EXPERIMENTAL

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MOLECULAR

PATHOLOGY

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124-133 (1992)

Ultrastructural Alterations in Mouse Capillary Blood Vessels after Experimental Injection of Venom from the Snake BotIwops asper (Terciopelo) LISELA

Jo& MARfA GUTI~RREZ, AND MICHAEL OVADIA~

MOREIRA,’

‘Unidad de Microscopia Universidad de Costa Department

GADI

BORKOW,~

Electrdnica and 21nstituto Clodomiro Picado, Facultad Rica, San Jo&, Costa Rica; and ‘George S. Wise Faculty of Zoology, Tel Aviv University, Ramat Aviv, Tel Aviv, Received

April

5, 1992, and in revised

form

de Microbiologia, of Life Sciences, Israel

July 20, 1992

Histological and ultrastructural alterations in capillary blood vessels were studied at various time intervals after im injection of 50 ug of Bothrops asper snake venom in mouse gastrocnemius muscle. Hemorrhage was observed as early as 5 min after envenomation, as abundant erythrocytes appeared in the interstitial space. Ultrastructural observations revealed two different patterns of pathological changes: in the majority of damaged capillaries, endothelial cells had blebs and cytoplasmic projections pinching off to the lumen. This phenomenon was observed together with a decrease in the number of pinocytotic vesicles, with endothelial cells becoming very thin. As an apparent consequence of this process, some endothelial cells had evident gaps in their continuity. In addition, basal laminae surrounding these capillaries were altered and discontinuous. Other endothelial cells underwent a morphologically different process of degeneration, characterized by swelling of the endoplasmic reticulum and of the cytosol. These cells had a diffuse appearance and their basal laminae were discontinuous or absent. No major changes in the intercellular junctions were noticed in damaged endothelial cells. Samples obtained 30 and 60 min after venom injection were devoid of normal capillaries in many areas, and only diffuse remnants of their structure were found. Many altered capillaries had platelet aggregates and fibrin, the latter also being observed in the interstitial space. It is concluded that B. asper venom induces rapid and drastic pathological effects on capillaries leading to hemorrhage per rhexis i.e., erythrocytes probably escape through gaps in damaged endothelial cells and not through widened inter0 1992 Academic Press, Inc. cellular junctions.

INTRODUCTION Hemorrhage is one of the most common consequences of envenomations induced by viperine and crotaline snakes around the world (Ohsaka, 1979; Ownby, 1982). This effect develops locally, at the site of venom injection, as well as systemically, and is responsible of permanent local tissue damage (Ownby, 1982) as well as of cardiovascular disturbances. In Central America, the large majority of snakebites are inflicted by Bothrops asper, an abundant crotaline species distributed in tropical rainforests (Bolafios, 1982). These envenomations are characterized by prominent local effects, i.e., myonecrosis , hemorrhage, and edema, which develop rapidly after venom injection (Gutierrez et al., 1980, 1984a). The pathogenesis of local hemorrhage induced by snake venoms has been studied experimentally with crude venoms (Ownby et al., 1974) and purified hemorrhagic toxins (Tsuchiya et al., 1974; Ohsaka et al., 1975; Ownby et al., 1978,199O; Ownby and Geren, 1987). With most of these venoms and toxins hemorrhage occurs per rhexis, i.e., erythrocytes escape through gaps in damaged endothelial cells (Ownby, 1982, 1990), although in the case of hemorrhagic toxins from the 124 0014-4800192 $5.00 Copyright 0 1992 by Academic Press, Inc. AU rights of reproduction in any form reserved.

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venom of Trimeresurus jlavoviridis, erythrocytes escape through widened intercellular junctions (Ohsaka, 1979), a mechanism named hemorrhage per diapedesis (Ownby, 1982). The goal of this work is to study the ultrastructural alterations in capillary vessels of mouse skeletal muscle after experimental injection of B. asper snake venom, in order to define the morphological basis of this model of hemorrhage . MATERIALS

AND METHODS

Venom. Venom was obtained from more than 50 adult specimens of B. asper collected in the Pacific region of Costa Rica and kept at the serpentarium of the Instituto Clodomiro Picado. Once collected venom was lyophilized and stored at - 30°C. Experimental

protocol. Groups of four Swiss-Webster mice (18-20 g body wt) were injected intramuscularly in the right gastrocnemius with 50 pg venom (dissolved in 50 ~1 of phosphate-buffered saline solution, pH 7.2, PBS). Control animals were injected with 50 ~1 of PBS under otherwise identical conditions. At several time intervals (5, 15, 30, and 60 min) groups of four mice were killed by cervical dislocation. Immediately, the injected gastrocnemius muscle was dissected out and tissue samples were obtained from different areas in order to have representation of the entire muscle. Samples were fixed for 2 hr in Karnovsky fixative (2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2). Then, posttixation was performed for 1 hr with 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.2. Samples were then dehydrated in ethanol and embedded in Spur-r resin. Thick (1 rJ.m) sections were stained with toluidine blue. Thin sections were obtained with glass knives, stained with uranil acetate and lead citrate, and observed on a H-7000 Hitachi electron microscope at the Electron Microscopy Unit, University of Costa Rica.

RESULTS Control animals. Muscle obtained from mice injected with PBS had a normal structure, both histologically and ultrastructurally. Capillary vessels had a continuous basal lamina, abundant pinocytotic vesicles, and an array of organelles corresponding to what has been described in normal skeletal muscle (Fig. 1). Envenomated animals. Hemorrhagic alterations were evident macroscopically as early as 5 min after venom injection, becoming more extensive and prominent at later time intervals. Light microscopic examination revealed widespread extravasation in muscle tissue, as abundant erythrocytes were observed in the interstitial space. Ultrastructural observations revealed different signs of degeneration in capillary endothelial cells at all time intervals studied. Some cells had cylindrical or rounded cytoplasmic projections, some of which were pinching off to the lumen of the vessel (Figs. 2 and 3). In these capillaries there were blebs and vesicles apparently filled with cytoplasmic contents in the lumen; concomitantly, endothelial cells became very thin (Figs. 2 and 3) and had a decreased number of pinocytotic vesicles. Initially, these cells had a normal structure and the basal lamina was continuous (Fig. 2). However, cells in an apparently more advanced degenerative stage showed prominent blebbing and their basal lamina was absent (Fig. 3). Endothelial cells undergoing this process of cytoplasmic blebbing and reduction in cell width also showed evident breaks in their continuity, giving rise

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FIG. 1. Electron micrograph of capillary vessel from a mouse injected with saline solution. Endothelial cells contain a normal array of organelles and pinocytotic vesicles. Basal lamina (BL) and intercellularjunction (arrow) also have a normal appearance. Portions of a skeletal muscle cell (M) and a pericyte (P) are observed (24,000~).

to gaps (Fig. 4). The majority of damaged capillaries presented this morphological pattern of endothelial cell degeneration. In these areas of rupture of endothelial continuity the basal lamina was evidently altered and discontinuous (Figs. 3 and 4). Other endothelial cells presented a different pattern of cellular damage, with swelling of endoplasmic reticulum and of the cytosol, with a reduction in the number of pinocytotic vesicles and degenerative changes in the rest of the organelles. In these cells the basal lamina was altered and the whole cellular structure had a diffuse appearance (Fig. 5). No alterations of intracellular junctions were observed even in cells going through an advanced degenerative stage, except that in some cells there was a decrease in the electrodensity of the junctions (Figs. 6 and 8). However, most intercellular junctions in damaged capillaries had a normal appearance. At 30 and 60 min it was difficult to find normal capillaries in affected areas, as most of them were altered, and in some areas there were only remnants of the structure of capillaries. In samples collected 15,30, and 60 min after venom injection abundant platelets were located in damaged capillaries (Fig. 7). In addition, fibrin was present in the capillary lumen of damaged vessels as well as in the extravascular space (Fig. 8). Abundant erythrocytes were located in the interstitial space surrounding capillary vessels; in some cases erythrocytes were evidently lysed. Moreover, an amorphous granular material, probably a consequence of plasma exudation, was also observed in the interstitial space.

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FIGS. 2-8. Electron micrographs of capillaries from mice injected with Bothrops asper venom. FIG. 2. Capillary vessel 5 min after venom injection. The basal lamina is present and the vessel has not lost its integrity, but there is a reduction in the thickness of endothelial cells. Notice several cytoplasmic blebs protruding toward the lumen (arrows) (14,000~).

DISCUSSION Our observations corroborate previous findings in that hemorrhage develops very fast after intramuscular injection of B. asper venom in mice (Gutierrez er al., 1980, 1984a). Despite the fact that this venom affects other vessels (Arce et al., 1991), in this study we focused our attention on capillary vessels, in order to define the morphological basis of extravasation at this level of the microvasculature. Understanding the basis of capillary damage by this venom is relevant not only to the study of hemorrhage, but also to the issue of skeletal muscle regeneration, as it has been proposed that poor regeneration is mainly due to the drastic disruption of capillary vessels in envenomated muscle (Gutierrez et al., 1984b; Arce et al., 1991). Results indicate that many endothelial cells lost cytoplasmic material by the formation of cytoplasmic projections or blebs, a process that continued with the pinching off of cytosol-filled vesicles to the vascular lumen. Simultaneously, the number of pinocytotic vesicles in these endothelial cells was reduced. As an apparent consequence of this blebbing process, endothelial cells became very thin and eventually had gaps or discontinuities in their cytoplasm through which erythrocytes and plasma might escape. Interestingly, in the first stages of this process the cells were not irreversibly damaged, as the plasma membrane was continuous and the rest of the organelles were not drastically affected. In addition, the basal lamina was affected only at more advanced stages of degeneration. The development of cytoplasmic protrusions and blebs which then burst is a common response

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FIG. 3. Endothelial cells in an advanced degenerative stage 30 min after envenomation. The basal lamina is absent and cells lack pinocytotic vesicles. In addition, prominent blebbing is observed and the cell has become extremely thin in some portions (arrows). Fibrin (F) is present in the interstitial space, and a portion of a normal muscle cell is observed (11,200~).

of endothelial cells in many types of injury (Mason and Balis, 1980). Similar changes were described by Ownby er al. (1974, 1978) when studying the pathogenesis of hemorrhage induced by Crotalus atrox venom and three purified hemorrhagins isolated from this venom. Besides the process described above, other endothelial cells showed a different degenerative pattern, characterized by swelling of the cytoplasm and particularly of the endoplasmic reticulum. Similar alterations have also been described after injection of C. utrox venom (Ownby et al., 1974), a partially purified hemorrhagin from Viperu palestime (McKay et al., 1970) and proteinase IV, a hemorrhagic component isolated from Crotulus horridus (Ownby and Geren, 1987). Such endothelial cell swelling is also a common response of endothelial cells to injurious agents (Mason and Balis, 1980). It is not clear from this study the basis of the difference between these two types of endothelial cell alteration, although it is suggested that they do not represent different stages of cell degeneration, but most likely different patterns of endothelial cell damage. Since this study was carried out with crude venom, it is necessary to investigate the pathogenesis of hemorrhage induced by purified toxins in order to clarify this issue. Intercellular junctions were apparently intact even in capillaries that were drastically affected. Thus, our observations clearly suggest that extravasation at the level of capillary vessels in muscle tissue injected with B. usper venom occurs per rhexis, since erythrocytes probably escape through gaps in damaged endothelial cells and not through widened intercellular junctions. It would be relevant to

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F IG. 4. Endothelial cell with drastic alterations 30 min after venom injection. The cell is very thin and its continuity has been lost in some portions where gaps are observed (arrow). Basal lamil la is absc:nt in some points and there is pyknosis of the endothelial cell nucleus (N) (21,000~).

F ‘IG, 5. Capillary vessel 15 min after venom injection. Endothelial cells are in an advanced de:genera1tive stage, as there is an absence of typical cellular structures, and only a diffuse material surrou nding an erythrocyte remains (19,600~).

MOREIRA

ET AL.

IQG. 6. Affected capillary vessel 5 min after envenomation. Endothelial cells have a diffuse app tearam :e and show evident signs of degeneration, with gaps in cytoplasmic continuity (arrow). Note : the we ‘sence of an intact intercellular junction (J), although with less electrodensity than in normal ves!sels. Ab undant debris are located in the capillary lumen (23,800~).

FIG. 7. Aggregation of platelets (P) in a capillary vessel 15 min after envenomation. endothelial cells are very thin and discontinuous in some places (15,200~).

Affected

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FIG. 8. Capillary vessel in a sample collected 15 min after venom injection. One endothelial cell is very thin and lacks pinocytotic vesicles whereas another cell has lost its integrity (arrow). Note a well-preserved intercellular junction (J) and the presence of fibrin (F) in the lumen (17,100~).

study if a similar mechanism takes place in venules and other sites in the microvasculature. In this regard, our observations agree with those of previous workers who studied hemorrhage induced by C. UPOX venom (Ownby et al., 1974) and purified hemorrhagic toxins from the venoms of C. arrox (Ownby et al., 1978), C. horridus (Ownby and Geren, 1987), and Agkistrodon bilineatus (Ownby et al., 1990). Biochemical studies have clearly demonstrated that hemorrhagic toxins isolated from snake venoms are zinc metalloproteinases capable of enzymatically degrading components of the basal lamina (Ohsaka et al., 1973; Bjarnason and Fox, 1988-1989; Bjamason et al., 1988; Baramova et al., 1989). On this basis, it has been proposed that degradation of basal lamina components might be the primary effect of these toxins on capillary vessels (Ohsaka et al., 1973; Bjarnason and Fox, 1988-1989; Baramova et al., 1989). In our study it was observed that the basal lamina was still continuous when endothelial cells were undergoing alterations, and only when these cells were in advanced degenerative stages was the basal lamina discontinuous or absent. However, the fact that basal lamina appeared structurally continuous does not necessarily mean that it was intact, since basal lamina is a complex extracellular network made of different components. Thus, despite an apparent ultrastructural continuity, some of these components may be affected by the action of the venom. It is necessary to investigate the causal relationship between alterations in individual basal lamina proteins and pathological changes in endothelial cells. In samples collected after 15 min, damaged capillaries were plugged with plate-

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lets and contained fibrin. Similar findings have been made with several purified hemorrhagic toxins (Ownby and Geren, 1987; Ownby et al., 1978, 1990). These changes may reflect the onset of hemostatic mechanisms which prevent further extravasation, although they might also be a consequence of the action of venom components on the coagulation system. B. asper venom contains a thrombin-like enzyme (Arag6n and Gubensek, 1978) responsible for the characteristic appearance of microthrombi and depletion of fibrinogen levels in envenomations (Barrantes et al., 1985; Chaves et al., 1989). Thrombi were also observed in larger vessels after experimental injection of a variety of snake venoms (Homma and Tu, 1970; Arce et al., 1991). Thus, B. asper venom affects muscle vasculature in a complex way, and more studies are required to understand the mechanisms behind these pathological alterations. ACKNOWLEDGMENTS The authors thank Dr. Olga Arroyo and the staff of the Electron Microscopy Unit, University of Costa Rica, for their support throughout this study. Thanks are also due to Javier Ndfiez and Jorge Sanabria for their technical assistance. This study was supported by AID (Grant DHR-5544-6-00-106400). J. M. Gutitrrez is a research fellow of the Costa Rican National Council for Science and Technology (CONICIT).

REFERENCES ARAG~N, F., and GUBENSEK, F. (1978). Characterization of thrombin-like proteinase from Bothrops asper venom. In “Toxins: Animal, Plant and Microbial” (P. Rosenberg, Ed.), pp. 107-111. Pergamon Press, Oxford. ARCE, V., BRENES, F., and GUTIBRREZ, J. M. (1991). Degenerative and regenerative changes in murine skeletal muscle after injection of venom from the snake Bothrops asper: A histochemical and immunocytochemical study. Znt. J. Exp. Puthol. 72, 21 l-226. BARAMOVA, E. N., SHANNON, J. D., BJARNASON, J. B., and Fox, J. W. (1989). Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases. Arch. Biochem. Biophys. 275,63-71. BARRANTES, A., So~fs, V., and BOLANOS, R. (1985). Alteraciones en 10s mecanismos de la coagulaci6n en el envenenamiento por Bothrops asper (terciopelo). Toxicon 23, 39W7. BJARNASON, J. B., and Fox, J. W. (19884989). Hemorrhagic toxins from snake venoms. J. ToxicoLToxin Rev. 7, 121-209. BJARNASON, J. B., HAMILTON, D., and Fox, J. W. (1988). Studies on the mechanism of hemorrhage production by five proteolytic hemorrhagic toxins from Crotulus afrox venom. Biol. Chem. HoppeSeyler 369, 121-129. BOLA~OS, R. (1982). Las serpientes venenosas de Centroamerica y el problema de1 otidismo. Primera parte. Aspectos zool6gicos, epidemiol6gicos y biomtdicos. Rev. Costarricense Ciencias Mbd. 3, 165-184. CHAVES, F., GUYIBRREZ, J. M., LOMONTE, B., and CERDAS, L. (1989). Histopathological and biochemical alterations induced by intramuscular injection of Bothrops asper (terciopelo) venom in mice. Toxicon 21, 1085-1093. GUTI~RREZ, J. M., ARROYO, O., and BOLAROS, R. (1980). Mionecrosis, hemorragia y edema inducidos por el veneno de Bothrops asper en rat6n blanco. Toxicon 18, 603-610. GUTIERREZ, J. M., OWNBY, C. L., and ODELL, G. V. (1984a). Pathogenesis of myonecrosis induced by crude venom and a myotoxin of Bothrops asper. Exp. Mol. Pathol. 40, 361-379. GUTI~RREZ, J. M., OWNBY, C. L., and ODELL, G. V. (1984b). Skeletal muscle regeneration after myonecrosis induced by crude venom and a myotoxin from the snake Bothrops asper (Fer-deLance). Toxicon 22, 719-731. HOMMA, M., and Tu, A. T. (1970). Morphology of local tissue damage in experimental snake envenomation. Br. J. Exp. Pathol. 52, 538-542. MASON, R. G., and BALIS, J. U. (1980). Pathology of the endothelium. In “Pathobiology of Cell Membranes” (B. F. Trump and A. U. Arstila, Eds.), Vol. II, pp. 425-471. Academic Press, New York. MCKAY, D. G., MOROZ, C., DE VRIES, A., CSAVOSSY, I., and CRUSE, V. (1970). The action of

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hemorrhagin and phospholipase derived from Vipera pnlestiruze venom on the microcirculation. Lab. Invest. 22, 387-399. OHSAKA, A. (1979). Hemorrhagic, necrotizing and edema-forming effects of snake venoms. In “Snake Venoms” (C. Y. Lee, Ed.), pp 480-546. Springer-Verlag, Berlin. OHSAKA, A., JUST, M., and HABERMANN, E. (1973). Action of snake venom hemorrhagic principles on isolated glomerular basement membrane. Biochim. Biophys. Acta 323,415-438. OHSAKA, A., SUZUKI, K., and OHASHI, M. (1975). The spurting of erythrocytes through junctions of the vascular endothelium treated with snake venoms. Microvasc. Res. 10, 208-213. OWNBY, C. L. (1982). Pathology of rattlesnake envenomation. In “Rattlesnake Venoms. Their Actions and Treatment” (A. T. Tu, Ed.), pp. 163-209. Dekker, New York. OWNBY, C. L. (1990). Locally-acting agents: Myotoxins, hemorrhagic toxins and dermonecrotic factors. In “Handbook of Toxinology” (W. T. Shier and D. Mebs, Eds.), pp 601455. Dekker, New York. OWNBY, C. L., and GEREN, C. R. (1987). Pathogenesis of hemorrhage induced by hemorrhagic proteinase IV from timber rattlesnake (Crotalus horridus horridus) venom. Toxicon 25, 517-526. OWNBY, C. L., KAINER, R. A., and Tu, A. T. (1974). Pathogenesis of hemorrhage induced by rattlesnake venom. An electron microscopic study. Am. J. Pathol. 76, 401-414. OWNBY, C. L., BJARNASON, J., and Tu, A. T. (1978). Hemorrhagic toxins from rattlesnake (Crotalus atrox) venom. Pathogenesis of hemorrhage induced by three purified toxins. Am. J. Pathol. 93, 201-218. OWNBY, C. L., NIKAI, T., IMAI, K., and SUGIHARA, H. (1990). Pathogenesis of hemorrhage induced by bilitoxin, a hemorrhagic toxin isolated from the venom of the common cantil (Agkistrodon bilineatus bilineatus). Toxicon 28, 837-846. TSUCHIYA, M., OHSHIO, C., OHASHI, M., OHSAKA, A., SUZUKI, K., and FUJISHIRO, Y. (1974). Cinematographic and electron microscopic analyses of the hemorrhage induced by the main hemorrhagic principle, HR-1, isolated from the venom of Trimeresurusflavoviridis. In Platelets, Thrombosis, Inhibitors (P. Didishein, T. Shimamoto, and H. Yamazaki, Eds.), pp. 439-446. Schattauer, Stuttgart.

Ultrastructural alterations in mouse capillary blood vessels after experimental injection of venom from the snake Bothrops asper (Terciopelo).

Histological and ultrastructural alterations in capillary blood vessels were studied at various time intervals after im injection of 50 micrograms of ...
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