- Email: [email protected]
Scanning electron microscopy: Morphology of aortic endothelium following injury by endotoxin and during subsequent repair
319
26 (1977) 319-328 0 Elsevier/North-Holland Biomedical Press
Atherosclerosis,
SCANNING ELECTRON MICROSCOPY: ENDOTHELIUM FOLLOWING INJURY SUBSEQUENT REPAIR
MORPHOLOGY BY ENDOTOXIN
OF AORTIC AND DURING
M.A. REIDY and D.E. BOWYER University of Cambridge, 1 QP (Great Britain)
Department
of Pathology,
Tennis
Court
Road,
Cambridge
CB.2
(Received 4 June, 1976) (Revised, received 13 September, 1976) (Accepted 17 September, 1976)
Summary
A single injection of endotoxin P45 Poly Serratia marcescens was used to induce endothelial injury in rabbits. The aortic endothelium was examined by Scanning Electron Microscopy (SEM), at various times after administration of endotoxin, using the technique of silver staining and pressure fixation. Within one hour after injection, some endothelial cells were curled-up and spindleshaped in appearance. Areas of aorta devoid of endothelial cover were occasionally observed and platelets were sometimes found adhering to these sites. Two and four weeks after initial injury no spindle-shaped cells were found. Instead, some endothelial cells were heavily stained with silver. Small denuded zones were still found and these were surrounded by brightly silver-stained cells. This study confirms that endotoxin rapidly causes endothelial injury and suggests that regenerating endothelial cells which were formed following injury are avidly stained by silver salts and appear as bright cells by SEM. Key words:
Aortic endothelium electron microscopy
-
Endothelial
injury
and repair
-
Endotoxin
-
Scanning
Introduction
It is widely held that disturbances in the integrity of arterial endothelium may lead to the development of atherosclerosis. Both loss of endothelium and more subtle changes in permeability of the intact endothelial layer, following The authors are grateful to May and Baker Ltd., Dagenham for financial assistance.
320
injury and during subsequent repair may permit an increased entry of blood constituents into the wall. Thus, lipoproteins and lipids may accumulate and smooth muscle cells may be stimulated to proliferate by lipoproteins [l] and platelet constituents [ 21. In order to study the importance of endothelial integrity following injury and during repair, various experimental techniques have been used to produce damage. These have included mechanical devices such as a brass rod [3], a catheter equiped with a cutting wire [4] or a balloon catheter [5] which can denude large areas of artery. The studies of Bjiirkerud [6] using a cutting catheter have shown that the type and persistence of the lesions produced depend upon the extent and severity of the initial injury and upon the rate of subsequent re-endothelialisation. If there was medial involvement with subsequent necrosis, then the lesions were severe and persistent. When the medial damage was minimal and the area of endothelial injury small, re-endothelialisation was rapid and the lesions often resolved completely. In the initiation of spontaneous atherosclerosis in man, it is unlikely that there would be mechanical damage to the artery, with medial involvement. Indeed there is little evidence to suggest that initial injury in vivo is other than endothelial. In order, therefore, to extend earlier studies of the role of endothelial injury and repair in atherogenesis we have sought more subtle forms of arterial injury, whose effect might be expected to be limited to endothelium. Methods which might be applicable include local alterations in pH [ 71, osmolarity [ 71, anoxia [ 71, administration of carbon monoxide [8], injection of vasoactive chemicals such as methoxamine [9], allylamine [lo], vasoactive amines [ll], anaphylatic shock [12], immune complex disease [13], EDTA [ 141, and endotoxin [ 151. In studying the effect of noradrenaline on endothelium, Christensen observed the morphological appearance of aortic endothelium using SEM [ 161. He demonstrated that seventeen days after initial injury the luminal surface contained endothelial cells with an unusual morphology. It was not clear, however, whether these cells were produced by the insult or were formed during repair. In this study we have used a single dose of endotoxin to injure arterial endothelium and then observed the morphological appearance of those cells at various times after injury by SEM. The use of SEM permits the observation of large areas of endothelium and our newly developed techniques for tissue preparation [17] minimise the production of misleading morphological artefacts. Methods and Materials Twenty Male New Zealand rabbits, approximately 2 kg/body weight, were used in this study. The animals were divided into 4 groups and subjected to the following procedure: Group 1: 5 animals were injected with sterile isotonic saline then killed immediately and their aortas stained and fixed for SEM. Group 2: 5 animals were injected with endotoxin P-45 Poly Serratia marcestens (200 E.cg/kg body weight) suspended in sterile isotonic saline. They were killed 1 h later and their aortas stained and fixed for SEM.
321
Group 3: 5 animals were injected with endotoxin P-45 Poly Serratia marcestens (200 pg/kg body weight) suspended in sterile isotonic saline. They were killed 14 days after endotoxin administration and their aortas stained and fixed for SEM. Group 4: 5 animals were injected with endotoxin P-45 Poly Serratia marcestens (200 pg/kg body weight) suspended in sterile isotonic saline. They were killed 28 days after endotoxin administration and their aortas stained and fixed for SEM. Preparation of aortas for SEM The methods are essentially those described previously by us [17,18]. Each animal was injected via an ear vein with Heparin (200 units/kg body weight) and killed by a blow on the head. A small opening was made in the thoracic cavity, the aorta was exposed and a femoral artery severed to allow the blood to drain. A plastic cannula was inserted into the aorta through which 30 ml of a 4.6% glucose solution, buffered with N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (HEPES, 20’ mM, pH 7.4) was introduced to wash out the residual blood. A 0.2% solution of silver nitrate in 4.2% glucose buffered with HEPES (20 mM) to pH 7.4, was then passed into the aorta for approximately 30 seconds. The aorta was flushed again with 30 ml of the 4.6% buffered glucose solution and then fixed with 2% phosphate buffered formalin, pH 7.4. By means of a three-way tap connected to the cannula, the above solutions were infused into the aorta at a constant pressure. The femoral artery was ligated and the aorta was left in situ to fix for 18 h at an approximate pressure of 100 mm Hg. The fixed vessel was carefully excised, pinned out onto cork boards and washed in distilled water. The tissue was then allowed to air-dry and stored in a dessicator. Approximately 2 cm* of tissue was mounted onto an SEM stub with high conductivity silver paint (Dag 915, Acheson Colloids Co., Prince Rock, Plymouth U.K.) and coated with gold in a Polaron Sputtering unit (Polaron Equipment Ltd., Watford, U.K.). The specimens were examined in a Stereoscan S600 at a beam voltage of 15 kV. Results The luminal surface of aortas from the control animals of Group 1, were lined by a continuous sheet of endothelial cells whose nuclei and silver stained boundaries were clearly seen (Fig. 1). The orientation of this and all other segments was such that the blood flow was from top to bottom; endothelial cells lie with their long axis parallel to the flow. One hour after the administration of endotoxin there was a pronounced change in the morphology of the endothelial cells. As is shown in Fig. 2, long rows of spindle shaped endothelial cells were seen on the luminal surface. The cells were curled-up and partially detached from the underlying tissue (Fig. 3). Between these areas of detaching cells normal endothelium was observed, similar to that in normal animals. In some areas the endothelium was completely detached from the arterial wall, leaving large denuded zones. As is shown in Fig. 4 the exposed tissue was occasionally partially covered by platelets.
322
Fig. 1. The luminal surface
Fig. 2. The luminal surface
of control
rabbit aorta stained with silver nitrate.
of rabbit aorta 1 h after endotoxin
administration.
X 600.
X 250.
Fig. 3. As in Fig. 2. X 600.
Fig. 4. The luminal surface of rabbit aorta 1 h after endotoxin to the exposed sub-endothelial tissue. X 250.
administration
showing platelets
adhering
324
Fig. 5. The luminal surface of rabbit aorta 4 weeks after endotoxin
Fig. 6. As in Fig. 5. X 600.
administration.
X 250.
325
Fig. 7. The luminal surface of rabbit aorta 4 weeks after endotoxin surrounded by brightly stained endothelial cells. X 600.
Fig. 8. The luminal surface clearly visible. X 1,200.
of rabbit
aorta 4 weeks
after endotoxin
administration.
administration.
The denuded
zone is
Several stomata
are
326
Two and four weeks after the endotoxin treatment, rows of endothelial cells with brightly stained boundaries were again found covering the luminal surface of the aortas, (Fig. 5). These cells gppeared to be of two types; those where the entire cell was heavily stained with silver and those where only the cytoplasm and the nuclear membrane were deeply stained (Fig. 6). Both types of cell were more numerous four weeks after endotoxin treatment and in some instances, the surface of these arteries were covered with very large bodies of up to 80 E.crn in length which had a prominently stained boundary. Zones denuded of endothelium were also present but no platelets were seen adhering to the surface of such areas. The borders of these regions were frequently surrounded by brightly stained cells. Fig. 7 shows a small denuded region situated in an area of normal endothelium which was surrounded by intensely silver-stained endothelial cells. Changes were also noted in the normal silver stained endothelial cells where there was an increase in the number of stomata as compared to the control tissue (Fig. 8). This finding was observed four weeks after endotoxin treatment. Discussion The results of this study show that within 1 h after endotoxin administration, aortic endothelial cells show signs of injury. The injured cells are spindlelike in appearance and appear to be partially detached from the arterial wall. In some areas cells have sloughed off leaving small denuded zones. We have previously noted endothelial cells with a similar morphology in aortic regions where the endothelium is subjected to high shear forces and their abnormal morphology was thought to be the result of cellular injury [19]. The possibility that the procedures used for staining, fixing and drying were responsible for the appearance of these unusual endothelial cells was considered. It was demonstrated however, that even when the perfusion was controlled to avoid overdistension, the silver stain was omitted and the arteries were dried by another procedure such as critical-point drying, the spindle-shaped cells were still found in high shear areas. Endotoxins are known to cause vascular injury by acting primarily at the endothelium [ 20-231. Extensive degeneration of cellular organelles have been observed in aortic endothelial cells soon after endotoxin administration [24], and loss of endothelium has been shown to follow [ 231. The precise mechanisms of this action is still unclear, although it is thought that endotoxins either have a direct toxic action on the endothelial cells or injure the endothelium by generating vasotoxic or vasactive substances in an intermediate reaction, involving granulocytes and platelets [25]. In this study platelets were seen adhering to the exposed intimal surface and presumably adhered to the exposed tissue after loss of endothelium. No evidence was found for adherence of platelets to the surface of intact endothelium. Two and four weeks after the single injection of endotoxin, the aortic surface was covered with brightly silver stained endothelial cells. Christensen [ 161 has reported similarly stained endothelial cells in rabbit aortas 17 days after vascular injury with nor-adrenalin. He concluded that these cells were either young regenerating endothelial cells or cells which had been damaged by the
321
noradrenalin. Likewise, after mechanically traumatising arteries, Gottlob and Zinner [26] also noted heavily silver-stained endothelial cells and thought them to be either newly formed cells, or else old damaged cells which were more permeable to silver salts. They could not, however, distinguish between them. Poole et al. [3], using Hiiutchen preparations of mechanically damaged aortas, found that endothelial cells bordering denuded zones were heavily stained with silver granules. These cells were considered to be newly formed endothelial cells. In this study, bright endothelial cells are found only 2 and 4 weeks after the onset of the injury and are not found after 1 h. Furthermore, they are often found surrounding denuded zones (Fig. 7) appearing as though new cells are growing in to cover these areas. It is not unreasonable therefore to suggest that these bright cells are newly formed or regenerating endothelial cells that are formed in response to the endotoxin-induced injury. In support of this concept, it is known that aortic endothelial cells show high mitotic index after endotoxin injection [ 27-301. Thus, this study has shown that the injection of a single dose of endotoxin injures a large number of aortic endothelial cells. Initially, platelets adhere to the exposed luminal surface but after two weeks large denuded zones show no platelet involvement. Two and four weeks after endotoxin treatment, young regenerating endothelial cells appear bright by SEM, presumably due to increased uptake of silver, and are found to cover large areas of the aortas. Acknowledgements We are grateful to Mr. K. Thurley and Mr. W. Moue1 of the Electron Microscopy Unit, Department of Anatomy, University of Cambridge, for their valuable help. We thank Dr. D.B. Cater for his kind gift of endotoxin, and Dr. P.F. Davies for his involvement with,these studies. References 1 Dzoga, K., Vesselinovitch. D.. Fraser, R. and Wissler. R.W.. The effect of lipoproteins on the growth of aortic smooth muscle cells in vitro, Amer. J. Path., 62 (1971) 32a. 2 Ross, R., Glomset. J., Kariya, B. and Harker, L., A platelet dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro, Proc. Natl. Acad. Sci., 71 (1974) 1207. 3 Poole, J.G.. Sanders, A.G. and Florey. H.W.. The regeneration of aortic endothelium. J. Path. Bact., 75 (1958) 133. 4 Bjb’rkerud, S.. Reactions of the aortic wall of the rabbit after superficial, longitudinal, mechanical trauma, Virchows Arch. Abt. A. Path. Anat., 347 (1969) 197. 5 Christensen. B.C. and Garbarsch, C., Repair in arterial tissue - A scanning electron microscopic (SEM) and light microscopic study on the endothelium of rabbit thoracic aorta following a single dilatation injury, Virchows Arch. Abt. A. Path. Anat.. 360 (1973) 93. initiated by mechanical trauma in normolipidemic rabbits, Athero6 Bjorkerud, S.. Atherosclerosis sclerosis, 9 (1969) 209. 7 Constantinides, P. and Robinson, M., Ultrastructural injury of arterial endothehum. Part I (Effects of pH, osmolsrity anoxia and temperature), Arch. Path., 88 (1969) 99. 8 Kjeldsen, K., Astrup, P. amd Wan&up, J., Ultrastructural intimal changes in the rabbit aorta after a moderate CO exposure, Atherosclerosis. 9 Herbertson, B.M. and Kellaway, T.D.. Bact., 80 (1960) 87. 10 Hennigar, G.R. and Katz, H.P., Effect rabbits.
Fed. Proc..
20 (1961)
92.
16 (1972) 67. Arterial necrosis
in the rat produced
of IV glucosamine-HCl
by methoxamlne.
on the aortas
of normal
J. Path.
and diabetic
328
11
Constantinides, P. and Robinson, M., Ultrastrtictural vasoactive amines), Arch. Path., 88 (1969) 106.
12
Wright, H.P. and Giacometti, N.J., Circulatory anaphylactic shock, Brit. J. Exp. Path., 53 (1972)
13
Cochrane.
C.G.. Mechanisms
injury of arterial
endothelial 1.
involved in the deposition
endothelium.
cells and arterial
of immune
complexes
Part 2 (Effects
endothelial
mitosis
in tissues, J. Exp.
of in
Med.,
134 (1971) 755. 14 Sheppard, B.L., Platelet adhesion in the rabbit abdominal aorta following the removal of endothelium with EDTA. Proc. Roy. Sot. Lond. B, 182 (1972) 103. 15 Gaynor. E., Bouvier. C.A. and Spaet, T.H., Circulating endothelial cells in endotoxin-treated rabbits, Clin. Res., 16 (1958) 535. 16 Christensen, B.C., Repair in arterial tissue - A scanning electron microscopic (SEM) and light microscopic study on the endothelium of the rabbit thoracic aorta following noradrenaliie in toxic doses, Virchows Arch. A. Path. Anat., 363 (1974) 33. 17 Davies, P.F. and Bowyer, D.E.. Scanning electron microscopy - Arterial endothelial integrity after fixation at physiological pressure, Atherosclerosis. 21 (1975) 463. 18 Davies, P.F., Reidy. M.A.. Goode, T.B. and Bowyer, D.E.. Scanning electron microscopy in the evalua19
20 21 22 23 24 25 26 27 28 29
30
tion of endothelial integrity of the fatty lesion in atherosclerosis. Atherosclerosis, 25 (1976) 125. Reidy, M.A. and Bowyer. D.E., Scanning electron microscopy of arteries - The morphology of aortic endothelium in haemodynamically stressed areas associated with branches, Atherosclerosis, 26 (1976) 181-194. McGrath, J.M. and Stewart, G.J., The effects of endotoxin on vascular endothelium, J. Exp. Med., 129 (1969) 833. Freudenberg, N. and Haublein, U., The effect of endotoxin shock on the aortic endothelium of young rats, Beitr. Path., 156 (1975) 1. Spaet. T.H.. Gaynor, E. and Bouvier, C., A vascular basis for the generalised Schwartzman reaction, Thromb. Diathes. Haemorrh., Suppl. 45 (1970) 157. Gaynor, E., Vascular lesions in endotoxemia, Adv. EXP. Med. Biol., 23 (1972) 337. Stewart, G.J. and Anderson, M.J., An ultrastructural study of endotoxin induced damage in rabbit mesenteric arteries, Brit. J. EXP. Path., 52 (1971) 75. Margaretten. W. and McKay. D.G., The role of the platelet in the generalised Schwartzman reaction, J. EXP. Med.. 129 (1969) 585. Gottlob. R. and Zinner. G., Uber die Regeneration geschadigter Endothelien nach hartem und weichem Trauma, Virchows Arch. A. Path. Anat., 336 (1962) 16. Evensen, S.A. and Shepro. D.. General&d Schwartzman reaction induced by hquoid in the rat Increased DNA-synthesis in aortic endothelium. Thromb. Diathes. Haemorrh., 30 (1973) 347. Gaynor, E., Increased mitotic activity in rabbit endothelium after endotoxin - An autoradiographic study, Lab. Invest., 24 (1971) 318. Gerrity, R.G., Richardson, M., Caplan, B.A., Cade, J.F., Hirsch, J. and Schwartz. C.J., Endotoxininduced endothelial injury and repair, Part 1 (Endothelial cell turnover in the aorta of the rabbit), EXP. Mol. Path., 23 (1975) 379. Gerrity, R.G., Richardson, M., Caplan, B.A., Cade. J.F.. Hirsch, J. and Schwartz, C.J., Endotoxininduced vascular endothelial injury and repair, Part 2 (Focal injury, en face morphology, [3Hlthymidine uptake and circulating endothelial cells in the dog), EXP. Mol. Path., 24 (1976) 59.
26 (1977) 319-328 0 Elsevier/North-Holland Biomedical Press
Atherosclerosis,
SCANNING ELECTRON MICROSCOPY: ENDOTHELIUM FOLLOWING INJURY SUBSEQUENT REPAIR
MORPHOLOGY BY ENDOTOXIN
OF AORTIC AND DURING
M.A. REIDY and D.E. BOWYER University of Cambridge, 1 QP (Great Britain)
Department
of Pathology,
Tennis
Court
Road,
Cambridge
CB.2
(Received 4 June, 1976) (Revised, received 13 September, 1976) (Accepted 17 September, 1976)
Summary
A single injection of endotoxin P45 Poly Serratia marcescens was used to induce endothelial injury in rabbits. The aortic endothelium was examined by Scanning Electron Microscopy (SEM), at various times after administration of endotoxin, using the technique of silver staining and pressure fixation. Within one hour after injection, some endothelial cells were curled-up and spindleshaped in appearance. Areas of aorta devoid of endothelial cover were occasionally observed and platelets were sometimes found adhering to these sites. Two and four weeks after initial injury no spindle-shaped cells were found. Instead, some endothelial cells were heavily stained with silver. Small denuded zones were still found and these were surrounded by brightly silver-stained cells. This study confirms that endotoxin rapidly causes endothelial injury and suggests that regenerating endothelial cells which were formed following injury are avidly stained by silver salts and appear as bright cells by SEM. Key words:
Aortic endothelium electron microscopy
-
Endothelial
injury
and repair
-
Endotoxin
-
Scanning
Introduction
It is widely held that disturbances in the integrity of arterial endothelium may lead to the development of atherosclerosis. Both loss of endothelium and more subtle changes in permeability of the intact endothelial layer, following The authors are grateful to May and Baker Ltd., Dagenham for financial assistance.
320
injury and during subsequent repair may permit an increased entry of blood constituents into the wall. Thus, lipoproteins and lipids may accumulate and smooth muscle cells may be stimulated to proliferate by lipoproteins [l] and platelet constituents [ 21. In order to study the importance of endothelial integrity following injury and during repair, various experimental techniques have been used to produce damage. These have included mechanical devices such as a brass rod [3], a catheter equiped with a cutting wire [4] or a balloon catheter [5] which can denude large areas of artery. The studies of Bjiirkerud [6] using a cutting catheter have shown that the type and persistence of the lesions produced depend upon the extent and severity of the initial injury and upon the rate of subsequent re-endothelialisation. If there was medial involvement with subsequent necrosis, then the lesions were severe and persistent. When the medial damage was minimal and the area of endothelial injury small, re-endothelialisation was rapid and the lesions often resolved completely. In the initiation of spontaneous atherosclerosis in man, it is unlikely that there would be mechanical damage to the artery, with medial involvement. Indeed there is little evidence to suggest that initial injury in vivo is other than endothelial. In order, therefore, to extend earlier studies of the role of endothelial injury and repair in atherogenesis we have sought more subtle forms of arterial injury, whose effect might be expected to be limited to endothelium. Methods which might be applicable include local alterations in pH [ 71, osmolarity [ 71, anoxia [ 71, administration of carbon monoxide [8], injection of vasoactive chemicals such as methoxamine [9], allylamine [lo], vasoactive amines [ll], anaphylatic shock [12], immune complex disease [13], EDTA [ 141, and endotoxin [ 151. In studying the effect of noradrenaline on endothelium, Christensen observed the morphological appearance of aortic endothelium using SEM [ 161. He demonstrated that seventeen days after initial injury the luminal surface contained endothelial cells with an unusual morphology. It was not clear, however, whether these cells were produced by the insult or were formed during repair. In this study we have used a single dose of endotoxin to injure arterial endothelium and then observed the morphological appearance of those cells at various times after injury by SEM. The use of SEM permits the observation of large areas of endothelium and our newly developed techniques for tissue preparation [17] minimise the production of misleading morphological artefacts. Methods and Materials Twenty Male New Zealand rabbits, approximately 2 kg/body weight, were used in this study. The animals were divided into 4 groups and subjected to the following procedure: Group 1: 5 animals were injected with sterile isotonic saline then killed immediately and their aortas stained and fixed for SEM. Group 2: 5 animals were injected with endotoxin P-45 Poly Serratia marcestens (200 E.cg/kg body weight) suspended in sterile isotonic saline. They were killed 1 h later and their aortas stained and fixed for SEM.
321
Group 3: 5 animals were injected with endotoxin P-45 Poly Serratia marcestens (200 pg/kg body weight) suspended in sterile isotonic saline. They were killed 14 days after endotoxin administration and their aortas stained and fixed for SEM. Group 4: 5 animals were injected with endotoxin P-45 Poly Serratia marcestens (200 pg/kg body weight) suspended in sterile isotonic saline. They were killed 28 days after endotoxin administration and their aortas stained and fixed for SEM. Preparation of aortas for SEM The methods are essentially those described previously by us [17,18]. Each animal was injected via an ear vein with Heparin (200 units/kg body weight) and killed by a blow on the head. A small opening was made in the thoracic cavity, the aorta was exposed and a femoral artery severed to allow the blood to drain. A plastic cannula was inserted into the aorta through which 30 ml of a 4.6% glucose solution, buffered with N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (HEPES, 20’ mM, pH 7.4) was introduced to wash out the residual blood. A 0.2% solution of silver nitrate in 4.2% glucose buffered with HEPES (20 mM) to pH 7.4, was then passed into the aorta for approximately 30 seconds. The aorta was flushed again with 30 ml of the 4.6% buffered glucose solution and then fixed with 2% phosphate buffered formalin, pH 7.4. By means of a three-way tap connected to the cannula, the above solutions were infused into the aorta at a constant pressure. The femoral artery was ligated and the aorta was left in situ to fix for 18 h at an approximate pressure of 100 mm Hg. The fixed vessel was carefully excised, pinned out onto cork boards and washed in distilled water. The tissue was then allowed to air-dry and stored in a dessicator. Approximately 2 cm* of tissue was mounted onto an SEM stub with high conductivity silver paint (Dag 915, Acheson Colloids Co., Prince Rock, Plymouth U.K.) and coated with gold in a Polaron Sputtering unit (Polaron Equipment Ltd., Watford, U.K.). The specimens were examined in a Stereoscan S600 at a beam voltage of 15 kV. Results The luminal surface of aortas from the control animals of Group 1, were lined by a continuous sheet of endothelial cells whose nuclei and silver stained boundaries were clearly seen (Fig. 1). The orientation of this and all other segments was such that the blood flow was from top to bottom; endothelial cells lie with their long axis parallel to the flow. One hour after the administration of endotoxin there was a pronounced change in the morphology of the endothelial cells. As is shown in Fig. 2, long rows of spindle shaped endothelial cells were seen on the luminal surface. The cells were curled-up and partially detached from the underlying tissue (Fig. 3). Between these areas of detaching cells normal endothelium was observed, similar to that in normal animals. In some areas the endothelium was completely detached from the arterial wall, leaving large denuded zones. As is shown in Fig. 4 the exposed tissue was occasionally partially covered by platelets.
322
Fig. 1. The luminal surface
Fig. 2. The luminal surface
of control
rabbit aorta stained with silver nitrate.
of rabbit aorta 1 h after endotoxin
administration.
X 600.
X 250.
Fig. 3. As in Fig. 2. X 600.
Fig. 4. The luminal surface of rabbit aorta 1 h after endotoxin to the exposed sub-endothelial tissue. X 250.
administration
showing platelets
adhering
324
Fig. 5. The luminal surface of rabbit aorta 4 weeks after endotoxin
Fig. 6. As in Fig. 5. X 600.
administration.
X 250.
325
Fig. 7. The luminal surface of rabbit aorta 4 weeks after endotoxin surrounded by brightly stained endothelial cells. X 600.
Fig. 8. The luminal surface clearly visible. X 1,200.
of rabbit
aorta 4 weeks
after endotoxin
administration.
administration.
The denuded
zone is
Several stomata
are
326
Two and four weeks after the endotoxin treatment, rows of endothelial cells with brightly stained boundaries were again found covering the luminal surface of the aortas, (Fig. 5). These cells gppeared to be of two types; those where the entire cell was heavily stained with silver and those where only the cytoplasm and the nuclear membrane were deeply stained (Fig. 6). Both types of cell were more numerous four weeks after endotoxin treatment and in some instances, the surface of these arteries were covered with very large bodies of up to 80 E.crn in length which had a prominently stained boundary. Zones denuded of endothelium were also present but no platelets were seen adhering to the surface of such areas. The borders of these regions were frequently surrounded by brightly stained cells. Fig. 7 shows a small denuded region situated in an area of normal endothelium which was surrounded by intensely silver-stained endothelial cells. Changes were also noted in the normal silver stained endothelial cells where there was an increase in the number of stomata as compared to the control tissue (Fig. 8). This finding was observed four weeks after endotoxin treatment. Discussion The results of this study show that within 1 h after endotoxin administration, aortic endothelial cells show signs of injury. The injured cells are spindlelike in appearance and appear to be partially detached from the arterial wall. In some areas cells have sloughed off leaving small denuded zones. We have previously noted endothelial cells with a similar morphology in aortic regions where the endothelium is subjected to high shear forces and their abnormal morphology was thought to be the result of cellular injury [19]. The possibility that the procedures used for staining, fixing and drying were responsible for the appearance of these unusual endothelial cells was considered. It was demonstrated however, that even when the perfusion was controlled to avoid overdistension, the silver stain was omitted and the arteries were dried by another procedure such as critical-point drying, the spindle-shaped cells were still found in high shear areas. Endotoxins are known to cause vascular injury by acting primarily at the endothelium [ 20-231. Extensive degeneration of cellular organelles have been observed in aortic endothelial cells soon after endotoxin administration [24], and loss of endothelium has been shown to follow [ 231. The precise mechanisms of this action is still unclear, although it is thought that endotoxins either have a direct toxic action on the endothelial cells or injure the endothelium by generating vasotoxic or vasactive substances in an intermediate reaction, involving granulocytes and platelets [25]. In this study platelets were seen adhering to the exposed intimal surface and presumably adhered to the exposed tissue after loss of endothelium. No evidence was found for adherence of platelets to the surface of intact endothelium. Two and four weeks after the single injection of endotoxin, the aortic surface was covered with brightly silver stained endothelial cells. Christensen [ 161 has reported similarly stained endothelial cells in rabbit aortas 17 days after vascular injury with nor-adrenalin. He concluded that these cells were either young regenerating endothelial cells or cells which had been damaged by the
321
noradrenalin. Likewise, after mechanically traumatising arteries, Gottlob and Zinner [26] also noted heavily silver-stained endothelial cells and thought them to be either newly formed cells, or else old damaged cells which were more permeable to silver salts. They could not, however, distinguish between them. Poole et al. [3], using Hiiutchen preparations of mechanically damaged aortas, found that endothelial cells bordering denuded zones were heavily stained with silver granules. These cells were considered to be newly formed endothelial cells. In this study, bright endothelial cells are found only 2 and 4 weeks after the onset of the injury and are not found after 1 h. Furthermore, they are often found surrounding denuded zones (Fig. 7) appearing as though new cells are growing in to cover these areas. It is not unreasonable therefore to suggest that these bright cells are newly formed or regenerating endothelial cells that are formed in response to the endotoxin-induced injury. In support of this concept, it is known that aortic endothelial cells show high mitotic index after endotoxin injection [ 27-301. Thus, this study has shown that the injection of a single dose of endotoxin injures a large number of aortic endothelial cells. Initially, platelets adhere to the exposed luminal surface but after two weeks large denuded zones show no platelet involvement. Two and four weeks after endotoxin treatment, young regenerating endothelial cells appear bright by SEM, presumably due to increased uptake of silver, and are found to cover large areas of the aortas. Acknowledgements We are grateful to Mr. K. Thurley and Mr. W. Moue1 of the Electron Microscopy Unit, Department of Anatomy, University of Cambridge, for their valuable help. We thank Dr. D.B. Cater for his kind gift of endotoxin, and Dr. P.F. Davies for his involvement with,these studies. References 1 Dzoga, K., Vesselinovitch. D.. Fraser, R. and Wissler. R.W.. The effect of lipoproteins on the growth of aortic smooth muscle cells in vitro, Amer. J. Path., 62 (1971) 32a. 2 Ross, R., Glomset. J., Kariya, B. and Harker, L., A platelet dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro, Proc. Natl. Acad. Sci., 71 (1974) 1207. 3 Poole, J.G.. Sanders, A.G. and Florey. H.W.. The regeneration of aortic endothelium. J. Path. Bact., 75 (1958) 133. 4 Bjb’rkerud, S.. Reactions of the aortic wall of the rabbit after superficial, longitudinal, mechanical trauma, Virchows Arch. Abt. A. Path. Anat., 347 (1969) 197. 5 Christensen. B.C. and Garbarsch, C., Repair in arterial tissue - A scanning electron microscopic (SEM) and light microscopic study on the endothelium of rabbit thoracic aorta following a single dilatation injury, Virchows Arch. Abt. A. Path. Anat.. 360 (1973) 93. initiated by mechanical trauma in normolipidemic rabbits, Athero6 Bjorkerud, S.. Atherosclerosis sclerosis, 9 (1969) 209. 7 Constantinides, P. and Robinson, M., Ultrastructural injury of arterial endothehum. Part I (Effects of pH, osmolsrity anoxia and temperature), Arch. Path., 88 (1969) 99. 8 Kjeldsen, K., Astrup, P. amd Wan&up, J., Ultrastructural intimal changes in the rabbit aorta after a moderate CO exposure, Atherosclerosis. 9 Herbertson, B.M. and Kellaway, T.D.. Bact., 80 (1960) 87. 10 Hennigar, G.R. and Katz, H.P., Effect rabbits.
Fed. Proc..
20 (1961)
92.
16 (1972) 67. Arterial necrosis
in the rat produced
of IV glucosamine-HCl
by methoxamlne.
on the aortas
of normal
J. Path.
and diabetic
328
11
Constantinides, P. and Robinson, M., Ultrastrtictural vasoactive amines), Arch. Path., 88 (1969) 106.
12
Wright, H.P. and Giacometti, N.J., Circulatory anaphylactic shock, Brit. J. Exp. Path., 53 (1972)
13
Cochrane.
C.G.. Mechanisms
injury of arterial
endothelial 1.
involved in the deposition
endothelium.
cells and arterial
of immune
complexes
Part 2 (Effects
endothelial
mitosis
in tissues, J. Exp.
of in
Med.,
134 (1971) 755. 14 Sheppard, B.L., Platelet adhesion in the rabbit abdominal aorta following the removal of endothelium with EDTA. Proc. Roy. Sot. Lond. B, 182 (1972) 103. 15 Gaynor. E., Bouvier. C.A. and Spaet, T.H., Circulating endothelial cells in endotoxin-treated rabbits, Clin. Res., 16 (1958) 535. 16 Christensen, B.C., Repair in arterial tissue - A scanning electron microscopic (SEM) and light microscopic study on the endothelium of the rabbit thoracic aorta following noradrenaliie in toxic doses, Virchows Arch. A. Path. Anat., 363 (1974) 33. 17 Davies, P.F. and Bowyer, D.E.. Scanning electron microscopy - Arterial endothelial integrity after fixation at physiological pressure, Atherosclerosis. 21 (1975) 463. 18 Davies, P.F., Reidy. M.A.. Goode, T.B. and Bowyer, D.E.. Scanning electron microscopy in the evalua19
20 21 22 23 24 25 26 27 28 29
30
tion of endothelial integrity of the fatty lesion in atherosclerosis. Atherosclerosis, 25 (1976) 125. Reidy, M.A. and Bowyer. D.E., Scanning electron microscopy of arteries - The morphology of aortic endothelium in haemodynamically stressed areas associated with branches, Atherosclerosis, 26 (1976) 181-194. McGrath, J.M. and Stewart, G.J., The effects of endotoxin on vascular endothelium, J. Exp. Med., 129 (1969) 833. Freudenberg, N. and Haublein, U., The effect of endotoxin shock on the aortic endothelium of young rats, Beitr. Path., 156 (1975) 1. Spaet. T.H.. Gaynor, E. and Bouvier, C., A vascular basis for the generalised Schwartzman reaction, Thromb. Diathes. Haemorrh., Suppl. 45 (1970) 157. Gaynor, E., Vascular lesions in endotoxemia, Adv. EXP. Med. Biol., 23 (1972) 337. Stewart, G.J. and Anderson, M.J., An ultrastructural study of endotoxin induced damage in rabbit mesenteric arteries, Brit. J. EXP. Path., 52 (1971) 75. Margaretten. W. and McKay. D.G., The role of the platelet in the generalised Schwartzman reaction, J. EXP. Med.. 129 (1969) 585. Gottlob. R. and Zinner. G., Uber die Regeneration geschadigter Endothelien nach hartem und weichem Trauma, Virchows Arch. A. Path. Anat., 336 (1962) 16. Evensen, S.A. and Shepro. D.. General&d Schwartzman reaction induced by hquoid in the rat Increased DNA-synthesis in aortic endothelium. Thromb. Diathes. Haemorrh., 30 (1973) 347. Gaynor, E., Increased mitotic activity in rabbit endothelium after endotoxin - An autoradiographic study, Lab. Invest., 24 (1971) 318. Gerrity, R.G., Richardson, M., Caplan, B.A., Cade, J.F., Hirsch, J. and Schwartz. C.J., Endotoxininduced endothelial injury and repair, Part 1 (Endothelial cell turnover in the aorta of the rabbit), EXP. Mol. Path., 23 (1975) 379. Gerrity, R.G., Richardson, M., Caplan, B.A., Cade. J.F.. Hirsch, J. and Schwartz, C.J., Endotoxininduced vascular endothelial injury and repair, Part 2 (Focal injury, en face morphology, [3Hlthymidine uptake and circulating endothelial cells in the dog), EXP. Mol. Path., 24 (1976) 59.