weighing about 250 g each (material was taken in December, during hibernation and in April, during the active period); and six mice, about 2 months old, weighing about 25 g. A lack of any comparative investigation between the different types of SM (pleura, peritoneum, pericardium and tunica
18
Materials and Methods
vaginalis testis) in the rat, cat, and human were addressed also. The investigation involved the serosal covering of the ovaries of newborn female guinea pigs (n = 15). The structure and arrangement of the ovary surface epithelium represents a suitable model of a classic multilayered organized mesothelium. The animals were deeply anesthetized ether by inhalation or intraperitoneal injection of Thiopental (50 mg/kg) or Nembutal (60 mg/kg), respectively and killed by decapitation. A median thoracotomy was carried out and the pleural cavity was opened. The serosal covering with underlying tissue of the heart, lung (apical and basal zones of the VP), and mediastinal, diaphragmatic and thoracic (intercostal zones and over ribs) PP were obtained. After median laparotomy the abdominal cavity was opened and the peritoneum removed from different parts of the diaphragm, liver, spleen, stomach, intestines, rectum, greater omentum, mesenteric duplicatures, abdominal wall, urinary bladder, uterus, uterine tube, broad ligament, surface epithelium of the ovary, testis, transitional areas between the broad ligament and the ovary, uterus, and the end of the uterine tube. All specimens were promptly removed, divided in two and trimmed into blocks of 3 × 1 × 1 mm for transmission electron microscopy (TEM), and 5 × 3 × 2 mm for scanning electron microscopy (SEM). The group of animal embryos comprised 35 pregnant Wistar rats aged between the ED13 and birth, 20 newborn rats, and a postnatal group of 25 animals. Embryos were examined at ED13–15, 14–21, 15–24, 16–26, 17–21, 18–20, 19–23, 21–22, and 21–24. The postnatal group was examined at PD 1, 5, 15, 30, and 45, each group consisting of five rats. The material was taken from the apical and basal regions of the lung and from the costal and the diaphragmatic part of the PP. 2.1.2 Human Human tissue was obtained from different sections of the visceral, costal and diaphragmatic pleura after thoracotomy from 21 patients from both sexes (13 male, 8 female; 12 of them smokers), aged 27–56 years (50 years, nine cases). Fourteen patients had lung carcinoma (without previous chemo- or radiotherapy), and there were three patients with echinococcosis, two with lung abscesses, and two with pleural mesothelioma. The samples (mean = 5) were taken from the right or left lung (in nine cases from the lower and in five cases from the upper lobes; in seven cases with pulmonectomy from both lobes). Samples of peritoneum with underlying tissue from the different sectors of stomach, liver, gall bladder, small intestine, greater omentum, appendices epiploicae, and abdominal wall were obtained after laparotomy from 15 patients of both sexes (nine male, six female), age 35–67 years (60 years, four cases). Five patients had pancreatic, gastric and rectal carcinoma (without previous chemo- or radiotherapy), two liver echinococcosis, five cholelithiasis, two liver abscesses and one metastasis after lung carcinoma.
Methods
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Different zones of the visceral and parietal sheets of tunica vaginalis testis were obtain from seven patients (four had seminoma, without previous chemo- or radiotherapy and three had teratoma testis) aged 21–44 years. The group of human fetuses was composed of 28 stillborn fetuses without lung disease, and abortions aged 9–36 GW, of both sexes. They were divided into the following groups: four aged 9–10 GW, six aged 11–13 GW, five aged 14–20 GW, five aged 21–24 GW, five aged 25–32 GW and three aged 33–36 GW. Samples from different parts of the visceral pleura together with the underlying lung tissue (from both lungs) and the PP of the thoracic wall were taken. All patients and parents of the human fetuses gave consent. 2.2 Methods 2.2.1 Transmission and Scanning Electron Microscopy The blocks were immersed for 1 h in 1% or 2% glutaraldehyde in 0.05 m or 0.1 m cacodylate buffer (pH 7.2). Some animals (ten rats, three rabbits, four cats) underwent preliminary perfusion fixation with 2% glutaraldehyde in 0.1 m cacodylate buffer (pH 7.2–7.4). They were perfused at low pressure through the right ventricle with an opened left atrium in order to obtain better fixation of the lung. Following a brief rinse in the same buffer, the pieces were postfixed for 1 h in 1% OsO4 also in the same buffer (pH 7.2). Following dehydration in graded ethanol and acetone, the blocks were embedded in Durcupan ACM (Fluka). The regions of interest were identified on semi-thin sections (1 µm), stained with different solutions of 1% Toluidine blue, 1% Methylene blue, Azure II or a stock solution of 1% Toluidine blue and 1% pyronin and examined using a light microscope to select appropriate areas for electron microscopy. Ultrathin sections (50–70 nm) were prepared on an LKB ultramicrotome. Specimens were counterstained with 2.5% uranyl acetate for 20 min, and 2.6% lead citrate for 20–30 min. The ultrathin sections were examined in a Hitachi U-11A and a Hitachi U-500 electron microscopes, operating at 50 kV. The blocks for SEM were immersed in 2.5% glutaraldehyde in 0.1 m cacodylate buffer (pH 7.4) for 24–36 h at 40 °C, depending on their respective size. The samples were dehydrated in a graded series of ethanol, acetone and transferred into amyl acetate. The blocks were critical point-dried with CO2 . They were then attached to stubs and sputter-coated with gold–paladium with their peritoneal surface exposed in a ‘Hochvakuum-Bedampfungsanlage’ HBA 120/2. The examination was carried out with an Opton 109 and a Philips 505 scanning electron microscopes. 2.2.2 Staining with Ruthenium Red Nine rats were anesthetized and perfused with 0.2% Ruthenium red (RR) solution prepared in Karnovsky’s aldehyde fixative (1% formaldehyde and 3% glutaralde-
20
Materials and Methods
hyde) in 0.1 m cacodylate buffer (pH 7.4) with CaCl2 , as described by Luft (1964; 1971). The pieces of tissue were immediately excised and immersed in the same fixative solution at room temperature (22 °C) for approximately 20 h. They were then washed with 0.1 m cacodylate buffer (pH 7.4) for 1 h and postfixed for 2 h in 1.5% OsO4 (pH 7.4) containing 0.05% RR. 2.2.3 Experiments with Horseradish Peroxidase Twenty male Wistar rats 60–120 days old and four young cats, each 1 kg in weight were used in the experiment. The rats received 10 mg horseradish peroxidase (HRP) (Merck) in 1 ml 0.9% NaCl solution by intrapleural application (n = 5 after 5 min and n = 5 after 10 min) as well as intracardiac injection (n = 5 after 5 min and n = 5 after 10 min). The cats received HRP by intrapleural injection (two animals with 20 mg in 2 ml 0.9% NaCl) and by intraperitoneal injection (two animals with 40 mg in 4 ml 0.9% NaCl), and the samples were taken 10 min after administration. The blocks (rat and cat pleura of the lung and of the diaphragm, and cat peritoneum of the spleen, intestine, uterus, anterior abdominal wall and diaphragm) were processed by the method of Cotran and Karnovsky (1968) without washing before fixation. The same experimental group involved the material of the ovary surface epithelium of the female newborn guinea pigs. These animals received 3 ml HRP solution at the same concentration as the rats. The tracer was injected in the hypogastric part of the peritoneal cavity (n = 5) and in the abdominal section of the aorta after ligation under the diaphragm (n = 5). All animal pieces were fixed by immersion in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 m cacodylate buffer (pH 7.4) for 4 h at room temperature. The vibratome sections 80 µm thick were kept overnight in 0.1 m cacodylate buffer (pH 7.4) at 4 °C and then incubated for 30 min in the dark at room temperature in 3-3 diaminobenzidine tetrahydrochloride and fresh H4 O4 in Tris/maleate buffer (pH 7.6). The samples were then washed four times in distilled water (10 min for each washing). Postfixation was accomplished with 1% OsO4 in 0.1 m cacodylate buffer (pH 7.4) for 1 h at 4 °C. Specificity controls were given by intrapleural injection (n = 2) and by intracardiac injection (n = 2) with 1 ml 0.9% NaCl without the addition of peroxidase and were incubated in a solution without diaminobenzidine. The material was studied by TEM, as described above. 2.2.4 Experimental Pathology In all experiments we used several survival periods, starting with a very short one, in order to study acute alterations. For example, survival periods of 5 and 8 days were chosen for experimental peritonitis because mesothelization and restoration of the subserosal granulation tissue occur around day 5 after damage, while almost complete recovery takes place after days 8–10 (Whitaker and Papadimitriou 1985; Isoda et al. 1987; Wittman et al. 1999). Unoperated and sham-operated young adult
Methods
21
rats, irrespective of sex, represented the control groups. In sham-operated animals, thoracotomy or laparotomy was performed and (a) closed immediately without injuring the organs, or (b) 1 ml 0.9% NaCl was applied. The sham-operated animals survived the same experimental periods. Samples were taken from the same organs and areas as those being used in the corresponding experimental conditions. 2.2.4.1 Hemothorax Thirty Wistar rats with the same characteristics as the normal animals underwent experimental hemothorax (EH). The rats were deeply anesthetized with ether inhalation, or by means of intraperitoneal injection of Thiopental (40 mg/kg). One milliliter autologous blood was obtained by transthoracic heart puncture and was injected immediately through the left infrascapular region into the pleural cavity. Animals were divided into six groups (five animals in each survival period). Samples from different parts of the visceral and parietal pleura of the left thoracic region were taken after 6 h and 1, 3, 5, 8, and 15 days and studied by TEM and by SEM. The contralateral side was intact and served as an additional control. 2.2.4.2 Pneumonectomy Fifteen young adult Wistar rats (five in each experimental period), as well as three control animals from both sexes were used after experimental pneumonectomy (PNT). The rats were deeply anesthetized and a thoracotomy of the left side was carried out and the pleural cavity was opened. The left principal bronchus and neighboring vessels were ligated and interrupted. In most cases the whole left lung was removed, but in two cases only the basal lobes of the left lung were resected. The survival intervals were: 24 h, 5 and 8 days. The two rats with lobotomy were sacrificed after 5 days. The animals were reanesthetized and sacrificed by cutting the carotid arteries. Material was obtained from both apical and basal regions of the right lung–VP with the underlying lung parenchyma, from costal (muscular regions of the thoracic wall) and from diaphragmatic (central and peripheries– muscular portions) parts of the parietal sheet from same side and were studied by TEM, as described above. 2.2.4.3 Peritonotis Caused by Pseudomonas aeruginosa Wistar rats (n = 20) for experimental peritonitis (EP) were deeply anesthetized with ether inhalation. Each rat received 1 ml of a suspension containing 107 live Ps. aeruginosa bacteria mixed with 5% bovine albumin solution in Ringers’s lactate by intraperitoneal injection. Five animals survived for 24 h. The remaining rats survived for 5 days (n = 7) and 8 days (n = 8). After ether inhalation, the serosal membranes were fixed by intraperitoneal injection with a solution containing 2.5%
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Materials and Methods
Fig. 1 Casts of paraffin, hardened in the left side of the pleural cavity after application of melted paraffin
glutaraldehyde in 0.1 m sodium cacodylate (pH 7.4) and sacrificed by cutting the carotid arteries. After a median laparotomy the peritoneum and the underlying tissue was removed from central and peripheral (muscular) portion of the diaphragm, liver, spleen, stomach, intestines, urinary bladder, uterus and anterior abdominal wall. Some of the blocks of material from the diaphragm were placed in the same fixative for 24–40 h at 4 °C. The material was examined by TEM and by SEM. 2.2.4.4 Application of Melted Paraffin Under deep ether anesthesia, 20 Wistar rats were injected slowly through the left infrascapular region into the pleural cavity with 5 ml melted paraffin (melting point −55 °C). The rats were divided into five groups (four animals in each survival period) and were left to recover for 24 h, 3, 5, 8, and 15 days. No postoperative treatment was carried out. After anesthesia and wide median thoracotomy, samples from different parts of both sheets of the left thoracic half were taken. Casts of paraffin, hardened in the left side of the pleural cavity are shown in Fig. 1. Blocks involved the visceral pleura with underlying lung tissue and parietal pleura of the mediastinum, intercostal spaces, and diaphragm of the compressed side. The ultrathin sections were examined by TEM.
General Organization of the Human and Animal Serosal Membranes
23
2.2.5 Injection with India Ink Six male Chinchila rabbits were used in a study of configuration of the blood and lymphatic vessels in the diaphragm and MS zones of the greater omentum and mediastinal pleura. The rabbits used for this purpose were 3–4 months old and weighed about 1 kg. They were anesthetized with ether after subcutaneous injection of papaverine hydrochloride (0.02 mg/g body weight) to prevent vasoconstriction. The rabbits were perfused through the left ventricle with 1 L 0.9% NaCl, and when the blood had been flushed out, in five animals the perfusion continued with 250 ml India ink solution, and in one rabbit 100 ml India ink was injected into the most proximal portion of the abdominal aorta. The lung, costal pleura, mediastinal pleura, heart, greater omentum and diaphragm were fixed in 4% neutral formaldehyde. These serosal structures were treated with Perl’s reagent for 1 h and then counterstained with safranin. MS and other serosal zones were trimmed out under a dissecting microscope, dehydrated in an ascending series of ethanol, cleared in xylene and mounted in Canada balsam. 2.2.6 Data and Image Analysis Statistical analyses were performed on electron micrographs at magnification 6,100 × enlarged in the printing process to 14,000 ×. Linear measures were obtained with the electron graphic calculator (Ted Pella Inc. ultrastructural Size Calculator). Morphometric analysis was performed using a microanalysis system (Olympus CUE-2) with primary magnification 7,600 ×. Data of the entire drawings of the mesothelial cells were entered in the computer program (Olympus CUE-2), recorded automatically and calculated. Statistical differences were examined by Student’s t-test. All values were presented as means ± standard error of the mean (SEM).
3 Results 3.1 General Organization of the Human and Animal Serosal Membranes The vertical arrangement of the SM, visible by TEM, is of a boundary mesothelial layer resting upon the BL, supported by an underlying submesothelial connective tissue layer (Fig. 2a). These three main components differ in structure and thickness in the parietal and visceral sheets, in various organs, in some specialized regions, as well as in investigated mammals and in man. An elastic membrane (EM) beneath the BL of the VP and over the spleen is seen in all of the animals investigated and in man (Fig. 2b). According to their distribution at the serosal
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Results
Fig. 2a, b Common organization of PP and VP in adult rats. a Diaphragm. Flat mesothelial cell (star), thin BL (arrows), and large collagen bundles (asterisks) in the submesothelial connective tissue layer. (×7,000). b Lung. Cubic mesothelial cell (star) over a thick BL (small arrows) and EM (large arrows). Pneumocyte type II (PII) from a peripheral alveolus. (×8,500)
surface, the cell shape, the microvillous covering and the organelle apparatus the mesothelial cells are divided into main cell types—cubic (cuboidal, high) and flat (squamous) cells, as well as numerous intermediate (transitional) cell forms. 3.1.1 Surface Relief of the Pleura, Peritoneum and Pericardium By SEM and, to a lesser extent, by TEM it is possible to examine the surface of the pleura, peritoneum and pericardium ‘en face’ and to observe their horizontal differences in the mesothelial cell arrangement, in various regions, organs, and in both serosal sheets. The apical portions of the mesothelial cell show heterogeneous images of their surface, depending on the sites of observation. The central portion of the cubic cells involves the nucleus and protrudes into the serosal cavity. The entire cell surface is coated with densely distributed microvilli. Uniform cubic cells cover the basal portion of the lung and the spleen (Fig. 3a). The peripheral zones are short and neighboring cells are separated by deep invaginations in the intercellular zones. Usually, the apical surface of the cubic cells is smaller than the apical surface of the flat cells. The mean apical surface area of the cubic cells is 13.0–37.6 µm2 , and that of the flat cells is 43.2–182.76 µm2 in the adult Wistar rats.
General Organization of the Human and Animal Serosal Membranes
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Same cells differ in size and in contour, and have a nonuniform microvillous coat (Fig. 3b). Cubic cells form a smooth surface with rich microvillous covering and indistinguishable cell boundaries in the remaining VP and in some sectors on the organs, where they are the basic cell type (Fig. 3c). The cubic cells characterize the visceral sheets of the three serosal cavities as follows: surface of the lung (the basal regions are covered with more prominent cubic cells with richer microvillous coat, that the apical areas), the heart, and of some organs in the peritoneal cavity (spleen, rectum, uterus, urinary bladder, large areas of the liver’s surface, distal portion of the uterine tube). They form large clusters in the costo-diaphragmal angle; in the vicinity to stomata regions at the diaphragm, costal pleura (lower intercostal spaces), anterior abdominal wall, over MS of the greater omentum, and broad ligament of the uterus. The cubic cells on the remaining investigated regions occur as small groups or are single between the flat cells. The central portion of the second main type, the flat mesothelial cells, protrude slightly into the serosal cavity (Fig. 3d). The peripheral zones are wider and the cell margins appear relatively flattened. The elongated finger-like cytoplasmic processes connect neighboring cells over considerable distance, but the intercellular zones remain unclear. Single flat cells of the visceral peritoneal sheets have clear boundaries (Fig. 3e) and their microvillous coat is poorly developed. The flat cell type extends to a considerably larger part of the SM than the cubic cells: this is the basic cell type for the parietal sheets of the pleura, peritoneum and pericardium, for some organs (large portions of the intestines, remaining areas of the liver, parts of the stomach, the proximal portion of the uterine tube, testis, the greater omentum, the broad ligament, the mesenteric duplicatures) and are located as single cells in the remaining sites. The intermediate forms of mesothelial cells show an irregular arrangement when compared to the two main cell types. Their long diameters are in different directions and form protrusions with variable size, more often with unclear boundaries (Fig. 3f). They contain a nonuniform microvillous covering. The mesothelial covering of the stomach and a portion of the intestines contain numerous intermediate cell forms. For some organs there is a lack of correspondence between data obtained by TEM and SEM. The significant differences and repeated regularities of the relief are observed on both serosal sheets, the various organs and regions over them. The surface contour is more rough on the visceral than on the parietal sheets. The view of the Fig. 3a–f Two main mesothelial cell types (cubic and flat), demonstrated by SEM. a Lung. Uniform cubic cells with extremely rich microvillous covering and rather unclear boundaries. (×200). b Lung. Cubic cells of different sizes and forms with nonuniform microvillous covering. (×150).c Lung. On the right are cells with clear boundaries, and on the left is shown a smooth pleural surface. (×200). d Costal pleura. Flat cells with unclear borders and scattered microvilli. (×3,000). e Testis. Only few cells (arrows) protrude from the smooth surface. (×2,000). f Stomach. Intermediate forms of mesothelial cells. They are irregularly arranged, with unclear boundaries and nonuniform microvillous covering. (×1,250)
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Results
General Organization of the Human and Animal Serosal Membranes
27
lung, diaphragm, greater omentum, uterine tube, uterus, broad ligament and ovary show impressive features and are distinguished immediately from other regions of the SM. The parallel, wide and low-growing furrows and folds form a lager part of the lung’s apical regions (Fig. 4a). Irregular, shallow furrows, scattered between wide and rounded evaginations occupy the basal regions of VP (Fig. 4b). The mesothelial covering of the VP are composed predominantly of typical cubic cells (Fig. 4c). Multiangular cubic cells with more prominent central portions and deeper invaginations in the intercellular zones characterize the VP. The relief of the diaphragm is smooth in its central portion and parallel ridges are separated by shallow folds in the periphery (muscular part) of its pleural and peritoneal surfaces (Fig. 4d). Elongated or, more rarely, polygonal flat cells with poorly defined borders, cover both of its sides (Fig. 4e). The microvilli are sparsely distributed and predominate at the lateral cell borders and in the intercellular clefts. The flat cells are sharply demarcated from the adjacent cubic mesothelial cells, which lie in large clusters in the costo-diaphragmal angle, in the vicinity of the stomata, and over the LL in the diaphragmatic peripheries (Sect. 3.1.5). The clusters of cubic cells show more complicated contours and take larger zones over the peritoneal side of the diaphragm. The peritoneum of the greater omentum forms large spherical protrusions, separated by relatively uniform deep clefts, as seen by SEM (Fig. 5a). The convex surfaces are covered by cells with sparsely distributed and short microvilli. Wide longitudinal folds, separated by deep parallel furrows, are present over the entire length of the uterine tube (Fig. 5b). The proximal portion of the uterine tube is covered by elongated and less prominent flat mesothelial cells with short, sparsely distributed microvilli. A gradual change from these cells at the proximal part, to cubic cells at the distal part, characterizes the mesothelial covering of the uterine tube. The cubic cells show an abundance of microvilli and rounded apical evaginations. Often coagulated material is deposited as large patches over its distal portion. The cubic cells on the distal portion of the uterine tube gradually change their shape toward high prismatic cells, which form an extremely thick multi-layered covering at the end of the uterine tube. The surface of the uterus shows relatively smooth wide folds with slightly irregular contours, separated by extremely deep and narrow crypt-like clefts, also visible by SEM (Fig. 5c). The microvillous covering shows a moderate density. Some regions have only sparsely distributed short microvilli, while central protrusions of the mesothelial cells are Fig. 4a–e The relief of the VP and PP. a Rat VP. Broad ridges, limited by parallel shallow furrows. (×200). b Rat VP. More prominent, irregular ridges and deep furrows. (×170). c Rat VP. Clear intercellular boundaries surround multiangular mesothelial cells. (×1,800). (From Michailova et al. 1989, with permission of Elsevier). d Rabbit PP. A smooth mesothelial relief. Parallel rows of mesothelial cells. (×650). e Higher of the central area in d showing the elongated cells with relatively few microvilli. Their central, protruded and broad portions are adjacent to the thin, peripheral sectors of the neighboring cells. (×2,500)
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General Organization of the Human and Animal Serosal Membranes
29
almost bare. Simple changes (as compared with changes towards the ovary and at the end of the uterine tube) from flat cells to cubic cells characterize the mesothelium of the transitional area between the broad ligament and the uterus. Large ridges with a complicated relief, limited by deep furrows with a variable direction, length and width are demonstrated on the surface of the broad ligament of the uterus (Fig. 5d). The shallow furrows, predominantly perpendicular to the deep furrows, constitute a finer relief over the ridges. Some of the clefts show extremely unusual contours—they contain invaginations from the first to the third order, and are variable in size. The mesothelial covering is composed predominantly of flat cells. The microvillous covering is poorly developed. The ovary surface of the newborn rat and guinea pig shows single deep crypt-like invaginations and numerous serous villi-like or papillae-like protrusions (Fig. 5e). Densely arranged cubic cells are separated by apparent intercellular clefts and are covered by numerous short uniformly distributed microvilli and rounded evaginations. The transitional area over the mesovarium involves gradual changes from flat cells of the broad ligament towards predominantly high prismatic or rounded (with a larger diameter) cubic cells of the ovary surface. The peritoneum of the spleen shows a sparsely undulating surface, which is composed of densely arranged typical cubic cells. The central apical portions are polygonal or rounded, followed by short peripheral zones, with borders forming deep invaginations. The cubic cells are covered by numerous and uniformly distributed microvilli over the central protruded portions and over the intercellular clefts. The peritoneal covering of the stomach show parallel folds separated by furrows. In some instances the furrows are bridged by obliquely running folds. An elongated shape with finger-like, frequently bifurcated processes characterizes the stomach mesothelial cells. Although the apical surfaces of these cells are also projecting towards the peritoneal cavity, they do not entirely resemble the spleen cubic cells. The mesothelial covering of the stomach have only sparsely distributed microvilli in some regions, allowing an assessment of the cell boundaries. Most of the mesothelial cells are considered to be an intermediate cell type (Fig. 3f). Wide zones of the liver peritoneum are characterized by cubic covering, richly coated with microvilli. However the central protrusions are not as pronounced as in the spleen. Their margins appear relatively flattened, but an abundance of long microvilli make the intercellular clefts indistinct. At the same time, zones with Fig. 5a–e The relief of the visceral sheet of the peritoneum. a Rat. Greater omentum. The spherical protrusions, surrounded by deep clefts. (×300). b Rat. Uterine tube. Wide folds, separated by longitudinal furrows (arrows). (×500). c Rat. Uterus. Extremely deep cleft (arrows). (×8,000). (From Michailova et al. 2004, with permission of Elsevier). d Rat. Broad ligament. Very deep (asterisks) and smaller furrows (arrows) divide into folds of difference size and shape. (×250). e Guinea pig. Ovary. Cubic cells cover a papilla-like evagination (asterisk) and a deep crypt-like invagination (arrow). (×1,200). (From Michailova et al. 1991, with permission of Elsevier)
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General Organization of the Human and Animal Serosal Membranes
31
equal size show mesothelial cells with an elongated shape in the central portion, which protrude weakly toward the peritoneal cavity. They extend long processes with poorly developed microvilli. Vast regions of the mesothelial covering of the liver are composed of intermediate cells. The intestinal peritoneal covering is composed of parallel orientated mesothelial cells, separated by long and shallow furrows. In contrast to the gastric mesothelium, their finger-like processes are more elongated but less prominent. The furrows are filled with sparsely distributed and short microvilli. The central portions of the cellular surfaces are almost bare. The mesothelial cell population is composed predominantly of the intermediate cell forms, together with some flat cells. The surface of the rectum forms zones with wide folds, separated by shallow concavities, as well as other zones with an almost smooth relief. Some regions have a dense microvillous covering while the neighboring regions have only sparsely distributed microvilli. The majority of the cubic cells have frequently bifurcated processes, extending from the central, slightly bulged portions. The remaining cells occupy the invaginations of the submesothelial layer, and the peritoneal surface appears smooth. The surface of the urinary bladder has almost a smooth appearance, richly covered with microvilli. Uniform, oval shaped mesothelial cells are separated by fine and unclear intercellular invaginations. Most of the covering cubic cells occupy the invaginations of the submesothelial layer and the peritoneal surface looks smooth. The covering of the testis forms rare and fine wide folds or appears smooth. The central slightly protruded portions and extensive smooth peripheral zones form relatively flattened intercellular zones. The uniformly distributed, poor microvillous coat makes the intercellular clefts indistinct. Small zones of the testis show elongated protrusions of the central portions of the mesothelial cells and finger-like peripheries (Fig. 3e). A smooth surface (costal pleura over the ribs) or less numerous extremely wide furrows and parallel, long ridges characterize the intercostal portion of the costal pleura, the central part of the diaphragm and peritoneum over the anterior abdominal wall (Fig. 3d). The surface of the visceral pericardial sheets shows zones with wide and shallow folds with an irregular contour, separated by furrows with the same characteristics. The majority of the cubic cells, with moderately microvillous covering, occupy the invaginations of the submesothelial layer. The neighboring cell zones show a smooth relief. 3.1.2 Cell Organelles, Vesicular System and Intercellular Junctions of the Cubic and Flat Mesothelial Cells According to cell shape and microvillous covering mesothelial cells are divided into cubic and flat types, as described above. Ultrastructural scrutiny shows that there are significant qualitative and quantitative differences between the common
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components in two main cell types, as well as numerous intermediate (transitional) cell forms. By TEM the typical cubic mesothelial cells have a ratio of vertical to horizontal diameter of between 2:1 and 5:1, and has a prominent nuclear portion as well as a short and thin periphery. The minimum and maximum vertical diameters (6.3–12.6 µm and 8.6–21.5 µm) of the cubic cells are larger than the same diameters (3.8–7.0 µm and 4.5–8.9 µm) of the flat cells. The rounded central apical portion, protruding towards the cavities and the thinnest cytoplasm in the regions of the intercellular zones, forms a markedly wavy border (Fig. 6a,d). Frequently the central apical portion has a particularly irregular shape and follows the form of the nucleus (Fig. 6b). There are clearly defined cell boundaries, which demarcate cells of different (ovary, rectum) or uniform size (spleen, urinary bladder, uterus). Often the cubic cells show a rounded and an oval apical contour (VP, spleen of rat, cat, rabbit), or more rarely their shape is prismatic (in guinea pig) and contain shallower invaginations (Fig. 6c). The central zones of the cubic cells of the urinary bladder, stomach, rectum, and large areas of the uterus are located in concavities of the underlying layer. The cubic cells have a large, rounded or ovoid nucleus with multiple and deep indentations (Fig. 6d), which occupies a central position—rarely it is placed eccentrically. Most of the cytoplasm is disposed around the nucleus. The well developed organelles form clusters or are distributed homogeneously in the perinuclear cytoplasm. The surrounding cytoplasm is moderately equipped with organelles in urinary bladder cubic cells. The nuclei of most cubic cells are electron-dark with a dense nuclear matrix. The heterochromatin content is mostly close to the nucleolemma. A single, large, spherical or irregular shaped nucleolus is often found in them. A conspicuous rough ER involves numerous flattened cisterna packed closely together and arranged in parallel close to the nucleus. In addition, the rough ER is always associated with a large number of free ribosomes and polysomes scattered between adjacent cisterna. With the electron-dense cytoplasm they form an increased electron opacity in single cubic cells (Fig. 6c). With regard to the electron density of the cytoplasm, the cubic cells (VP of rat, cat, and guinea pig, rat visceral pericardial sheet) are found in two varieties: electronlucent and electron-dense, the latter being small in number. The complexity of the Fig. 6a–h Cubic and flat mesothelial cells, demonstrated by TEM. a Rat. Spleen. Deep invagination, filled with microvilli between two cubic cells (C). (×8,700). b Ground squirrel during hibernation. Lung. The cytoplasm of a cubic cell follows the nuclear shape. Thin BL (arrows) over EM (EM). (×3,800). c Guinea pig. Lung. Electron-dense (D) and electronlucent (L) cubic cells. (×6,200). d Rat. Lung. Nucleus with undulated nucleolemma and rich organelle apparatus in a cubic cell. Cilium (arrow). (×9,500). e Rat. Ovary. Interdigitations (arrows) between rounded cells. Electron-dense granules in the basal cytoplasm (empty arrows). (×5,000). f Rat. Anterior abdominal wall. Flat mesothelial cells (F) with short, scant microvilli. Large collagen bundle (stars). (×3,800). g Rat. Intestines. Flat cell (F). Thin BL (arrows). (×5,000). (From Michailova 1995, with permission of Swets & Zeitlinger). h Rat. Stomach. Intermediate form of mesothelial cell in invagination (empty arrows) of the submesothelial layer. (×7,500)
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nuclear relief, as well as the degree of indentation of the nuclei and the amount of the heterochromatin condensations are more marked in these electron-dense cells. Numerous ribosomes and polysomes and dilated cisterna of rough ER are observed in the cytoplasm of the latter cells. The Golgi complex is, as a rule, well developed and is commonly seen as stacks of flattened and dilated sacs, or saccules, surrounding the nucleus of the cubic cells. In many cubic cells several (three or four) Golgi complexes form a large Golgi zone, occupying an extensive portion of the perinuclear cytoplasm. Other widely distributed organelles are mitochondria of variable size and shape, which are often observed throughout the cytoplasm, adjacent to the nucleus or are particularly abundant along the opposed cell junction membranes, where they form groups. Striking peculiarities are accumulations of small homogeneous electron-dense granules and large heterogeneous membrane-bound bodies (Fig. 6e). Such cells characterize the transitional areas of the lesser pelvis, as well as the ovary covering. These bodies comprise a fine electron-dark matrix in which dense particles, electron-lucent globules and pleomorphic deposits of varied nature are included. Primary lysosomes and vacuoles with a heterogeneous content are also seen. The second type is the flat cell, with a considerably elongated form, that occupy larger areas of the mesothelial surface (Fig. 6f). The central (nuclear) zone protrudes insignificantly into the serosal cavities, so that the boundary row is only slightly folded toward the cavities. Their borders with adjacent flat cells are rarely encountered. In TEM preparations the flat cells appear elongated, with slightly convex central zones containing the fusiform-like nucleus with single invaginations, surrounded by a scant cytoplasm (Fig. 6g). These nuclei are electron-lucent with a light nuclear matrix of euchromatin with only small patches of heterochromatin located over the nucleolemma. A nucleolus is a rare finding in their nuclei and is encountered in serial sections. Few cisterna with poor ribosomal covering comprises the rough ER. The Golgi apparatus takes an extremely small portion of the perinuclear cytoplasm making its identification in the flat cell difficult. The mitochondria are few or moderate—isolated or in discrete groups. The mesothelial cells have well developed vesicular systems, but the cubic cells have a more prominent vesicular system than the flat cells. The number, location and involvement of different components depend on the physiological condition of the cells. The vesicular system is composed of microvesicles, vacuoles, vesiculovacuolar complexes and irregular formations by the fusion of several microvesicles, which represent the multivesicular bodies. Two main types are evident: a small membrane-bound vesicle–the microvesicle 20–25 nm in diameter–which is the main component of the vesicular system, and a larger membrane-bound vesicle or vacuole (50–150 nm in diameter), which are fewer in number. By SEM they appear as depressions, shallow round pits of different size or form irregular invaginations of the apical mesothelial plasmalemma, located next to the microvilli. Most of these are associated with the cell surface, mainly with the apical surface; a lesser number are associated with neighboring cells, and fewest of them have basal positions. The vesicles occupy the adjacent cytoplasm in the same manner. Some of them
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show different internal structures (homogenous with different electron density, or a granulo-filamentous content). The intermediate cells comprise a considerable percentage of the mesothelial cells and are second in number after the characteristic basic cell type for an organ or a region. They follow in number the flat cells in all SM. Discrimination of the intermediate cells is often not easy, and observations of serial sections are needed to identify the largest vertical diameter. Often their nuclear portion is located in invaginations of the submesothelial layer (Fig. 6h). Regarding their organelle apparatus, apical membrane specializations and vesicular system, they occupy an intermediate position between the cubic and flat cells. For some organs (for example the stomach and intestines) intermediate form the major portion of the mesothelial covering (as seen by TEM). Some cell components (cytofilaments, LB, lipid droplets, glycogen accumulations) are seen as occasional findings in normal mesothelial cells. Cytofilament bundles are usually visible in cat spleen cubic cells (Fig. 7a). They are extremely rare in flat cells. In some cubic cells they form a densely packed net in the apical cytoplasm, as well as in zones with intercellular contacts. Identical in thickness, concentric membranes over an electron-dense center, or without it, form LB (Fig. 7b). They are observed as single findings in the cytoplasm or in the vicinity of the microvilli of cubic cells of the lung (in all investigated species), stomach, spleen, liver and uterine tube. Lipid droplets and small accumulations of glycogen might be observed more often in the cubic than in the flat cells of the rat’s liver and spleen. Rarely single or small groups (two to three cells) of cells with degenerative changes are seen between the remaining mesothelial cell in the SM of all animal species and in the human (Fig. 7c). Their nuclei are rounded, with a homogeneous content. The nucleolemma is disrupted and surrounded by an electron-lucent zone. The organelle content is poor and vacuolized. Few ribosomes and single cisterna of rough ER are present. Mitochondria appear swollen, with few cristae. The apical membrane is often interrupted and shows single evaginations. The contacts between the degenerative and adjacent unchanged mesothelial cells are
Fig. 7a–g Internal structure of the mesothelial cells and intercellular junctions. a Cat. Spleen. Large cytofilament bundle (arrows) in the cubic cell. (×5,500). b Rat. Lung after treatment with RR. LB (arrow) in the cytoplasm. (×12,000). c Human. Lung. Cubic (C) cell and a cell with degenerative alterations (arrow). (×4,200). (From Michailova 1997, with permission of Swets & Zeitlinger). d Rat. Spleen. Macrophage-like mesothelial cells with vacuoles and lysosomes (stars). Intercellular dilatation (arrows). (×4,700). e Human. Lung. Single adherent contacts (arrows) over complicated digitations (asterisks). (×18,000). (From Michailova 1997, with permission of Swets & Zeitlinger). f Rat. Lung. Occludens contacts (small arrows) and adherens contacts (large arrows) over shallow digitations (stars). (×15,000). (Michailova and Wassilev 1988, with permission of Elsevier). g Rat. Stomach. Large, round intercellular dilatation (asterisk), filled with microvilli. (×21,000)
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altered (specialized contacts are rare or absent). Some of these cells are completely detached from the mesothelial layer. SEM observations show relatively ‘bare’ cells or cells with single stubs. Often the underlying BL and EM of the VP have wide interruptions and altered structure. Usually there is a broad electron-lucent zone between the degenerative mesothelial cells and the BL. The rat spleen peritoneum and cat parietal pericardium include the groups of macrophage-like mesothelial cells of variable shape (Fig. 7d). They have a centrally located nucleus, of which the nucleolemma depicts an irregular contour. Large groups of primary lysosomes and vacuoles with various electron densities or with heterogeneous content are present. These cells emit numerous cytoplasmic evaginations of variable size. The dilated spaces and lack of specialized contacts characterize their intercellular spaces. The nature and structural relationship of cell-to-cell apposition is an important feature of the composition of these boundary tissues. Since the mesothelial layer displays a monolayer arrangement, these specific areas assume a far greater significance for maintaining integrity. All specialized contacts are dispersed, different in size and shape, and different in their interdigitations. Single overlappings are the most frequent contacts between mesothelial cells (Fig. 7e). The most commonly encountered junctions are tight macular junctions, zonular types, or occludens junctions, which provide a complete seal or form a plaque-like area. Intercellular gap (nexus) junctions are also present. The occludens and, more rarely, adherens junctions, are usually located close to the apical aspect of the intercellular channels (Fig. 7f). Contacts are observed in zones of the cell bodies of neighboring cells, or adjacent cells communicate with their numerous peripheral processes. Single maculae occludens predominate in the apical zones of the shallow interdigitations between the flat cells, followed by extremely rare zonulae adherentes. More numerous different types of tight junctions are located over the longer cell processes of the cubic cells, as compared with these in the flat cells. Desmosomes (maculae adherentes) are disposed in the deepest portion of the intercellular zones. They are relatively few, poorly developed and are only seen as usual findings of the interdigitations between cubic cells. The mesothelial intercellular zones of the spleen are characterized by rounded intercellular spaces. Apically they are limited by thin cell processes, and basally are opened toward the BL. At certain points along the intercellular channels, dilatations occasionally occur, giving the appearance of large lake-like spaces (Fig. 7g). Some of these dilatations have heterogeneous content and microvilli protrude into them. They are limited apically by occludent types of contacts and basally by the cytoplasmic processes without specialized junctions, or the cell processes make space and the dilations remain opened towards the underlying BL. Numerous vesicles, as well as multivesicular bodies, lie in close proximity to the intercellular spaces, or are directly exocytosed in them. Within the mesothelium there are certain zones of specific arrangement. Occasional findings are single serosal villi of the VP, the pericardium, the spleen and the uterus (Fig. 8a) which are covered by mesothelial cells, situated on BL, and in some cases have a slim core of connective tissue, with or without blood capillaries. A characteristic feature of the basal regions of the VP of cat, rabbit and guinea
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pig is the large groups of extremely high prismatic (cylindrical) mesothelial cells (Fig. 8b); these are often found in contact and with a multi-layered arrangement in certain sectors and have abundant and long microvilli over the entire apical surface. Rarely, extremely short sectors of the visceral mesothelium (lung, heart, spleen, stomach, rectum, uterus) or wider zones (ovary) show a multi-layered arrangement, covered with an exclusively rich microvillous coat. The epithelial cells of the ovary of the newborn guinea pig form a layer, which is very variable in the manner of its arrangement. Several rows of cells produce long multi-layered zones, cryptae-like invaginations and papillae-like evaginations as consistent findings (Figs. 6e, 8c). The epithelial cells from these sectors show different size and shape (round, oval, cubic or irregular). Most of them have processes (some of them prolongate in the subepithelial layer). Intercellular dilatations and, more rarely, interdigitations of different sizes and irregular in form embrace the cell boundaries over long distances. Over them are located single, short specialized (occludens-like or adherens-like) contacts. Specialized contacts are more numerous near to the apical part of the intercellular spaces. The remaining epithelial covering shows a monolayer arrangement of high prismatic cells. Some epithelial cells have a thicker apical than basal portion and form intercellular dilatations near the BL. The apical and the lateral plasmalemmas in the sectors with multi-layered arrangement have short microvilli-like and are/or variable sized, rounded evaginations. Single cells have one cilium in a supranuclear position. Small groups (three to five) cells have numerous ciliae containing a 9+1 arrangement of tubules. The large nuclei of the epithelial cells are usually round or oval and are centrally situated. The nucleus is markedly euchromatic and almost without heterochromatin condensations. The cytoplasm is poor in cell organelles. The cubic cells of the distal portion of the uterine tube show an abundance of microvilli, rounded apical evaginations and numerous undulations of the basal plasmalemma. They gradually change their shape toward high prismatic cells, which form extremely thick multi-layered covering on the end of uterine tube (Fig. 8d). The latter mucosa-like cells show different electron density (electron-lucent and -dense), an abundance of longer microvilli, numerous ciliae, and uniform, large electron-dense granules in the apical cytoplasm. Dilated cisternae of rough ER are distinguished in large percentage of them. Another specialized feature is the Fig. 8a–e Features in the mesothelial covering. a Cat. Lung. Serosal villus. Collagen bundles (stars) in the core. (×3,000). (From Michailova 1996, with permission of Elsevier). b Cat. Lung. On the left are shown high prismatic mesothelial cells (asterisk) and on the right multilayered mesothelium (star) is seen. (×2,7000). c Newborn guinea pig. Ovary. Dilatations (asterisks) between superficial cells of a multi-layered sector. (×2,000). d Rat. Uterine tube. Apex (empty arrow) of a fimbria. Groups of electron-dense granules (stars) and extremely rich microvillous covering. (×2,200). e Rat. Uterus. Cubic cells over thin BL (arrows) form crypt-like invagination with narrow lumen (star). (×3,700). (From Michailova et al. 2004, with permission of Elsevier)
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crypt-like invaginations with extremely narrow lumina; these are observed on the uterus (Fig. 8e). Similar invaginations characterize zones of the greater omentum and broad ligament, where their depth and width are larger. 3.1.3 Membrane Specializations The higher transmission and scanning electron microscopic magnifications demonstrate numerous apical membrane specializations with the presence of microvilli (Fig. 9a). The average measurements of the microvilli in the rat are 0.05–0.08 µm in diameter and from 0.5 to 3.5–4.0 µm in length. The microvilli have either smooth or relatively rough surfaces. They become thinner towards their endings, but sometime exhibit bulbous terminations. Two or more microvilli may start from one stem or from evaginations of the apical membrane, and other kinds of branching occur. TEM images show the very simple internal structure of the microvilli. Their core represents an extension of the cell cytoplasm and has the same electron density. Only cytofilaments located parallel to the long axis and extending to the tips are found. The microvilli are more densely arranged on the visceral than on the parietal sheets. They are especially numerous on organs with active movement in the body cavities (lung, heart), with volume changes (spleen, ovary) and on organs that have a prominent reservoir functions (rectum, uterus, urinary bladder, zones of the stomach). Numerous, fine coagulated particles are located over the richly microvillous border of both sides of the muscular portion of the diaphragm, basal regions of the lung, and the spleen (Fig. 9b). In close vicinity to stomata openings larger, irregular patches of the same coagulated material are visible (Sect. 3.1.5). On cubic cells microvilli are more numerous and longer than those on the flat cells, which have few and short microvilli. The peripheries of the cells (along the intercellular borders) may manifest a more densely packed microvillous arrangement, oriented perpendicularly to the cell surface. The mesothelial covering of the lung, the spleen, and the parietal zone of the SM is usually uniform. The remaining investigated organs show significant variations of the microvillous densities on different portions of their surfaces: there are large differences between adjacent cells, and also between different areas of the same cell (Fig. 9c). The central zone of single or groups of cells appear relatively bare, showing the stubs of microvilli on their central regions (Fig. 9d). The degenerating cells have few microvilli with interrupted plasmalemma. The additional apical membrane specializations are single ciliae, different evaginations or invaginations, pits of microvesicles, membrane structures or LB in contact as well as in the vicinity of the apical plasmalemma (Fig. 10a). Single, isolated ciliae about 5.0–10.0 µm in length, and approximately 0.2 µm in diameter, are observed on the parietal, and more so on the visceral sheets of the pleura, peritoneum and pericardium. Usually cilia arise from the central (nuclear) zone or may be surrounded by the components of the Golgi apparatus (Fig. 10b). Frequently cilia of the VP arise from the depressions of the apical cytoplasm. The internal structure
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Fig. 9a–d Microvillous covering. a Cat. Visceral pericardial sheet. Numerous microvilli with local thickenings (large arrow) and bifurcation (empty arrow). Membrane profiles (small arrows). (×14,000). b Rat. Lung. Different microvillous covering over adjacent cells. (×400). c Rat. Spleen. Coagulated particles (arrows) over the rich microvillous coat. (×800). (From Michailova 2004, with permission of Springer Verlag). d Rat. Lung. Short stubs of microvilli (arrows) over the central portions of two cells. (×300)
of the cilia shows a 9+0 microtubular organization, and a basal corpuscle near to its ablumenal end. The apical cytoplasm forms evaginations and invaginations, which are more numerous in cubic, and which vary in shape and size. The VP of guinea pig has flower-like evaginations over the central portion of the cubic cells,
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Fig. 10a–g Apical membrane specializations. a Rat. Lung. Microvilli (empty arrow), cytoplasmic evagination (star), cilium (arrow). (×17,000). b Rat. Stomach. Cilium (arrow) in vicinity of the Golgi apparatus (asterisk). (×7,000). c Guinea pig. Lung. Two flower-like evaginations, surrounded by microvilli. (×4,000). d Mouse. Uterine tube. The apical plasmalemma is continuous over the external membrane of a LB (arrow). (×12,000). e Newborn guinea pig. Ovary. Round evaginations (arrows). (×2,300). f Rat. Lung treated with RR. Microvilli cover a large protrusion (asterisk), filled with microvesicles. (×10,000). g Cat. Visceral pericardial sheet. Strip-like structure (arrows) over granulo-filamentous material between the microvilli. (×5,500)
usually between microvilli (Fig. 10c). The rounded evaginations are seen more frequently than are other types of evagination. Some organs are characterized by individual features of apical membrane specializations. Wide regions of the spleen covering possess rounded or oval mesothelial cells similar to macrophages. They are distinguished by pseudopode-like evaginations of the entire membrane. At the uterine tube, numerous vesicle-like profiles are produced from single or double membrane in the vicinity of the microvilli. Common external membrane and matrix with variable electron density characterize complex LB (Fig. 10d). Complex LB are filled with vesicles which are variable in size, content and membrane thickness, and that emerge from evaginations of apical cytoplasm or are in close proximity to microvilli. The apical mesothelial plasmalemma continues as an external membrane of the LB. Some of them consist of single multilamellar structures in a regular concentric or parallel arrangement,
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and with different membrane thickness. The superficial epithelium of the ovary has constant superficial membrane specializations—numerous short plump or oval evaginations, groups of ciliae and rare microvilli (Fig. 10e). Cubic cells of the VP, of the covering of the spleen, uterus, urinary bladder and rectum show exclusively large protrusions (Fig. 10f). They are covered by microvilli and their cytoplasm has filaments, vesicles and few organelles. Small areas of the pericardium and the spleen peritoneum are covered with strip-like formations (Fig. 10g). Under the linear membrane structure and between microvilli is a fine granulo-filamentous material. Staining with RR reveals a thin amorphous film that appears filamentous or granular in nature at high magnifications. It is distributed over the microvilli, and the cell surface between them, microvesicles connecting the apical membrane or filling the apical cytoplasm, and the intercellular spaces. This covering appears thicker on the visceral than on the parietal serosal sheets. Mainly from this layer (surface glycocalix) arise numerous radiating fine strands, which interconnect with each other the adjacent microvilli, plasmalemma, pits of microvesicles, or complicated vesicular complexes. These fine strands and membrane-like, amorphous and osmiophilic material correspond to the same fine structures, seen by SEM, but are better visible with RR-staining (Fig. 7b and 10f). 3.1.4 Basal Lamina, Elastic Membrane and Submesothelial Connective Tissue Layer The BL of the human and rat’s VP is well formed and is located immediately below the mesothelium (Fig. 11a). The BL is lacking under more of the investigated portions of the greater omentum and is difficult to distinguish in the mesenteric duplicatures (Fig. 11b). The thick, homogenous BL of the rat’s VP has an electrondense internal structure. The BL, as well as the EM are two to three times thicker at the basis of the lung, and there are more numerous and considerable local enlargements. The BL of the VP in all investigated species is folded, bifurcated, and is rarely interrupted in brief sections. In its stratified portions there are single fibers or small collagen bundles. Blood capillaries and peripheral alveoli may be in immediate contact with BL. Between the mesothelial basal plasmalemma and rabbit’s BL of the VP are single fibers or fine collagen bundles (Fig. 11c). The BL is unevenly thick in the VP of guinea pig, an average of 600 nm, while in the VP of the ground squirrel during hibernation there is a wide electron-lucent portion and a thin, breaking electron-dense portion (Figs. 6b, 11d). The BL of the PP of both pericardial and peritoneal sheets is identical. It is thin with a wide electron-lucent superficial part, and a thin, electron-dense deep part. The spleen BL is thick, electron-dense, and is similar to the BL of the VP. In the region of stomata, the BL contains large interruptions. The BL and EM under the mesothelial cells with degenerative alterations are thin, electron-lucent and interrupted or absent. In some places both structures show fibrous or nonhomogenous internal structure.
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The EM of the VP and spleen peritoneum is an obligatory component, while the PP has EM-like structures in the submesothelial layer (Fig. 11a,d). A perforated EM of the VP shows a folded course, inconstant thickness (from 700 nm in rat to 950 nm in guinea pig), interruptions, and evaginations toward the mesothelium and/or toward the submesothelial layer. In some short sectors of the lung basis, the EM is duplicated. In its bifurcated zones there are collagen bundles or cell processes. The core of the EM is composed of an electron-lucent amorphous material, while electron-dense parallel microfibrils form its peripheries. More often the EM is in direct contact with underlying tissue. Between the BL and EM are found collagen fibers/bundles or small cytoplasmic processes of fibroblasts. Our investigation has shown that VP from ground squirrel during hibernation repeats the general pattern of the other animals described above. The VP during the active period shows small remnants of elastic fibers beneath the mesothelial BL. In long sectors there is a well defined thick EM (Fig. 11e), located in the submesothelial layer separating it into two parts. The cells (connective tissue and extravasal) are predominant components of the superficial part of the submesothelial layer, while large collagen bundles and single elastic fibers form its deep part. The thick EM of the spleen is followed by several parallel EM-like structures in the connective tissue layer (Fig. 11f). Beneath the BL is a layer of connective tissue, which extends and forms interstitial septae of different sizes and shapes. The thick connective tissue layer of the PP is made up of fibroblasts/fibrocytes, single macrophages and mastocytes, large collagen bundles parallel to the surface, and elastic fibers or EM-like structures in some areas. These components form a dense three-dimensional network, building the thick submesothelial layer of the PP (diaphragmatic and costal) and of the anterior abdominal wall, as well as the roof and the floor of the areas with LL, in all investigated species. The sole structure in which cells predominate in the parietal sheet is the mediastinal pleura of the cat (Fig. 11g). In the submesothelial layer of the visceral sheet of the SM, the cellular elements are more prominent. Fibroblasts/fibrocytes are the most common cells and are closely associated with collagen and/or elastic fibers. Aggregates of macrophages and mastocytes are of-
Fig.11a–h BL, EM and connective tissue layer. a Rat. Lung. Thick BL (empty arrows) over EM (EM). Mastocyte (large arrow), macrophage (small arrow), type II pneumocyte (PII) and blood capillaries (stars). (×3,000). (From Michailova and Wassilev 1988, with permission of Elsevier). b Rat. Greater omentum. Flat cells (F) cover a fine core of a connective tissue (stars). BL is lacking. (×3,200). c Rabbit. Lung. Collagen fibers (arrows) between BL (empty arrows) and EM. (×7,000). d Guinea pig. Lung. Thick BL (BL) and EM (EM) under cubic cells (C). (×3,800). e Ground squirrel in the activated period. Lung. Thick BL (thin arrows). Deep EM (thick arrows) in the submesothelial layer. (×2,700). f Rat. Spleen. Several parallel EM (EM). (×2,600). g Cat. Mediastinal pleura. Flat cells over thin BL (arrows). Cells and large collagen bundles (stars). (×6,000). h Cat. Visceral pericardial sheet. A group of blood capillaries (stars) in the submesothelial layer. (×3,000)
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ten seen in this layer. Single cells or groups of extravasal cells (predominantly neutrophilic and eosinophilic leukocytes) as well as bundles of collagen fibers of various sizes, and cut at different angles, are observed. These usual components differ between species. The thin connective tissue layer of the rat’s VP shows more numerous blood capillaries, cells (mainly fibroblasts, macrophages, mastocytes, and single neutrophilic and eosinophilic leukocytes), a moderate number of collagen bundles and short elastic fibers. The guinea pig VP shows an extremely thick submesothelial layer (Fig. 11d). Its main components are the large collagen bundles and few fibroblasts. The connective tissue components, described above, are densely packed in wide areas in the visceral pericardium and the testicular peritoneum. The cells and fibers are organized into a loosely arranged layer in the stomach, rectum, uterus, and urinary bladder. The collagen bundles predominate in the thick submesothelial layer of the visceral sheets of the stomach, rectum and urinary bladder, whereas the cells characterize the same layer of the uterus. In the small intestine small collagen bundles are arranged a narrow band, otherwise the mesothelial covering is located directly on the smooth muscle layer. In lung a significant number of elastic fibers is scattered between the cells and blood vessels, and are more abundant than collagen fibers in large portions of the submesothelial layer. Elastic fibers are numerous in the spleen and liver connective tissue layer, while the remaining organs contain single fibers. On the other hand, large parts of the spleen and liver have an extremely thin layer containing only small collagen bundles and single elastic fibers separating the mesothelium and the specific organ tissue. A thick network of numerous elastic fibers, EM-like structures and groups of smooth myocytes are distinguished in the spleen submesothelial layer. Rare, small bundles or scattered collagen fibers, and single cells form the loosely organized connective tissue layer in the greater omentum, mesenteric duplicatures and broad ligament. The boundary between this layer and its connective tissue matrix is difficult to discriminate. In the submesothelial layer of the broad ligament there are groups of lipocytes and smooth muscle cells. Numerous blood capillaries and few arterioles and venules are observed in the submesothelial layer. The small blood vessels and capillaries of the heart are of smaller size and show thick, continuous endothelial cells (Fig. 11h). They form a superficial (close to the mesothelium) and a deep network in the lower portion of the submesothelial layer. Frequently on the VP the blood vessels are in the vicinity of the BL or EM and have a thin endothelium similar to the capillaries of the lung parenchyma. Some capillaries of the greater omentum have an endothelium with a significant number of microvesicles, corresponding to the rich vesicular system in the overlying mesothelium. Groups of blood capillaries are located in the superficial part of the submesothelial layer and show an interrupted electron-dense endothelium in the ovary. The India ink injections demonstrate some characteristic features of the blood supply of investigated organs. Blood vessels of different sizes are arranged in a dense irregular network in the rabbit’s VP (Fig. 12a). Blood vessels produce a parallel set in the costal pleura (Fig. 12b). Numerous arterio-venous anastomoses characterize the visceral pericardial sheet (Fig. 12c). A quadrangular network of
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large, parallel blood vessels and their perpendicular offshoots distinguish the both sides of the diaphragm (Fig. 12d). Constant finding are the wide lymphatic vessels, which cross, and in rare cases follow the large blood vessels (Fig. 12e,f). The endothelium of the blood capillaries in the investigated SM is of a continuous type and is situated on a BL (Fig. 12g,h). Single lymphatic capillaries with small lumens are constant findings in the submesothelial layer of some organs (uterus, ovary, testis). The lymphatic endothelium is highly stretched, electron-lucent and with poor organelle content. Some endothelial cells have a prominent cytofilamentous system (Fig. 12i). Interdigitations with rare specialized contacts occur between the adjacent endothelial cells. Their BL is not well defined. In the human VP small lymphatic vessels may predominate—a sign of ‘thick’ type VP (Fig. 12j). They are located in the immediate vicinity of the blood capillaries or the BL, and are rarely deep in the submesothelial layer. The lymphatic capillaries have round lumens with pale staining contents. Discrete bundles of thin unmyelinated and more rarely myelinated fibers are also seen in the SM (Fig. 12g). They are more numerous in the visceral sheets and are visible in close contact with the mesothelial BL (in the heart) or with the blood vessels in the submesothelial layer (in the lung, heart, stomach, rectum, urinary bladder, uterus and broad ligament). In the vicinity of the cell accumulations of the greater omentum they are more numerous. In other organs we observed only few, as well as occasional nerve elements. 3.1.5 Stomata On the muscular portion of the pleural side of the diaphragm, in proximity to the costo-diaphragmal angle and in the lower intercostal spaces there are groups of gaps in the mesothelial layer called stomata. They are much wider than the thin intercellular clefts normally present between adjacent mesothelial cells and most likely correspond to the mesothelial openings of channel-like stomata. An extensive microvillous coat covers their lumens. Single or groups of free blood cells Fig. 12a–j Vessels in the submesothelial layer. a–f Rabbit. a Lung. Dense network of small blood vessels. (×12). b Costal pleura. Parallel blood vessels. (×24). c Visceral pericardial sheet. Arterio-venous anastomoses (arrows). (×16). d–f Peritoneal side of the diaphragm. d A quadrangular network of large, parallel blood vessels and their perpendicular offshoots. (×24). e The blood vessels (arrows) are crossed by wide lymphatic vessels (stars). (×24). f The lymphatic vessels follow the course of the blood vessels. (×24). g Human. Lung. Unmyelinated axon and a single myelinated axon (arrows) in the vicinity of a blood capillary (star). (×7,800). (From Michailova 1997, with permission of Swets & Zeitlinger). h Rat. Uterus. Abundant microvesicles in the endothelium of blood capillaries (stars). (×5,200). i Rat. Broad ligament of the uterus. Thin electron-lucent endothelium of a large lymphatic capillary (stars). ( ×5,000). j Human. Lung. A cytofilament bundle (arrow) in the lymphatic endothelial cell. BL is lacking. (×21,000). (From Michailova 1997, with permission of Swets & Zeitlinger)
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frequently occupy these openings and thereby occlude them. Large coagulated particles are located in vicinity of stomata (Fig. 13a,b). Clusters of cubic cells form high projections near to them, but there are also single or small groups of flat cells (Fig. 14a). Both cell types are richly supplied with microvilli (Fig. 14b). Between the adjacent mesothelial cells of the liver peritoneum, deep tunnel-like formations extend from their apical surface towards the underlying tissue (Fig. 14c). Most of the cubic cells are located in invaginations of the connective tissue layer and form a relatively smooth surface supply with abundant microvilli. The peritoneal surface the anterior abdominal wall also has stomata (Fig. 14d). Their round openings are surrounded by flat mesothelial cells, coated by moderate number of microvilli. Adjacent stomata are frequently separated by thin cytoplasmic bridges of mesothelial cells, thus giving the impression of a clustered distribution. Free cells are attached to the boundaries of stomata, or are directly located within the openings, thereby occluding their lumens. Numerous stomata openings are encountered on the muscular portion of the diaphragmatic peritoneum, forming round or oval gaps between diverging cytoplasmic flaps of the cuboidal mesothelial cells, seen by SEM. Some peritoneal stomata appear as pocket-like slits or valve-shaped openings formed by overlapping short and bifurcated processes of the neighboring mesothelial cells. Most of the peritoneal stomata of the rabbit’ diaphragm are surrounded by extremely flat mesothelial cells (Fig. 14e). The mesothelial cell borders
Fig. 13a, b Stomata of the pleural side of the diaphragm in the ground squirrel during hibernation. a Group of stomata with coagulated material in their vicinity. (×600). b Detail from a, the region indicated by an arrow. (×2,000). (From Michailova 2004, with permission of Springer Verlag)
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do not overlap and therefore allow a direct view into the underlying connective tissue. Over the peritoneal covering of the greater omentum, a detailed identification of the cell boundaries is difficult. Short microvilli are sparsely distributed and thereby round or oval openings with clear boundaries are visible in clusters. In occasional cases the cuboidal mesothelial cells form small groups in their vicinity. A few stomata are also seen on the broad ligament of the uterus. By TEM, the diaphragm shows large groups of cubic cells over the LL in normal tissue. The components (cells and fibers) of the connective tissue layer form the roof and the floor of the areas with LL. The LL are located in the vicinity of the mesothelial layer or more rarely close to the muscular tissue of the diaphragm. More often, the thin intercellular zones of the mesothelium are located over the extremely fine interdigitations of the underlying endothelium of the LL (Fig. 14f). Fine collagen bundles, or single collagen fibers are dispersed between them. In rare cases direct contacts between mesothelium and endothelium of the LL on the common BL (or without BL) are seen. Only a few stomata in untreated animals and in sham-operated rats are observed, as typical spaces between mesothelial cells and direct contact between the mesothelium and the endothelium of LL. The majority of stomata in normal conditions are not really opened and are visible only on serial sections. Under peritoneal stomata, as a rule, the BL is absent, allowing direct access to the underlying loosely arranged collagen fibers, or to the lymphatic endothelium. A typical stomata involves direct contact between the mesothelial and endothelial cells of the LL, sharing a common BL. The lymphatic endothelium is thin and rests on a fine, incomplete BL. The endothelial electronlucent cytoplasm forms rare small microvilli and evaginations. It has few small bundles of cytofilaments and a rich vesicular system. The endothelial cells make contact through interdigitations with occasional maculae adherens over them. The LL are located parallel to the surface. They form cistern-type extremely flat lumens, in most cases are closed or have complicated contours. They form groups in the organs described above (PP—diaphragmatic and costal portions, peritoneal side of the diaphragm, liver, anterior abdominal wall, greater omentum, broad ligament). A complicated serosal relief covers these sectors. Extravasal cells, unmyelinated and rare myelinated nerve fibers are in their vicinity.
Fig. 14a–f Stomata. a Rat. Flat mesothelial covering surrounds a large cluster (asterisk) of cubic cells in vicinity of costo-diaphragmatic angle (arrows). (×200). b Rabbit. Costal pleura. Group of stomata openings (arrows). (×2,000). c Rat. Liver. Tunnel-like invaginations (asterisks) open between adjacent mesothelial cells. (×2,700). d Rat. Anterior abdominal wall. Large invagination is occupied by erythrocytes and (E) lymphocytes (asterisks). (×3,200). e Rat. Peritoneal side of the diaphragm. Adjacent mesothelial cells delimit a round stomata opening with fibrous structures in its depth. (×4,000). f Rat. Space (large arrow) between two mesothelial cells (M). Fine digitations (empty arrow) of the endothelium, a lymphatic vessel (Lv). Common BL (small arrows) between mesothelial and endothelial cells. ( ×5,500). (From Michailova et al. 1999, with permission of Elsevier)
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3.1.6 Milky Spots Deep furrows and large ridges cover human MS (Fig. 15a). Most of the mesothelial cells are cubic cells and are arranged in clusters over the MS (Fig. 15b). In other places the extremely thin and long processes of flat mesothelial cells are located over them (Fig. 15c). Small groups (two to four) or single macrophage-like mesothelial cells are also found, as covering cells. They have numerous pseudopode-like evaginations and abundant secondary lysosomes. In normal omentum, stomata are seen only occasionally. Actually, a space between two neighboring mesothelial cells is commonly observed, but these are not typical stomata openings. The interruptions of the mesothelial covering and its direct connection with underlying endothelium of the large lymphatic vessels as stomata are recognized only on serial ultrathin sections by means of TEM. The BL is absent over MS. The human MS are located in vicinity of the mesothelium or in the superficial part of the submesothelial layer, and more rarely in the deep adipose tissue. The most numerous free cells are the macrophages, followed by lymphocytes and mast cells. The observed cell populations are mixed in human MS, as well as those in the rat (Fig. 15d). Also in normal human MS, there is a significant component of connective tissue cells as perivascular fibroblasts. Discrete collagen bundles surround the vessels. Single or small groups of lipocytes are associated with small blood capillaries or are intermingled between the remaining free cells of MS. The free cells are commonly found around blood capillaries, while in some cases large clusters of them are to be observed in conjunction with arterioles (Fig. 15e). Endothelial cells with a well developed vesicular system rest over the BL and form blood capillaries with rounded lumens. Few discontinuous capillaries are seen. Large lymphatic vessels of the cysterna-type are a constant finding in vicinity of the mesothelium. Extremely flat lumens (some of them practically closed) of the lymphatic vessels are located with their wide diameter parallel to the peritoneal surface of the greater omentum. The lymphatic endothelium is thin, electron-lucent and displays a rich vesicular system. BL is lacking. The blood supply of the omental MS in rabbit was examined after injection of Indian ink. Networks of vessels within the MS with ovoid, round or irregular form were seen. Four types of capillary formations, according to the classification of Kanazawa et al. (1979) could be distinguished: intramembranous, sessile marginal, peduncular, and less vascularized (Fig. 16a,b). The pedunculated type was observed only in occasional cases if the classification above was followed strictly . Usually two arterioles supply a single MS and more rarely there are three to five arterioles. Often extra-omental MS are supplied by more than one arteriole (Fig. 16c). The afferent arterioles most commonly divide dichotomously, and a capillary network is formed from these branches. The capillaries display dilated portions located in the central zones of the MS. As a rule, the capillaries have markedly convoluted course and make numerous anastomoses. The rat’s omental MS are located closer to the mesothelium as compared with human ones. Long and thin processes of the
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Fig. 15a–e Human MS of the greater omentum. a Deep furrows (arrows) between folds (stars) over MS. (×4,200). b Cubic cell (C) over a group of blood capillaries (Bc), lymphatic vessel (arrows) and lymphocyte (Lc). (×3,000). c Extremely flat mesothelial covering (empty arrows) over two lymphocytes (arrows) and blood capillary (Bc). (×3,800). d Mastocytes (small arrows), group of macrophages (large arrow) and blood capillary (Bc). (×3,200). e Lymphocytes (small arrows) in proximity of muscle cells (large arrows) of a blood vessel (Bv). (×1,500)
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cubic mesothelial cells cover most of MS (Fig. 16d). The mesothelial cells display numerous fine evaginations, filled with microvesicles and groups of mitochondria. The main vascular components are the blood capillaries, whilst the lymphatic capillaries are scant. MS without lymphatic vessels are observed. Most of lymphatic capillaries of the rat display rounded lumens, electron-lucent endothelium and lack BL. The endothelial cells of the blood capillaries are electron-dense, unlike the electron-lucent endothelial cells of the remaining part of the greater omentum (Fig. 16e). The capillaries are of the continuous type and the endothelial cells are surrounded by a BL (Fig. 16f). An extensive vesicular system characterizes their endothelial cells. There are relatively few fenestrated capillaries. Densely packed continuous blood capillaries and smaller cell clusters characterize the rat’s MS. Around the blood vessels, there are rounded cells, rich in ribosomes, and with a large nucleus. They appear to be immature fibroblasts, or mesenchymal cells. The free cells in some MS are densely distributed and surround exclusively thin intercellular spaces. In other MS the cells are located sparsely over a net of fibrocytes, fine collagen bundles and single lipocytes. The types of free cells and their number differ significantly in the neighboring MS. The cell populations are presented by macrophages, followed by lymphocytes and mast cells. In the inactive MS of the rat leukocytes are seen also, mainly neutrophils, but there are also occasional eosinophils. Single or small groups (three to five cells) of erythrocytes are observed also. Neutrophilic leukocytes, macrophages and groups of mast cells predominate in other cell clusters. Bundles of thin unmyelinated or occasional myelinated fibers are located in vicinity of the vessels and the cells. There are MS-like structures located in the deep portions of the broad ligament of the uterus. The surface of the broad ligament forms large ridges with a complicated shape, limited by deep furrows like those of the human greater omentum, over MS. The majority of the mesothelial cells are of the flat cell type. Small clusters of cubic cells are seen between the flat cells. These MS-like cell accumulations are composed of macrophages, small groups of mastocytes, lipocytes, lymphocytes and neutrophilic leukocytes. Few fibroblasts and small collagen bundles surround the free cells. Lymphatic vessels are more often to be seen than blood vessels (Fig. 12i). Small groups of blood capillaries and large lymphatic vessels with extremely complicated contours and flat lumina comprise the vessel component of MS. The thin electron-lucent lymphatic endothelium rests on an incomplete BL. The neighboring lymphatic capillaries are separated by thin septae. Single endothelial valve-like formations are identified in them. Different types of contacts between mesothelial cells and protrusions of the endothelium of lymphatic vessels are observed. Bundles of thin unmyelinated and occasional myelinated nerve fibers are often observed. MS-like structures are visible also on the mediastinal pleura of the cat. The main cell types are fibroblasts, macrophages, neutrophilic and eosinophilic leukocytes, and lymphocytes. The blood and lymphatic capillaries take approximately equal parts in their vessel supply. The boundaries of the MS-like structures of the mediastinal pleura are unclear, because a small number of free cells invade also the neighboring connective tissue.
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Fig. 16a–f Rabbit and rat MS of the greater omentum and extra-omental MS. a,b Rabbit. Greater omentum. a The intramembranous type of blood vessel network. (×24). b Sessile marginal type of blood vessels. (×32). c Rabbit. Mediastinal pleura. Four main arteries (arrows) supply a single MS. (×12). (d–f) Rat. Greater omentum. d BL is lacking under the cubic cell (C). Small collagen bundles, fibroblasts (small arrows), mastocyte (large arrow) and blood vessel (Bv). (×4,800). e Numerous microvesicles in the endothelium of the blood capillaries (Bc). (×7,500). f The BL (small arrows) of a blood capillary (star). Fibroblast (large arrow) and macrophage (asterisk). (×5,500)
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3.2 Development of the Pleura 3.2.1 Rat Prenatal and Postnatal Development At ED13 and ED14 the surface of the lung, the thoracic wall and the diaphragm are occupied by undifferentiated spindle-like or irregular shaped cells, devoid of any kind of apical membrane evaginations (Fig. 17a). They have a centrally located large nucleus. Numerous ribosomes and polysomes, single cisternae of rough ER, rare, small mitochondria and few vesicles are found in the cytoplasm. Extremely thin cytoplasmic processes overlap, but specialized contacts are absent in the boundary layer. At this period BL is still not formed. The thick underlying layer is arranged by undifferentiated mesenchymal cells. Their cytoplasmic processes surround large intercellular spaces, and there are no fibers around them. Single blood vessels of the capillary type and without BL are seen in vicinity of the terminal bronchiolar spaces, in which high prismatic shaped cells are arranged in a monolayer over the BL. At ED15 the boundary layer is built of uniform flat cells (Fig. 17b). These cells have single, short villus-like evaginations of the apical surface (Fig. 17c). They appear in close proximity of the intercellular spaces. In this stage for first time are observed individual cilium (Fig. 17d). Single interdigitations are found between the covering cells. These cells show large, elongated nuclei with little heterochromatin. The granular ER is presented by separate cisternae with electrondark content. The cytoplasm shows numerous ribosomes, polysomes and small number of vesicles of various size. The Golgi complex has four to five short and parallel membrane profiles and a small number of vesicles. Specific to this period is the emergence of the BL. An electron-lucent band separates the mesothelial row from thin, interrupted BL, which has a granulo-filamentous structure. The underlying layer is composed from undifferentiated mesenchymal cells and single blood vessels, located in deep portion of this layer. At the ED16 the pleural surface shows single folds, surrounded by individual furrows. For the first time cubic mesothelial cells appear (Fig. 17e). The central portion of the cubic cells incorporates a large, rounded or oval nucleus and protrudes Fig. 17a–f Prenatal development of the rat. ED14–16. a ED14. Elongated cells form the superficial row. The BL is lacking (arrows). Large spaces (stars) between the mesenchymal cells. (×2,600). (From Michailova and Wassilev 1991, with permission of Elsevier). b–d ED15. b Uniform flat mesothelial cells (F). (×3,000). c Villous evagination (large arrow) in the vicinity of the intercellular spaces (stars). Thin, interrupted BL (small arrows). (×11,000). d Cilium (arrow) in an invagination of a mesothelial cell, over the nucleus (N). (×26,000). e,f 16 ED. f Immature occludens-like contacts (empty arrows) in the apical portions of the interdigitations (stars). Electron dense bodies (arrows). (×17,000). (From Michailova and Wassilev 1991, with permission of Elsevier). e Cubic (C) cell with a single microvillus (arrow) and a flat (F) cell. (×5,300)
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towards the pleural cavity. The peripheries of these cells remain thin and form invaginations of the mesothelial layer in the intercellular zones. The flat mesothelial covering with scattered single cubic cells, or small groups (three to five) of them, characterizes this stage. There are rare, short microvilli with fine, single filaments in their cytoplasmic matrix, as well as individual cilia in the proximity of the nucleus. The microvilli are more numerous and longer in the zones of the neighboring mesothelial cells. The vesicles (their number is larger than in the earlier period) show different size and content (electron-lucent or fine granular matrix), and have an intracytoplasmic position. The adjacent cells are connected by interdigitations with a more complex route, or contact spaces are occupied with isolated annular protrusions and single overlappings. The intercellular dilated spaces of different size, which apically may be limited by immature occludens-type contacts, are not rare findings. Interesting features of this stage (ED15 and ED16) are groups of three to ten electron-dense membrane-bound bodies in the peripheral mesothelial cytoplasm or in close proximity to the intercellular contacts (Fig. 17f). Their cross section is in the range 150–200 nm, while their length varies within broad limits, due to the different angles of section. Their usual shape is rounded or oval. Some bodies are elongated and one of their ends is rounded, while the others become gradually thinner. Mainly longitudinally orientated microtubules packed densely in an electron-dark matrix, or an electron-light core with few microtubules are observed. The same body can show sectors with differences in electron density and microtubular arrangement. Electron-dense fibrous structure in a homogenous osmiophilic matrix characterizes some bodies. Small collagen bundles are seen under the BL and in the vicinity of the underlying cells. At ED15 and ED16 the initial acini are lined with nondifferentiated high, prismatic epithelial cells, arranged in one layer on the BL. Their apical plasmalemma forms microvilli and the cytoplasm contains numerous glycogen granules. The adjacent epithelial cells are connected with tight junctions and desmosomes. The subsequent period (ED17–19) is characterized by the parallel development of apical membrane specializations and the vesicular system. Parallel with the flat cells in the same period there are cubic cells, the nuclear portion of which have extensive protrusions toward the cavity. The cubic cells form the larger portion of the VP (Fig. 18a). The flat cells are the usual covering of the PP at the end of this period—ED19. The cubic cells contain a richer and structurally complicated organelle apparatus. A significant portion of the intercellular spaces is occupied by complex digitations with local expansions, an occludens type of contact in the apical portion, and an adherence type distally. Such an arrangement is typical for adults. The microvilli show greater length, diameter, density, and more cytofilaments in their core and in the apical cytoplasm. Single or rare, short microvilli appear on the prominent central region. A significantly larger number of long microvilli in the concave intercellular sections and evaginations as well as invaginations of various shapes and sizes are observed. Membrane formations are visible in the vicinity of the apical membrane. More numerous (up to 16) electrondark bodies persist in this stage, located in the peripheral cytoplasm. They are
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larger than those of the previous period, and their cross section reaches about 250 nm. More frequently, the bodies show intense homogeneous electron density, or microtubules are disposed in electron-dark matrix. An abundance of these bodies is observed in the multi-layered sectors of the lung basal mesothelium. Cubic cells and multi-layered mesothelial cells in a multi-layered arrangement characterize the large zones of the basis and the edges of the lung (Fig. 18b,c). The cubic cells in these sectors have considerably electron-dense cytoplasm. They are joined by more numerous specialized contacts than the ordinary monolayer organized mesothelium. During the same period the underlying cells increase in number and differentiate into various types of connective tissue cells—fibroblasts, macrophages and exclusively rare extravasal cells (Fig. 18d,e). More numerous collagen bundles become visible. The number of blood vessels, usually capillaries, in the vicinity of the mesothelial layer increases. At ED17 the cell lining of the future air spaces begins its typical differentiation. The glycogen granules form congestions, among which single or grouped light vesicles, or multivesicular bodies may be observed. A feature of this period is the emergence of single LB in the cytoplasm, which defines the cells as immature alveolocytes type II (prealveolocytes type II). At ED18 all epithelial cells contain a great number of LB and may be characterized as type II prealveolocytes. The epithelial cells of the future alveoli are differentiated into two types at ED19. The flat cells (type I) show centrally located nucleus and a thinned periphery, rich in glycogen, but there is a lack of multivesicular bodies and LB. The second cell type (type II) preserve their high prismatic shape and the number and size of the LB are increased as compared with the previous period. These cells have outgrowths of the basal cytoplasm which pass through openings of the BL and contact the underlying cells. At the end of the prenatal period (ED20 and ED21) two cell types are observed: flat (poor in organelles, with rare, short microvilli); and cubic cells (with well developed rough ER, extensive Golgi complex, vesicular system, and numerous, long microvilli). Regarding the electron density of the cytoplasm, electron-lucent and electron-dark cells are found, the latter being fewer in number (Fig. 18f). The electron-dark cells have an increased quantity of ribosomes and polysomes, they are elongated and have local enlargements with an osmiophilic content containing cisternae of rough ER. The intercellular spaces are made up of apically located
Fig. 18a–f Prenatal development of the rat. ED17–20. a–c ED17. a Cubic mesothelial cells (c). (×2,000). (From Michailova and Wassilev 1991, with permission of Elsevier). b Extremely high cubic cells. Densely packed submesothelial layer (stars). (×1,500). c Multi-row mesothelial section. Single collagen fibers (arrows) under the BL. (×2,300). d ED18. Mesothelial cell (M) over blood capillary (white asterisks) and terminal portions of the lung parenchyma (black asterisk). (×2,400). (c,d from Michailova and Wassilev 1991, with permission of Elsevier). e ED19. Numerous cisternae of rough ER in two cubic cells (C). Blood capillary (asterisk). (×2,800). f ED20. Electron-lucent (L) and electron-dense (D) cells. Thin collagen bundles under the BL (arrows). (×5,000)
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specialized junctions, while the most of the remaining intercellular cell membranes form deep digitations. The BL becomes thicker at the end of the prenatal period. The groups of connective tissue cells are increased and larger collagen bundles are seen in the submesothelial layer. More numerous small blood vessels (predominantly capillaries) and terminal spaces of the growing lung parenchyma are in contact with the mesothelial BL. A typical finding for this stage is the presence of mature pneumocytes type I and type II. The apical membrane specializations, mostly the microvilli, increase markedly in number and size, and change their filamentous content in the late prenatal period (ED 19, ED 20, ED 21), and in the early postnatal life (PD1, PD5), parallel with the forming of the vesicular system, and following the development of the intercellular junctions. At this time there is also extensive increase of the microvesicles, most of which are connected with plasmalemma, or are located in the contact zones of the cytoplasm, while relatively few are found in the rest of the cytoplasm in the same early postnatal periods. The cytofilaments are located predominantly in the apical cytoplasm and close to the intercellular plasmalemma. At PD1 cubic cells (most of them located in the invaginations of the submesothelial layer) are the predominant cell type of the VP (Fig. 19a). The visceral BL is thicker than in the previous period as is also thicker than the BL of the PP. The flat cells cover the PP almost entirely (Fig. 19b). A wide electron-lucent zone characterizes thin parietal BL. Cubic cells over the lymphatic vessels in the PP are rare findings. At PD15 the mesothelial cells show well developed organelles, numerous microvesicles and microvilli. The electron-lucent part of the BL is lacking in some small places. Single small elastic fibers appear under the BL of the VP (Fig. 19c). Superficial alveoli are found in close proximity or in contact with the BL of the VP. More numerous collagen bundles are disposed under the BL of the PP (Fig. 19d). Elastic fibers are occasional findings in the thin submesothelial layer of the PP. Development of the microvilli and the vesicular system continues until PD30, when it is difficult to find any differences from mature animals (Fig. 19e). During the same period elastic fibers are more numerous and thicker than in previous period. Thin, interrupted EM with electron dense peripheries or well developed EM is observed at PD45 (Fig. 19f,g). The connective tissue and extravasal cells, collagen fibers/bundles, and the blood capillaries form a thick submesothelial layer in some sectors. Elastic fibers remain fewer in number in long sectors, than these in the adult rats. Larger number of alveoli and blood capillaries are in contact with the BL or EM. 3.2.2 Human Prenatal Development The VP of fetuses aged 9–10 GW is represented by a mesothelium, BL and a submesothelial mesenchymal layer. The covering cells show a smooth relief with short sectors of stratified arrangement of two or three cell rows (Fig. 20a). The cells are elongated and of two different electron densities: light and dark. The apical
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Fig. 19a–g Postnatal development of the rat. 1–45PD. a,b 1PD. a Lung. Cubic cell over a thin BL (arrows) in a submesothelial invagination. (×4,500). b Costal pleura. Flat cells (F). (×2,700). c, d 15PD. c Lung. Cubic cell (C) over a electron-dense BL (empty arrows). Short and thin elastic fibers (arrows) under the BL and in the submesothelial layer. Superficial alveolus (asterisk). (×3,000). d Diaphragm. Flat cell (F) over a thin BL (arrows). Collagen bundles (small stars). Striated muscle fibers (large stars). (×3,200). e 30PD. Numerous microvesicles in a cubic cell (C). Thick elastic fibers (arrows) under the BL. (×4,200). (From Michailova and Wassilev 1991, with permission of Elsevier). f,g 45PD. f Cubic cell (C). Numerous elastic fibers (arrows) with electron-dense peripheries. Collagen bundles (asterisks) and blood capillary (star). (×3,600). g EM (arrows) under the BL. (×3,200)
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plasmalemma runs a smooth course with occasional short microvillous evaginations. The basal mesothelial plasmalemma follows an undulated course. The large elongated nucleus with single folds is centrally located and contains a small amount of heterochromatin, attached to the nucleolemma. Single vesicles of various sizes and content are observed in an organelle-poor cytoplasm. Specific for this stage are glycogen accumulations, lipid droplets and single electron-dense granules. Between the mesothelial cells, simple interdigitations with apical zonula occludens-like contacts are found (Fig. 20b). The BL has a superficial electronlucent part, and a thin deep electro-dense part. The latter is partly broken up, with thinner regions and a finely granulo-filamentous structure, or is entirely absent in wide region of the PP. The thick submesothelial layer is composed of mesenchymal cells (Fig. 20c) which have a large rounded nucleus surrounded by organelle-poor cytoplasm that projects long processes. These cells surround large intercellular spaces with scattered short, thin collagen fibers. The single blood vessels and distal airways are few in number and situated far from the mesothelium. The beginning of the period 11–13 GW is characterized by flat elongated cells. Regions of the VP have a cubic covering at 13 GW, while flat cells remain the predominant cell type in the PP. The central (nuclear) portions of the cubic cell type protrude towards the pleural cavity, or occupy the invaginations of the submesothelial layer (Fig. 20d,e). Long and thin cytoplasmic processes run parallel to the pleural surface and form the peripheral zones of most of the cubic cells. The nucleolemma of the large, rounded nucleus forms single, shallow invaginations in the cubic cells. An unfolded nucleolemma covers the elongated nuclei of the flat cells. The organelle content of both cell types is poor. The visceral BL maintains the same appearance as the previous period. A set of single, thin and short collagen fibers is disposed under the BL. The parietal BL can be distinguished only with difficulty. The connective tissue layers of both sheets are composed of spindle-like fibroblasts (Fig. 20f). They surround smaller intercellular spaces than those of the previous stage, and arrange a thick layer with few blood vessels. The number and size of the collagen fibers is increased and they form more numerous bundles than in the previous stage. Fig. 20a–g Prenatal development of the human. 9–20 GW. a–c 9–10 GW. a Mesothelial cell with glycogen accumulations (asterisks) and an envagination (arrow) towards the submesothelial layer. (×7,000). (From Michailova 1996, with permission of Elsevier). b Interdigitations between electron-lucent (L) and electron-dense (D) mesothelial cells. Thin BL (arrows). (×6,500). c Large spaces (stars) between the mesenchymal cells. Blood capillary (Bc). (×2,200). d–f 11–13 GW. d Protruded nuclear portions of cubic cells (C). Fine collagen fibers (stars) under a thin BL. (×4,000). e The nuclei of the cubic cells, located in invaginations of the submesothelial layer. (×2,700). (d,e from Michailova 1996, with permission of Elsevier). f Collagen bundles (empty arrows) and elongated fibroblasts (arrows). (×4,200). g 14–20 GW. On the right, the superficial zone with larger intercellular spaces and small collagen bundles in shown, and on the left a dense network of fibroblasts and larger collagen bundles. (×1,800)
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At 14–20 GW, the shape and arrangement of the mesothelial cells are still the same as the end of the previous period. The scant perinuclear cytoplasm remains with poorly developed organelles in both cell types. The microvesicles predominate over the other intracytoplasmic membrane structures. The microvilli are more numerous than at the previous stage. Single cubic cells have particularly rich microvillous covering and individual cilia in the vicinity of the nucleus. The interdigitations run a more complicated course, and short adherent-type contacts are scattered along them. The BL is thicker, forming single evaginations toward the submesothelial layer which are not followed by a mesothelial cytoplasm. The parietal BL is characterized by large interruptions. Fine collagen bundles retain the same kind of arrangement under the BL. The submesothelial layer in fetuses more than 17 GW may be divided into three zones: superficial, sparsely distributed fibroblasts, surrounding large intercellular spaces with fine collagen bundles; middle, densely packed fibroblasts, single extravasal cells, smaller intercellular spaces with larger collagen bundles; deep, an abundance of extravasal cells, lesser number of fibroblasts, small intercellular spaces with smaller collagen bundles and numerous blood capillaries (Fig. 20 g). At 21–24 GW, the cubic mesothelial cells predominate over the VP and the flat cells are basic type for the PP (Fig. 21a). This distribution is preserved until the end of the prenatal period. The ribosome, polysome and cisterna content of the rough ER is increased and the cytoplasm shows greater electron density in both cell types. The intercellular spaces are occupied with interdigitations with a more complicated course and adherence type contacts are rarely found along them. The electrondense part of the visceral BL is wider than at the previous stage, while in the PP it remains extremely thin. The arteries of the submesothelial layer are arranged as a mesh throughout the pleura, accompanied by lymphatic vessels. The number of superficial blood capillaries in the submesothelial layer is increased and they are smaller than the deep capillaries. They have a thick, continuous endothelium, with numerous microvillous evaginations of the apical membrane, and electron-dense bodies in the cytoplasm. The endothelial cells are located on a thin, interrupted BL. An interesting finding is the larger lymphatic capillaries with lightly staining luminal content in the submesothelial layer of the VP (Fig. 21b). They may be seen in vicinity of the mesothelial BL. Their endothelium is extremely thin, interrupted, organelle-poor, and forms large evaginations resembling valves. The lymphatic vessels have no BL. At 25–32 GW, the both pleural sheets resemble those of the previous period (Fig. 21c). The microvesicles increase their number and form complicated groups in the cubic mesothelial cells (Fig. 21d). Most of the microvesicles are in contact with the apical and basal membranes. The microvilli are more numerous and longer, as compared to the previous stage, especially in the peripheral zones of the cells, and are more numerous on the VP. The interdigitations run a complicated course, with zonulae occludens located apically, followed by zonulae adherens. In some places large intercellular dilations are visible, limited only apically with specialized contacts (Fig. 21e). The cytofilaments produce bundles and are free in
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Fig. 21a–g Prenatal development of the human VP. 21–36 GW. a,b 21–24 GW. a A cubic cell (C). Thin BL (arrows). (×3,000). (From Michailova 1996, with permission of Elsevier). b Lumen of a lymphatic vessel (Lv) in the vicinity of a flat cell (F). Small collagen bundles (stars). (×3,800). c–e 25–32 GW. c Cubic cells (C). Large collagen bundles (stars) in the thick submesothelial layer. (×3,000). d Group of microvesicles (asterisk). Thin BL (arrows). (×11,000). e Microvilli in the vicinity of intercellular contacts (small arrows). Cilium (large arrow). (×6,300). (c–e from Michailova 1996, with permission of Elsevier). f,g 33–36 GW. f Two cubic cells (C). Elastic fibers under the BL (arrows). (×3,000). g Blood capillary (large arrow) and peripheral alveoli (stars) in the vicinity of the mesothelium (small arrows). (×2,800)
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the cytoplasm, or are in close contact with the apical and intercellular membranes. The elastic-like fibers with a microfibrillar periphery and an amorphous center may be seen under the BL. The thick submesothelial layer is composed of fibroblasts, a few macrophages and extravasal cells, as well as larger collagen bundles, and numerous blood capillaries. Few large lymphatic vessels are a consistent finding in the VP. In some places this layer continues to run directly into the connective tissue of the underlying tissue interstitium. At 33–36 GW, both cell types have more numerous and longer microvilli, compared to the previous stage. The multi-layered mesothelium is not visible. The organelle content (especially the Golgi complexes and cisterna of the rough ER), microvillous covering, and vesicular system appear to be richer in the cubic cells, as compared to the flat ones (Fig. 21f). The BL is thicker in the VP and forms long sectors with an electron-dense structure. The frequently interrupted EM may be observed under the visceral BL, together with single isolated short and thin elastic fibers in the submesothelial tissue. The connective tissue layer generally remains markedly thick in both the pleural sheets. In some points the peripheral alveoli are in close contact with visceral BL and the submesothelial layer becomes extremely thin (Fig. 21g). Small lymphatic vessels and blood capillaries are usual findings in the submesothelial layer of the VP. Large collagen bundles and lymphatic capillaries with extremely flat lumens, orientated parallel to the pleural surface, predominate in the PP. 3.3 Horseradish Peroxidase Transfer Across the Pleura and Peritoneum Within 5 min of the application of HRP to the pleural cavities of the rat and the cat, reaction product (RP) is detected on the mesothelial apical membrane and on its microvilli (Fig. 22a). Electron-dense material is visualized in vesicles connected with the apical plasmalemma and also in some vesicles in the abundant cytoplasm. The superficial areas of the intercellular spaces are likewise filled with RP. In the 10th postinjection minute, an obviously increased number of lysosomes and phagosomes in the mesothelial cells of all investigated organs is encountered (Fig. 22b). At the same time the tracer is found within vesicles presenting in variable cytoplasmic positions, some of them appose the basal and intercellular plasmalemma (Fig. 22c). Labeled vesicles are found in large amounts in the peripheral zones of the mesothelial cells, and are rather fewer in their central and perinuclear parts. The vesicles have variable electron density and in some cases the tracer is deposited on the membrane only. RP is also observed in composite vesicular complexes presenting complicated forms, as well as within vesiculo-vacuolar formations. A nonhomogenous distribution of RP is observed in the intercellular spaces (Fig. 22d). At some spaces they are dilated and come into contact with marked vesicles. On account of RP accumulation, the mesothelial BL displays marked electron density and is in direct contact with the deep zones of the intercellular spaces. RP is found in the peripheral areas of the thickened EM, in the
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intercellular spaces of the connective tissue layer, and within the alveolo-capillary BL (Fig. 22f). The peripheries of the EM and the elastic fibers in the submesothelial layer of the spleen are electron-dense. In single capillaries, RP is present in the lumen and in the vesicles of the endothelium. At 10 min after injection of HRP into the right ventricle of the rat, RP is found in the lumen of the most of the capillaries in the submesothelial layer of the VP (Fig. 23a). Here numerous vesicles of the endothelium, and its BL, are labeled. Not infrequently, adjacent vesicles disclose a varying degree of tracer accumulation. Reaction product is observed as free granules, forming groups in the cytoplasm— in the core, or bound to the terminal lamella of LB of the type II pneumocytes. The apical plasmalemma of the type II pneumocytes is labeled irregularly with HRP. The plasmalemma, large vacuoles, different membrane bound structures, and numerous phagosomes are labeled in the alveolar macrophages. The periphery of the EM and the mesothelial BL are also marked, with the latter showing a pronounced electron density (Fig. 23b). The EM in the neighboring areas is free of RP (Fig. 23c). At some points it is considerably thickened, making contact with areas of the intercellular spaces, which are filled with RP and often substantially dilated (Fig. 23d). Isolated mesothelial vesicles also contain RP. They are connected with the basal plasmalemma or are located within the adjoining cytoplasm zones. Within 10 min of HRP injection into the peritoneal cavity, RP is detected irregularly on the apical membrane, microvilli-like, and other forms of evaginations of the newborn guinea pig surface epithelium. Electron-dense material is visualized in the microvesicles connected with the apical plasmalemma, and also in those located in the underlying cytoplasm. The microvesicles exhibit variable electron density and at some points the tracer is deposited on the membrane alone. RP is also observed in vesicular and vesiculo-vacuolar formations. Some of the intercellular spaces are filled with RP only in the superficial parts, but most of them contain the marker throughout their entire lengths. Some of the intercellular spaces show RP in a nonhomogeneous distribution (Fig. 23e). The BL exhibits marked electron density. RP is not found in the lumen or endothelium of the blood capillaries of the underlying layer. At 10 min after HRP injection into the abdominal aorta, RP is
Fig. 22a–f Rat and cat pleura and peritoneum after HRP. a Rat. Lung. RP on the apical membrane, microvilli and microvesicles (arrows). (×3,200). (From Mikhaylova and Vasilev 1988, with permission of Springer Verlag). b Cat. Diaphragmatic peritoneum. Group of lysosomes (Ly). (×8,000). (From Michailova 1996, with permission of Elsevier). c Cat. Spleen. RP in microvesicles (stars) and in intercellular spaces (arrows). (×6,300). d Rat. Lung. RP in microvesicles (stars), in the intercellular spaces (arrows) and BL (empty arrows). (×24,000). (From Wassilev et al. 1985, with permission of Elsevier). e Cat. Lung. Electrondense peripheries (arrows) of the EM (EM). (×15,000). f Rat. Lung. RP in the collagen bundles (asterisk), alveolo-capillary BL (small arrows), lumen (empty arrow) of a blood capillary and in activated macrophage (large arrow). (×6,000). (e, f From Mikhaylova and Vasilev 1988, with permission of Springer Verlag)
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found in the lumens of most blood capillaries in the deep part, and in few of those in the superficial zone of the subepithelial layer. RP is present in the endothelial microvesicles. Microvesicles located in the immediate vicinity show a varying degree of tracer accumulation. In some places, the BL of the capillaries accumulates the RP. Electron-dense material is visible between the collagen bundles and on the periphery of the elastic fibers in the underlying layer (Fig. 23f). The BL of the ovary surface epithelium is also labeled. The cells of the multi-layered epithelium appear as one of two types according to their density (electron-lucent and electron-dense). Some of the intercellular spaces between these cells preserve an electron-lucent view and the usual contacts, but others form round dilatations. Most of the intercellular spaces are filled nonhomogeneously with RP and are markedly dilated. The neighboring spaces show different degrees of homogeneous electron density. Others are filled homogeneously with electron-dense material, or have round electron-lucent membrane bound profiles. Vesicles, or single vacuoles in the cells of the surface epithelium, contain RP also. 3.4 The Injured Serosal Membranes and Their Response 3.4.1 Experimental Hemothorax Six hours following EH the monolayer mesothelium of the both pleural sheets shows an abundance of long microvilli and a more extensive vesicular system, than that of control animals (Fig. 24a). Numerous cubic cells have a prominent lysosomal system. Single, or groups of, primary or secondary lysosomes occupy the cytoplasm in the vicinity of the intercellular spaces (Fig. 24b). The vesicles, vacuoles, membrane-bound profiles and dilated intercellular zones contain a homogeneous electron-dense product (Fig. 24c). Specialized contacts are lacking in the extremely widened intercellular spaces. Membrane-bound formations of varying size and irregular form are filled with granulo-filamentous material. The BL and periphery of the EM show a conspicuous electron density. Fig. 23 a–d Rat pleura after HRP and e, f superficial epithelium of guinea pig after HRP. a RP in the BL (small arrows), in the lumens (large arrows) of blood capillaries and in the macrophageal lysosomes (stars). Blood capillary (Bc) without RP. (×2,700). b RP in the peripheries of elastic fibers (small arrows), in the collagen bundle (large arrow) and lumens (asterisks) of blood capillaries. (×4,500). (From Savov and Michailova 1991, with permission of Elsevier). c RP is lacking over the EM and the lumen (stars) of a large blood vessel. (×2,700). (From Kikhaylova and Vasilev 1988, with permission of Springer Verlag). d RP in the dilated intercellular spaces (large arrows) and in the microvesicles (small arrows). (×20,000). e RP and vacuoles in the dilated intercellular spaces (long arrows). Electrondense microvesicles (short arrows). (×4,000). f RP and large vacuoles in a wide dilation (large arrows). Electron-dense material in the submesothelial layer (small arrows). (×2,400)
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The two pleural sheets display a multi-layered arrangement over the BL 24 h after EH. The mesothelial cytoplasm shows widened cisternae of rough ER, secondary lysosomes and an abundance of vesicles (Fig. 24d). The superficial cells are connected by extremely thin peripheral zones, where specialized contacts are rarely observed. The mesothelial cells with degenerative changes in cytoplasm and organelles form an unusually thin covering, separated from underlying cells (Fig. 24e). The extravasal cells (exclusively neutrophilic granulocytes) are observed in a superficial position to the pleural cavity. Fibroblast-like cells with thin cytoplasmic processes, cells with lymphocyto-plasmocytic or with monocytomacrophageal characteristics are seen over the BL (Fig. 24f). An electron-dense material irregularly fills the widened intercellular spaces between these cells, as well as between them and the boundary mesothelium. In these places specialized contacts are lacking. Connective tissue and extravasal cells are located between the interrupted BL and the remnants of the EM (Fig. 24g). The multiplication of the cells described above, and wider intercellular spaces in the connective tissue of the both pleural sheets, lead to an increase of thickness of their submesothelial layer. The furrows and ridges over two pleural sheets are more prominent than those in control animals by day 3 post-EH. Extremely highly activated mesothelial cells, with several times greater vertical than horizontal diameter, form deep invaginations over broader regions of the VP, than of the PP (Fig. 25a). The activated cells have a lobulated nucleus, numerous parallel cisternae of rough ER, several Golgi units, groups of lysosomes, mitochondria, an abundance of microvesicles, and clusters of electron-dense granules. More numerous and longer microvilli cover the activated cells. Single occludens or adherens types of contacts are seen in the apical portion, while the following intercellular dilatations are often opened toward the BL (Fig. 25b). The thicker BL has a homogeneous electron-dense appearance, irregular course and interruptions. Remnants of EM are inconstantly present. Small collagen bundles and single elastic fibers of variable course slightly increase the thickness of the submesothelial layer. Fig. 24a–g EH. a–c Six hours after EH. a Lung. Activated cell with numerous vesicles, vacuolus (star) and lysosomes (arrows). The EM is disrupted. (×5,500). b Lung. Group of lysosomes (large arrow) in the vicinity of electron-dense intercellular dilatations (small arrows). (×9,000). (a, b From Michailova 2004, with permission of Elsevier). c Costal pleura. Large dilatation (arrows) with electron-dense content. (×12,600). d–g Twenty-four hours after EH. d Lung. Dilated cisterna of rough ER in the mesothelium (m). Electron-dense material in widened intercellular spaces (arrows) under the mesothelium (m) and between the underlying cells. (×8,400). e Lung. Extremely thin mesothelium (small arrows) over two neutrophilic leukocytes (N) and cytoplasmic fragments (large arrow). (×9,000). f Lung. Thin mesothelium over lymphocyte-like cell (asterisk). Neutrophilic leukocytes (N) over the BL (arrows) and between it and EM (EM). (×4,200). g Lung. Profile with heterogeneous content (large arrow) in the mesothelium. Neutrophilic leukocytes (N). Macrophage-like cells (stars). Cell between BL (small arrows) and EM (EM). (×8,000). (d–g From Michailova 2004, with permission of Elsevier)
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Fig. 25a–g EH. a–c Three days after EH. a Lung. Numerous microvilli in deep invaginations between activated cells (C). Electron-dense granules (arrows). (×3,000). (From Michailova 2004, with permission of Elsevier). b Diaphragm. Bundles of cytofilaments (arrows), numerous microvesicles and microvilli in intercellular dilatation (asterisks). (×10,000). c Five days after EH. Lung. LB (arrow). (×9,000). (From Michailova 2004, with permission of Springer Verlag). d–f Eight days after EH. d Lung. Group of LB. LB with thin and concentric (small arrow), with thick and parallel (large arrow), and with thick and radial (empty arrow) arranged membranes. (×8,000). (From Michailova 2004, with permission of Elsevier). e Lung. Elastic-like structures (arrows) in the mesothelial cytoplasm. Thicker EM (EM). (×10,000). f Lung. EM (EM) with double course. (×6,000). g Fifteen days after EH. Costal pleura. Large collagen bundles (stars) and single, short elastic fibers (arrow) closely packed in the thick submesothelial layer. (×2,600). (f,g From Michailova 2004, with permission of Elsevier)
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Activated mesothelial cells remain a predominant cell type in the boundary layer 5 days after EH. The LB form small groups and are located near to the intercellular spaces, or in them. The electron-light center of LB is enveloped by few, fine concentric membranes (Fig. 25c). Vesicle-like profiles, surrounded by common external membranes, form complex LB, in which groups of vesicles are separated by single or several membranes. In only a few cases membranes have a perpendicular position and form a net in the vicinity of microvilli. At the same time, a single or a small number of typical LB are located in vicinity of the microvilli. The mesothelial cells possess large apical evaginations, which are occupied by cytofilament bundles and clusters of vesicles on the day 8 after EH. Groups (5–15) of LB are visible near to microvilli and more often over the intercellular zones of the mesothelial layer (Fig. 25d). Concentric, parallel, radiating or irregular arrangement of the membranes form spheres, cylinders, rounded, ovoid, or irregular shaped LB of different sizes. The mean diameter of the LB after 8 days of EH is significantly increased (13.54±0.6µm) over that of the control animals (4.95±0.17µm). Membranes of LB show considerable variation in their number and thickness. Single balloon-like structures over microvilli are visible; their external membrane covers a homogenous core with moderate electron density. Larger and oval coagulated particles are disposed over the apical portion of the most cubic cells. Elastic-like structures of irregular form, and probably without membrane envelopes, are observed in the mesothelial cytoplasm (Fig. 25e). The BL of two pleural sheets shows a homogeneous electron-dense structure and forms local interruptions. The EM is several times thicker in the longer areas of the PP, as compared with those of the control animals. EM with an irregular course builds protrusions (toward the mesothelium, or toward the submesothelial layer) and bifurcations. It shows changes in the electron-density, structure (homogeneous or filamentous) and building of its peripheries. Double EM is visible over areas of both pleural sheets (Fig. 25f). The submesothelial layer is increased in volume due to the more numerous collagen bundles and elastic fibers (more extensive in the PP). The lung surface displays deeper furrows and ridges at day 15 after EH, than the control animals. Cubic and flat cells cover the VP and PP, respectively. The BL is thinner than that in the normal rats. Numerous cells (fibroblasts, macrophages, mastocytes, single lymphocytes, neutrophilic and eosinophilic granulocytes) and an increased number of collagen bundles, are loosely organized in the VP and are densely packed in the PP (Fig. 25g). Short elastic fibers are dispersed between collagen bundles, or form elastic membrane-like structures in the submesothelial layer. Newly formed blood capillaries are surrounded by fine collagen bundles. Alterations of stomata and the underlying lymphatic vessels are not observed. Components of the submesothelial layer mentioned above form wider and deeper septae between the underlying tissue in the PP than in the VP. Areas of different lengths in both pleural sheets show identical structure, as in the control animals.
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3.4.2 Healing after Pneumonectomy Twenty-four hours following PNT both pleural sheets (more often of the VP) have longer sectors with the multi-layered arrangement than in the control animals. Extravasal cells, predominantly neutrophlic leukocytes are located under the superficial mesothelium (Fig 26a). In some areas erythrocytes and neutrophilic leukocytes form a boundary band over the mesothelium, which is observed in all investigated periods. The mesothelial cells have numerous and longer microvilli and a more extensive vesicular system than the control animals. The mean length of the microvilli is significantly longer than in the control group (t = 10.08; P < 0.001). Some of them show an increased number of primary and secondary lysosomes. The mesothelial cells communicate with a lower number of occludens contacts. The mean number of occludens contacts is significantly decreased 24 h after PNT compared with the control group (t = 7.8; P < 0.01). The interdigitations and adherens junctions are substituted by small intercellular dilatations. Numerous mesothelial cells are connected by extremely thin peripheral zones which lack specialized contacts (Fig. 26b). The multiplication of the extravasal cells and wide intercellular spaces in the connective tissue layer of both pleural sheets leads to its increase in thickness. The lumens of the submesothelial and of the peripheral lung blood capillaries are filled mainly with erythrocytes. Five days after PNT the superficial layer is formed by several rows in almost all areas of the VP and in some parts of the PP. The remaining mesothelium keeps the monolayer arrangement, which is difficult to distinguish from that of the control animals. The superficial cells in the multi-layered zones possess the features of the mesothelium (Fig. 26c). The cell populations under the superficial cells show morphologic characteristics which are different from those of the typical mesothelial cells. The first type of these cells has an abundant rough ER, which occupies a considerable volume of the electron-dense cytoplasm (Fig. 26d). The second type represents fibroblast-like cells, which show long cytoplasmic processes with single, thin, short collagen fibers or extremely fine bundles in their vicinity. The third cell type has well-developed lysosomal and vesicular systems in the electron-lucent cytoplasm. Some of the cells have cytofilament bundles or clusters of electron-dense granules in the cytoplasm. The extravasal cells above the BL decrease in number. The visceral BL has an interrupted appearance, a superficial electron-lucent and a deep electron-dense part and at this time it appears as a wide, homogenous electron-dense structure. The parietal BL shows extremely thin electron-dense part. The submesothelial layer is increased in volume due to connective tissue cells, a small number of extravasal cells and large intercellular spaces. The mean thickness of the submesothelial layer of the VP (25.9±2.4µm) and PP (72.9±3.7µm) is significantly higher at 5 days after PNT than in the control group of the VP (13.8±2.5µm) and of the PP (33.3±2.6µm). There are numerous small collagen bundles (more extensive in the PP), which are organized loosely
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Fig. 26a–e Pleura after PNT. a,b One day after PNT. a Diaphragmatic pleura. Neutrophilic leukocyte (N) in multi-layered sectors. Large mesothelial evagination (arrow). (×9,000). b Costal pleura. Extremely thin mesothelial sector (large arrow) over a thin, interrupted BL (small arrows). (×5,500). c–e Five days after PNT. c Lung. Multi-layered sector built from: activated mesothelium (m), electron-lucent cells (stars), cytoplasmic fragment with lysosomes (large arrow), collagen fibers (small arrows) and neutrophilic leukocytes (N). (×4,500). d Lung. Mesothelial cells with numerous cisternae of rough ER. Collagen bundles under a thin BL (arrows). (×4,500). e Diaphragmatic pleura. Neutrophilic leukocytes and erythrocytes cover the thin mesothelium (small arrows). Collagen bundles around group of blood capillaries (large arrows). (×1,900)
and have different directions. Single short elastic fibers are seen in the PP. More numerous blood capillaries, which form groups, are seen in the submesothelial layer of both pleural sheets (Fig. 26e). Eight days after PNT the lung surface shows more numerous furrows and ridges, compared with control animals. The superficial layer form several rows in broad regions of the VP and in short sectors of the PP. It is difficult to find clear mor-
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phologic differences in some multi-layered sectors, compared to the day 5. Typical features for this period are the multi-layered areas, which have only cells with mesothelial characteristics. They surround intercellular dilatations and contain microvilli of the neighboring cells, or more rarely granulo-filamentous material. Superficial cells of these sectors have rich microvillous coverings. Characteristic for this period are the extremely high ‘activated’ mesothelial cells. They have more numerous microvilli, abundant dilated cisterna of rough ER, prominent Golgi zones, numerous lipid droplets, and a rich vesicular system (Fig. 27a). Some mesothelial cells show degenerative changes in their organelle apparatus. Collagen bundles of different sizes and with variable orientation of fibers are located in multi-layered sectors (Fig. 27b). Electron-dense BL in the VP is thicker, with local thickenings and bifurcations, while the parietal BL remains extremely thin. The EM is inconsistently present in the VP. It is thickened several-fold with protrusions or local bifurcations, and has a double-layered structure in some zones. The submesothelial layers in both pleural sheets remain thicker. Predominant elements in them are the large collagen bundles, long elastic fibers and newly formed blood vessels (Fig. 27c,d). These components are more numerous and have a looser arrangement in some sectors of the PP. The PP shows groups of LL, which are located in close contact with each other and in the vicinity of the mesothelium. In some places the overlying mesothelium produces broad intercellular spaces, stomata, where the mesothelium makes contact with the underlying lymphatic endothelium and the BL is lacking. The numbers of pleural villi increase on the VP. In most cases they have a thick connective tissue core (Fig. 27e). 3.4.3 Postinflammatory Changes of the Peritoneum with Special Reference to Stomata At 24 h after EP the number and the length of the microvilli and other evaginations of the peritoneal mesothelial cells are more numerous than in the control animals. Extremely large evaginations of the apical cytoplasm with abundant vesicles in them are observed. Microvesicles, vesicular complexes and large vacuoles with a homogenous electron-lucent content are increased, too. Primary, and more numerous secondary lysosomes form groups (Fig. 28a). Some of them have a large diameter. Only single adherents or more rarely occludens contacts are seen apically, while the large distal part of the intercellular spaces form round dilatations. They are often opened to underlying tissue. The mesothelial basal plasmalemma displays an undulating course, with numerous cytoplasmic processes towards the BL, and thus the submesothelial layer. In some places the evaginations of the mesothelial cytoplasm are not followed by the smooth BL and between them there are large electron-empty spaces (Fig. 28b). The mesothelial cells with interrupted apical membranes, a small number of microvilli, dilated cisternae of rough ER and Golgi apparatus, and degenerative changes in the nucleus are seen far more frequently than in the control animals (Fig. 28c). The BL of the two serosal sheets shows a superficial electron-lucent part and a deep, extremely thin, interrupted,
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Fig. 27a–e Pleura 8 days after PNT. a Costal pleura. Activated mesothelium (m). Thick submesothelial layer with cells, large collagen bundles, elastic fibers (arrows) and blood capillary (star). (×6,000). b–d Lung. b Two large collagen formations (large arrows), small collagen bundles (small arrows) and electron-dense granules (empty arrow) in a multilayered mesothelium. (×7,500). c Thin collagen bundles (small arrows) in multi-layered mesothelium (m). Septa-like structures (large arrows). (×2,200). d Multi-row mesothelium (m). Collagen bundles (arrows) under the thin BL and around the blood capillary. (×2,000). e Diaphragm. Thin mesothelium covers a collagen core of a pleural villus. (×3,800)
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electron-dense part. Numerous furrows and ridges are observed over the surface of the visceral sheet of the peritoneum. The central portions of most of the mesothelial cells are located in the invaginations of the submesothelial layer and occupy the bottom of the furrows. The larger portions of the surface of the small intestines and the stomach have the rounded folds (Fig. 28d). The number of extravasal cells (exclusively neutrophilic leukocytes) increases and the intercellular spaces become larger in the connective tissue layer. Five days after EP furrows and ridges persist over the surface of the visceral serosal sheet. Some cells show degenerative changes, as in the previous period. The activated mesothelial cells have a rich microvillous cover and vesicular system. Vacuoles (3–5 µm in size) with homogeneous, electron-lucent or heterogeneous granulo-filamentous content, and more rarely with irregular membrane profiles, occupy a large portion of the cytoplasm (Fig. 28e). The usual findings of this period are single giant rounded vacuoles, which occupy almost the entire cytoplasm of the mesothelial cells (Fig. 28f). Other constant features are the LB. They are located under the mesothelial basal plasmalemma, or under the BL, as well as in the superficial part of the connective tissue layer (Fig. 29a). Single or small groups of LB are disposed in the basal and lateral cytoplasm or in the intercellular dilatations. An electron-light center, surrounded by small number of thin, concentric or parallel membranes with regular periodicity builds the LB in this period. Intercellular dilations, round or irregular in form, are closed apically with single occludens or adherens contacts and are opened towards the BL. Some contacts are completely disrupted. Single cells lose the junctions with neighboring cells and with the BL (Fig. 29b). The central portions of these cells protrude towards the cavity and only the cell peripheries are located close to the BL. The mesothelial basal plasmalemma and the BL show the features of the previous period described. Long newly formed elastic fibers are found under the thin BL and/or in the submesothelial layer as a deep EM-like structure in the liver, the stomach and the intestines. The part of the submesothelial layer over the EM-like structure is loosely organized as compared with the underlying connective tissue. The most interesting features in the Fig.28a–f EP. a–d One day after EP. a Spleen. Group (large arrows) of vacuoles and lysosomes. Round intercellular dilatation (small arrow). Wide spaces between the collagen bundles (stars) in the submesothelial layer. (×6,300). b Liver. Large spaces (stars) between the mesothelial cells (M) and the extremely thin, interrupted BL (arrows). (×5,700). c Stomach. On the left is shown homogenous material and microvilli (arrows) under a mesothelial cell (star) with degenerative alterations. On the right there is an activated cubic cell (C). (×6,300). d Intestine. Deep furrows (large arrows). The mesothelial basal plasmalemma is disconnected from the BL (small arrows). Wide spaces (stars) under the BL. Large collagen bundles (asterisks). (×4,500). e,f Five days after EP. e Stomach. Membrane bound electronlucent spaces (arrows) in the cytoplasm and between the cells. Loosely arranged over and densely packed collagen fibers under the EM-like structure (arrows). (×7,700). f Spleen. Giant vacuole (large star) in the mesothelial cell. Intercellular dilatations (small stars). (×5,000)
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connective tissue layer are the extremely large rounded or oval formations that are surrounded by a membrane or by collagen bundles which enclose and finely project into them (Fig. 29c). These structures show homogeneous content with variable electron density or a heterogeneous matrix with a granulo-filamentous structure. There are also membrane profiles that separate the matrix. Under the BL large, electron-lucent spaces are seen, that are surrounded by large collagen bundles. The fibers in them are short, with different orientations and they are arranged loosely around few fibroblasts. Single, large collagen bundles have densely packed fibers. The LL increase their size and are located in close proximity to the diaphragmatic mesothelium (Fig. 29e). Different types of direct contacts between mesothelium and protrusions of endothelium of the LL are observed, with or without BL at day 5 after EP. Extremely thin mesothelium on BL covers a fine endothelial intercellular zone without BL. Mesothelial interruptions are disposed over flattened processes of the neighboring endothelial cells of LL. Various types of contacts are seen: thin mesothelium cover the fine protrusions of the lymphatic endothelium; thick, funnel-shaped contacts between the two cell populations with a common BL; contacts between processes of the mesothelial and endothelial cells over a common BL (Fig. 29e,f,g). More numerous typical stomata are visible than in controls. Eight days after EP long mesothelial sectors show a rich microvillous coat. Large electron-lucent spaces are formed between the mesothelial basal plasmalemma and the BL, or under the latter. Some mesothelial cells interrupt their connections with BL and with the adjacent cells. In some areas a cellular sloughing at different stage is seen, as in the previous period. Groups of isolated LB (two to five) or larger formations, package with common external membrane, and several centers of concentric lamellae are located in the intercellular spaces (Fig. 30a). A homogenous material with moderate electron-density fills the intercellular dilatations and surrounds an electron-lucent halo around lamellar formations. The mean diameters of the LB 8 days after EP and the control group show insignificant differences. The mesothelial cells with abundant microvilli and filled with vesicles cover the serosal villi of the stomach (Fig. 30b). A connective tissue core is lacking in such villi. A larger amount of granulo-filamentous material, that is also more rough in comparison with that Fig. 29a–g Five days after EP. a Stomach. LB (arrow) under the mesothelial basal plasmalemma. Membrane profile (star). (×9,000). (From Michailova 2004, with permission of Springer Verlag). b Spleen. Large spaces (stars) under the mesothelial cells. Only the intercellular zone (large arrow) is located close to remnants of BL. Continuous, newly formed EM (small arrows). (×4,800). c Stomach. Activated mesothelial cells. Large collagen bundles (stars). Electron-lucent spaces (arrows) in the submesothelial layer. (×5,000). d–g Diaphragm. d LL (star) in vicinity of cubic cells (C). Cilium (arrow). (×4,500). e Contact (arrows) between intercellular zone of cubic mesothelium (C) and an endothelial protrusion of a LL (star). (×6,800). f Tunel-like contact (arrows) between cubic mesothelium (C) and endothelium of a LL (star). (×4,000). g Processes of mesothelial (M) and endothelial (E) cells of a LL over a common BL (arrows). (×6,500). (e–g From Michailova 2001, with permission of Elsevier)
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in control animals, is observed over a strip-like formation and between microvilli. The strip-like structure is thicker than in untreated animals and covers the cubic mesothelium of the diaphragm, stomach and spleen. The deep EM mentioned above, which divides the connective tissue layer into a loosely organized superficial part and a densely packaged underlying part, becomes significantly thicker (Fig. 30c,d). Fibroblasts and a smaller number of extravasal cells are surrounded by wider spaces, in which abundant, newly formed small collagen bundles with a different orientation, and extremely disarranged and short elastic fibers are seen. The submesothelial layer is thicker and loosely organized; it extended as thick septae deep into the underlying tissue of all the organs investigated. An intriguing feature is the appearance of new LL. They are covered with clusters of cubic mesothelial cells. The LL are located in close proximity to the mesothelium on the both sides of the diaphragm, the anterior abdominal wall and the liver (Fig. 30e). The mesothelial covering over the MS in the greater omentum is built by extremely activated mesothelial cells. They have abundant dilated cisternae of rough ER, prominent Golgi zones, a rich vesicular system and more numerous microvilli. Some mesothelial cells show degenerative changes in their organelle apparatus. The MS are disposed closer to the mesothelium, as compare with those in normal rats. They increase in number and in size. The connective tissue component (fibroblasts, collagen fibers/bundles, and lymphocytes) decreases in abundance and often is lacking. Most MS are composed of a large number of free cell accumulations separated by wider intercellular spaces (Fig. 30f). The activated macrophages form large groups and are filled with secondary lysosomes. The superficial position of the macrophages is observed far more often when compared with the healthy animals. The quantity of lymphocytes appears to be only slightly changed. They occupy a superficial position under the mesothelial cells, or together with plasmocytes they form small groups in the center of the MS. The number of the neutrophilic leukocytes is increased and they are arranged in clusters without other cells types. The perivascular cells are lacking around the more numerous small blood capillaries, i.e., there is a formation of new vessels. Few units of mast cells and eosinophilic leukocytes are located in vicinity of blood capillaries. The number, shape and size of the lymphatic capillaries, as well as the appearance of the stomata are comparable with those observed in the MS of untreated rats. Fig. 30a–f Eight days after EP. a Stomach. To the left is shown a group of LB, and to the right complex LB (arrows) in the intercellular spaces. (×5,000). (From Michailova 2004, with permission of Springer Verlag). b Stomach. Large portion of a peritoneal villus. (×4,000). c Spleen. Thin BL (empty arrow). Numerous collagen bundles (asterisks) and elastic fibers (arrows) in a thick submesothelial layer. (×3,800). d Small intestines. A large collagen bundle (asterisk). Deep EM-like structure (arrows). (×3,300). e Liver. Large spaces (asterisks) and collagen bundles under the mesothelium (M). Extremely thin endothelium of the LL (LL). (×7,700). f Greater omentum. Portion of a MS. Fibroblast (F), part of a macrophage (arrow), erythrocytes (E), neutrophilic leukocytes (N) and a blood capillary (star). (×2,700)
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Eight days after EP an amorphous, continuous and folded covering of coagulated material, as seen by SEM, is located over large areas of cubic mesothelium and, as usual, over cubic cells in the vicinity of stomata (Fig. 31a). Thin peripheral zones of cubic mesothelial cells form broad intercellular openings, where the mesothelium contacts the underlying endothelium of LL by means of single digitations (Fig. 31b). These form new channel-shaped openings, stomata, as interruptions of the mesothelial layer. Some stomata are observed to form groups (Fig. 31c). The mean size of the stomata (10.35±0.45µm) is significantly increased 8 days after EP, as compared with the control group (2.65±0.29µm). The majority of the LL show oval-shaped contours and forms groups. The mean horizontal (155.57±31.97µm) and vertical (50.59±7.36µm) diameters of the LL in the same time interval are larger compare with the horizontal (74.08±9.23µm) and vertical (12.37±2.45µm) diameters of normal animals. The great proportion of the mesothelio-endothelial channels are made only from mesothelial cells, while the endothelium participates in formation of the part of the channel located in the submesothelial layer. In some cases, endothelial cells of LL are extended toward the peritoneal cavity and form the larger portion of the channels. The BL is lacking at the stomata, but often the contacting edges of the mesothelial and the endothelial cells are located over a common BL. The cubic mesothelial cells overlying the LL form numerous apical and basal evaginations (Fig. 31d). The cytoplasm of the mesothelium and the underlying lymphatic endothelium display rich vesicular systems. Small valve- or bridge-like structures are identified in close proximity to stomata (Fig. 31e). They are formed by cytoplasmic processes or by nuclear portions of the endothelial cells of LL. Tall endothelial cells with apical evaginations and electron-dark bodies form the wall of LL (Fig. 31f). More rarely, the cytoplasmic processes of mesothelial peristomatal cells overlap each other and close stomata. On the opposite side of the LL, and in single cases in the vicinity of stomata, lymphatic endothelium forms typical valves with connective tissue core (Fig. 31i). Common septae separate the neighboring LL (Fig. 31j). They contain a core of fine connective tissue. Usually the septae are covered with extremely thin endothelium on the common BL. In some places, the nuclear portions of the endothelial cells display prominent protrusions over a thicker connective tissue core.
Fig. 31a–j Diaphragmatic stomata after EP. a Uninterrupted coat over the cubic cells. (×1,500). (From Michailova 2004, with permission of Springer Verlag). b Stomata (long arrow). Contact (short arrows) between cubic cells (C) and endothelial cells (E) of the LL (star). (×7,700). c Group of stomata openings (arrowheads). (×4,800). d Apical (arrows) and basal (empty arrows) evaginations of the mesothelium over LL (asterisk). (×5,700). e Cytoplasmic process (empty arrow) and nuclear portions (arrows) of endothelial cells in the vicinity of stomata. (×8,000). (d,e From Michailova 2001, with permission of Elsevier). f Electron-dense bodies (arrows) in the tall endothelial cells of a LL (star). (×2,500). g Endothelial cells cover the valve of LL. (×3,000). (From Michailova 2001, with permission of Elsevier). h A septa between two LL (stars). (×5,000)
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3.4.4 Pleura after Application of Melted Paraffin At the first day after application of melted paraffin, both pleural sheets show only small sectors with preserved structure—the usual monolayer mesothelial arrangement. The number of the microvilli, evaginations, microvesicles and lysosomal system are more extensive of the mesothelial cells of the VP in these sectors. Different membrane-bound profiles in the vicinity of the microvillous border are seen. During the same time a large portion of the mesothelial cells possess numerous dilated cisterna of rough ER and vacuolized mitochondriae. Single extravasal cells (exclusively neutrophilic leukocytes) are located between the mesothelial cells and in the superficial part of the submesothelial layer. Degenerative alterations in the nucleus and organelles of the mesothelial cells characterize some areas of the both pleural sheets. Other larger zones are entirely devoid of the mesothelial covering and the BL or the EM remains in a superficial position towards the cavity (Fig. 32a). Free-floating mesothelial cells are visible near to the stripped surface. More numerous areas on the VP as compared with the PP have short zones of a multi-layered mesothelium (Fig. 32b). The superficial mesothelial cells in these regions are covered with numerous microvilli and show abundant dilated cisterna of rough ER. The underlying cells emit long cytoplasmic processes, and are surrounded by large angulated intercellular spaces. Microvilli are lacking over the deeply located cells of these sectors. The BL is difficult to distinguish over wide areas, while the undulated and extremely thick EM is the most prominent feature of the VP. Three days after melted paraffin application only short sectors of the PP have a monolayer building from activated cells filled with numerous dilated cisterna of rough ER. The layer over the BL shows a multi-layered arrangement in the larger sectors of the VP. Two different cell types may be distinguished between the superficial cells in the multi-layered areas of the VP (Fig. 32c). The majority of these cells is rounded, covered with microvilli and has an internal structure similar to that of the mesothelial cells. Few cells resemble macrophages. They have numerous primary or secondary lysosomes, vacuoles with heterogeneous content and different shaped evaginations. Spaces of variable size are formed between the superficial cells and they do not form a continuous row. The rounded underlying cells possess well developed rough ER or, more rarely, macrophage-like structures Fig. 32a–e One, three and five days after application of melted paraffin. a,b One day. Lung. a BL is lacking. Remnants of EM (stars). Free mesothelial cell (M) in the pleural space. (×4,500). b Large intercellular spaces (stars) under the mesothelial cells. (×2,600). c Three days. c Costal pleura. Mesothelial cells (m). Rounded cells with well developed rough ER form the underlying layer. (×2,000). d,e Five days. d Costal pleura. Elongated cells with microvilli form a superficial layer. BL is lacking. Parallel rows of fibroblasts (arrows). (×4,000). e Lung. Long processes (large arrows) of mesothelial cell over the thin, newly formed BL (small arrow). (×5,500)
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border the large intercellular spaces. They form several rows, as a thick layer under the free superficial cells. The BL has a smooth course over the underlying EM. Extravasal cells (almost exclusively neutrophilic leukocytes, and far more rarely eosinophilic leukocytes) and large intercellular spaces form the thicker submesothelial layer. Collagen bundles of variable length, different patterns of packaging and a defined course are located in the large spaces between connective tissue and the extravasal cells of the same layer. At day 5 after application of melted paraffin the superficial mesothelial cells have an elongated shape, form thin intercellular spaces, and microvilli predominantly cover the apical surface (Fig. 32d). Their basal processes contact (without specialized junctions) to each other and to BL and form a multi-layered mesothelial covering. The underlying fibroblast-like cells form a densely packed thick layer. Single or small groups (three to five) of elongated mesothelial cells emit basal processes, which are attached to neighboring cells and to the thin BL. These mesothelial cells have well developed organelles (Fig. 32e). Eight days after melted paraffin application sectors of the VP show the multilayered sectors of different lengths. These cells surround rounded spaces of variable size and filled with microvilli, in contrast to the previous period (Fig. 33a). During the same time interval other sectors of both pleural sheets show cubic mesothelial cells with well defined organelles, which contact with denuded cells from the mesothelium and the BL areas (Fig. 33b). Short collagen fibers over the thick EM are located in the VP. Numerous small collagen bundles form a thicker submesothelial layer, as compared with the previous period. Fifteen days after melted paraffin application, small collagen fibers are located between the cells of the multi-layered sectors (Fig. 33c). Large protrusions characterize both pleural sheets (Fig. 33d). They have an exclusively fine connective tissue core composed of single fibroblasts and short, thin collagen fibers. In addition there are activated mesothelial cells with degenerative changes over them. It is difficult to distinguish the BL in these sectors. The EM is located deep and does not participate in these formations. In longer pleural sectors of the VP, as compared with the PP, the BL is lacking or is thinner. The EM under the mesothelium is several times thicker in the larger areas of the VP and of the diaphragmatic pleura. Single collagen fibers are located between the mesothelium and the EM of the VP. Fig. 33a–e Eight and fifteen days after application of melted paraffin. a,b Eight days. Lung. a The multi-layered sector with large intercellular spaces (stars) filled with numerous microvilli. (×2,500). b To the left are two cubic cells (C) over thin BL (empty arrows) and to the right mesothelial covering and BL are lacking (large arrow). Thin collagen fibers (stars) over the EM (small arrows). (×2,400). c,d Fifteen days. c Lung. Collagen fibers (small arrow) between mesothelial cells (stars) with multi-layered. Thick elastic fibers (large arrows). (×2,800). d Diaphragm. Mesothelial cells over loosely organized core form a large pleural evagination (E). EM-like structure (arrowheads). (×2,400). e Costal pleura. Rounded spaces (stars) and deep EM (arrows) in the thick submesothelial layer. (×2,000)
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More numerous cells (fibroblasts, macrophages, mastocytes, single lymphocytes, neutrophilic and eosinophilic granulocytes), and an increased number of small collagen bundles are loosely organized in the VP submesothelial layer. Numerous short elastic fibers with different orientations are visible in the both pleural sheets. Few cells, wide-meshed collagen bundles, and elastic fibers arranged parallel with the surface characterize the submesothelial layer of the PP. Components of the submesothelial layer mentioned above protrude as wider and deeper septae between underlying tissue in the PP, as compared with those in the VP. The thick constant EM with an undulating course and considerable continuity is disposed in the submesothelial layer of the costal and mediastinal PP (Fig. 33e). The superficial portion of the PP shows few cells, small collagen bundles and larger spaces, while the deep part has numerous cells, larger collagen bundles and smaller intercellular spaces. Large electron-empty spaces (with a microvillous covering in a few cases) are observed in both parts. Clear alterations of stomata and the underlying LL are not observed. There is no region that resembles the pleura of the untreated animals.
4 Discussion 4.1 Normal Structures of the Serosal Membranes 4.1.1 General Organization The present data confirm the general organization of the SM in man and experimental animals as previously described separately for the pleura, peritoneum, pericardium, and tunica vaginalis testis (Baradi and Rao 1980; Albertine et al. 1982; 1984; Fentie et al. 1986; Gotloib and Shostack 1987) in different species. We observed three basic types of relief over SM (Michailova et al. 1986; 1989; 1991; 1999; 2004; Michailova and Vassilev 1988a; 1988b; 1990b; Michailova 1995b; 1996b; 1997a; 1997b; 2001b; Michailova et al. 1986; 1989; 1991; 1999; 2004). The most common type forms a smooth surface and extends over the parietal sheets of the serosal cavities. It is most characteristic over both sides of the diaphragm, the costal pleura, the anterior abdominal wall, and the mesenteric duplicatures. The same pattern of smooth relief (extremely rare and broad folds alternating with shallow and wide furrows) is observed over the larger portions of some organs (heart, intestines, liver, stomach, rectum, urinary bladder and testis), regardless of their cubic mesothelial covering. A specific relief with significant individual differences at several organs (lung, greater omentum, uterine tube, uterus, ovary) characterize the second type of surface contour. The third type of surface is characterized by numerous complicated furrows and ridges, covered with cubic mesothelium (around the stomata, over LL and MS); the remaining surface of the organ is smooth. The main components (mesothelial layer, BL and connective tissue layer) of the SM in human and experimental animals are repeated in all of the organs observed.
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At the same time, they present significant quantitative and qualitative variations in the pleura, peritoneum and pericardium, in the parietal and visceral sheets, on the organs and regional diversities of the same organ, which are observed consistently (Wassilev et al. 1998; Michailova et al. 1999; 2004; Michailova 1995b; 1995c; 2001a; 2001b). These differences concern one or all of the main components of the SM. Our data support the presence of the EM under the BL as a fourth constant component of the serosal covering of the VP and the spleen peritoneum in human and animals. The described multi-layered sectors of the mesothelial layer, deep cryptae-like invaginations, papillae-like evaginations and serosal villi (Michailova et al. 1986; 1991; 2004; Michailova 1996b; 1996d; 1997b; Michailova and Takeva 1997) may be considered as highly specialized zones increasing the surface of the visceral sheet (lung, visceral sheet of the pericardium, distal end of the uterine tube, uterus, ovary) in normal situations. The mesothelial cells without other components of the SM form the multi-layered sectors, cryptae-like and papillae-like formations. All main components of the SM build the villi. The lack of blood vessels in their connective tissue core may be considered as a sign of immaturity. Blood vessels are rarely found in the smaller villi. The multi-layered arrangement of the mesothelium is reported by Whitaker et al. (1982a) only in embryonic material. Villi were mentioned in occasional light microscopic observations (Baron 1949). Sokolova et al. (1978) and Vasilieva et al. (1986) characterize them as reactive serosal structures after pathologic conditions. We observed an increased number of villi over both sheets in the late stages of PNT, EP and after application of melted paraffin (Michailova 1996c; 2001d). 4.1.2 Cubic and Flat Mesothelial Cells Some reports describe the mesothelium as a monolayer of simple squamous-like or pavement-like cells, which forms an universal covering (Holt 1970; Ivanova 1970; Gotloib et al. 1983; Digenis 1984; Fentie et al. 1986; Junqueira et al. 1987; Gotloib and Shostack 1987; Tao 1988; Slater et al. 1991; Ettarh and Carr 1996; Herrick and Mutsaers 2004). Several authors continued to consider the finding of cubic cells as an artificial result of lung collapse or of temporary swelling, as well as a consequence of pathological exposures, or as features of underlying tissue (Wang 1974; Leeson 1977; Dodson et al. 1983; Moalli et al. 1987; Bucher and Wartenberg 1989). Mutsaers (2002) defines a different enzymatic profile of the flattened resting and cubic activated mesothelial cells, which indicates a wide spectrum of functional activities. His observations support the concept that flat resting cells can become metabolitically activated cubic mesothelial cells. Our data on pleura, peritoneum and pericardium of man, rat and cat, as well as detailed observations on the pleural mesothelium in rabbit, guinea pig, ground squirrel and mouse established the existence of two types (flat and cubic) of mesothelial cells in normal conditions (Michailova and Vassilev 1988a; 1988b; 1990b; Michailova et al. 1989; 1999; 2004; Michailova 1994; 1995a; 1995b; 1996b; 1906d; 1997a; 2001a;
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2001b; Wassilew et al. 1998). The clear differences in size, configuration, organelle content and membrane specializations allow us to distinguish these two basic cell types in the SM (pleura, peritoneum, pericardium and tunica vaginalis testis) of man and experimental animals. In addition, the validity of this distinction is supported by their constant distribution in both serosal sheets, in individual organs, and in their different regions. The regional variations of the underlying structures and their correspondence with a different mesothelial coat support our view for the co-existence of two main cell types. The present observations of intimate contacts between the two cell types in one and the same section (seen by both techniques—TEM and SEM), as well as numerous intermediate cell forms and the rare degenerative cells confirm their presence. One or both mesothelial cell types characterize the sheets, organs or regions and consists of a larger portion of its covering, while the second main type represents a relatively minor portion and appears as single or small aggregates of cells. Most often the intermediate cell forms are second in frequency after the typical cells, while degenerative cells are very few present. In view of the larger surface occupied by the flat cells (parietal sheets of the SM, greater omentum, broad ligament of the uterus, mesenteric duplicatures, large areas of the covering of the intestines and of the liver, the proximal portion of the uterine tube, and the testis), we suggest that this cell type is more uniform and widespread. Delicate and gradual changes in organelles, granules, membrane specializations and cell contours from flat in the broad ligament of the uterus, toward cubic cells in the neighboring organs (surface epithelium of the ovary, uterus and uterine tube), characterize the transitional areas between them (Michailova 1997b; Michailova et al. 2004). The flat mesothelial cells have poor organelle apparatus, a scant microvillous coat and are less active. They represent a covering with more universal functions, mainly barrier and supportive (protective) as well as transport capabilities. The cubic mesothelial cells, are far more characteristic of the visceral sheets of the SM. They have well developed organelles, electron-dense granules, a rich vesicular system, numerous and longer microvilli (Baradi and Hope 1964; Kluge and Hovig 1967; Barberini et al. 1977; Tsilibarry and Wissig 1977; Mariassy and Wheeldon 1983; Michailova et al. 1995a; 1996b; 1999; 2004). They represent a large fraction of the covering of more mobile organs (lung and heart), of those with reservoir function (stomach, rectum, urinary bladder, uterus and distal portion of the uterine tube), as well as of organs subjected to volume change (spleen, ovary, portions of the liver). The cubic cells form large clusters over highly specialized regions around stomata, over LL and MS (Michailova et al. 1989; 1999; Wassilew et al. 1998; Michailova 2001e; Michailova and Usunoff 2004). The cubic, rounded or high prismatic cells constitute the multi-layered sectors, papillae-like evaginations, cryptae-like invaginations and the end of the uterine tube. In this context, it is difficult to explain the zones of flat-cell covering at the liver and the proximal portion of the uterine tube, as well as the numerous intermediate cell forms of the stomach, intestines and urinary bladder, observed by TEM. A greater number of microvilli forms a frictionless interface for the free movements of op-
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posing linings and provides a protective barrier for the underlying mesothelium (Andrew and Porter 1973). This may explain their presence on the surface of motile organs, hollow reservoir organs, as well as on organs changing their volume considerably. The cytoplasmic organization of the cubic cells suggests an enhanced synthetic and secretory potential. The mesothelial cells participate in formation of some components of the serosal fluid, surfactant, glycosoaminoglycans of the cell coat, cytoskeletal proteins, coagulation proteins, ECM components of the BL, the EM, and the connective tissue layer (Raftery 1981; Wu et al. 1982; Harvey and Almot 1983; La Rocca and Rheinwald 1984; Rennard et al. 1984; Ohashi et al. 1988; Owens et al. 1994; 1996; Dobbie 1996; Michailova 2001c; to cite only a few). In all probability, the existence of a prominent cytoskeleton in the cubic cells, especially under experimental conditions, and numerous complicated interdigitations or intercellular dilatations support their cubic contour and might be considered as important factors about the maintenance of the cell shape (Hama 1960; Mariassy and Wheeldon 1983; Alavi et al. 1985). The presence of intermediate cell forms, as well as the quantitative differences, invites the speculation that the two cell types reflect different stages of differentiation of a primary mesothelial cell, or are probably further subdivided from it. We suppose that the initial mesothelial cells are the flat ones. Moreover, in ontogenesis, the first to appear are the flat mesothelial cells. The cubic cells evolve from small patches and produce the cubic covering through the intermediate forms, while a few degenerating cells are an expression of the last stage of mesothelial life. The common origin (Sadler 1990) and the ability to change into activated mesothelial cells in experimental conditions (Tomashefski et al. 1985; Whitaker and Papadimtriou 1985; Fotev et al. 1987; Moalli et al. 1987; Michailova 1996c; 2001d; 2001e; 2004a; 2004b) support the present view. The commonly observed degenerating mesothelial cells promote the assumption on their continuous physiological ‘cloth changing’ role (Legrand et al. 1972; Krause and Leeson 1975; Ivanova and Puzirev 1977; Dodson and Ford 1985; Michailova 1995a; 1997a; Mutsaers 2004). The described macrophage-like mesothelial cells on the spleen and over the MS of the greater omentum might be considered to have mainly a phagocytic function, and enhance the active barrier capabilities of the mesothelium. The presence of electron-lucent and electron-dark mesothelial cells may be due to species differences (the VP of the guinea pig shows numerous electron-dark mesothelial cells), or as a sign of a higher differentiation of the basic cell type. Legrand et al. (1972) described mesothelial cells of different electron density in the recovery mesothelial layer. 4.1.3 Membrane Specializations and Lammelar Formations In this paragraph, we will discuss the lamellar formations–especially the LB–as apparently the most important mesothelial products in the protecting layer (Chen and Hills 2000), that preserve the space of the serosal cavities. The density of
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microvilli–the most conspicuous membrane specialization–follows the distribution of cubic and flat mesothelial cells. The latter are discussed in Sect. 4.1.2. We would like to discuss only some aspects of our ideas about them. Our results show that SEM data reveal denser populations of microvilli on the SM, than the TEM images. The cubic cells possess a more representative microvillous coat than the flat cells. These data explain the richer and longer microvillous covering of the visceral, than of the parietal serosal sheets. The most prominent microvilli possess the cubic cells of the mobile organs (the visceral pericardial sheet has the richest microvillous border), as well as parts of them (basal regions of the lung), organs with reservoir functions, and organs subjected to volume changes. Another reason for a larger density of microvilli may be connected also with active transport, determined by the submesothelial structures (over the LL and MS). Microvilli are more numerous and longer in the periphery than in the central portion of the mesothelial cell. Significant differences might be observed between the microvillous border of adjacent cells. Few mesothelial cells have only stubs of microvilli or they are completely lacking. The number of microvilli expressed on each cell changes under different physiological conditions (Madison et al. 1979). The microvillous concentrations of recovery mesothelial cells suggest that their differences reflect their functional adaptation (Wang 1974). These changes may be associated with alterations in the surface charge of the negatively charged glycocalyx, or may be due to trapping of protecting molecules from the serosal fluid by microvilli (Fedorko and Hirsch 1971; Andrews and Porter 1973; Baradi and Rao 1976; Gotloib et al. 1988; Mutsaers et al. 1996). The microvillous border, tightly connected with the glycocalyx, shows several peculiarities, which are visible with RR staining. The RR displays a thicker and a more rough covering on the VP than on the PP (Michailova and Wassilew 1988). The glycosoaminoglycans are demonstrated with RR within the vesicles of the apical mesothelial cytoplasm too. The mesothelial cells possess a well-developed vesicular system, which represents another main membrane specialization together with the rich microvillous border. It involves: vesicles (most are microvesicles), vacuoles (a few large and single giant vesicles occupy the majority of mesothelial cytoplasm), and multivesicular bodies. The vesicles are located mainly along the luminal cell surface often contacting the plasmalemma. The concentration of the vesicles in both serosal sheets, in both mesothelial cell types (cubic and flat), and in single cells differ and correspond to the number of microvilli. Different membrane formations, short strip-like structures and few or single LB in the cytoplasm and in the vicinity of the microvilli, are constant components of the peritoneal, pleural and pericardial mesothelium in normal conditions (Michailova 1996b; 2004a; 2004b). Dobbie and Anderson (1996) observed three to four foci of osmiophilic lamellar formations in ‘each and every mesothelial cell’ with relatively high density. The majority of the lamellar structures in routine fixation are visualized as various membrane profiles, different from the typical LB. We propose that most of them in normal conditions represent initial LB. It is difficult to explain the small number, structural variety and location of these
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primary lamellar formations with only differences in the routine fixation or in specific lipid-fixing technique (Futaesaku et al. 1972). In agreement with our findings, Hjelle et al. (1991) describe predominantly nonlamellar ‘lipid inclusions’ in rat peritoneal mesothelial cells. A study by Dobbie and Lloyd (1989) presents multivesicular bodies in serial sections and define that LB have their origin in them. The strip-like structures, seen by TEM, appear as another constant image of linear membrane formations. Occasional investigations (Jonecko 1990; Peng et al. 1994; Michailova 1995b; 1996b; Ettarh and Carr 1996) reported ‘serosal fluid trapped as linear amorphous material’, ‘patchy depositions of amorphous or crystallized fibrin’, ‘superficial liquid film’ or ‘strip-like band’. According to Hills (1992) the entire sheep VP is covered with several lipid monolayers. The absorption of exogenous material enhances the peritoneal lining of surfactant in its lubrication (Chen and Hills 2000; Hills 2000). Attached layers of surface-active phospholipids possess an ability to influence permeability, consistent with their hydrophilic nature (Hills 1989). The present data demonstrate a simultaneous finding of two main membrane formations (Michailova 1995b; 1996b; 2004a; 2004b). The first represent typical LB, as well as other rounded membrane profiles and coagulated particles. Our data show that these structures are widespread over larger serosal surfaces and correspond to ‘ball-and-roller bearings that constantly form and reform’ in the hypothesis of Dobbie and Anderson (1996). The second membrane formation is the strip-like structure–uninterrupted covering as observed by SEM. The original concept of Hills (1992) describes such structure as an oligolamellar surfactant and defines it as a main membrane formation. The significant variations of the mesothelial covering (Michailova 1995a; 1995b; 1995c; 1996b; Wassilev et al.1998; Michailova et al.1999; 2004) suggest the differences of the membrane profiles as well as their TEM and SEM images of the SM, the parietal and visceral serosal sheets, and the individual organs. The correlation between the TEM and SEM images in some cases is incomplete and it is difficult to explain the correspondence only with the different electron microscopic techniques (Ettarh and Carr 1996). The TEM results present membrane formations smaller in number and size, as compared with the corresponding images visible by SEM. The significant enlargement of the number of LB and of the length of striplike structures, as well as their SEM equivalent are observed after EH and EP (Michailova 2004a; 2004b). The LB under experimental conditions undergo considerable changes in size, shape, position toward the mesothelium, as well as arrangement, structure or thickness of their membranes. The two main formations (round and linear) are exhibited differently after EH and EP. Round lamellar profiles and oval coagulated particles are features after EH, while strip-like structures and continuous covering occur after EP. Newly secreting mediators (Mutsaers 2002) of activated cells for healing process in response to endotoxins, cytokines, asbestos or other instilled agents (Boylan et al. 1992; Antony et al. 1992; 1995; Owens and Grimes 1993; Peng et al. 1994) supports the assumption for a production of new generation of membrane formations. In agreement with our results
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concerning a great diversity of lamellar profiles after different experimental conditions, Müller et al. (1998) postulate that the surfactant system depends on the size, solubility, chemical reactivity, concentration and duration of exposure of different agents. In response to pressure and to movement of the surrounding structures, Zasadzinski et al. (1988) describe constant change in the fine morphology of LB. The increased concentration of albumin and fibrinogen is followed by lamellar profiles as ‘rod-like structure with open ends into spherical structures with loss of their open ends’ (Park et al. 1998). The submesothelial position of the LB after EP becomes possible due to the extremely thin and incomplete BL, which is in contrast with the findings of Dobbie and Anderson (1996) that LB probably never pass this ‘intact, formidable’ barrier. The same location of the LB suggests the possibility that they have an other origin, from cells of the submesothelial layer. The LB leave the mesothelial surface by exocytosis. More numerous LB are observed in the apical and lateral cytoplasm (Michailova 2004a; 2004b). The extracellular location is over intercellular spaces, in close proximity of the microvilli, and rarely, in the vicinity of the BL. We observed fusion of the mesothelial plasmalemma with the external membrane of the LB or of other lamellar formations. The findings of Dobbie (1996) and Chailley-Heu et al. (1997) provide evidence that the mesothelium is specialized for biosynthesis and secretion of lubricant surfactant. According to the present data, obtained experimentally, the preferable site for the exocytosis of LB is the intercellular space. This finding confirms the hypothesis for a safe defense against the frictional damage and a barrier to both protein leakage and pathogen invasion by spanning cell junctions (Hills 2000). The validity of this hypothesis is justified by the larger coagulated particles visible by SEM in the vicinity of stomata. The more numerous intercellular contacts between the cubic mesothelial cells, and especially by the activated cells, suggest more places for exocytosis of the LB, compared to the nonactivated cells and the flat cells. 4.1.4 Mesothelial Basal Lamina Our study confirms the existence of BL as a constant main component of all SM. Some descriptions (Felix and Dalton 1956; Von Hayek 1960; Barrett 1970; Nagaishi 1972; Ishihara et al. 1980) reject or do not mention the existence of BL. The BL of the human and animal VP, except in the ground squirrel, and the peritoneum of the spleen, is thick and electron dense, with significant regional variations of its contour and thickness (Michailova and Vassilev 1988a; 1988b; 1990b; Michailova 1995a; 1995c; 1996b; 1997a). We have demonstrated a thin BL with a wide superficial electron-lucent part and a thin electron-dense part in the human and animal PP, in both sheets of the peritoneum, in the pericardium and in tunica vaginalis testis (Michailova 1995a; 1995b; 2001a; 2001b; Michailova et al. 1999; 2004). The BL could not be distinguished in the mesenteric duplicatures and in the entire animal as well as in the majority of the human greater omentum in
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normal conditions. The BL is lacking (after application of melted paraffin) or shows significant structural alterations after EH, EP and PNT (Michailova 1996c; 2001d; 2001e; 2004a; 2004b). Similar changes of the BL are described following chronic injury (Martinez-Hernandes and Amenta 1983; Gotloib et al. 1988; Davila and Crouch 1993). Reduplication of the BL in continuously ambulatory peritoneal dialysis patients is a result of nonenzymatic glycosylation of protein (Dobbie 1990). Metastatic tumor cells are able to attach to the BL, degrade and penetrate it, while in diabetes the BL is thicker, with a double course, and more porous (Martin et al. 1983). Bornfield et al. (1984) established the epithelial origin of the BL. Common organization of the BL consists of a three-dimensional network of irregular, fuzzy strands, composed of at least five substances: collagen IV, laminin, heparan sulfate proteoglycan, entactin, and fibronectin (Laurie et al. 1983; Leblond and Inoue 1989). As reviewed in Sect. 1.6, the mesothelial cells are able to produce these substances. Niedbala et al. (1986) declared that the human mesothelial cells–ECM interactions are important for maintaining normal morphology, behavior, proliferation, repair and transformation of the mesothelial cells in vitro. Our observations of significant variations in the thickness and structure of the BL under the cubic and the flat cell covering, as well as its alteration beneath degenerative cells in normal and experimental conditions, are strongly in favor of its mesothelial origin. Our data about pleural development confirm the same assumption (Michailova and Savov 1991; Michailova and Vassilev 1991; Michailova 1995e; 1996a). 4.1.5 Elastic Membrane The presence of the EM is a matter of debate. One group of authors favors the extreme opinion of its absence in pleura (Wang 1974; Wiener-Kronish and Matthay 1988; Sahn 1988). The second group (Nagaishi 1972; Obata 1978; Pinchon et al. 1980; Herbert 1986) has established the presence of two EM (superficial and deep). Thus, the present findings are in disagreement with both opinions. Our data support the existence of EM under the BL as fourth constant component of the human and animal VP (Wassilev et al. 1986; Michailova and Vassilev 1988a; 1988b; 1990b; Michailova 1996b; 1997a; 2001d). The EM, as well as the dense network of elastic fibers in the submesothelial layer continues in the interalveolar septa, and builds a strong elastic sac of the VP. The material taken during the active period of the ground squirrel’s VP shows large sections with a deep EM, separating the underlying layer into a superficial cellular part and a deep fibrous part. This deep EM might be viewed as a compensatory mechanism in connection with elevated respiratory activity during that period, and is in accordance with its role in the act of breathing. Some observations (Albertine et al. 1982; Oldmixon and Hoppin 1984; Mariassy and Wheeldon 1983; Sorokin 1983; Stamenovic 1984; Humphrey 1987) point out the greater amount of collagen and elastic fibers, compared with the lung. This is probably in conjunction with retaining the form of the lung, with limitation of the lung’s expansion and, most importantly, the participation of the lung movement
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together with the chest wall. Pinchon et al. (1980) described numerous elastic fibers that form membranes in the rat’s VP. The EM under the BL, and several EM-like structures in the submesothelial layer, are obligatory components of the spleen serosal covering (Michailova 1994; 1995a; 1996b; Michailova et al. 1999). The existence of the elastic formations in the spleen could be associated with its greater mobility (intraperitoneal organ), as well as with its volume change. The EM-like structures probably participate in the superficial part of the spleen capsule. An interesting finding is a deep EM, located in the submesothelial layer of the visceral sheet of the peritoneum after EP. Single studies report EM-like structures in the peritoneum (Ivanova 1970) and in the pericardium (Zioupos et al. 1994). Knudsen (1991) described ‘lamina elastica peritonei’ just beneath the BL of both sheets of the peritoneum in man. It is most prominent in organs with volume changes, such as the intestines. It is less prominent or discernible in static organs such as the uterus; the stomach is in an intermediate category. Parsons et al. (1983) describe a three-dimensional elastic reticulum of the parietal peritoneum as a ‘network without free ends’ intermingled with large bundles of banded collagen is present under the BL. 4.1.6 Submesothelial Connective Tissue Layer In this paragraph, a summary is given of the most significant differences in the submesothelial layer of the observed SM. Some investigations (Holt 1970; Lesson and Lesson 1981; Albertine et al. 1984; Digenis 1984; Fentie et al. 1986; Gotloib and Shostack 1987; Pfeiffer et al. 1987; Sahn 1988; Tao 1988; Wiener-Kronish and Mathay 1988; Simionescu et al. 1993) have described the submesothelial layer as simply organized connective tissue, making no distinction between parietal and visceral sheets. Other studies describe the submesothelial tissue as a complicated structure, consisting of several layers with prominent variations in width, and in the arrangement of the cells, fibers, and vessels (Miller 1947; Von Hayek 1960; Nagaishi 1972; Wang 1974; Obata 1978; Ishihara et al. 1980; Pinchon et al. 1980; Albertine et al. 1982; Mariassy and Wheeldon 1983; Herbert 1986; Vladutiu 1986). The features of the blood and lymphatic vessels are discussed according to their role in the transport through the SM in Sects 4.1.7 and 4.3. In the submesothelial layer of the visceral sheet of the SM, the cellular elements are more prominent. The fibroblasts are the most common cells and are closely associated with collagen and/or elastic fibers. Macrophages and mastocytes are often found together with single extravasal cells (predominantly neutrophil and eosinophilic leukocytes), and form larger clusters in the visceral, than in the parietal sheet. In the lesser pelvis, the cells are scattered uniformly and predominate over the single fibers and small collagen bundles (Michailova et al. 2004). Smooth myocytes are observed in the connective tissue layer of the spleen, stomach, rectum, urinary bladder, and in the broad ligament of the uterus. Bundles of collagen fibers of various sizes and with different orientations direction are observed in the visceral sheets
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of the SM. Large bundles are located parallel with the surface, and are densely packed in the parietal sheets, and in the visceral sheet of the pericardium and the testis peritoneum (Michailova 1995a; 1995b; 1995c; 1996b; 2001a; 2001b). Smaller collagen bundles with a different course compound a thick, loosely organized layer on the stomach, rectum, urinary bladder, or form a narrow band of the intestines (Michailova 1995a). Short fibers, rare small bundles, single cells and large intercellular spaces arrange the core of the mesenteric duplicatures and a large portion of the greater omentum (Wassilew et al. 1998; Michailova et al. 1999; Michailova and Usunoff 2004). The elastic fibers are more abundant than the collagen fibers in the submesothelial layer of the spleen and form a thick network or EM-like structures. Numerous elastic fibers are visible in broad areas of the lung, the pleural side of the diaphragm and anterior abdominal wall, while in the remaining regions they are single. The endothelium of the blood capillaries of the investigated SM is of a continuous type and is situated on a BL. Few blood capillaries of the greater omentum and ovary show features of fenestration. The endothelium of the lymphatic vessels is highly stretched and the BL is not well formed. The ultrastructural characteristic of the lymphatic vessels appear the same as reported in other organs, and in other species (Leak 1980; Gnepp 1984; Nakamura et al. 1994). Small lymphatic vessels are usual findings in the submesothelial layer of the intestines, rectum, uterus, urinary bladder and testis, while in other serosal coverings they are few. The LL are discussed according to their connections with stomata in Sect. 4.1.7. Discrete bundles of thin unmyelinated and, more rarely, myelinated nerve fibers are also seen. The latter, most probably, represent sensory fibers. They are more numerous in some species (cat), and are commonly observed in the organs of the lesser pelvis. The nerve fibers are disposed in close contact with BL (heart) or with the blood vessels in the submesothelial layer (lung, heart, stomach, rectum, urinary bladder, uterus). For decades, only a few studies (Dwinnell 1966; Nagaishi 1972; Becade et al. 1976; Amenta et al. 1982; 2002; Allen et al. 1984; Sancesario et al. 1984; Wedekind 1997; Artico et al. 1998; Lynn and Blackshaw 1999; Berthoud et al. 2001) appeared on the nerve fibers of the SM, and are concerned with their structure (myelinated and unmyelinated), nature (sensory and autonomic), and their position (close to the blood vessels). Only recently a comprehensive description of the sensory innervation of the peritoneum appeared (Tanaka et al. 2002). These authors studied the distribution of sensory neurons innervating the peritoneum in rats by means of the retrograde axonal transport of Fluoro-gold. The tracer was applied to parietal peritoneum, diaphragm, mesentery, mesocolon, serosal covering of the stomach, small intestine, colon, liver, spleen, kidney, urinary bladder, and uterus. Tanaka et al. (2002) examined the spinal ganglia bilaterally from C7 to S6 and the nodose ganglia. In cases where the tracer was applied to the anterior abdominal wall, labeled neurons were found only in the ipsilateral spinal ganglia. A small number of cells in nodose and cervical dorsal root ganglia were labeled after placing the Fluoro-gold on the central part of the diaphragm. When the tracer was injected in the peripheral parts of the diaphragm, the nodose ganglia
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were negative and spinal ganglia from Th6 to Th12 were positive. Many neurons in the nodose ganglia in addition to stomata in the spinal ganglia Th4 –Th13 were labeled when the tracer was placed on the peritoneum lining the stomach, small intestine, or cecum. After applying the tracer onto the colon, labeled cells were observed in the dorsal root ganglia Th13 –L2 and L5 –S1 . Ganglion cells in the nodose and spinal ganglia Th5 –Th13 were positive when Fluoro-gold was applied to the mesentery. Interestingly, Tanaka et al. (2002) did not observe labeled neurons in any ganglia when the tracer was applied to the peritoneum covering the spleen, kidney, uterus, urinary bladder and liver. On the other hand, Collins et al. (1999) found retrogradely labeled neurons in the nodose ganglia following Fluoro-gold and pseudorabies virus injections in the uterus. Thus, the results of these authors run counter to the common belief that the visceral sensory fibers of the vagus nerve do not reach the organs of the lesser pelvis. Miller (1947), McLaughlin et al. (1961) and Nagaishi (1972) made a qualitative comparison, and classified the VP as thick (sheep, cow, pig, horse and man) or thin (dog, cat and monkey). Our data confirm the statement that the human VP belongs to the thick type (Vassilev et al. 1986; Wassilew et al. 1986; Michailova 1995c; 1997a). The present results demonstrate considerable variations in the pleural thickness, which according to strict criteria make the differentiation of the pleura into ‘thick’ or ‘thin’ type difficult. The only reliable criterion for the statement that human VP is ‘thick’ is the extensive lymphatic network, reported presently. There are broad communications between the superficial and deep lymphatic networks (Albertine et al. 1982; Gnepp 1984). It is hard to discriminate the blood vessels ultrastructurally–the capillaries of the submesothelial layer of the VP in human and in experimental animals–from those of the underlying, superficial lung parenchyma. These vessels have been classified as a ‘thick’ bronchial type (Nagaishi 1972) or as a ‘thin’ pulmonary type (von Hayek 1960; Wiener-Kronish and Matthay 1988), or as arising from both vascular systems (Vladutiu 1986). The lack of lymphatic vessels or their occasional finding in the investigated animals (mouse, rat, guinea pig, ground squirrel, rabbit, cat) confirms the classification of their VP as a ‘thin type’ (Michailova and Vassilev 1988a; 1988b; 1990b; Michailova 1996b). 4.1.7 Stomata in Normal Conditions and Postinflammatory Changes There are significant differences of the serosal covering around stomata and over LL, when compared with its common organization. These structures are located over both sides of the diaphragm, the costal pleura of the lower intercostal spaces, the parietal peritoneum of the anterior abdominal wall, and over the greater omentum (Michailova 1996b; 2001a; 2001e; Michailova et al. 1999; Michailova and Usunoff 2004). Our data extend the location of stomata and LL also over the serosal covering of the rat’s liver and on the broad ligament of the uterus (Wassilew et al. 1998; Michailova et al. 2004). According to our results, these zones involve
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cubic mesothelium, underlying LL, their BL, different types of contacts between both mesothelial and endothelial cells, and surrounding connective tissue, with or without opening–stomata–as highly differentiated structural complexes and we nominate them ‘lymphatic units’. The typical stomata images seen by TEM involve only contact zones of both cell populations (mesothelial and endothelial), which form openings in the mesothelial layer. In contrast with these data are the descriptions of other authors (Tsilibary and Wissig 1987; Miura et al. 2000; Li and Li 2004) that stomata have three components: cuboidal mesothelial cells, submesothelial connective tissue, containing many foramina, and endothelium of the LL. According to present observations, connective tissue only surrounds the contact zones, but does not participate in the forming of stomata. Stomata are constant features seen by SEM, but are occasional findings observed by TEM (mentioned also by Masada et al. 1992 and Oya et al. 1993), and are visible only in serial sections in untreated animals. Different types of contacts between cubic mesothelial and underlying endothelial cells of LL, with common BL or without BL, are usual findings, visible by TEM, but they do not form the openings in the mesothelial layer in the control animals. The LL display a constant appearance of collapsing (closed) cisterna-like pattern of their lumens under normal conditions. They are flat and are arranged parallel with toward the serosal surface. The lymphatic endothelium builds LL with a complicated contour in the greater omentum and in the broad ligament of the uterus. Most numerous LL are located beneath the cubic mesothelium, or in the middle and rarely in the deep parts of the submesothelial layer. Some authors (French et al. 1960; Leak 1976; Leak and Rahil 1978; Tsilibarry and Wissig 1987; Abu-Hujleh et al. 1995; Li et al. 1997) regard only the subperitoneal terminal lymphatic vessels of the diaphragm, the roof of which had a stomata as ‘lacunae’. We define as ‘lymphatic lacunae’ all large lymphatic vessels with shape and location described yet (see above), with or without stomata, in all places with lymphatic units. Some structures (mesothelium, endothelium of LL, their BL and surrounding connective tissue) after EP are interrupted and structurally modified to build new stomata and LL. Larger stomata and rounded LL are more numerous after EP in the peritoneal side of the diaphragm and the anterior abdominal wall (Michailova 2001e). Wang (1975) also claimed that in diseases such as chest tuberculosis and tumors, additional pleural stomata might be formed. Lindic et al. (1993) reported similar results after continuous ambulatory peritoneal dialysis. After PNT, the LL are enlarged on the pleural side of the diaphragm and their number does not change significantly (Michailova 2001d), but after EH the number and the structure of the LL do not change (Michailova 2004a). The LL are located near the mesothelium after EP and the tall endothelial cells, peristomatal evaginations, valves, and septae increase significantly in number and change their structure. This high degree of structural complexity in experimental conditions leads us to conclude that lymphatic units are stable, but on the other hand, dynamic structures with the possibility for quantitative and qualitative changes according to different experimental conditions. Numerous studies have attempted to find the reasons for
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rapid morphologic changes in stomata and the LL. Some authors (Casley-Smith 1964; Bettendorf 1979) propose that opening and closing of stomata depends upon expiration and inspiration. These reports for the first time stress the importance of diaphragmatic movement, emphasizing the role of valve-like endothelium, which is thought to prevent reflux of fluid in the respiratory cycle. But it is difficult to reconcile the present data (see also Michailova 2001d; 2001e) with the statement above. According to Tsilibarry and Wissig (1987) and Li (1992), the differences in the image of stomata depend upon pressure in the peritoneal cavity. Our results show a high degree of differentiation and possibility for fast changes in elements of lymphatic units, in response to increased abdominal pressure by EP (Michailova 2001e). The structural changes in them in the late period (day 8) after EP are stable and enhance the drainage function of the diaphragm. Somewhat in contrast with present results, a previous study of pathologic and chronic ascites (Vipond et al. 1990) reported that at early stages peritoneal absorption is normal, but later it decreases, which suggests insufficiency of the subsequent structural changes. Tsilibarry and Wissig (1983) suggested that the patency of the stomata could vary in response to fluid pressure, relaxations, and closing the diaphragm. Miserocchi et al. (1982) and Miserocchi (1991) reported that the peritoneal stomata can undergo stretch-related change. Negrini et al. (1991) suggest that the stomata geometry is affected by the diaphragmatic tension or by myofibroblastic conversion of the mesothelial cells (Yang et al. 2003). We do not observe more numerous actin-like filaments in the mesothelial and endothelial cells of LL around stomata, as described by others (Leak 1976; Tsilibarry and Wissig 1983). These authors suggest that such filaments are responsible for formation of stomata. We do not think that only the contractile filaments in both cell populations are responsible for these complicated structural changes in the lymphatic units. Levine and Saltzman (1988) found a prominent submesothelial fibrosis of the diaphragm after EP. The present results do not confirm the increased number of collagen and elastic fibers, surrounding stomata and LL. Dobroszynska et al. (1999) suggested that nitric oxide and endothelin play a role in the mechanisms regulating the tone of peritoneal stomata. Similarly, Li and Li (2004) think that nitric oxide can increase lymphatic absorption by opening pleural stomata. The variability of the pattern of the lymphatic regions might be due to the relatively slow process of fixation, different methods of investigation or differences between the species studied (Shinohara et al. 1989; Oya et al. 1993; Shinohara 1997). We feel that it is not possible from the present data to propose if mesothelial or endothelial cells play an initiating role in the structural changes of the lymphatic units in experimental conditions. Wang et al. (1997) suggest a primary role of the cubic mesothelial cells and their secretion of chemotactic substances for formation of the LL. Some changes of membrane specializations of the mesothelium at day 8 after EP could be interpreted only as adaptive mechanisms to prevent disruption of the mesothelial layer by the increased pressure. There is also an entirely different hypothesis based on ontogenetic observations (Shao et al. 1998) that stressed the primary role of the endothelium of LL by the formation of stomata. According to
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the same results, the endothelium projected ‘many bulges that subsequently became elongated and contacted with pores among mesothelial cells’. This is also in agreement with the data of Nakatani et al. (1996) for the overlying cubic mesothelial cells, which receive some stimuli from the endothelium of LL that results in alteration of the mesothelial shape. The presence of newly formed types of contacts between the mesothelium and the lymphatic protrusions (at day 5 after EP), as well as electron-dark granules in tall endothelial cells (at day 8 after EP) are in agreement with the data reviewed above for the initiating role of the endothelium. Our data show parallel and rapid changes after EP of the cubic mesothelium and the endothelium, and suggest their equivalent role in new formation of stomata and the LL. The lymphatic units in the abdominal cavity play a radical role by its drainage under experimental conditions, where the diaphragm probably represents the main pathway. Typical stomata are occasional findings in the broad ligament of the uterus, seen by SEM, and are visible by TEM only in serial sections in the normal situation (Michailova et al. 2004). In contrast to these data, Li et al. (1997) postulate, ‘lymphatic stomata of the pelvic peritoneum are stable structures and are normally present’. The data about numerous, large LL with extremely complicated relief and closed lumina in the submesothelial layer of the broad ligament of the uterus, suggest that it is the most specialized region of the serosal covering in the lesser pelvis. We conclude that they represent a preferable pathway for the draining of the caudal part of the peritoneal cavity. It is possible that some clusters of free cells (mastocytes, macrophages and lymphocytes) and nerve fibers (unmyelinated and rare myelinated) in the vicinity of the LL are related with the same process of rapid removal of fluid, or they play a role of MS-like structures. 4.1.8 Human and Animal Milky Spots The MS are located mainly on the greater omentum, but also on other peritoneal regions, as well as on the pleura and pericardium. They are composed of cellular aggregations of mesenchymal cells around blood capillaries and are covered incompletely with mesothelial cells (for extensive reviews, see Lieberman-Meifert and White 1983; Shimotsuma et al. 1993; Michailova and Usunoff 2004). We examined the ultrastructure of omental and extraomental MS in man and rat, and we studied also the vascularization of MS in the rabbit’s greater omentum and mediastinal pleura. Kanazawa et al. (1979) described four types of capillary formations in MS in mouse. We identified these types also in the rabbit omental MS but with a somewhat different sequence. In our material, most common were the ‘intramembranous’ and the ‘sessile marginal’ types, whilst the ‘pedunculated’ type was encountered extremely rarely. Most often, two arterioles supply a single MS, but also three to four sources may be distinguished, especially in the mediastinal MS. The capillaries display dilated portions in the central part of the MS and/or they have a convoluted course, and make anastomoses.
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The mesothelial covering of the MS consists of the two basic cell types, with predominance of cubic cells. In the recently described MS over the broad ligament of the uterus (Michailova and Usunoff 2004), the mesothelial covering is composed mainly of flat cells, and between them there are small clusters of cubic cells. In agreement with previous studies (Dux 1990; Shimotsuma et al. 1989; 1991; 1993; Cui et al. 2002) we observed fairly often a replacement of the mesothelial cells by macrophage-like cells. Moreover, also in normal human omental MS, we found small groups (three to five) of such cells, and not only when following serial sections, but also in a single favorable ultrathin section. Since the report of Mironov et al. (1979), several authors declared the stomata as a common finding in the MS (see especially Cui et al. 2002). Actually, a space between neighboring mesothelial cells is very often observed over the MS but we would like to stress that only a small percent of these openings represent ‘true’ stomata. Such are rare in normal MS, and are recognized only on serial ultrathin sections. As reported repeatedly (Felix 1961; Carr 1967; Hodel 1970; Beelen et al. 1980; Cranshaw and Leak 1990; Cui et al. 2002; Michailova and Usunoff 2004), the mesothelium of the MS lacks a BL that facilitates the cellular migration. Generally, the human MS are located deeper in the submesothelial connective tissue layer than the MS in the rat (Michailova and Usunoff 2004). Numerous studies (reviewed in Shimotsuma et al. 1993; Michailova and Usunoff 2004; see also Sect. 1.4) reported the cellular composition of the MS. The authors unanimously agree that the most common cells are the macrophages followed by lymphocytes (Krist et al. 1995). This is also in agreement with the present data. On the other hand, it is a common belief that the macrophages are located in the periphery and the lymphocytes, in the center of the MS (see especially Shimotsuma et al. 1989; 1993). Only rarely could we confirm this statement. Both in human and animal MS, we observed macrophages and lymphocytes mixed in one and the same cluster of free cells. The third cell type in the MS are the mastocytes. According to Shimotsuma et al. (1993) in normal MS they account for 6.1%. We have not counted precisely the different cell types but we feel that the number of mast cells is somewhat higher (for example, see Fig. 15d), in all rat MS presently examined, including the recently described (Michailova and Usunoff 2004) MS-like formations on the broad ligament of the uterus. Connective tissue components: perivascular fibroblasts, rounded fibroblast-like cells, collagen and elastic fibers were commonly observed by us, and especially in the extraomental MS. On the another hand, lipocytes are a constant component in the omental MS, whilst in the broad ligament of the uterus they form very small groups, or are single, and lipocytes are lacking in the MS of the mediastinal pleura. The MS of healthy rats also contains small clusters of neutrophils and occasional eosinophilic leukocytes and erythrocytes. Since Hodel (1970), several studies insisted that the blood capillaries in the MS are fenestrated (Takemori 1979a; Cranshaw and Leak 1990; Takemori et al. 1994). We think that this finding is overestimated. In all MS presently examined, we found that the majority of the capillaries are of the continuous type, and the fenestrated
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capillaries are relatively few. On the other hand, we noticed a distinct difference between the capillaries in the MS and in the remaining greater omentum. As a rule, the endothelial cells of the MS in the rat are considerably more electron-dense than the ‘usual’ omental capillaries. Another characteristic feature, especially by the human omental MS is that we often observed clusters of free cells not only adjacent to blood capillaries, but also surrounding small arterioles, containing smooth muscle cells in their tunica media. We found that the blood capillaries are the main vessel component of the MS in the rat, and rounded lymphatic capillaries are few or are absent. Only in the broad ligament, are the lymphatic capillaries the predominant vessels. In the human MS, the lymphatic vessels are numerous and of the LL type. As a rule, BL is lacking under the lymphatic endothelium. In agreement with Krist et al. (1995), we also encountered a significant innervation of the MS by unmyelinated and (more rarely) myelinated fibers. In agreement with numerous studies (Beelen et al. 1980; Liebermann-Meifert and White 1983; Cranshaw and Leak 1990; Kremli and Mamontov 1990; Van Vugt et al. 1992; 1996; Weinberg et al. 1992; Broche and Tellado 2001; Cui et al. 2002), we found that the MS react dramatically to inflammation, e.g., by our experiments with EP. In rats infected with Pseudomonas aeruginosa we encountered a clear increase of the size of the MS, and they were located closer to the mesothelial layer. The MS are covered by extremely activated mesothelial cells, and more numerous macrophage-like cells. The connective tissue components decrease in number, and often are lacking (to recall, the reduced number of lipocytes by inflammation4 was noticed already by Seifert 1920). By EP, the MS are composed almost exclusively of free cells, separated by electron-empty intercellular spaces. The activated macrophages form large groups as a main cell accumulation, but the number of lymphocytes and erythrocytes appear to be only slightly increased. Zareie et al. (2001) found a clear increase of mast cells by peritoneal dialysis and we observed a significant increase of mastocytes also by EP. As a rule, the number of neutrophil and eosinophilic leukocytes is increased, and we observed them in clear clusters, that do not contain other cell types. By EP, the number of blood capillaries increase significantly, while there is no obvious change of number and size of lymphatic vessels. 4.2 Prenatal and Postnatal Development of the Pleura The main components (mesothelium, BL and connective tissue layer) of the human (between 4GW and 7GW, according to Lee and Olak 1994), and rat pleura are formed early in prenatal life, but they do not attain their final differentiation until birth. It is well known that the lung and the chest remain immature during prenatal life (Davies and Reid 1970; Krause and Leeson 1975; Scheuermann et al. 1988; Reid 1984). The development of the different areas of the VP proceeds asynchronously, which could be explained by similar changes in the lung as well as a different manner of maturation of the parietal sheet. The processes of differ-
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entiation advance from the center to the periphery of the lung for the bronchus tree, whereas the alveolar differentiation follows a distal–proximal sequence and is asynchronous between the lobes (Kotas et al. 1977; Zeltner et al. 1990). Burri’s classification of the stages of prenatal lung development (1984) could be a model for staging fetal differentiation of the pleura: early stage (until 17GW), and late stage up to birth. According to Otto-Verberne et al. (1988) this time interval (16GW) is labeled with the transformation of the type II and type I pneumocytes in the pseudoglandular period and its beginning is between 4GW and 7GW (Lee and Olak 1994). We divided the pleural components of the human and the rat into two basic groups: first, mesothelial layer and underlying BL, which undergoes epithelial development; and second, submesothelial mesenchyme, which differentiates as a connective tissue layer. The mesothelial and BL changes start early in gestation and continue throughout the early postnatal period (Michailova 1995d; 1995e; 1995f; 1996a; Michailova and Savov 1991). The submesothelial layer differentiates predominantly during the second half of fetal development (after GW17 for man, and after ED18 for rat). The EM develops after GW25 of prenatal life for man and after birth for rat (after PD5). The development of the submesothelial connective tissue probably follows a different pattern in the early stage from the subalveolar connective tissue. The submesothelial connective tissue forms a thick layer in both serosal sheets in rat in the early stages (until ED17). Fukuda et al. (1983) demonstrated a relatively late differentiation of the lung epithelium, although the septal subalveolar connective tissue develops early. Our findings of the appearance of the cubic cells after the flat mesothelial cells (after 11GW for man and at ED16 for rat), a great number of cubic cells at the late stage, and an intimate contact between them show that the existence of both cell types is already evident through the prenatal life (Michailova and Vassilev 1990a; Michailova and Savov 1991; Michailova 1995d; 1995e; 1996a). Fetal ‘breathing’ begins after 20GW (Perelman et al. 1981), i.e., significantly later than the first appearance of the cubic mesothelial cells. The cubic cell type, as a basic type of the VP, is not an artificial result of the moving lung (Dodson et al. 1983). From ED17 to ED20 the respiratory movements increase (Rosenkrans et al. 1983) and the rat pleural surface shows adequate changes, forming cubic mesothelial covering, multi-layered sectors, high-prismatic mesothelial cells in the lung basis and blood capillaries located nearer to the mesothelium. Whitaker et al. (1982a) described the double cell layer of the VP of the 4.5 GW embryo as a rare finding. The mesothelium of both pleural sheets descends from one and the same undifferentiated cell type, spindle cells, followed first by flat, and then by cubic cells. The barrier function develops early. It is most important and is carried out by the initial spindle covering cells, as well as by flat cells. The transport capability follows and gradually becomes more complex. At the end of the prenatal period the PP is built mainly from flat cells and the VP is covered predominantly by cubic cells. The morphology of the intercellular spaces in early stages (11 GW in human and ED14 in rat) suggests that permeability starts through them. The primary mesothelial cells contact each other with a few shallow interdigitations. Short immature occludens junctions are
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located in the apical portion of the deeper interdigitations in the next stage (14– 20 GW in the human and ED17 in the rat), and form a complicated layer, which fulfils the initial barrier and transport function. Suzuki and Nagano (1979) define three types of tight junctions in the mesothelium of the mouse embryo, which are permeable. The first villous evaginations of the apical plasmalemma are located in the vicinity of the intercellular zones (9–10 GW in human and ED15 in rat). The vesicular system and the microvilli, as a morphological substrate for a transcellular mesothelial transport, increase rapidly and changes in their distribution in the mesothelial cells occur during the late fetal stage. They finish their development in the postnatal life. Ukeshima et al. (1986) demonstrated a parallel increase only in the microvilli and microvesicles. Our results show that the furrows and ridges of the pleural surface, the undulations between the cells, and the membrane specializations (microvilli, microvesicles and intercellular contacts), undergo simultaneous changes in the later stages. The differentiation of the intercellular contacts appears before the formation of the microvilli and vesicular systems. This explains why the transcellular transport develops later than the intercellular pathway. The development of the cytoskeleton in the mesothelial cells in the late fetal stage suggests an additional mechanism of intercellular transport (DiBona and Schafer 1984). Our findings of glycogen accumulations and lipid droplets in the mesothelium (9–10 GW in human and ED18 in rat), probably suggest a temporary formation of some products in these cells. Whitaker et al. (1982a) observed large amounts of glycogen in the mesothelium between ED25 and ED37, and its amount was reduced to only small deposits towards term. Vessels are practically absent in the submesothelial connective tissue during the early stage and development of the pleura is unrelated to vasculogenesis. Thus, the present data suggest that the mesothelial cells remain the sole source of the secretion that lubricates the organ surface at this stage. The further mesothelial changes (mainly in the cubic cells) involve well developed organelles and electron-dark granules, representing a substrate for secretion. According to these data, groups of osmiophilic bodies of different sizes and different internal structures appear from ED16 to ED18. They resemble the Weibel-Palade bodies in the endothelium (Nikolov and Vankov 1984), and probably play the same role (Michailova and Vassilev 1990a; Michailova and Vassilev 1991). The data on the secretion possibilities of the mesothelial cells (Haidar et al. 1990) and the results from cell cultures (LaRocca and Rheinwald 1984), as well as the assumption of Bornfield et al. (1984) that BL arises from the epithelial cells, are in agreement with our suggestion that the BL has a mesothelial origin. Obata (1978) explained the differences in the BL in both pleural sheets by variations in the mesothelial covering. The fibroblasts and collagen fibers at the beginning of the second half of prenatal life form a thick submesothelial layer. Jackson et al. (1990) explain the greater rate of collagen synthesis in prenatal life, as compared to the adult, by faster lung growth during gestation. At the end of fetal life (33–36 GW in human, ED18–20 in rat) the submesothelial layer becomes thinner in some areas. The thinning of the connective tissue may be explained by the rapid penetration into the subme-
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sothelial layer of respiratory saccules by transitional ducts (Perelman et al. 1981). According to Duncker (1990), in the human there is no saccular phase, and from the beginning of the peripheral differentiation there are alveoli—bronchioli alveolares. The elastic fibers, as well as the EM under the BL, appear during the late period of human fetal life (see also Michailova 1995d; 1995f; 1996a). The amount of elasticity in the pleura increases continuously after 25 GW, parallel with the lung development. Our findings are in agreement with the study of fetal lung by Collet and Des Biens (1974) showing the appearance of elastic fibers during the saccular stage, or in areas corresponding to the primitive alveolar zones, according to the immunohistochemical investigation by Cossar et al. (1993). Several observations describe the formation of the elastic fibers in earlier glandular (Shibahara et al. 1981) or canalicular (Fierer 1976) stages of the developing lung. Our data on the rat pleura (Michailova and Vassilev 1991; Michailova 1995d) support the results of Krause and Leeson (1977), in which the rat’s EM differentiates after birth (from PD5 to PD30). Most probably, collagen and elastic fibers increase and arrange in a manner that corresponds to the changes in lung parenchyma (Nomura et al. 1998). The breathing lung plays a major role in the development of the elements of connective tissue layer and especially of the elastic fibers in the submesothelial layers and EM of the VP. We have not found, in the human and in the rat, that the fibroblasts close to the mesothelium resemble myofibroblasts and smooth muscle cells. The latter become associated with several types of extracellular components in the connective tissue and are especially important for the process of elastogenesis (Collet and Des Biens 1974; Fukuda et al. 1983). The process of blood vascularization in the VP follows the same process as in the lung in the late stages. The invasion of capillaries into the terminal airways of the developing lung starts at the canalicular stage (Burri and Weibel 1977), and according to Perelman et al. (1981) the capillaries penetrate the terminal spaces. Our data (Michailova 1995d; 1995; 1996a) show that the blood vessels enter the submesothelial layer from the lung at 19–20 GW in the human and at ED18 of the rat, and appear earlier than the lymphatic vessels. The human fetuses (after 21 GE) show an extensive lymphatic network of small capillaries in the submesothelial layer, while they are completely lacking in the rat. The fetal human pleura seems to be moderately thick and extends into deeper interlobular connective tissue septae. The latter feature and a dense lymphatic network suggest that the human VP is formed as ‘thick type’ and rat VP is formed as ‘thin type’ in the prenatal period. 4.3 Transport Across the Serosal Membranes 4.3.1 General Pathways The complicated relief, the intercellular invaginations, the undulating cell membrane, as well as the microvilli, evaginations and invaginations of the apical plasmalemma, ciliae, and extensive vesicular system, determine the transcellular trans-
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port of both mesothelial cell types (Michailova and Wassilew 1988; Mikhaylova and Vasilev 1988; Savov and Michailova 1991). The microvilli and the microvesicles are the main structures which support this pathway through the mesothelium. A correlation exists between the number of microvilli and microvesicles, and both structures are markedly dynamic. The latter differences are manifested by the ‘activated’ cells in experimental conditions, and by changes of the developing mesothelium. Some investigations postulated that the quantity of microvilli and microvesicles is either a constant structural variation, or it is a subject to physiological fluctuation (Mariassy and Wheeldon 1983; Dodson and Ford 1985). The ultrastructural basis of the intercellular pathway is the moderate number and short specialized contacts (occludens and adherens), predominantly located on the apical portions of the interdigitations, as well as the different in size dilatations, frequently opened toward the BL. Two zones may be demarcated in the mesothelial cells: a central (metabolic) zone, comprising the nucleus; and a significant number of organelles, and a peripheral (transport) zone. It is clear that flat cells are not only more numerous, but have also a larger transport zone, and thus are a major participant in the mesothelial transport. The main type of transport through the cubic mesothelium is intercellular, while across the flat covering, transcellular transport appears to be more significant. The flat cells, despite their smaller transport capabilities (rare and short microvilli, poorly or moderately developed vesicular system, and smaller number of intercellular contacts), cover a much larger surface of the SM. The more prominent relief, an abundance of microvilli, the richer vesicular system, the internal cytoskeleton similar to that in resorptive epithelia (Simionescu and Simionescu 1977), as well as the more numerous intercellular contacts between cubic cells suggest a faster and more active participation in the transport processes. The clusters of cubic cells over the LL are very active in fluid exchange, as reported in several investigations (Casley-Smith 1967; Leak and Rahil 1978; Bettendorf 1979; Tsilibarry and Wissig 1982; 1987; Fukuo et al. 1990; Wassilew et al. 1998; Michailova 2001e). Transcellular transport may be considered as basic, especially the transport of substances from the pleural and peritoneal cavities to the underlying structures. This conclusion is supported by the mesothelium enzyme content (Raftery 1973c; 1976; Marsan and Cayphas 1974; Chalet et al. 1976; Adnet et al. 1978; Clausen et al. 1979; Whitaker et al. 1980b; 1982c; Zawieja et al. 1992; see also Sect. 1.5), and by numerous studies using contrast substances (Gosselin and Berndt 1962; Fukata 1963; Cotran and Karnovsky 1968; Cotran and Nicca 1968; Fedorko et al. 1971; Digenis 1984; Alavi et al. 1985; Tsilibarry and Wissig 1987; Fukuo et al. 1988; Gotloib and Shostack 1992; Agostoni and Zocchi 1998). Experimental studies (Levine 1985; Jonecko 1990; Konig et al. 1990) provide evidence for an active mesothelial transport, rather than passive diffusion. On the other hand, the intercellular mechanism is considered more important for the resorption of fluids and substances toward the cavities (Casley-Smith 1967; Cotran and Karnovsky 1968; Baradi and Ryans 1976; Kim et al. 1979; Telvi et al. 1979; Gotloib et al. 1988; Mikhaylova and Vasilev 1988; Payne et al. 1988). The particle size, chemical nature, molecular weight, and mode of the application determine the different capabilities for mesothelial
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transport (Gotloib and Shostack 1987). Zakaria and Rippe (1993) and Negrini et al. (1994) propose models of the peritoneal permselectivity, including pores with different diameters. We consider the multi-layered mesothelial sectors and serosal villi as specialized zones for transport with a more complicated and longer route, involving inter- and transcellular transport (Michailova and Takeva 1997). The flat covering displays a lower density of intercellular spaces per unit area than the cubic cells. The intercellular pathway is basic for the visceral sheets, and the transcellular transport is developed mainly in the parietal sheets, but both serosal sheets represent highly permeable membranes. Despite numerous investigations with contrast materials, it is very difficult to speculate on the relative transport significance of all components of the visceral and parietal sheets. For the movement of water through the SM, an osmotic or hydrostatic pressure gradient is obligatory (Kim et al. 1979), with a very important role of different pressures in the blood and lymphatic vessels (Miserocchi et al. 1982). Several morphologic investigations are devoted to the differences in the vessels in both the serosal sheets as well as in one and the same sheet (Albertine at al. 1982; 1984; Mariassy and Wheeldon 1983; Vladutiu 1986). The effect of body movement (Lee and Olak 1994), the pressure differences between the vessels and the serosal cavities (Payne et al. 1988), the different pressure in the systemic and the pulmonary blood circulation (Corrin and Addis 1990), are other important factors in serosal transport. Our data support the significant ultrastructural variation in the vessels supplying the visceral and parietal sheets. We point out that the abundance of blood vessels is a sign of their more important transport role in the visceral sheet, while the lymph vessels (the LL) are the main paths of transport through the parietal sheet. The barrier between the serosal cavities and the lumina of the submesothelial vessels is represented by the mesothelial layer, the endothelial cells, their BL and the intercellular spaces (between cells and fibers) of the submesothelial layer. The fine structure of the mesothelial and endothelial cells suggest their transcellular and intercellular transport possibilities. They are permeable two-way barriers across which both inter- and intracellular passage takes place. Henderson (1973) and Digenis (1984) suggested that only small parts of peritoneum over the vessels participate in solute exchange. The significant ultrastructural variations of the SM in the species studied, in the both serosal sheets and in various organs, suggest different mechanisms of fluid exchange. Apparently, organ and region transport is a local phenomenon. Our findings of the correspondence of an extensive vesicular system in the mesothelium and the underlying endothelium of the blood capillaries in the diaphragm and in the greater omentum confirm this suggestion. Words (1984) describes heterogeneous density and significant variations in the ultrastructure of the peritoneal microvasculature. A single study (Gotloib et al. 1985) demonstrated the existence of fenestrated capillaries in the peritoneum of the human and the rabbit. The blood capillaries in the animal VP show a thin, continuous endothelial wall, and lymphatic vessels are absent, in contrast to numerous small lymphatic vessels in the human VP. The LL and stomata represent a main pathway for fluid exchange in the organs with lymphatic units (discussed in
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Sect. 4.1.7). Some organs (heart, lung) possess a densely packed network of blood capillaries as well as numerous arterio-venous anastomoses. A system of probably equal portions of blood and lymphatic capillaries occupies large areas in the organs of the lesser pelvis (rectum, uterus, urinary bladder, ovary, testis). A fenestrated endothelium characterizes the blood capillaries, located in close proximity to the surface epithelium of the ovary. The differences between the sheets (parietal and visceral), individual organs and their regions discussed above does not allow us to propose general principles of the transport in the entire SM. 4.3.2 Transport of Horseradish Peroxidase Through the Pleura and Peritoneum The present study reveals the possibilities for two-way HRP transport across the pleura and the peritoneum (see also Michailova and Wassilew 1988; Mikhaylova and Vasilev 1988; Savov and Michailova 1991). Upon intrapleural and intraperitoneal injections of the HRP, active transcellular transport via the mesothelium appears as a basic pathway. The complex relief of the pleural and peritoneal surfaces is a vast contact area promoting the initial retention of the tracer and its subsequent uptake by the vesicular system. The increased number of microvesicles, the composite vesicular complexes and the vesiculo-vacuolar formations indicate that this is a form of active transport controlled by the mesothelial cells in the changeable conditions after application of the tracer. This is further corroborated by the quantitative differences documented, which are attributable to the varying functional state of individual cells. The present results lead to the conclusion that the most probable route of the tracer from the mesothelial vessels towards the serosal cavities is are the mesothelial intercellular spaces. The presence of HRP in some microvesicles in the mesothelial cells does not exclude the possibility for a concomitant transcellular transport to exist. After intraperitoneal injection of HRP the transcellular route is the basic pathway at the start of transport across the surface epithelium of the ovary, which continues through the intercellular spaces (Michailova and Takeva 1997). On the other hand, after injection of HRP in the aorta, the intercellular transport through the surface epithelium is the most prominent route for the tracer. The RP in the distal dilated parts of the intercellular spaces might be explained by the absence of specialized contacts over them. Thus, the present results reveal the possibilities for two-way HRP transport from and to the peritoneal cavity across the multi-layered surface epithelium of the ovary. The RP retained in the BL follows the line of least resistance across the dilated intercellular spaces of the mesothelium, which are free of specialized contacts. Here, a likely explanation is the difference between blood pressure in the pulmonary vessels and the changing pressure within the serosal cavities, especially in the pleural cavity (Miserocchi and Agostoni 1980). RP accumulation in the BL and the EM indicate their involvement in the transport processes as barrier structures in the SM (Herbert 1986). The labeling of alveolar macrophages, type II pneumocytes and in interstitial spaces between cells, fibers and vessels of the submesothelial
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layers, is an expression of the functional and structural relationship between the SM and the underlying tissue. 4.4 Injured Serosal Membranes and Their Recovery 4.4.1 Early Alterations Prominent mesothelial changes characterize the early experimental period (6 h and 1 day after EH, PNT, EP and after application of melted paraffin) consisting of an increased number of microvilli, microvesicles and alterations in the intercellular zones (decrease of specialized contacts, multiplication of dilatations and openings towards the BL). We consider these changes as a first compensatory mechanism for an increase of the serosal surface, and they may be interpreted as initial, unspecific membrane reaction to irritants (Michailova 1996c; 2001d; 2001e; 2004a; 2004b). The tendency towards enlargement of the serosal surface is preserved in the late stages (3, 5, 8 and 15 days), which is achieved through more complicated relief (furrows and ridges), the serosal villi, the exclusively high activated mesothelial cells, multi-layered sectors, newly formed large intercellular dilatations covered with microvilli, undulations of the apical and the basal mesothelial plasmalemma, and an increase in the number of membrane specializations. These structures may be considered more stable in the late period and as a sign of compensatory surface growth, enabling a more extensive transport and secretion. After the initial retention, which is promoted from the larger surface, the material after EH and EP is located in vesicles, vacuoles or in different membrane profiles. Using the transcellular route the same products pass the mesothelial cytoplasm. Their presence in the intercellular dilatations suggests the simultaneous possibility for intercellular transport. It is difficult to define the preferred manner of transport in experimental conditions. During the early period, the mesothelium undergoes an activation of the lysosomal system, which is closely related to its changeable enzyme content and newly formed phagocytic possibilities (Visser et al. 1995; 1996; Agostoni and Zocchi 1998). Similar alterations toward high metabolic and catabolic functions occur in the submesothelial connective tissue cells (Davila and Crouch 1993). Several observations (Mohr 1971; Baradi and Campbell 1974; Forteza-Vila et al. 1977; Korten et al. 1988; Jonecko 1990; Wittman et al. 1994; Bloechle et al. 1998; Wieczorowska-Tobis et al. 2002) after application of different irritants, demonstrated early mesothelial changes with a destructive character, similar to some of our findings at 1 day after EH and EP (Michailova 1996c; 2001e; 2004a; 2004b). They described the clotting and atrophy or vanishing of the microvilli, broadening of the intercellular spaces, ‘cell shedding’, desquamation and separation of the mesothelial cells from each other, and from the destructed BL. The mesothelium mostly becomes destroyed and forms openings in the ‘rough fibrous layer’. We presently report also such data. Large electron-empty spaces are located between
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the processes of the mesothelial cells and thin, interrupted BL. The number of mesothelial cells with degenerative changes increases and connections between them and with underlying BL are interrupted in broad areas in all investigated regions by EP. The zones of the intercellular contacts are located only in the vicinity of the BL and newly formed EM, while the central portions are detached from them. Most drastic is the alteration after application of melted paraffin, where the serosal covering looses the mesothelial layer and the BL in large sectors. An extreme decrease of the volume capacity in the pulmonary vessels (after PNT) and an increased pressure (Agostoni 1972; Wiener-Kronish and Matthay 1988; Wittmann et al. 1994) in the serosal cavities (after EH, EP or application of melted paraffin) may explain other group of changes. These alterations involve: congestion of the capillaries, fluid accumulations, large interruptions in the mesothelium, widened intercellular spaces, and an abundance of extravasal cells (exclusively neutrophilic leukocytes) in the layer over and under the BL. The early vascular or transudative phase involves a massive influx of leukocytes initiating the inflammatory response by realizing ‘a battery of chemokines, cytokines and prostaglandins’ (Antony et al. 1993; 1995; Antony 2003). Mesothelial cells participate in initiating, resolving and controlling serosal inflammation and recovery by secreting various pro-, antiand immunomodulatory mediators (Topley and Williams 1994; Mutsaers 2004). The synthesis of membrane glycoproteins, proteoglycans, uronic acid and noncollagen proteins are increased in the initial period (Wittmann et al. 1994). In the same time coagulations and fibrin depositions represent critical factors for the following serosal regeneration (Buckman et al. 1976; Duffy and diZerega 1994; Holmdahl et al. 1997). If fibrin is not quickly removed, a fibroblastic invasion takes place and serves as progenitor and basis for the adhesions. The mesothelial cells have both procoagulant and fibrinolytic activity, which are depressed by serosal damage (Raftery 1981; Montz et al. 1987; Vipon et al. 1990; Holmdahl 1997). 4.4.2 Late Alterations The beginning of the late ‘resolution’ period (from day 3 after application of melted paraffin and EH, and day 5 after EP and PNT) coincides with the appearance of activated cells. Our results support the double origin of the cell precursors involved in serosal recovery in this time, as described for the peritoneum (Gotloib and Shostack 1987). The initial recovery cells after EH, EP and PNT probably derive from adjacent or apposing uninjured mesothelial cells (Wheeldon et al. 1983; Whitaker and Papadimitriou 1985; Mutsaers 2002). At the same time interval after application of melted paraffin both pleural sheets possess multi-layered sectors formed from free mesothelial and macrophageal cells without junctions between them. These cells probably come from the same free-floating cells (Eskeland and Kjaerheim 1966; Ryan et al. 1973; Watters and Buck 1973; Whitaker and Papadimitriou 1985; Foley-Comer et al. 2002). The first recovery cells, regardless of their origin, proliferate and accomplish re-epithelization as an activated mesothelium
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(Moalli et al. 1987; Peng et al. 1994; Holmdahl et al. 1997; Michailova 2004a; 2004b). The activated cells form monolayer or multi-layered sectors, which surround intercellular dilatations (Michailova 1996c; 2001d). Our results demonstrate the large group of electron-dense granules, cytofilament bundles, elastic-like formations in the mesothelial cytoplasm, as well as numerous newly formed extracellular membrane profiles. Hills (1992) defines this lamellar material expanded over the SM as an anti-inflammatory and face protection layer (Dobbie 1996; Chen and Hills 2000). The activated mesothelium or neighboring cell populations have enhanced possibilities to produce other substances in this stage, as well as the same ones reviewed in the early stage (Montz et al. 1987; Wittmann et al. 1994; Hott et al. 1994; Baumann et al. 1996; Holmdahl et al. 1997; Warn et al. 2001). The secretory products of the new populations of activated mesothelial cells in this late period might be considered as substances to ensure complete serosal healing, to keep the homeostasis of the cavities and to prevent adhesions until day 3 and day 5 after experiments. Richards and Curtis (1984) define similar changes as processes of ‘fibrilogenesis’, which might be arrested, or reversed. Duffy and diZerega (1994) and Mutsaers et al. (2000) describe large number of recovery mesothelial cells in the wound center by 3–4 days after injury. Five days after PNT three new cell populations appear along with typical superficial mesothelial cells in the multi-layered sectors. The most numerous cells display a considerable secretory potential. Few cells undergo activation of the lysosomal system. Fibroblast-like cells arrange the third cell population around which fine collagen fibers are located. The collagen fibers and bundles between the mesothelial cells appear larger and with specific arrangement 8 days after application of melted paraffin. They form thin longitudinal bands between superficial cells or over incomplete BL after PNT at the same time interval. Irregular, interrupted and clumped elastic fibers and EMlike structure are constant findings in all organs after EP (day 8). Neutrophils may also play a role for similar elastolysis and abnormal resynthesis (Uriarte et al. 1993). The morphology of the deep EM as well as elastic fibers, the collagen accumulations, and more numerous blood capillaries in the submesothelial layer suggest their recent formation. The changes in the same period have diffuse character, and extend from the submesothelial layer deep within the underlying parenchyma as thick septa, which ensure an incomplete serosal restoration and appear to be stable. Data about the collagen bundles between mesothelial cells, newly formed serosal villi, several times thicker EM, significant in size collagen aggregates, and newly formed blood capillaries in the submesothelial layer in all experiments suggest the participation of a new cell population. These alterations suggest that ‘secondary recovery cells’ probably arise from the submesothelial precursor cells in the late stage. Our suggestion is in accordance with other reports about ‘submesothelial fibroblasts’ (Swanwick et al. 1973; Renvall et al. 1987; Jimenez-Heffernan et al. 2004), ‘activated resident fibroblasts’ (Friemann et al. 1993), ‘transformed perivascular cells’ (Duffy and diZerega 1994), ‘subpleural mesenchymal cells’ (Isoda et al. 1987), or ‘submesothelial stem cells’ (Wittmann et al. 1994). Other authors have described the presence of cells with epithelial-like characteristics in the subserosal layer
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(Davila and Crouch 1993) or a multipotential subserosal cell (Bolen et al. 1986; 1987). Maybe, such healing cells are mainly responsible for the late ‘fibrosis’, observed by Richards and Curtis (1984), and confirmed by our results on the day 8 (EP, PNT) and the day 15 (EH, after application of melted paraffin). According to the last article, the second stage involves products of ‘other proteins apart from collagens and irreversible fibroblast growth’. The secretion of the mesothelial cells in the late period might be associated with other extracellular substances: different collagen types, elastin, laminin, fibronectin and proteoglycans (Rennard et al. 1984; Herbert 1986; Friemann et al. 1993; Owens and Grimes 1993; Milligan et al. 1995) for the newly formed fibrous structures in the mesothelial and connective tissue layers. The mesothelial transition from epithelial to mesenchymal phenotype and the probable existence of mesothelial progenitor cells (Herrick and Mutsaers 2004) suggests their wide plasticity and expands the possibilities for likely partners in the late period.
5 Summary The coelomic cavities are covered with serosal membranes (SM): peritoneum, pleura, pericardium and tunica vaginalis testis. The present review compiles data on their normal structure, development and involvement in pathologic processes. We add also our results on the ultrastructure of the parietal pleura, peritoneum and pericardium and visceral sheets of the different organs as well in transitional areas between them in man and experimental animals (rat, cat, rabbit, guinea pig, mouse, ground squirrel). By transmission and electron microscopy (TEM, SEM) we distinguish three basic types of relief on both serosal sheets, organs and their different regions. We provide a comprehensive description of the main components of the SM involving: mesothelium, an underlying basal lamina (BL), and a submesothelial connective tissue layer. The clear differences in the size, configuration, organelle content and membrane specializations allow us to distinguish two basic cell types (flat and cubic), numerous intermediate forms and rare degenerative cells. The flat cells are more uniform and widespread over the parietal sheets and some organs. The cubic cells characterize more mobile organs (lung, heart), with hollow reservoir function (stomach, rectum, urinary bladder, uterus and distal portion of the uterine tube) and organs changing their volume considerably (spleen, areas of the liver, ovary). They are located around stomata, over lymphatic lacunae (LL), milky spots (MS), as well as serosal villi, papillaelike evaginations, and criptae-like invaginations. The cubic cells possess more representative organelle apparatus and microvillous coat, corresponding to the number of microvesicles. Two extracellular membrane formations (lamellar bodies and strip-like) and numerous initial lamellar profiles characterize the normal mesothelium. An elastic membrane is a constant component of the serosal covering of the lung and spleen. The presently reported extensive lymphatic network
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in the human visceral pleura is the sole reliable criterion for the statement that it belongs to the ‘thick type’, while all observed animals have a ‘thin’ type VP. The mesothelium and underlying structures of the SM represent a highly permeable bidirectional membrane with significant differences in the organ and region transport as a local phenomenon after horseradish peroxidase application. Stomata are constant features seen by SEM, but are occasional findings observed by TEM over both sides of the diaphragm, lower intercostal spaces, anterior abdominal wall and greater omentum in untreated animals. Our data extend the location of stomata over the liver and broad ligament of the uterus. We strictly defined and nominated the main structures of the lymphatic regions as lymphatic units, stomata, and LL. Several different types of vascularization of omental and extraomental (mediastinal pleura and lesser pelvis) MS are observed after India ink application. Human and animal differences in their location, mesothelial covering, the vessel (blood and lymphatic) supply, free and connective tissue cells and their arrangement are discussed. The mesothelium and the BL changes start early in the gestation and continue throughout the postnatal period. Both cell types (flat and cubic) are already evident through prenatal life. The submesothelial layer of the pleura differentiates during the second half of fetal development (after week 17 of gestation for man and after embryonic day 18 for rat) and corresponds to the changes in the lung parenchyma. The elastic membrane develops after the week 25 of gestation in man and after birth in rat. The alterations of the SM after experimental hemothorax, pneumonectomy, Pseudomonas aeruginosa peritonitis and melted paraffin application suggest the existence of early, reversible, and late definite periods. Prominent changes in the mesothelium and BL characterize the first period. They involve an increased number of membrane specializations, an activation of the lysosomal system, alterations in the intercellular spaces, influx of fluid, and extravasal cells. Some of the alterations have a destructive character. The activated cells, multi-layered mesothelium and altered lamellar bodies mark the beginning of the second period. The late changes involve predominantly the components of the submesothelial layer and extend deep within the underlying tissue by septae. The enlargement of the serosal surface and the thickening of the submesothelial layer is due to the processes of fibrilogenesis, vasculogenesis and recently formed serosal villi. Parallel and rapid changes of cubic mesothelium and the underlying lymphatic endothelium are followed by newly arranged stomata and LL. A drastic increase of the number and the size of the MS, as well as changes in the location and cell composition after peritonitis are described. The present data suggest that late alterations over the entire SM are irregular and asynchronous, showing significant morphologic differences in the various experiments and in both serosal sheets. Some of them are diffuse in character, the final ones appear to be stable and ensure incomplete serosal restoration.
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Subject Index
adhesion, serosal
13
bodies, lamellar – by application of melted paraffin 88 – by hemothorax 70, 118 – by inflammation 107 – development 59 – review 4 chemokines and cytokines 115 ciliae 38, 40, 43, 110
11, 15, 97,
development of – human pleura 19, 61, 107 – rat pleura 70, 107 – serosal cavities 2 fibrinolysis 13, 15, 115 fibroblasts, fibrocytes, fibroblast-like cells 8, 44, 59, 65, 72, 76, 84, 90, 100, 106, 109, 116 hematopoiesis, extramedullary
8
innervation of – milky spots 8, 54, 107 – pleura, pericardium, peritoneum 47, 50, 101 – serosal adhesions 15 lamina, basal – by hemothorax 70 – by inflammation 78 – by peritoneal dialysis 99 – by pneumonectomy 76 – development 56, 63 – normal ultrastructure 43, 98 – of milky spots 54
leukocytes – by application of melted paraffin 90, 93 – by hemothorax 70 – by inflammation 15, 80 – by pneumonectomy 76 – in milky spots 54 lymphocytes 8, 54, 75, 84, 92, 105, 106 macrophages – by application of melted paraffin 93 – by hemothorax 70 – by inflammation 15, 84, 107 – in normal milky spots 8, 54, 106 – in normal submesothelial tissue 44, 100 – role in repair 14 mastocytes 8, 44, 75, 100, 105, 106 matrix, extracellular – by application of melted paraffin 93 – by hemothorax 70 – by inflammation 82 – by pneumonectomy 76 – development 59 – normal ultrastructure 13, 44, 101 membrane, elastic – by hemothorax 70 – by inflammation 84, 100 – development 61, 108 – normal ultrastructure 44, 92 mesothelial cells – activated 72, 75, 78, 80, 84, 90, 93, 111, 116 – degenerative 35, 40, 43, 72, 78, 95, 99, 114 – free-floating 14, 88, 115 – intermediate 35, 94
144 – macrophage-like 35, 42, 52, 88, 95, 106 – precursor cells 14, 116, 117 – prismatic 38 – regional differences 27, 35, 93 microvilli – by hemothorax 72 – by inflammation 16, 78, 82 – by pneumonectomy 78 – development 56 – normal ultrastructure 25, 40, 49, 96 – review 4 milky spots – by inflammation 16, 84 – extraomental 8, 54, 106 – normal structure and ultrastructure 52, 105 – review 7 myocytes, smooth 46, 100
Subject Index relief of serosal membranes
24, 92
stomata – by inflammation 78, 86, 107 – by peritoneal dialysis 103 – extradiaphragmatic 6, 50 – in milky spots 52, 86 – normal ultrastructure 47, 102 – relation with lymphatic lacunae 50, 103 – review 5 vessels, blood and lymphatic – experimental injections 23 – fenestrated capillaries 7, 54, 106, 112, 113 – in milky spots 7, 52 – in pleura 46 – lymphatic lacunae 50, 86, 103 – role in transport 112 villi, serosal 78, 82, 93, 114, 116
