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Lymphatic absorption from the peritoneal cavity: Regulation of patency of mesothelial stomata
MICROVASCULAR
RESEARCH
25, 22-39 (1983)
Lymphatic Absorption from the Peritoneal Cavity: Regulation of Patency of Mesothelial Stomata’ EFF-IE C. TSILIBARY~ AND STEVEN
L. WISSIG~
Department of Anatomy, University of California, San Francisco, California 94143 Received
March 4, 1982
Absorption of fluid from the peritoneal cavity is carried out by terminal lymphatics, called lacunae, located in the diaphragm. Lacunae communicate with the peritoneal cavity via stomata1 openings between mesothelial cells that lead into channels formed by juxtaposed processes of mesothelial and lacunar endothelial cells. We examined the patency of stomata under conditions of drug-induced relaxation or contraction of the diaphragm, increased intraabdominal pressure, and experimental ascites caused by intraperitoneal infection with Toxoplasma gondii. We observed numerous patent stomata when the diaphragm is relaxed and when intraabdominal pressure is raised. When the diaphragm is contracted we observed either few of no patent stomata. Ascites accompanying peritoneal parasitic infection resulted in “hypertrophy” of mesothelial cells and fewer patent stomata. We then examined the means for regulating the patency of stomata and the conformation of endothelial flaps that extend across the lumen of channels. Using the technique of decoration of actin with the Sl subfragment of myosin, we identified numerous actin filaments in the cytoplasm of mesothelial and endothelial cells that border stomata and form channels. Treatment in situ of the diaphragm with cytochalasin D resulted in pronounced deformation of mesothelial cells overlying lymphatic lacunae and endothelial cells that line channels. The deformation was reversed after cytochalasin D was washed away with n-saline. Our findings indicate that the patency of mesothelial stomata can vary in response to changing conditions in the peritoneal cavity. They also indicate that maintenance of the normal conformation of stomata and channels and perhaps control of the patency of stomata as well rely upon actin components in the cytoplasm of mesothelial and endothelial cells.
INTRODUCTION
Fluid in bulk, particles, and cells are removed from the peritoneal cavity by large terminal lymphatics, called lacunae, located beneath the mesothelium of the peritoneal surface of the diaphragm. The absorbed material enters the lacunae via openings called stomata between lateral borders of the mesothelial cells that overlie lacunae, i.e., lacunar mesothelial cells (Yoffey and Courtice, 1970). The ’ This study was supported by National Institutes of Health Research Grant HL-04512 and Patent Funds of the Graduate Division, University of California, San Francisco. * Present address: Department of Pathology, Harvard Medical School, Childrens’ Hospital Medical Center, 300 Longwood Avenue, Boston, Massachusetts 02115. 3 To whom all correspondence should be addressed. 22 M)26-2862/83/010022-18$03.00/O Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
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stomata can accomodate objects as large as frog erythrocytes which are 23 pm in diameter (Allen, 1936). Along the margin of stomata, mesothelial and lacunar endothelial cells are joined to one another so that they line a channel that leads from the peritoneal cavity into the lumen of the lacuna (Wang, 1975; Leak and Rahil, 1978; Tsilibary and Wissig, 1979a). The stoma may be bridged by filamentous processes of the mesothelial cells. Flattened processes from the endothelial cells lining the channels frequently protrude across the luman of the channel below the stoma, creating a tortuous pathway. It has not yet been established whether stomata are stable openings or are openings whose patency is altered in response to changing physiologic and pathologic conditions within the peritoneal cavity. The principal aim of the first part of our study was to determine whether the patency of stomata could be altered by experimental manipulation. The specific manipulations we used were drug-induced contraction and relaxation of the diaphragm and an increase in intraabdominal pressure. In the second part of our study, we verified the identification of fine fibrils in the cytoplasm of endothelial and mesothelial cells bordering the stoma and channel as actin filaments. In addition we examined the effects of cytochalasin D (CD), a drug that depolymerizes actin filaments (Godman and Miranda, 1978; Brown and Spudich, 1979, 1981) on the structure of stomata and channels. In the final portion of the study we examined the impact of chronic ascites stemming from infection of the peritoneal cavity with Toxoplasma gondii on the lacunar mesothelium. Previous physiological studies of pathologic and experimental chronic ascites had shown that at early stages peritoneal absorption is normal, but later it falls (Bangham, 1953; Itani, 1959). The effect of ascites on the morphology of the mesothelium was not examined. MATERIALS
AND METHODS
(A) We anesthetized eight male Sprague-Dawley rats, weighing 40-60 g with an intravenous injection of sodium pentobarbital(35 mg/kg body wt). We injected four rats intravenously with 0.1 ml carbachol solution (carbamylcholine chloride, Sigma Chemical Company, St. Louis, MO., 0.3 mg/kg body wt, in n-saline) to induce contraction of the diaphragm and four rats with 0.1 ml succinylcholine solution (succinylcholine chloride, Sigma Chemical Company, 0.75 mgikg body wt, in n-saline) to induce relaxation of the diaphragm (Goodman and Gilman, 1975). In both groups of rats respiration ceased after 5-20 set, and we fixed the diaphragm with an intraperitoneal injection of 2% glutaraldehyde (Ladd Research, Burlington, Vt.) and 1% acrolein (Polysciences, Inc., Warrington, Pa.) in 0.1 M sodium cacodylate, pH 7.4, warmed to 37”. Fifteen minutes later, we excised the diaphragm, cut anterolateral areas containing lacunae into 5-mm squares, and placed the squares in fixative overnight at 4”. The next day, the tissue was treated with 1.5% partially reduced osmium tetroxide for 90 min at room temperature (Karnovsky, 1971). We dehydrated the tissue with ethanol and dried it by the critical-point method (Bomar Instrument Corporation, Fort Wayne, Ind.). We mounted the specimens on aluminum stubs with the peritoneal surface exposed, coated them with a layer of gold 20 nm thick in a Hummer V Sputter coater (Technics, Inc., Alexandria, Va.), and examined them with a scanning
24
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electron microscope (Model S-150, Cambridge Instrument Co., Inc., Ossining, N.Y.). We examined a total surface area of approximately 90 mm2 of each diaphragm. (B) We injected 10 male C3H mice weighing approximately 30 g and 2 male Sprague-Dawley rats weighing 30-50 g intraperitoneally with 0.1-0.2 ml n-saline in order to increase intraabdominal pressure. We anesthetized them 5-10 min later with ether and examined the peritoneal surface of their diaphragms with the scanning electron microscope by the method described in Section A. We treated another group of 10 male Sprague-Dawley rats, 40-50 g body wt , similarly except that, instead of administering n-saline by intraperitoneal injection, we made a small midventral incision in the abdominal wall to prevent any increase in intraabdominal pressure, and then introduced 0.1-0.2 ml n-saline into the peritoneal cavity. We gently held the small incision open for the next 5-10 min. The diaphragm was then fixed. The fixative used in this set of experiments contained 2% glutaraldehyde and 1% paraformaldehyde (Matheson Scientific, Chicago, Ill.) in 0.1 M sodium cacodylate, pH 7.4, with 0.006% succinylcholine chloride added to prevent initiation of the contraction of the diaphragm by the fixative. The specimens of diaphragm were examined with the scanning electron microscope according to the procedure described in Section A. (C) We injected a male Swiss albino mouse weighing 30 g intraperitoneally with 0.1 ml India ink in n-saline in order to label lacunae of the diaphragm. Ten minutes later, we anesthetized the mouse with ether and made a small incision in the midventral abdominal wall. We dripped a 50% solution of glycerol in modified salt solution (MSS) on the peritoneal surface of the diaphragm for 5 min, followed by 25% glycerol for 5 min, and then 5% glycerol for 5 min (Ishikawa et al., 1969). We excised the diaphragm, cut 0.5-mm square pieces from areas that contained lacunae, and incubated the pieces in a standard salt solution (SSS) containing 1-2 mg/ml SI subfragment of myosin for 5 hr at 4” (Ishikawa et al., 1969) in order to decorate actin filaments. We then fixed the pieces in 1% glutaraldehyde and 0.2% tannic acid in 0.1 M (mono-dibasic) sodium phosphate buffer, pH 7.0, for 30 min (Begg et al., 1978). We then placed the pieces in 2% glutaraldehyde and 1% paraformaldehyde in 0.1 M sodium phosphate (monodibasic) buffer, pH 7.4, overnight at 4”. The next morning the pieces were transferred to 1.5% osmium tetroxide in sodium phosphate buffer, pH 6.0, for 90 min at 4” (Maupin-Szamier and Pollard, 1978). The pieces were then rinsed in distilled water and stained en bloc in 2% aqueous uranyl acetate for 90 min at room temperature. They were dehydrated in acetone for 40 min and propylene oxide for 30 min and were embedded in Epon 812. Thin sections of the specimens were cut with a Sorvall MT-2 ultramicrotome. The sections were stained with 0.6% lead citrate for 5-10 min and were examined with a Siemens 1 electron microscope operated at 80 kv. (D) We anesthetized nine male Sprague-Dawley rats, 40-50 g body wt, with an intravenous injection of sodium pentobarbital. After making a small incision in the midventral abdominal wall, we flooded the peritoneal cavity with a solution at 4-40 $I4 CD (Aldrich Chemical Company, Milwaukee, Wise.) in n-saline for 10-30 min. We then fixed and processed specimens of the diaphragm for examination with the scanning and transmission electron microscopes as described in Sections A and C. We exposed the diaphragms of two male Sprague-Dawley rats, 40-
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50 g body wt, to 40 E,LMCD in n-saline for 20 min as described above. We replaced this solution with n-saline for 15 min in one rat and 30 min in the other. We then fixed the diaphragms for examination with the scanning and transmission electron microscopes. (E) We injected three male C3H mice, 30 g body wt, with 0.1 ml Hanks’ medium containing Toxoplasma gondii in order to induce experimental ascites (Nichols and O’Connor, 1981). We anesthetized an infected mouse with ether 30 set, 5 min, and 48 hr later and fixed the diaphragm with an intraperitoneal injection of the glutaraldehyde-formaldehyde solution as described in Section B. Fifteen minutes later we collected specimens of diaphragm for examination with the scanning electron microscope according to the procedure described in Section A. Specimens were also collected for examination with the transmission electron microscope. After treatment with partially reduced osmium tetroxide, these were rinsed with 0.05 M sodium maleate buffer, pH 5.2, for 30 min at 4” and were stained en bloc with 1.5% uranyl acetate in the same buffer for 90 min at 37”. We then dehydrated the specimens with absolute acetone for 40 min and propylene oxide for 30 min, and embedded them in araldite (British Araldite, Grade CY-212, Polysciences, Inc.). Thin sections of the specimens were examined by electron microscopy as described in Section C. RESULTS In all animals injected intravenously with carbachol prior to fixation, the mesothelial surface of the diaphragm was crenellated, i.e., was thrown up into tight folds with their long axis running at right angles to the long axis of muscle fibers (Fig. 1). The folded appearance of the surface indicated that the underlying muscle cells were in a contracted state at the time of fixation. In the diaphragm of these animals patent stomata were either extremely few in number4 or totally absent (Figs. 1 and 2). In diaphragms from four rats injected intravenously with succinylcholine, the mesothelial surface of the diaphragm was smooth, indicating that the underlying muscle was in a relaxed state at the time of fixation (Fig. 3). Numerous patent stomata were observed in areas of lacunar mesothelium (Figs. 3 and 4). In the animals that received an intraperitoneal injection of 0.1 ml n-saline prior to fixation of the diaphragm, numerous patent stomata were invariably observed between lacunar mesothelial cells (Fig. 5). In contrast, in animals in which the same volume of fluid was introduced into the peritoneal cavity but in which a small incision was made in the abdominal wall and was held open, stomata were totally absent from the lacunar mesothelium cells (Fig. 6). The cytoplasm of lacunar mesothelial and endothelial cells that border stomata and channels usually contains abundant microfilaments which resemble actin (Fig. 7). In the mesothelial cells, the microfilaments are frequently observed in 4 We did not attempt to make quantitative measurements of stomata per unit surface area in this study. Because of the tendency of the lacunar surface area to form undulating folds, it was not possible to measure accurately the area of their surface or to visualize stomata at the top, bottom, or far side of the curvatures. When we report differences in the number of stomata qualitatively in the different experimental groups of this study, we report only those differences that were distinctive and highly consistent.
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FIGS. 1, 2. Lacunar mesothelium from the diaphragms of two rats injected intravenously with carbachol prior to sacrifice. Under low power (Fig. l), the surface of the diaphragm appears crimped into tight folds running at right angles to the long axis of the underlying contracted muscle fibers. Patent stomata cannot be detected under low (Fig. I) or high power (Fig. 2). 750x (Fig. 1); 2900x (Fig. 2).
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FIGS. 3, 4. Lacunar mesothelium from two rats injected intravenously with succinylcholine prior to sacrifice. Lacunar mesothelium forms low ridges running parallel to underlying muscle fibers and is bordered on either side by flat nonlacunar mesothelium (Fig. 3). Patent stomata are seen under low power (Fig. 3) and, in more detail, under high power (Fig. 4) between adjacent mesothelial cells. 900x (Fig. 3); 2400x (Fig. 4).
TSILIBARY AND WISSIG
FIG. 5. Lacunar mesothelium of an animal that received an intraperitoneal injection of n-saline 5-10 min prior to sacrifice. Numerous stomata are observed between lacunar mesothelial cells. 1250x. FIG. 6. Lacunar mesothelium of an animal in which n-saline was introduced into the peritoneal cavity after a midventral incision was made in the abdominal wall to prevent a rise in intraabdominal pressure. Patent stomata are not seen between mesothelial cells. 1700x.
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FIG. 7. Channel in a mouse injected intraperitoneally with colloidal carbon to label lacunae prior to sacrifice. Carbon particles are seen in the peritoneal cavity (PC) and lumen of the channel (CH). The mesothelial stoma is not present in the plane of section. A band of microfilaments (mf) stretches across the base of the mesothelial cell (MC), and microfilaments (mf) are also seen in the lymphatic endothelial cell (EC) forming the wall of the channel. Submesothelial connective tissue (CT). 56,000 x .
cell processes bordering the stomata and organized as a bundle that parallels the base of these cells. In lacunar endothelial cells, the microfilaments are prominent in processes that extend across the lumen of the channel. In specimens extracted with glycerol and subsequently treated with the Sl fragment of myosin, filaments in the cytoplasm of mesothelial and endothelial cells were decorated with the Sl fragment in the familiar arrowhead configuration, signifying that the filaments are in fact composed of actin (Figs. 8 and 9). Following treatment with CD, the morphology of lacunar mesothelial cells and lymphatic endothelial cells, examined with the SEM and TEM, was markedly distorted. The degree of distortion was more or less the same in specimens treated with 4 or 40 pM CD for lo-30 min. The shape of the mesothelial cells was flattened and highly irregular (Fig. 10). Microvilli had disappeared from their
30
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FIG. 10. Lacunar mesothelium of a rat treated with CD for 20 min prior to fixation. The mesothelial cells are flattened, and their microvilli have disappeared. A single cilium (arrows) is observed on the apical surface of individual mesothelial cells. The lateral borders of the cells are extensively retracted, preventing identification of stomata. Fibrillar connective tissue underlying the mesothelium is exposed to view. 3000 x .
surface. The stomata were enlarged and distorted. In addition, the lateral borders of lacunar mesothelial cells had pulled apart from one another exposing underlying collagenous fibers to view (Fig. IO), a condition that is never encountered in untreated specimens. In thin sections examined with the TEM, actin filaments were no longer visible in the cytoplasm of mesothelial and endothelial cells in stomata1 regions. Clumps of more or less homogeneous material of moderate electron density appeared in the cytoplasm of the cells. Some of the clumps were located in protruberances from the cut surface (Fig. 11). Although the outline of the cells was distorted, intracellular organelles appeared normal (Fig. 11). Nonlacunar mesothelium was unaffected by the treatment with CD. After CD was washed out of the peritoneal cavity and replaced with n-saline for 15-30 min, we observed that the lacunar mesothelial cells, examined with the SEM, had recovered their normal domed shape. The borders between adjacent mesothelial cells were sealed, and collagenous fibers of the underlying connective tissue were no longer exposed to view (Fig. 12). The stomata returned to their normal configuration. However, even after the CD had been washed FIGS. 8, 9. A lacunar mesothelial cell (Fig. 8) and a lacunar endothelial cell (Fig. 9) from a glycerol-extracted diaphragm “stained” with Sl subfragment of myosin. Actin filaments decorated with the Sl subfragment are seen in the microvilli (MV) and cytoplasm of the mesothelial cell and in the perinuclear cytoplasm of the endothelial cell. Peritoneal cavity (PC). Basement membrane (BM). Nucleus (NU). 58,000~ (Fig. 8); 66,000x (Fig. 9).
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FIG. 11. Lacunar mesothelium (MC) and endothelium (EC) of a rat injected intraperitoneally with CD 20 min prior to sacrifice. Actin filaments normally seen in the cytoplasm of these cells are replaced by clumps of amorphous material, much of which is located in abnormal protruberances of the cell surface (arrows). The shape of mesothelial and endothelial cells is distorted. 32,900~
away for 30 min, normal numbers of microvilli were not yet restored to the surface of the mesothelial cells. In thin sections examined with the TEM, the channels formed by mesothelial and endothelial cells had returned to their normal configuration, and actin filaments were normal in appearance and topology in both mesothelial and lymphatic endothelial cells (Fig. 13). In mice infected by an intraperitoneal injection of Toxoplasma go&ii 30 set and 5 min prior to fixation, the lacunar mesothelium appeared essentially normal. Stomata were observed between adjacent lacunar mesothelial cells. With the SEM, Toxoplasma were observed as small, free cells scattered over the surface of the lacunar mesothelium. After 48 hr of infection, the peritoneal cavity was markedly dilated with ascitic fluid. With the SEM, we observed that the surface of the lacunar mesothelium was markedly altered. Instead of their normal, smoothly domed shape, the mesothelial cells resembled “fried eggs” (Figs. 14 and 15). The center of the cell was thickened and cuboidal in height, and was encircled by a narrow rim of flattened cytoplasm (Fig. 15). The surface of the flattened rim of cytoplasm was relatively smooth, but the central, thickened portion of the cell was coated with numerous microvilli. Their number was much higher than is seen on normal lacunar mesothelial cells. Stomata between lacunar
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FIG. 12. Lacunar mesothelium of a rat treated intraperitoneally with CD for 20 min. CD was subsequently washed out of the peritoneal cavity with n-saline for 15 min. Mesothelial cells (MC) have recovered their normal configuration although they still lack their normal population of microvilli. The underlying connective tissue is no longer exposed between mesothelial cells. A stoma shown in the field (arrow) appears normal. 3500 x .
mesothelial cells were sparse in number (Fig. 14). In thin sections, we observed that occasional mesothelial cells contained one or more Toxoplasma enclosed within a large membrane-limited vesicle. The rest of the cytoplasm of the cell appeared healthy and undamaged. The lacunar mesothelial cells that were not invaded by parasites showed no sign of injury or necrosis. As a matter of fact, the opposite appeared to be the case. Each cell appeared to have undergone a form of “hypertrophy.” It seemed to contain more mitochondria and vesicles of endoplasmic reticulum than normal, and the Golgi apparatus seemed to be enlarged (Fig. 16). The patency of mesothelial stomata under the above mentioned experimental conditions is summarized in Table 1. DISCUSSION Stomata with their associated channels, which connect the peritoneal cavity with the lumen of the lymphatic lacunae of the diaphragm, are structurally differentiated units. For example, the lateral borders of mesothelial cells and lymphatic endothelial cells join at the rim of stomata and form a continuous cellular lining for the channel (Wang, 1975). The submesothelial connective tissue at the site of a stoma is interrupted and in addition, is structurally modified to accommodate the channel. For example, both the mesothelium and endothelium lack a basement membrane at the site of channel. The connective tissue im-
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FIG. 13. TEM micrograph from the diaphragm of the same rat as in Fig. 12. The appearance of mesothelial (MC) and endothelial (EC) cells is normal, and actin filaments (mf) reappear along the base of mesothelial cells and in endothelial cell processes. The arrows point to intermediate filaments which are normally present in both types of cells. 31,500x
mediately adjacent to the channel contains, as its principal fibrillar component, masses of microfibrils, not the bundles of collagenous fibers found elsewhere in submesothelial connective tissue (Bettendorf, 1978; Tsilibary and Wissig, 1979a). The endothelium lining the channel extends many flattened processes across the lumen of the channel. Finally, the cytoplasm of the mesothelial and endothelial cells at the sites of stomata and channels contains abundant fine filaments (Leak, 1976; Tsilibary and Wissig, 1979a; 1979b). This high degree of structural complexity of the stomata and channels leads us to conclude that they are stable structural units whose number and location are fixed in the short run. Although the number and location of stomata and channels are presumably stable on a short-term basis, it had not been demonstrated whether or not the patency of stomata is subject to regulation depending upon changing conditions within the peritoneal cavity. In order to test this, we used two experimental maneuvers. We examined how many stomata were patent, first, when the muscle of the diaphragm was either contracted or relaxed, and, second, when fluid pressure within the peritoneal cavity was increased. We observed by scanning electron microscopy that the number of patent stomata is correlated with drug-induced contraction or relaxation of the muscle of the diaphragm. When the muscle of the diaphragm is stimulated to contract
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FIGS. 14, 15. Lacunar mesothelium from a mouse injected intraperitoneally with Toxoplasma 48 hr prior to sacrifice. Mesothelial cells have an increased number of microvilli. The increased number are confined to the rounded central region of the cell and are absent from its flattened perimeter. A few organisms (T) are seen on the mesothelial surface. Either no stomata (Fig. 14) or few stomata (Fig. 15) are observed. 1500x (Fig. 14); 2500x (Fig. 15).
go&ii
with carbachol, stomata are very few in number (Figs. 1 and 2). Conversely, when the diaphragm is stimulated to relax with succinylcholine, large numbers of patent stomata are present (Figs. 3 and 4). In animals in which we injected fluid into the peritoneal cavity, a maneuver that presumably increases intraabdominal pressure, stomata were invariably abundant (Fig. 5). It seems that increased pressure rather than the presence of fluid is responsible for this response of stomata, because in animals which received a similar amount of fluid in the peritoneal cavity, but in which an incision was made in the abdominal wall to
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FIG. 16. Lacunar mesothelial cell from the same animal as in Figs. 14 and 1.5.Many organelles of the mesotheliai cell, e.g., endoplasmic reticulum, mitochondria, Golgi apparatus, lysosomes, etc., are present in greater than normal size and number. The apical surface of the cell shows increased numbers of microvilli. 17,400X
prevent any increase in intraabdominal pressure, stomata were either sparse or totally absent (Fig. 6). These findings clearly indicate that the patency of stomata, under defined experimental conditions, is subject to regulation. The physiologic significance of the regulation, as well as the steps involved in the process of reflex control, remain to be defined. A number of physiological studies have shown that the rate of fluid absorption from the peritoneal cavity is augmented by respiratory movements of the diaphragm (Allen and Vogt, 1937; Courtice and Steinbeck, 1950; Courtice and Morris, 1953; Allen and Raybuck, 1960). In addition, Allen and Vogt (1937) observed that intraperitoneally injected tracers which are taken up by the lacunae do not escape from the lacunae when the diaphragm is excised and pressure is exerted on its peritoneal surface. Both of these findings imply that the pathway
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TABLE 1
EFFECTS OF VARIOUS EXPERIMENTAL CONDITIONS ON PATENCYOF MESO~HELIALSTOMATA
Experimental condition Intravenous injection of carbachol (contraction of the diaphragm) Intravenous injection of succinylcholine (relaxation of the diaphragm) Intraperitoneal injection of fluid accompanied by increase in intraabdominal pressure. Intraperitoneal introduction of fluid without increase in intraabdominal pressureb Experimental ascites from infection with Toxoplasma gondii
Relative number of stomata” -
++++
+++ -
5
’ Scaled by SEM examination from essentially none (- ) to maximum numbers (+ + + +). b Increase in intraabdominal pressure was prevented by an open incision in the abdominal wall.
leading from the peritoneal cavity to the lumen of a lacuna, i.e., the stoma and its associated channel, is protected by a valving mechanism to prevent regurgitation of absorbed fluid. Several workers (Allen, 1936; Bettendorf, 1978) have proposed that mesothelial stomata, opening passively as the diaphragm stretches during expiration and closing passively as the diaphragm contracts during inspiration, act as valves. Casley-Smith (1964) has suggested that the endothelial processes that protrude across the lumen of the channel serve this purpose. Until this time, however, experimental evidence assigning a valving function to either the mesothelium or endothelium has not been reported. Our findings suggest that both the mesothelial processes which form the opening and the endothelial flaps criss-crossing the channel may act as valves under different conditions. We have shown that mesothelial stomata open and close with drug-induced relaxation and contraction of the diaphragm. Therefore, it is possible that the patency of stomata could be similarly altered during the respiratory cycle, although not necessarily by a passive process. Whether the patency of stomata does, in fact, change during the respiratory cycle is a difficult point to establish because fixation of the diaphragm with conventional methods is, first, a relatively slow process and, second, necessitates major alterations of the environment within the peritoneal cavity. Such alterations in themselves might initiate reflex changes in patency of the stomata. Our observations that the stomata remain patent when intraabdominal fluid pressure is raised suggests that their patency in this situation may be an adaptive response to expedite removal of fluid from the peritoneal cavity. Their protracted
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patency could, however, lead to regurgitation of fluid from the lacunae when the diaphragm contracts. Regurgitation would be averted if, in this situation, the endothelial flaps stretching across the channel assumed responsibility for acting as valves. At this point, it is not possible to decide whether either or both the mesothelium and endothelium serve as a valving mechanism to prevent reflux of fluid in the passageway from the peritoneal cavity to the lacunae. There may actually be a division of labor between the two structures with patency of the stomata regulating rate of flow and the processes of endothelial cells acting as valves to prevent reflux. We then examined the structural mechanism for regulating patency of stomata and conformation of endothelial flaps. We observed, as have others (Leak, 1976) that, at the sites of stomata and their channels, both mesothelial and endothelial cells contain actin-like filaments with a diameter of 5-6 nm (Fig. 7). We confirmed that these filaments are actin by the method of arrowhead decoration with the S1 subfragment of myosin (Figs. 8 and 9). Furthermore, we were able to depolymerize the filaments by exposing the mesothelial and endothelial cells to CD (Figs. 10 and 11). In the latter situation, the conformation of both types of cells was severely altered by selective disruption of actin filaments. The cells were not permanently affected by exposure to CD because, after the drug was washed out with n-saline, the cells returned to their normal morphology (Figs. 12 and 13). These findings indicate that the actin filaments located in the cytoplasm of mesothelial and endothelial cells adjacent to stomata and channels are components of a cytoskeletal framework that maintains the conformation of the passageway between the peritoneal cavity and the lymphatic lacunae. Finally, we examined the changes that occur in the lacunar mesothelium in the presence of severe ascites caused by intraperitoneal infection with Toxoplasma go&ii. After infection with the parasite for 48 hr, stomata were largely absent (Fig. 14) even though the peritoneal cavity was filled with abundant ascitic fluid and intraabdominal pressure had presumably increased. A complex reaction of mesothelial cells to the parasitic infection was observed. The mesothelial cells were not damaged by the protozoon, although occasional cells were parasitized. In contrast, a presumed hypertrophy of the cells was observed (Figs. 14-16). Specifically, most cytoplasmic organelles, i.e., the endoplasmic reticulum, the Golgi apparatus, mitochondria, and lysosomes were increased in size or number (Fig. 16) and the surface of mesothelial cells was dotted with vastly increased numbers of microvilli (Figs. 14 and 15). This complex response might be initiated by parasitism of mesothelial cells and underlying tissues, as well as by alteration in the amount and composition of peritoneal fluid. At this time, we do not know what the significance of this type of reaction by the mesothelium is. The observations indicate that the mesothelium has the capacity to respond to a chronic pathologic situation in ways that have not previously been recognized. In conclusion, our observations show that the patency of stomata can vary in response to changing conditions in the peritoneal cavity. Contractile components of mesothelial and endothelial cells seem to be responsible for maintaining the conformation of both the stomata and underlying channels and presumably also for effecting changes in patency of stomata. Finally, we have observed that the mesothelium responds by a previously underscribed form of hypertrophy to chronic ascites caused by infection with the parasite Toxoplasma gondii.
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ACKNOWLEDGMENTS The authors are indebted to Dr. Roger Cooke, who provided the subfragment 1 of myosin, to Ms. Simona Ikeda for technical assistance, to Mr. Dave Akers for assistance with photography, and to Ms. Alana Schilling for typing the manuscript.
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glass particles from the peritoneal cavity. Brit. J. Exp. Puthol. 34, l-11. BEGG,D. A., RODEWALD, R., AND REBHUN, L. I. (1978). The visualization of actin filament polarity in thin sections. J. Cell Biol. 79, 846-852. BETTENDORF, U. (1978). Lymph flow mechanism of the subperitoneal diaphragmatic lymphatics. Lymphology 11, 111-116. BROWN, S. S., AND SPUDICH, J. A. (1979). Cytochalasin inhibits the rate of elongation of actin filament fragments. J. Cell Biol. 83, 657-662. BROWN, S. S., AND SPUDICH, J. A. (1981). Mechanism of action of cytochalasin: Evidence that it binds to actin filament ends. J. Cell Biol. 88, 487-491. CASLEY-SMITH, J. R. (1964). Endothelial permeability-the passage of particles into and out of diaphragmatic lymphatics. Quart. J. Exp. Physiol. 49, 365-383. COURTICE, F. C., AND MORRIS,B. (1953). The effect of diaphragmatic movement on the absorption of protein and of red cells from the pleural cavity. Aust. J. Exp. Biol. Med. Sci. 31, 227-234. COURTICE, F. C., AND STEINBECK, A. W. (1950). The lymphatic drainage of plasma from the peritoneal cavity of the cat. Aust. J. Exp. Biol. Med. Sci. 28, 161-169. GODMAN, G. C., AND MIRANDA, A. F. (1978). Cytochalasins-biochemical and cell biological aspects, Chapter 12. In ‘Cellular Contractility and the Visible Effects of Cytochalasin” (S. W. Tanenbaum, ed.), pp. 279-429. Elsevier/North-holland, New York. GOODMAN, L. W., AND GILMAN, A. (1975). The pharmacological basis of therapeutics. 5th ed. pp. 434-439, 575-588, McMillan, New York. ISHIKAWA, H., BISCHOFF, R., AND HOLTZER, H. (1969). Formation of arrowhead complexes with heavy meromyosin in a variety of cell types. J. Cell Biol. 43, 312-328. ITANI, K. (1959). Studies on the peritoneal absorption of particulate material. Arch. Jupon. Chirurg. 28, 802-824. KARNOVSKY, M. J. (1971). Use of ferrocyanide-reduced osmium tetroxide in electron microscopy.
“Proceedings of the 1lth Annual Meeting of the American Society for Cell Biology.”
p. 146a.
LEAK, L. V. (1976). Permeability of peritoneal mesothelium: A TEM and SEM study. J. Cell Biol. 70, 423a. LEAK, L. V., AND RAHIL, K. (1978). Permeability of the diaphragmatic mesothelium: the ultrastructural basis for stomata. Amer. J. Anat. 157, 5.57-594. MAUPIN-SZAMIER, P., AND POLLARD, T. D. (1978). Actin filament destruction by osmium tetroxide.
J. Cell Biol. 77, 837-852. NICHOLS, B. A., AND O’CONNOR, R. G. (1981). Penetration of mouse peritoneal macrophages by the
protozoon Toxoplasma gondii. Lab. Invest. 44, 324-335. TSILIBARY, E. C., AND WISSIG, S. L. (1977). Absorption from the peritoneal cavity: SEM study of mesothelium covering the peritoneal surface of the mouse diaphragm. Amer. J. Anat. 149, 127-133. TSILIBARY, E. C., AND WISSIG, S. L. (1979a). Structural plasticity in the pathway for lymphatic drainage from the peritoneal cavity. Microvasc. Res. 17, Sl44. TSILIBARY, E. C., AND WISSIG, S. L. (1979b). Cytochalasin D modifies the cell surface and actin
distribution of cells in vivo. J. Cell Biol. 83, 328a. WANG, N.-S. (1975). The preformed stomas connecting the pleural cavity and the lymphatics in the parietal pleura. Amer. Rev. Resp. Dis. 111, 12-20. YOFFEY, T. M., AND COURTICE, F. C. (1970).“Lymphatics, Lymph and the Lymphomyeloid complex.”
Chapter 4. “Lymph Flow from Regional Lymphatics.” Cavities.” pp. 295-319, Academic Press, New York.
Section XV. “The Pleural and Peritoneal
RESEARCH
25, 22-39 (1983)
Lymphatic Absorption from the Peritoneal Cavity: Regulation of Patency of Mesothelial Stomata’ EFF-IE C. TSILIBARY~ AND STEVEN
L. WISSIG~
Department of Anatomy, University of California, San Francisco, California 94143 Received
March 4, 1982
Absorption of fluid from the peritoneal cavity is carried out by terminal lymphatics, called lacunae, located in the diaphragm. Lacunae communicate with the peritoneal cavity via stomata1 openings between mesothelial cells that lead into channels formed by juxtaposed processes of mesothelial and lacunar endothelial cells. We examined the patency of stomata under conditions of drug-induced relaxation or contraction of the diaphragm, increased intraabdominal pressure, and experimental ascites caused by intraperitoneal infection with Toxoplasma gondii. We observed numerous patent stomata when the diaphragm is relaxed and when intraabdominal pressure is raised. When the diaphragm is contracted we observed either few of no patent stomata. Ascites accompanying peritoneal parasitic infection resulted in “hypertrophy” of mesothelial cells and fewer patent stomata. We then examined the means for regulating the patency of stomata and the conformation of endothelial flaps that extend across the lumen of channels. Using the technique of decoration of actin with the Sl subfragment of myosin, we identified numerous actin filaments in the cytoplasm of mesothelial and endothelial cells that border stomata and form channels. Treatment in situ of the diaphragm with cytochalasin D resulted in pronounced deformation of mesothelial cells overlying lymphatic lacunae and endothelial cells that line channels. The deformation was reversed after cytochalasin D was washed away with n-saline. Our findings indicate that the patency of mesothelial stomata can vary in response to changing conditions in the peritoneal cavity. They also indicate that maintenance of the normal conformation of stomata and channels and perhaps control of the patency of stomata as well rely upon actin components in the cytoplasm of mesothelial and endothelial cells.
INTRODUCTION
Fluid in bulk, particles, and cells are removed from the peritoneal cavity by large terminal lymphatics, called lacunae, located beneath the mesothelium of the peritoneal surface of the diaphragm. The absorbed material enters the lacunae via openings called stomata between lateral borders of the mesothelial cells that overlie lacunae, i.e., lacunar mesothelial cells (Yoffey and Courtice, 1970). The ’ This study was supported by National Institutes of Health Research Grant HL-04512 and Patent Funds of the Graduate Division, University of California, San Francisco. * Present address: Department of Pathology, Harvard Medical School, Childrens’ Hospital Medical Center, 300 Longwood Avenue, Boston, Massachusetts 02115. 3 To whom all correspondence should be addressed. 22 M)26-2862/83/010022-18$03.00/O Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
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stomata can accomodate objects as large as frog erythrocytes which are 23 pm in diameter (Allen, 1936). Along the margin of stomata, mesothelial and lacunar endothelial cells are joined to one another so that they line a channel that leads from the peritoneal cavity into the lumen of the lacuna (Wang, 1975; Leak and Rahil, 1978; Tsilibary and Wissig, 1979a). The stoma may be bridged by filamentous processes of the mesothelial cells. Flattened processes from the endothelial cells lining the channels frequently protrude across the luman of the channel below the stoma, creating a tortuous pathway. It has not yet been established whether stomata are stable openings or are openings whose patency is altered in response to changing physiologic and pathologic conditions within the peritoneal cavity. The principal aim of the first part of our study was to determine whether the patency of stomata could be altered by experimental manipulation. The specific manipulations we used were drug-induced contraction and relaxation of the diaphragm and an increase in intraabdominal pressure. In the second part of our study, we verified the identification of fine fibrils in the cytoplasm of endothelial and mesothelial cells bordering the stoma and channel as actin filaments. In addition we examined the effects of cytochalasin D (CD), a drug that depolymerizes actin filaments (Godman and Miranda, 1978; Brown and Spudich, 1979, 1981) on the structure of stomata and channels. In the final portion of the study we examined the impact of chronic ascites stemming from infection of the peritoneal cavity with Toxoplasma gondii on the lacunar mesothelium. Previous physiological studies of pathologic and experimental chronic ascites had shown that at early stages peritoneal absorption is normal, but later it falls (Bangham, 1953; Itani, 1959). The effect of ascites on the morphology of the mesothelium was not examined. MATERIALS
AND METHODS
(A) We anesthetized eight male Sprague-Dawley rats, weighing 40-60 g with an intravenous injection of sodium pentobarbital(35 mg/kg body wt). We injected four rats intravenously with 0.1 ml carbachol solution (carbamylcholine chloride, Sigma Chemical Company, St. Louis, MO., 0.3 mg/kg body wt, in n-saline) to induce contraction of the diaphragm and four rats with 0.1 ml succinylcholine solution (succinylcholine chloride, Sigma Chemical Company, 0.75 mgikg body wt, in n-saline) to induce relaxation of the diaphragm (Goodman and Gilman, 1975). In both groups of rats respiration ceased after 5-20 set, and we fixed the diaphragm with an intraperitoneal injection of 2% glutaraldehyde (Ladd Research, Burlington, Vt.) and 1% acrolein (Polysciences, Inc., Warrington, Pa.) in 0.1 M sodium cacodylate, pH 7.4, warmed to 37”. Fifteen minutes later, we excised the diaphragm, cut anterolateral areas containing lacunae into 5-mm squares, and placed the squares in fixative overnight at 4”. The next day, the tissue was treated with 1.5% partially reduced osmium tetroxide for 90 min at room temperature (Karnovsky, 1971). We dehydrated the tissue with ethanol and dried it by the critical-point method (Bomar Instrument Corporation, Fort Wayne, Ind.). We mounted the specimens on aluminum stubs with the peritoneal surface exposed, coated them with a layer of gold 20 nm thick in a Hummer V Sputter coater (Technics, Inc., Alexandria, Va.), and examined them with a scanning
24
TSILIBARY
AND
WISSIG
electron microscope (Model S-150, Cambridge Instrument Co., Inc., Ossining, N.Y.). We examined a total surface area of approximately 90 mm2 of each diaphragm. (B) We injected 10 male C3H mice weighing approximately 30 g and 2 male Sprague-Dawley rats weighing 30-50 g intraperitoneally with 0.1-0.2 ml n-saline in order to increase intraabdominal pressure. We anesthetized them 5-10 min later with ether and examined the peritoneal surface of their diaphragms with the scanning electron microscope by the method described in Section A. We treated another group of 10 male Sprague-Dawley rats, 40-50 g body wt , similarly except that, instead of administering n-saline by intraperitoneal injection, we made a small midventral incision in the abdominal wall to prevent any increase in intraabdominal pressure, and then introduced 0.1-0.2 ml n-saline into the peritoneal cavity. We gently held the small incision open for the next 5-10 min. The diaphragm was then fixed. The fixative used in this set of experiments contained 2% glutaraldehyde and 1% paraformaldehyde (Matheson Scientific, Chicago, Ill.) in 0.1 M sodium cacodylate, pH 7.4, with 0.006% succinylcholine chloride added to prevent initiation of the contraction of the diaphragm by the fixative. The specimens of diaphragm were examined with the scanning electron microscope according to the procedure described in Section A. (C) We injected a male Swiss albino mouse weighing 30 g intraperitoneally with 0.1 ml India ink in n-saline in order to label lacunae of the diaphragm. Ten minutes later, we anesthetized the mouse with ether and made a small incision in the midventral abdominal wall. We dripped a 50% solution of glycerol in modified salt solution (MSS) on the peritoneal surface of the diaphragm for 5 min, followed by 25% glycerol for 5 min, and then 5% glycerol for 5 min (Ishikawa et al., 1969). We excised the diaphragm, cut 0.5-mm square pieces from areas that contained lacunae, and incubated the pieces in a standard salt solution (SSS) containing 1-2 mg/ml SI subfragment of myosin for 5 hr at 4” (Ishikawa et al., 1969) in order to decorate actin filaments. We then fixed the pieces in 1% glutaraldehyde and 0.2% tannic acid in 0.1 M (mono-dibasic) sodium phosphate buffer, pH 7.0, for 30 min (Begg et al., 1978). We then placed the pieces in 2% glutaraldehyde and 1% paraformaldehyde in 0.1 M sodium phosphate (monodibasic) buffer, pH 7.4, overnight at 4”. The next morning the pieces were transferred to 1.5% osmium tetroxide in sodium phosphate buffer, pH 6.0, for 90 min at 4” (Maupin-Szamier and Pollard, 1978). The pieces were then rinsed in distilled water and stained en bloc in 2% aqueous uranyl acetate for 90 min at room temperature. They were dehydrated in acetone for 40 min and propylene oxide for 30 min and were embedded in Epon 812. Thin sections of the specimens were cut with a Sorvall MT-2 ultramicrotome. The sections were stained with 0.6% lead citrate for 5-10 min and were examined with a Siemens 1 electron microscope operated at 80 kv. (D) We anesthetized nine male Sprague-Dawley rats, 40-50 g body wt, with an intravenous injection of sodium pentobarbital. After making a small incision in the midventral abdominal wall, we flooded the peritoneal cavity with a solution at 4-40 $I4 CD (Aldrich Chemical Company, Milwaukee, Wise.) in n-saline for 10-30 min. We then fixed and processed specimens of the diaphragm for examination with the scanning and transmission electron microscopes as described in Sections A and C. We exposed the diaphragms of two male Sprague-Dawley rats, 40-
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50 g body wt, to 40 E,LMCD in n-saline for 20 min as described above. We replaced this solution with n-saline for 15 min in one rat and 30 min in the other. We then fixed the diaphragms for examination with the scanning and transmission electron microscopes. (E) We injected three male C3H mice, 30 g body wt, with 0.1 ml Hanks’ medium containing Toxoplasma gondii in order to induce experimental ascites (Nichols and O’Connor, 1981). We anesthetized an infected mouse with ether 30 set, 5 min, and 48 hr later and fixed the diaphragm with an intraperitoneal injection of the glutaraldehyde-formaldehyde solution as described in Section B. Fifteen minutes later we collected specimens of diaphragm for examination with the scanning electron microscope according to the procedure described in Section A. Specimens were also collected for examination with the transmission electron microscope. After treatment with partially reduced osmium tetroxide, these were rinsed with 0.05 M sodium maleate buffer, pH 5.2, for 30 min at 4” and were stained en bloc with 1.5% uranyl acetate in the same buffer for 90 min at 37”. We then dehydrated the specimens with absolute acetone for 40 min and propylene oxide for 30 min, and embedded them in araldite (British Araldite, Grade CY-212, Polysciences, Inc.). Thin sections of the specimens were examined by electron microscopy as described in Section C. RESULTS In all animals injected intravenously with carbachol prior to fixation, the mesothelial surface of the diaphragm was crenellated, i.e., was thrown up into tight folds with their long axis running at right angles to the long axis of muscle fibers (Fig. 1). The folded appearance of the surface indicated that the underlying muscle cells were in a contracted state at the time of fixation. In the diaphragm of these animals patent stomata were either extremely few in number4 or totally absent (Figs. 1 and 2). In diaphragms from four rats injected intravenously with succinylcholine, the mesothelial surface of the diaphragm was smooth, indicating that the underlying muscle was in a relaxed state at the time of fixation (Fig. 3). Numerous patent stomata were observed in areas of lacunar mesothelium (Figs. 3 and 4). In the animals that received an intraperitoneal injection of 0.1 ml n-saline prior to fixation of the diaphragm, numerous patent stomata were invariably observed between lacunar mesothelial cells (Fig. 5). In contrast, in animals in which the same volume of fluid was introduced into the peritoneal cavity but in which a small incision was made in the abdominal wall and was held open, stomata were totally absent from the lacunar mesothelium cells (Fig. 6). The cytoplasm of lacunar mesothelial and endothelial cells that border stomata and channels usually contains abundant microfilaments which resemble actin (Fig. 7). In the mesothelial cells, the microfilaments are frequently observed in 4 We did not attempt to make quantitative measurements of stomata per unit surface area in this study. Because of the tendency of the lacunar surface area to form undulating folds, it was not possible to measure accurately the area of their surface or to visualize stomata at the top, bottom, or far side of the curvatures. When we report differences in the number of stomata qualitatively in the different experimental groups of this study, we report only those differences that were distinctive and highly consistent.
26
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FIGS. 1, 2. Lacunar mesothelium from the diaphragms of two rats injected intravenously with carbachol prior to sacrifice. Under low power (Fig. l), the surface of the diaphragm appears crimped into tight folds running at right angles to the long axis of the underlying contracted muscle fibers. Patent stomata cannot be detected under low (Fig. I) or high power (Fig. 2). 750x (Fig. 1); 2900x (Fig. 2).
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FIGS. 3, 4. Lacunar mesothelium from two rats injected intravenously with succinylcholine prior to sacrifice. Lacunar mesothelium forms low ridges running parallel to underlying muscle fibers and is bordered on either side by flat nonlacunar mesothelium (Fig. 3). Patent stomata are seen under low power (Fig. 3) and, in more detail, under high power (Fig. 4) between adjacent mesothelial cells. 900x (Fig. 3); 2400x (Fig. 4).
TSILIBARY AND WISSIG
FIG. 5. Lacunar mesothelium of an animal that received an intraperitoneal injection of n-saline 5-10 min prior to sacrifice. Numerous stomata are observed between lacunar mesothelial cells. 1250x. FIG. 6. Lacunar mesothelium of an animal in which n-saline was introduced into the peritoneal cavity after a midventral incision was made in the abdominal wall to prevent a rise in intraabdominal pressure. Patent stomata are not seen between mesothelial cells. 1700x.
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FIG. 7. Channel in a mouse injected intraperitoneally with colloidal carbon to label lacunae prior to sacrifice. Carbon particles are seen in the peritoneal cavity (PC) and lumen of the channel (CH). The mesothelial stoma is not present in the plane of section. A band of microfilaments (mf) stretches across the base of the mesothelial cell (MC), and microfilaments (mf) are also seen in the lymphatic endothelial cell (EC) forming the wall of the channel. Submesothelial connective tissue (CT). 56,000 x .
cell processes bordering the stomata and organized as a bundle that parallels the base of these cells. In lacunar endothelial cells, the microfilaments are prominent in processes that extend across the lumen of the channel. In specimens extracted with glycerol and subsequently treated with the Sl fragment of myosin, filaments in the cytoplasm of mesothelial and endothelial cells were decorated with the Sl fragment in the familiar arrowhead configuration, signifying that the filaments are in fact composed of actin (Figs. 8 and 9). Following treatment with CD, the morphology of lacunar mesothelial cells and lymphatic endothelial cells, examined with the SEM and TEM, was markedly distorted. The degree of distortion was more or less the same in specimens treated with 4 or 40 pM CD for lo-30 min. The shape of the mesothelial cells was flattened and highly irregular (Fig. 10). Microvilli had disappeared from their
30
TSILIBARY
AND
WISSIG
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FIG. 10. Lacunar mesothelium of a rat treated with CD for 20 min prior to fixation. The mesothelial cells are flattened, and their microvilli have disappeared. A single cilium (arrows) is observed on the apical surface of individual mesothelial cells. The lateral borders of the cells are extensively retracted, preventing identification of stomata. Fibrillar connective tissue underlying the mesothelium is exposed to view. 3000 x .
surface. The stomata were enlarged and distorted. In addition, the lateral borders of lacunar mesothelial cells had pulled apart from one another exposing underlying collagenous fibers to view (Fig. IO), a condition that is never encountered in untreated specimens. In thin sections examined with the TEM, actin filaments were no longer visible in the cytoplasm of mesothelial and endothelial cells in stomata1 regions. Clumps of more or less homogeneous material of moderate electron density appeared in the cytoplasm of the cells. Some of the clumps were located in protruberances from the cut surface (Fig. 11). Although the outline of the cells was distorted, intracellular organelles appeared normal (Fig. 11). Nonlacunar mesothelium was unaffected by the treatment with CD. After CD was washed out of the peritoneal cavity and replaced with n-saline for 15-30 min, we observed that the lacunar mesothelial cells, examined with the SEM, had recovered their normal domed shape. The borders between adjacent mesothelial cells were sealed, and collagenous fibers of the underlying connective tissue were no longer exposed to view (Fig. 12). The stomata returned to their normal configuration. However, even after the CD had been washed FIGS. 8, 9. A lacunar mesothelial cell (Fig. 8) and a lacunar endothelial cell (Fig. 9) from a glycerol-extracted diaphragm “stained” with Sl subfragment of myosin. Actin filaments decorated with the Sl subfragment are seen in the microvilli (MV) and cytoplasm of the mesothelial cell and in the perinuclear cytoplasm of the endothelial cell. Peritoneal cavity (PC). Basement membrane (BM). Nucleus (NU). 58,000~ (Fig. 8); 66,000x (Fig. 9).
32
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AND WISSIG
FIG. 11. Lacunar mesothelium (MC) and endothelium (EC) of a rat injected intraperitoneally with CD 20 min prior to sacrifice. Actin filaments normally seen in the cytoplasm of these cells are replaced by clumps of amorphous material, much of which is located in abnormal protruberances of the cell surface (arrows). The shape of mesothelial and endothelial cells is distorted. 32,900~
away for 30 min, normal numbers of microvilli were not yet restored to the surface of the mesothelial cells. In thin sections examined with the TEM, the channels formed by mesothelial and endothelial cells had returned to their normal configuration, and actin filaments were normal in appearance and topology in both mesothelial and lymphatic endothelial cells (Fig. 13). In mice infected by an intraperitoneal injection of Toxoplasma go&ii 30 set and 5 min prior to fixation, the lacunar mesothelium appeared essentially normal. Stomata were observed between adjacent lacunar mesothelial cells. With the SEM, Toxoplasma were observed as small, free cells scattered over the surface of the lacunar mesothelium. After 48 hr of infection, the peritoneal cavity was markedly dilated with ascitic fluid. With the SEM, we observed that the surface of the lacunar mesothelium was markedly altered. Instead of their normal, smoothly domed shape, the mesothelial cells resembled “fried eggs” (Figs. 14 and 15). The center of the cell was thickened and cuboidal in height, and was encircled by a narrow rim of flattened cytoplasm (Fig. 15). The surface of the flattened rim of cytoplasm was relatively smooth, but the central, thickened portion of the cell was coated with numerous microvilli. Their number was much higher than is seen on normal lacunar mesothelial cells. Stomata between lacunar
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FIG. 12. Lacunar mesothelium of a rat treated intraperitoneally with CD for 20 min. CD was subsequently washed out of the peritoneal cavity with n-saline for 15 min. Mesothelial cells (MC) have recovered their normal configuration although they still lack their normal population of microvilli. The underlying connective tissue is no longer exposed between mesothelial cells. A stoma shown in the field (arrow) appears normal. 3500 x .
mesothelial cells were sparse in number (Fig. 14). In thin sections, we observed that occasional mesothelial cells contained one or more Toxoplasma enclosed within a large membrane-limited vesicle. The rest of the cytoplasm of the cell appeared healthy and undamaged. The lacunar mesothelial cells that were not invaded by parasites showed no sign of injury or necrosis. As a matter of fact, the opposite appeared to be the case. Each cell appeared to have undergone a form of “hypertrophy.” It seemed to contain more mitochondria and vesicles of endoplasmic reticulum than normal, and the Golgi apparatus seemed to be enlarged (Fig. 16). The patency of mesothelial stomata under the above mentioned experimental conditions is summarized in Table 1. DISCUSSION Stomata with their associated channels, which connect the peritoneal cavity with the lumen of the lymphatic lacunae of the diaphragm, are structurally differentiated units. For example, the lateral borders of mesothelial cells and lymphatic endothelial cells join at the rim of stomata and form a continuous cellular lining for the channel (Wang, 1975). The submesothelial connective tissue at the site of a stoma is interrupted and in addition, is structurally modified to accommodate the channel. For example, both the mesothelium and endothelium lack a basement membrane at the site of channel. The connective tissue im-
34
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FIG. 13. TEM micrograph from the diaphragm of the same rat as in Fig. 12. The appearance of mesothelial (MC) and endothelial (EC) cells is normal, and actin filaments (mf) reappear along the base of mesothelial cells and in endothelial cell processes. The arrows point to intermediate filaments which are normally present in both types of cells. 31,500x
mediately adjacent to the channel contains, as its principal fibrillar component, masses of microfibrils, not the bundles of collagenous fibers found elsewhere in submesothelial connective tissue (Bettendorf, 1978; Tsilibary and Wissig, 1979a). The endothelium lining the channel extends many flattened processes across the lumen of the channel. Finally, the cytoplasm of the mesothelial and endothelial cells at the sites of stomata and channels contains abundant fine filaments (Leak, 1976; Tsilibary and Wissig, 1979a; 1979b). This high degree of structural complexity of the stomata and channels leads us to conclude that they are stable structural units whose number and location are fixed in the short run. Although the number and location of stomata and channels are presumably stable on a short-term basis, it had not been demonstrated whether or not the patency of stomata is subject to regulation depending upon changing conditions within the peritoneal cavity. In order to test this, we used two experimental maneuvers. We examined how many stomata were patent, first, when the muscle of the diaphragm was either contracted or relaxed, and, second, when fluid pressure within the peritoneal cavity was increased. We observed by scanning electron microscopy that the number of patent stomata is correlated with drug-induced contraction or relaxation of the muscle of the diaphragm. When the muscle of the diaphragm is stimulated to contract
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FIGS. 14, 15. Lacunar mesothelium from a mouse injected intraperitoneally with Toxoplasma 48 hr prior to sacrifice. Mesothelial cells have an increased number of microvilli. The increased number are confined to the rounded central region of the cell and are absent from its flattened perimeter. A few organisms (T) are seen on the mesothelial surface. Either no stomata (Fig. 14) or few stomata (Fig. 15) are observed. 1500x (Fig. 14); 2500x (Fig. 15).
go&ii
with carbachol, stomata are very few in number (Figs. 1 and 2). Conversely, when the diaphragm is stimulated to relax with succinylcholine, large numbers of patent stomata are present (Figs. 3 and 4). In animals in which we injected fluid into the peritoneal cavity, a maneuver that presumably increases intraabdominal pressure, stomata were invariably abundant (Fig. 5). It seems that increased pressure rather than the presence of fluid is responsible for this response of stomata, because in animals which received a similar amount of fluid in the peritoneal cavity, but in which an incision was made in the abdominal wall to
36
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FIG. 16. Lacunar mesothelial cell from the same animal as in Figs. 14 and 1.5.Many organelles of the mesotheliai cell, e.g., endoplasmic reticulum, mitochondria, Golgi apparatus, lysosomes, etc., are present in greater than normal size and number. The apical surface of the cell shows increased numbers of microvilli. 17,400X
prevent any increase in intraabdominal pressure, stomata were either sparse or totally absent (Fig. 6). These findings clearly indicate that the patency of stomata, under defined experimental conditions, is subject to regulation. The physiologic significance of the regulation, as well as the steps involved in the process of reflex control, remain to be defined. A number of physiological studies have shown that the rate of fluid absorption from the peritoneal cavity is augmented by respiratory movements of the diaphragm (Allen and Vogt, 1937; Courtice and Steinbeck, 1950; Courtice and Morris, 1953; Allen and Raybuck, 1960). In addition, Allen and Vogt (1937) observed that intraperitoneally injected tracers which are taken up by the lacunae do not escape from the lacunae when the diaphragm is excised and pressure is exerted on its peritoneal surface. Both of these findings imply that the pathway
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TABLE 1
EFFECTS OF VARIOUS EXPERIMENTAL CONDITIONS ON PATENCYOF MESO~HELIALSTOMATA
Experimental condition Intravenous injection of carbachol (contraction of the diaphragm) Intravenous injection of succinylcholine (relaxation of the diaphragm) Intraperitoneal injection of fluid accompanied by increase in intraabdominal pressure. Intraperitoneal introduction of fluid without increase in intraabdominal pressureb Experimental ascites from infection with Toxoplasma gondii
Relative number of stomata” -
++++
+++ -
5
’ Scaled by SEM examination from essentially none (- ) to maximum numbers (+ + + +). b Increase in intraabdominal pressure was prevented by an open incision in the abdominal wall.
leading from the peritoneal cavity to the lumen of a lacuna, i.e., the stoma and its associated channel, is protected by a valving mechanism to prevent regurgitation of absorbed fluid. Several workers (Allen, 1936; Bettendorf, 1978) have proposed that mesothelial stomata, opening passively as the diaphragm stretches during expiration and closing passively as the diaphragm contracts during inspiration, act as valves. Casley-Smith (1964) has suggested that the endothelial processes that protrude across the lumen of the channel serve this purpose. Until this time, however, experimental evidence assigning a valving function to either the mesothelium or endothelium has not been reported. Our findings suggest that both the mesothelial processes which form the opening and the endothelial flaps criss-crossing the channel may act as valves under different conditions. We have shown that mesothelial stomata open and close with drug-induced relaxation and contraction of the diaphragm. Therefore, it is possible that the patency of stomata could be similarly altered during the respiratory cycle, although not necessarily by a passive process. Whether the patency of stomata does, in fact, change during the respiratory cycle is a difficult point to establish because fixation of the diaphragm with conventional methods is, first, a relatively slow process and, second, necessitates major alterations of the environment within the peritoneal cavity. Such alterations in themselves might initiate reflex changes in patency of the stomata. Our observations that the stomata remain patent when intraabdominal fluid pressure is raised suggests that their patency in this situation may be an adaptive response to expedite removal of fluid from the peritoneal cavity. Their protracted
38
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patency could, however, lead to regurgitation of fluid from the lacunae when the diaphragm contracts. Regurgitation would be averted if, in this situation, the endothelial flaps stretching across the channel assumed responsibility for acting as valves. At this point, it is not possible to decide whether either or both the mesothelium and endothelium serve as a valving mechanism to prevent reflux of fluid in the passageway from the peritoneal cavity to the lacunae. There may actually be a division of labor between the two structures with patency of the stomata regulating rate of flow and the processes of endothelial cells acting as valves to prevent reflux. We then examined the structural mechanism for regulating patency of stomata and conformation of endothelial flaps. We observed, as have others (Leak, 1976) that, at the sites of stomata and their channels, both mesothelial and endothelial cells contain actin-like filaments with a diameter of 5-6 nm (Fig. 7). We confirmed that these filaments are actin by the method of arrowhead decoration with the S1 subfragment of myosin (Figs. 8 and 9). Furthermore, we were able to depolymerize the filaments by exposing the mesothelial and endothelial cells to CD (Figs. 10 and 11). In the latter situation, the conformation of both types of cells was severely altered by selective disruption of actin filaments. The cells were not permanently affected by exposure to CD because, after the drug was washed out with n-saline, the cells returned to their normal morphology (Figs. 12 and 13). These findings indicate that the actin filaments located in the cytoplasm of mesothelial and endothelial cells adjacent to stomata and channels are components of a cytoskeletal framework that maintains the conformation of the passageway between the peritoneal cavity and the lymphatic lacunae. Finally, we examined the changes that occur in the lacunar mesothelium in the presence of severe ascites caused by intraperitoneal infection with Toxoplasma go&ii. After infection with the parasite for 48 hr, stomata were largely absent (Fig. 14) even though the peritoneal cavity was filled with abundant ascitic fluid and intraabdominal pressure had presumably increased. A complex reaction of mesothelial cells to the parasitic infection was observed. The mesothelial cells were not damaged by the protozoon, although occasional cells were parasitized. In contrast, a presumed hypertrophy of the cells was observed (Figs. 14-16). Specifically, most cytoplasmic organelles, i.e., the endoplasmic reticulum, the Golgi apparatus, mitochondria, and lysosomes were increased in size or number (Fig. 16) and the surface of mesothelial cells was dotted with vastly increased numbers of microvilli (Figs. 14 and 15). This complex response might be initiated by parasitism of mesothelial cells and underlying tissues, as well as by alteration in the amount and composition of peritoneal fluid. At this time, we do not know what the significance of this type of reaction by the mesothelium is. The observations indicate that the mesothelium has the capacity to respond to a chronic pathologic situation in ways that have not previously been recognized. In conclusion, our observations show that the patency of stomata can vary in response to changing conditions in the peritoneal cavity. Contractile components of mesothelial and endothelial cells seem to be responsible for maintaining the conformation of both the stomata and underlying channels and presumably also for effecting changes in patency of stomata. Finally, we have observed that the mesothelium responds by a previously underscribed form of hypertrophy to chronic ascites caused by infection with the parasite Toxoplasma gondii.
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ACKNOWLEDGMENTS The authors are indebted to Dr. Roger Cooke, who provided the subfragment 1 of myosin, to Ms. Simona Ikeda for technical assistance, to Mr. Dave Akers for assistance with photography, and to Ms. Alana Schilling for typing the manuscript.
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J. Physiol. 119, 776-782. BANGHAM, A. D. (1953). The effect of inflammation and other factors on the movement of radioactive
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Chapter 4. “Lymph Flow from Regional Lymphatics.” Cavities.” pp. 295-319, Academic Press, New York.
Section XV. “The Pleural and Peritoneal