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Scanning (SEM) and transmission (TEM) electron-microscopic studies on post-ischemic endothelial lesions following recirculation
219
Atherosclerosis, 24 (1976) 219-232 @ Elsevier Scientific Publishing Company, Amsterdam -Printed
in The Netherlands
SCANNING (SEM) AND TRANSMISSION (TEM) ELECTRONMICROSCOPIC STUDIES ON POST-ISCHEMIC ENDOTHELIAL FOLLOWING RECIRCULATION
LESIONS
GABRIELLA ELEMkR, T. KERkNYI and H. JELLINEK with the technical assistance of Mrs. K. Kovacs, Z. Sarbdy, Mrs. M. Vbg6 and Mrs. E. Wallinger 2nd Department
of Pathology,
Semmelweiss
Medical
University,
Budapest
(Hungary)
(Received 1st September, 1975) (Accepted 3rd October, 1975)
Summary
The endothelial surface of the vessel wall was studied after various periods of recirculation following transitory mechanical hypoxia. The acute changes consisted of partial or total destruction of the endothelium in certain areas. Later on, the regeneration (division and process-formation) of endothelial cells took place over the damaged areas. These events were demonstrated both by transmission and scanning electron microscopy. When severe vessel damage occurred, the subendothelial matrix, collagen and elastic fibres, and the stomata were also set free. Blood cells anchored to these areas were later covered by endothelium. Regeneration was practically complete after 10 days; previously only some small endothelial processes had shown evidence of regenerative activity. In spite of the extensive damage to the endothelium, no occluding thrombus formation was seen. The phagocytes functioned only in removing debris, and the thrombocytes in facilitating endothelial overgrowth. Key words:
Arterial hypoxaemia - Endothelialdamage regeneration - Hypoxaemia
- Endothelial
lesions
- Endothelial
Introduction Vascular damage caused by hypoxia has been studied by several authors [ 1, 4,9,12,14,16,23]. However, few scanning electron-microscopic examinations of such lesions are available [16]. Nelson et al. [16] examined vessels in which the ischemic state was not followed by recirculation. The present scanning and transmission electron microscopic studies (SEM and TEM) were performed on
220
vessels in which recirculation ischemia.
took place for various periods following
transitory
Material and Methods Twelve inbred albino rats, weighing 150-200 g, were used. The animals were anaesthetized with ether, and after laparotomy a l-l.5 cm part of the infrarenal segment of abdominal aorta was exposed, and circulation was temporarily arrested for one hour by means of a double ligature. The ligatures were then removed, and the animals were sacrificed after a period of one or two hours’ or one or two days’ recirculation. Untreated animals served as controls. The aorta of the anaesthetized rats was perfused for 5 min with Kamovsky fixing solution at 120--130 mm Hg pressure. The fixing solution contained 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 N sodium cacodylate buffer (pH 7.4). Specimens were collected from the middle third of the double-ligated aortic segment for TEM and SEM. The specimens for transmission electron microscopy underwent additional immersion fixation for 3-4 h in sodium cacodylate buffer containing 2% paraformaldehyde and 2.1% glutaraldehyde. This was
Fig.
1. Scanning
electron
micrograph
of endothelial
surface
of normal
aorta.
X 3,000.
221
followed by overnight rinsing in buffer solution, and by 90-min post-fixation in 1% 0~0~ in Palade buffer (pH 7.4). The tissue blocks were then treated with 2% uranyl acetate, dehydrated in step-graded ethanol and embedded in Epon. The specimens for SEM were fixed in 2.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3), and were subsequently treated with 1% 0~0, at 4°C. Dehydration was carried out at room temperature in step-graded aceton series (starting with 20%). After a final series comprising 3 changes of absolute aceton, the specimens were dried in a calcium chloride desiccator at room temperature Finally the specimens were mounted with Dotite D 550 type conductile cement into a copper plate or grid. The mounted specimens were coated first with carbon (200 A), then with gold vapour (200 A). A JEM 100B electron microscope was used for both scanning and transmission electron micrographs. Results The SEM of normal aorta revealed the structure of the luminal surface to be slightly uneven and undulating (Fig. 1). The first changes following hypoxia were the crater-like bulgings and processes of the endothelial cells (Fig. 2). In some areas the undulating structure disappeared, depending on the degree of damage rather than on its duration. In such border areas, the endothelial cells
Fig. 2. Scanning electron micrograph. dicated by arrows. X 3.000.
Note bulgings and process
formation
on the endothelial
surface,
in-
Fig. 3. Scanning electron micrograph of damaged endothelial surface. The remaining endothelial cell (E) has extending processes (P); cross- (PC) and longitudinally (Plkut surfaces can be clearly seen by transmission electron micrographs. IEL = internal elastic lamina; L = lumen. X 3.000; X 24,000.
had already begun to migrate over the damaged areas after one hour (Figs. 3,4, 5). At the edges of the damaged areas, the appearance of different numbers of thrombocytes (Figs. 4 and 5) and leukocytes were observed. SEM revealed uneven surfaces, i.e. the ground substance of the subendothelium, in these damaged areas (Fig. 6). In these areas thrombocytes and endothelial cell processes were also visible under TEM (Fig. 6). In areas with total destruction of the intimal surface, the denuded ground substance and the bridge- or process-like remains of collagen fibres could be recognized. Occasionally completely denuded elastic fibres and traces of their stomata were seen in the form of deep craters or holes. In such areas, small groups of thrornbocytes were anchored (Fig. 7). The occasional appearance of lymphocytes and macrophages was noted (Fig. 8). In these same areas, debris of destroyed cells may also appear, and SEM and TEM may show leukocytes moving across the stoma of an elastic fibre. In the TEM micrograph, a histiocyte-like cell could be seen above the newly-regenerated endothelial cell (Fig. 8). After two hours, early regenerative changes could already be seen in SEM
223
Fig. 4. Free subendothelial ground substance (G), with thrombocyte (T) clearly visible under scanning electron micrograph between the processes of the endothelial cell (E). Transmission electron micrograph of some area: on the left, thrombocyte (T), on the right, endothelial cell ~rocesss (E) are clearly visible. IEL = internal elastic lamina; L = lumen. X 10,000; X 24,000.
with the appearance of swollen endothelial cells whose surfaces were covered with numerous spike-like processes. These cells bulged deeply into the lumen. exhibited a mitotic The same cells - when viewed under TEM - occasionally morphology (Fig. 9).
224
Fig. 5. The scanning electron micrograph (top) shows deterioration of aortic endothelial surface, in which thrombocytes (T) and between them endothelial processes (Ep) are seen. The transmission electron micrograph (bottom) shows the endothelial cell (E) on the right, with thrombocytw (T) above it. IEL = internal elastic lamina: L = lumen. X 10,000; X 24.000.
225
Fig. 6. Scanning electron micrograph showing deterioration of luminal surface of aorta. The injury involved all layers as far as the internal elastic lamina (IEL) or collagen fibres (C). Holes (arrow) - stomata, collagen fibres (C) and endothelial cell processes (EP) -are clearly visible in the transmission electron micrograph. IEL = internal elastic lamina: SM = smooth muscle cell. X 10.000: X 24.000.
226
Fig. 7. Scanning electron micrograph showing deterioration of luminal surface of the aorta. Necrotic cell debris (N), cell processes (P) and thrombocytes (T) are seen in this area. with ducts occasionally reaching to the elastic fibres and stomata (arrow). X 6.000.
The leukocytes and other phagocytic cells viewed under SEM became subendothelial as a result of superposition of migrating endothelial cells. At some places a phagocytic cell could be seen in a ditch-like cavity, partly covered by an endothelial cell. With TEM, one could clearly trace the formation of a new endothelial layer with typical intercellular junctions overlaying the phagocytic cell (Fig. 10). At a later stage, the regenerating endothelial cells were seen to cover most of the destroyed and denuded areas. The deep ditches had disappeared, along with the stomata and subendothelial structures, until by the tenth day a “restitutio ad integrum” had taken place. The even, undulating structure as seen in the control preparations was only missing in a few areas, where a minor perturbation of the undulating structure was seen (Fig. 11).
227
Fig. 8. Leukocytes (L) and histiocytic elements (H) in the damaged area can also be easily identified under the transmission electron micrograph (leukocyte on the left, histiocyte on the right); the leukocyte is just passing acro~ a stoma of the internal elastic lamina (IEL). E = newly-formed row of endothelial cells. X 12,000; X 6,000; X 18,000.
228
Fig. 9. Onset of regeneration, marked by appearance of swollen endothelial cells with processes (E). Such cells show mitotic activity, as can be seen from the upper transmission electron micrograph. The cell retained in a bay of the internal elastic lamina (IEL) is in the division phase: note processes. which assist it in migration across the surface. Cross- and longitudinally-cut surfaces of cell processes (P) are seen in the bottom picture. L = lumen. X 9,600; X 10,000; X 24,000.
229
230
Fig. 11. After 10 days, the regular structure seen in the controls is only missing in some areas. ning electron micrographs some newly regenerated areas with multiple endothelial cell processes (arrow). x 3,000.
In scanare seen
Discussion The effects of acute ischemia on the intima have so far only been studied by SEM by Nelson and coworkers [ 161; their experimental method of producing hypoxia only permitted the study of acute endothelial reactions, but could not be used for the examination of damage to the vessel wall which developed at a later stage. We believe that hypoxia of the vessel wall, which probably plays an important role in many types of vascular lesions, is always a temporary condition, whereas the reparative processes are of long duration. Accordingly, it seemed worthwhile to study the morphology of the vessel wall after varying periods of recirculation. This experimental model appeared to simulate the pathomechanism of in vivo hypoxic vascular injury more closely than that of other models. From our observations, it appears that the crater-like structures observed by Nelson et al. [ 161 may well be artificially punctured intra-cytoplasmic vacuoles or desquamation of endothelial cells, resulting from the mechanical effect of
231
subendothelial fluid accumulation. In the present studies, we also found focal desquamation of endothelial cells from the first elastic lamella and anchoring of thrombocytes, leukocytes and macrophages into the denuded subendothelial surface. All these changes were visible under SEM and TEM, while no thrombus formation could be seen in the severely damaged denuded areas. The cells from the blood which attached themselves to the damaged surface became covered by migrating endothelial cells during the regenerative process; or, as shown by TEM, actively migrated into deeper layers of the vessel wall. Their presence was linked with the first phase of regeneration following vascular injury. The processes of intact cells remaining in the marginal parts first began to extend over the denuded areas; later, an almost orderly pattern of endothelial structure made its appearance. The origin of the endothelial bridges [18,19,20,21] could not be unequivocally explained from the transmission electron migrographs, but there is reason to believe that they may have been part of those endothelial cells which developed processes in response to cell injury. The scanning electron micrographs in this study accord well with the findings of Frost [5,6,7], Fuchs [8], Geissinger [lo], Moseley et al. [15], Riede et al. [ 171, Shimamoto [18,19,20,21], Weber [24,25] and Webster [26] , who studied the luminal surface of the aorta under different conditions. Thus it appears that the lesions found can be regarded as a nonspecific endothelial response to various kinds of vascular wall injury. References 1 Astrup, A.. Some physiological and pathological effects of moderate carbon monoxide exposure. Brit. Med. J.. 4, (1972) 447-452. 2 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) 93-106. 3 Davies, P.F. and Bowyer. D.E., Scanning electron microscopy - Arterial endothelial integrity after fixation at physiological pressure, Atherosclerosis, 21 (1975) 463469. 4 Elemer. G., Kerenyi, T. and JeIIinek, H., Effect of temporary hypoxia on permeability of rat aorta, Path. Europ.. 10 (1975) 123-128. 5 Frost. H., EM der Aorta abdominahs. Med. Tribune, 3 (1970) 84. 6 Frost, H.. Endothelschiidigungen und Abscheidungen van Elementen des strdmenden Blutes aIs initiales Geschehen in der Pathogenese der Arteriosklerose. Verh. Dtsch. Ges. Inn. Med., 78 (1972) 11391145. 7 Frost, H., Investigations into the pathogenesis of arteriosclerosis - Drug prophylaxis. In: T. Shimamoto (Ed.), Atherogenesis II, Excerpta Medica, Amsterdam, 1972, pp. 32-51. 8 Fuchs. U., Geppe, G.. Lobe, J., Mauffe, W. and Riethling, A.K., Labelled serum albumin in the aortic wall after short-term blood pressure increase by angiotensin II. Arterial Wall. 2 (1974) 187-195. cell boundaries after staining 9 Garbarsch. C. and CoIlatz-Christensen, B., SEM of aortic endothelial with silver nitrate. Angiologfa, 7 (1970) 365-373. 10 Geissinger, H.D., The use of silver nitrate as a stain for SEM of arterial intima and paraffin sections of kidney, J. Microsc.. 95 (1972) 471481. 11 Groniowski, J., Biczowska, W. and Walski, M.. Scanning electron microscopic observation on the surface of vascular endothelium, Fol. Histochem. Cytochem., 9 (1971) 243-246. 12 Helin, P. and Lorenzen, I.B.. Arteriosclerosis in rabbit aorta induced by systemic hypoxia, Angiology. 20 (1969) l-12. 13 Jones, R.J. (Ed.). Atherosclerosis: Proceedings of the 2nd International Symposium, Springer-Verlag. Berlin, 1970. in smooth muscle cells of vascular type - Small ar14 Kerenyi, T. and JeIlinek, H.. Fibrin deposition teries under temporary conditions of hypoxia, Exp. Molec. Path., 17 (1972) l-5. 15 Moseley. H.S., Connell, R.S. and Krippaehne. W.W.. Healing of the canine aorta after endarterectomy - A scanning electron microscopic study, Ann. Sum.. 180 (1974) 329-335.
232
16
Nelson, E.. Sunaga. T., Shimamoto, endothelium. Arch. Path., 99 (1975)
17
Riede, U.N. and Villiger. W., OberflPchenfeinstruktur cus. Virch. Arch. Abt. B Zellpath., (1970) 294300.
18 19
T., Kawamura. 125-132.
T., Rennels,
M.L. and Hebel, R., Ischemic
der Aortenwand
beim arteriosklerotischen
carotid Ul-
Shimamoto. T. and Numano. F., Atherogenesis I. Excerpta Medica, Amsterdam, 1969. Shimamoto, T., Numano, F. and Addison. G.M., Atherogenesis II, Excerpta Medica. Amsterdam. 1973. 20 Shimamoto. T., Yamashita. Y. and Sunaga. T., Scanning electron microscopic observations of endothehal surface of heart and blood vessels. The discovery of intracellular bridges of vascular endothelium, Proc. Jap. Acad., 45 (1969) 507-511. 21 Shimamoto, T. and Sunaga. T., The contraction and blebbing of endothelial cells accompanied by acute infiltration of plasma substances into the vessel wall and their prevention. In: T. Shimamoto (Ed.). Atherogenesis II, Excerpta Medica, Amsterdam, 1972, pp. 3-32. 22 Smith, U., Ryan, J.W., Miche. D.D. and Smith, D.S., Endothelial projections as revealed by scanning electron microscopy, Science, 173 (1971) 925-926. 23 Sunaga, T., Yamashita, Y. and Numano. F., Luminal surface of normal and atherosclerotic artery observed by scanning electron microscopy, Proc. Jap. Acad., 45 (1969) 627-631. 24 Weber, G., Fabbrini, P. and Resi. L.. On the presence of a Concanavalin-A reactive coat over the endothelial aortic surface and its modifications during early experimental cholesterol atherogenesis in rabbits, Virchows Arch. Abt. A Path. Anat., 359 (1973) 299307. 25 Weber, G.. Fabbrini, P. and Resi, L., Scanning and transmission electron microscopy observations on the surface lining of aortic intimal plaques in rabbits on a hypercholesterolic diet, Virchows Arch. A. Path. and Histol., 364 (1974) 325-331. 26 Webster, W.S.. Bishop, S.P. and Geer. J.C., Experimental aortic intimal thickening, Part 2 (Endothelialization and permeability), Amer. J. Path., 76 (1974) 265-284.
Atherosclerosis, 24 (1976) 219-232 @ Elsevier Scientific Publishing Company, Amsterdam -Printed
in The Netherlands
SCANNING (SEM) AND TRANSMISSION (TEM) ELECTRONMICROSCOPIC STUDIES ON POST-ISCHEMIC ENDOTHELIAL FOLLOWING RECIRCULATION
LESIONS
GABRIELLA ELEMkR, T. KERkNYI and H. JELLINEK with the technical assistance of Mrs. K. Kovacs, Z. Sarbdy, Mrs. M. Vbg6 and Mrs. E. Wallinger 2nd Department
of Pathology,
Semmelweiss
Medical
University,
Budapest
(Hungary)
(Received 1st September, 1975) (Accepted 3rd October, 1975)
Summary
The endothelial surface of the vessel wall was studied after various periods of recirculation following transitory mechanical hypoxia. The acute changes consisted of partial or total destruction of the endothelium in certain areas. Later on, the regeneration (division and process-formation) of endothelial cells took place over the damaged areas. These events were demonstrated both by transmission and scanning electron microscopy. When severe vessel damage occurred, the subendothelial matrix, collagen and elastic fibres, and the stomata were also set free. Blood cells anchored to these areas were later covered by endothelium. Regeneration was practically complete after 10 days; previously only some small endothelial processes had shown evidence of regenerative activity. In spite of the extensive damage to the endothelium, no occluding thrombus formation was seen. The phagocytes functioned only in removing debris, and the thrombocytes in facilitating endothelial overgrowth. Key words:
Arterial hypoxaemia - Endothelialdamage regeneration - Hypoxaemia
- Endothelial
lesions
- Endothelial
Introduction Vascular damage caused by hypoxia has been studied by several authors [ 1, 4,9,12,14,16,23]. However, few scanning electron-microscopic examinations of such lesions are available [16]. Nelson et al. [16] examined vessels in which the ischemic state was not followed by recirculation. The present scanning and transmission electron microscopic studies (SEM and TEM) were performed on
220
vessels in which recirculation ischemia.
took place for various periods following
transitory
Material and Methods Twelve inbred albino rats, weighing 150-200 g, were used. The animals were anaesthetized with ether, and after laparotomy a l-l.5 cm part of the infrarenal segment of abdominal aorta was exposed, and circulation was temporarily arrested for one hour by means of a double ligature. The ligatures were then removed, and the animals were sacrificed after a period of one or two hours’ or one or two days’ recirculation. Untreated animals served as controls. The aorta of the anaesthetized rats was perfused for 5 min with Kamovsky fixing solution at 120--130 mm Hg pressure. The fixing solution contained 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 N sodium cacodylate buffer (pH 7.4). Specimens were collected from the middle third of the double-ligated aortic segment for TEM and SEM. The specimens for transmission electron microscopy underwent additional immersion fixation for 3-4 h in sodium cacodylate buffer containing 2% paraformaldehyde and 2.1% glutaraldehyde. This was
Fig.
1. Scanning
electron
micrograph
of endothelial
surface
of normal
aorta.
X 3,000.
221
followed by overnight rinsing in buffer solution, and by 90-min post-fixation in 1% 0~0~ in Palade buffer (pH 7.4). The tissue blocks were then treated with 2% uranyl acetate, dehydrated in step-graded ethanol and embedded in Epon. The specimens for SEM were fixed in 2.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3), and were subsequently treated with 1% 0~0, at 4°C. Dehydration was carried out at room temperature in step-graded aceton series (starting with 20%). After a final series comprising 3 changes of absolute aceton, the specimens were dried in a calcium chloride desiccator at room temperature Finally the specimens were mounted with Dotite D 550 type conductile cement into a copper plate or grid. The mounted specimens were coated first with carbon (200 A), then with gold vapour (200 A). A JEM 100B electron microscope was used for both scanning and transmission electron micrographs. Results The SEM of normal aorta revealed the structure of the luminal surface to be slightly uneven and undulating (Fig. 1). The first changes following hypoxia were the crater-like bulgings and processes of the endothelial cells (Fig. 2). In some areas the undulating structure disappeared, depending on the degree of damage rather than on its duration. In such border areas, the endothelial cells
Fig. 2. Scanning electron micrograph. dicated by arrows. X 3.000.
Note bulgings and process
formation
on the endothelial
surface,
in-
Fig. 3. Scanning electron micrograph of damaged endothelial surface. The remaining endothelial cell (E) has extending processes (P); cross- (PC) and longitudinally (Plkut surfaces can be clearly seen by transmission electron micrographs. IEL = internal elastic lamina; L = lumen. X 3.000; X 24,000.
had already begun to migrate over the damaged areas after one hour (Figs. 3,4, 5). At the edges of the damaged areas, the appearance of different numbers of thrombocytes (Figs. 4 and 5) and leukocytes were observed. SEM revealed uneven surfaces, i.e. the ground substance of the subendothelium, in these damaged areas (Fig. 6). In these areas thrombocytes and endothelial cell processes were also visible under TEM (Fig. 6). In areas with total destruction of the intimal surface, the denuded ground substance and the bridge- or process-like remains of collagen fibres could be recognized. Occasionally completely denuded elastic fibres and traces of their stomata were seen in the form of deep craters or holes. In such areas, small groups of thrornbocytes were anchored (Fig. 7). The occasional appearance of lymphocytes and macrophages was noted (Fig. 8). In these same areas, debris of destroyed cells may also appear, and SEM and TEM may show leukocytes moving across the stoma of an elastic fibre. In the TEM micrograph, a histiocyte-like cell could be seen above the newly-regenerated endothelial cell (Fig. 8). After two hours, early regenerative changes could already be seen in SEM
223
Fig. 4. Free subendothelial ground substance (G), with thrombocyte (T) clearly visible under scanning electron micrograph between the processes of the endothelial cell (E). Transmission electron micrograph of some area: on the left, thrombocyte (T), on the right, endothelial cell ~rocesss (E) are clearly visible. IEL = internal elastic lamina; L = lumen. X 10,000; X 24,000.
with the appearance of swollen endothelial cells whose surfaces were covered with numerous spike-like processes. These cells bulged deeply into the lumen. exhibited a mitotic The same cells - when viewed under TEM - occasionally morphology (Fig. 9).
224
Fig. 5. The scanning electron micrograph (top) shows deterioration of aortic endothelial surface, in which thrombocytes (T) and between them endothelial processes (Ep) are seen. The transmission electron micrograph (bottom) shows the endothelial cell (E) on the right, with thrombocytw (T) above it. IEL = internal elastic lamina: L = lumen. X 10,000; X 24.000.
225
Fig. 6. Scanning electron micrograph showing deterioration of luminal surface of aorta. The injury involved all layers as far as the internal elastic lamina (IEL) or collagen fibres (C). Holes (arrow) - stomata, collagen fibres (C) and endothelial cell processes (EP) -are clearly visible in the transmission electron micrograph. IEL = internal elastic lamina: SM = smooth muscle cell. X 10.000: X 24.000.
226
Fig. 7. Scanning electron micrograph showing deterioration of luminal surface of the aorta. Necrotic cell debris (N), cell processes (P) and thrombocytes (T) are seen in this area. with ducts occasionally reaching to the elastic fibres and stomata (arrow). X 6.000.
The leukocytes and other phagocytic cells viewed under SEM became subendothelial as a result of superposition of migrating endothelial cells. At some places a phagocytic cell could be seen in a ditch-like cavity, partly covered by an endothelial cell. With TEM, one could clearly trace the formation of a new endothelial layer with typical intercellular junctions overlaying the phagocytic cell (Fig. 10). At a later stage, the regenerating endothelial cells were seen to cover most of the destroyed and denuded areas. The deep ditches had disappeared, along with the stomata and subendothelial structures, until by the tenth day a “restitutio ad integrum” had taken place. The even, undulating structure as seen in the control preparations was only missing in a few areas, where a minor perturbation of the undulating structure was seen (Fig. 11).
227
Fig. 8. Leukocytes (L) and histiocytic elements (H) in the damaged area can also be easily identified under the transmission electron micrograph (leukocyte on the left, histiocyte on the right); the leukocyte is just passing acro~ a stoma of the internal elastic lamina (IEL). E = newly-formed row of endothelial cells. X 12,000; X 6,000; X 18,000.
228
Fig. 9. Onset of regeneration, marked by appearance of swollen endothelial cells with processes (E). Such cells show mitotic activity, as can be seen from the upper transmission electron micrograph. The cell retained in a bay of the internal elastic lamina (IEL) is in the division phase: note processes. which assist it in migration across the surface. Cross- and longitudinally-cut surfaces of cell processes (P) are seen in the bottom picture. L = lumen. X 9,600; X 10,000; X 24,000.
229
230
Fig. 11. After 10 days, the regular structure seen in the controls is only missing in some areas. ning electron micrographs some newly regenerated areas with multiple endothelial cell processes (arrow). x 3,000.
In scanare seen
Discussion The effects of acute ischemia on the intima have so far only been studied by SEM by Nelson and coworkers [ 161; their experimental method of producing hypoxia only permitted the study of acute endothelial reactions, but could not be used for the examination of damage to the vessel wall which developed at a later stage. We believe that hypoxia of the vessel wall, which probably plays an important role in many types of vascular lesions, is always a temporary condition, whereas the reparative processes are of long duration. Accordingly, it seemed worthwhile to study the morphology of the vessel wall after varying periods of recirculation. This experimental model appeared to simulate the pathomechanism of in vivo hypoxic vascular injury more closely than that of other models. From our observations, it appears that the crater-like structures observed by Nelson et al. [ 161 may well be artificially punctured intra-cytoplasmic vacuoles or desquamation of endothelial cells, resulting from the mechanical effect of
231
subendothelial fluid accumulation. In the present studies, we also found focal desquamation of endothelial cells from the first elastic lamella and anchoring of thrombocytes, leukocytes and macrophages into the denuded subendothelial surface. All these changes were visible under SEM and TEM, while no thrombus formation could be seen in the severely damaged denuded areas. The cells from the blood which attached themselves to the damaged surface became covered by migrating endothelial cells during the regenerative process; or, as shown by TEM, actively migrated into deeper layers of the vessel wall. Their presence was linked with the first phase of regeneration following vascular injury. The processes of intact cells remaining in the marginal parts first began to extend over the denuded areas; later, an almost orderly pattern of endothelial structure made its appearance. The origin of the endothelial bridges [18,19,20,21] could not be unequivocally explained from the transmission electron migrographs, but there is reason to believe that they may have been part of those endothelial cells which developed processes in response to cell injury. The scanning electron micrographs in this study accord well with the findings of Frost [5,6,7], Fuchs [8], Geissinger [lo], Moseley et al. [15], Riede et al. [ 171, Shimamoto [18,19,20,21], Weber [24,25] and Webster [26] , who studied the luminal surface of the aorta under different conditions. Thus it appears that the lesions found can be regarded as a nonspecific endothelial response to various kinds of vascular wall injury. References 1 Astrup, A.. Some physiological and pathological effects of moderate carbon monoxide exposure. Brit. Med. J.. 4, (1972) 447-452. 2 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) 93-106. 3 Davies, P.F. and Bowyer. D.E., Scanning electron microscopy - Arterial endothelial integrity after fixation at physiological pressure, Atherosclerosis, 21 (1975) 463469. 4 Elemer. G., Kerenyi, T. and JeIIinek, H., Effect of temporary hypoxia on permeability of rat aorta, Path. Europ.. 10 (1975) 123-128. 5 Frost. H., EM der Aorta abdominahs. Med. Tribune, 3 (1970) 84. 6 Frost, H.. Endothelschiidigungen und Abscheidungen van Elementen des strdmenden Blutes aIs initiales Geschehen in der Pathogenese der Arteriosklerose. Verh. Dtsch. Ges. Inn. Med., 78 (1972) 11391145. 7 Frost, H., Investigations into the pathogenesis of arteriosclerosis - Drug prophylaxis. In: T. Shimamoto (Ed.), Atherogenesis II, Excerpta Medica, Amsterdam, 1972, pp. 32-51. 8 Fuchs. U., Geppe, G.. Lobe, J., Mauffe, W. and Riethling, A.K., Labelled serum albumin in the aortic wall after short-term blood pressure increase by angiotensin II. Arterial Wall. 2 (1974) 187-195. cell boundaries after staining 9 Garbarsch. C. and CoIlatz-Christensen, B., SEM of aortic endothelial with silver nitrate. Angiologfa, 7 (1970) 365-373. 10 Geissinger, H.D., The use of silver nitrate as a stain for SEM of arterial intima and paraffin sections of kidney, J. Microsc.. 95 (1972) 471481. 11 Groniowski, J., Biczowska, W. and Walski, M.. Scanning electron microscopic observation on the surface of vascular endothelium, Fol. Histochem. Cytochem., 9 (1971) 243-246. 12 Helin, P. and Lorenzen, I.B.. Arteriosclerosis in rabbit aorta induced by systemic hypoxia, Angiology. 20 (1969) l-12. 13 Jones, R.J. (Ed.). Atherosclerosis: Proceedings of the 2nd International Symposium, Springer-Verlag. Berlin, 1970. in smooth muscle cells of vascular type - Small ar14 Kerenyi, T. and JeIlinek, H.. Fibrin deposition teries under temporary conditions of hypoxia, Exp. Molec. Path., 17 (1972) l-5. 15 Moseley. H.S., Connell, R.S. and Krippaehne. W.W.. Healing of the canine aorta after endarterectomy - A scanning electron microscopic study, Ann. Sum.. 180 (1974) 329-335.
232
16
Nelson, E.. Sunaga. T., Shimamoto, endothelium. Arch. Path., 99 (1975)
17
Riede, U.N. and Villiger. W., OberflPchenfeinstruktur cus. Virch. Arch. Abt. B Zellpath., (1970) 294300.
18 19
T., Kawamura. 125-132.
T., Rennels,
M.L. and Hebel, R., Ischemic
der Aortenwand
beim arteriosklerotischen
carotid Ul-
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