Vascular injury and thrombosis: A scanning electron microscopic study

Vascular injury and thrombosis: A scanning electron microscopic study

HRO~03OSIS RESEARCH Printed in the United States VASCULAR INJURY AND THROMBOSIS : :ol . pp . 699-^,U6, i974 e_oamon Press, T--c . A SCANNING E...

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HRO~03OSIS RESEARCH Printed in the United States

VASCULAR INJURY AND THROMBOSIS :

:ol .

pp .

699-^,U6,

i974

e_oamon Press, T--c .

A SCANNING ELECTRON MICROSCOPIC STUDY

G .E . Stoner, and G .M . Chisolm, Department of Material Sciences, University of Virginia, Charlottesville, Va . 22901 S . Srinivasan, T .R . Lucas, and P .N . Sawyer Electrochemical and Biophysical Laboratories, Department of Surgery, State University of New York Downstate Medical Center, Brooklyn, New York 11203

(Received 16 .11 .1973 ; in revised form Accepted by Editor A .L . Copley)

.4 .1974,

ABSTRACT Injury to the blood vessel wall and atherosclerosis enhance the possibility of thrombus depostion . A knowledge of the changes in vascular morphology and the extent of thrombus deposition produced by injury is essential in elucidating the mechanism of this reaction . In the present work, scanning electron microscopic technique was used to examine the canine blood vessel wall at or near a site of injury caused by vascular clamping . Proximal to the clamping, the endothelial folds in normal vessels are clearly visible . The intima is completely severed at the site of clamping . At a distance of lmm distal to the injury, the vascular wall is completely covered with a layer of thrombi, composed mainly of platelets, fibrin and trapped erythrocytes . Thus, injury to the blood vessel wall destroys the endothelial structure and triggers thrombus deposition .

INTRODUCTION A knowledge of the three dimensional structure of the blood vessel wall under normal and abnormal conditions is essential in characterizing changes in vascular morphology and understanding the extent and nature of thrombosis brought about by injury and disease . The ability of the healthy intima to prevent thrombus deposition is a poorly understood process

1-3 , which is

disrupted by injury and vascular diseases like atherosclerosis . The purpose of this initial study, is to examine the endothelium near a limited vascular injury using a scanning electron microscope (SEM) . 699



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METHODOFPROCEDURE A young 7 Kg dog was anesthetized with an intravenous dose of sodium pentabarbital (approximately 25 mg/kilogram body weight) . Approximately 6cm of the left femoral artery was exposed and clamped with a hemostat for sixty seconds . The clamp was released allowing normal blood flow . The chest of the animal was opened and the pericardial membrane was cut . About 200 ml of isotonic saline was then injected slowly into the left heart and the right heart was pierced to allow blood to flow out . In this manner, the vascular tree was hemodiluted to clear most of the blood . Following this, the left heart was similarly perfused with diluted formalin to initiate tissue fixation in vivo .

Following death the 6cm segment of the left femoral artery, 3 cm

proximal and 3 cm distal to the hemostat injured area, was removed and placed in 10% formalin-in-saline for 24 hours final fixation . The vessel was then removed, dried, mounted and coated with gold for SEM observation in a manner described elsewhere 4,5 . RESULTS AND DISCUSSION

Proximal to the clamping of the artery the normal . femoral endothelium is seen in Figure 1 .

Fig . 1 : Endothelial folds of left femoral artery at a distance of 1 millimeter proximal to injury (850X) .



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The longitudinal

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ridges first observed in SEM and labeled "endothelial

folds" 7-13 are characteristic of by Shimamoto 6 and later corroborated by others arteries and vena cava of all mammalian species observed to date (rabbits, monkeys, humans, dogs, rats, and guinea pigs) . It is also interesting to note that the width and length of those folds (approximately 15-17M) was found to be approximately the same among the species . The site of clamping with the hemostat is shown in Figures 2a and b .

Fig .

2(a) :

Left femoral artery at site of hemostat-induced injury (130X) .

As can be seen, the intima is completely severed along both outer edges of the clamp exposing subendothelial tissue . Figure 2(b) shows that the extent of the injury is probably restricted to the intima endothelium . Figures 3(a) and (b) show the wall at a distance of one millimeter distal to the injury (as compared to Figure 1 which is one millimeter proximal) . Here, the wall is almost_ completely covered with a layer of thrombus composed mainly of platelet, fibrin strands and RBCs aggregates adhering to the vessel wall . It is

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Fig . 2(b) :

Left femoral artery at site of hemostat-induced injury (850X) .

Fig . 3(a) :

Platelet thrombi adhering to endothelium at a distance of one millimeter distal to injury (700X) .



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3(b) :

Platelet thrombi

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adhering to endothelium at a distance of one

millimeter distal to injury (2800x) .

of particular interest to observe the one "clean" portion in the figure . This little "island" of endothelium was the only such area in the vicinity of 0-1 centimeter distal to the injury . (In Figure 4, at 2 cm, distal, the entire vessel is again completely normal) . This seems to indicate that alteration in vascular homeostasis which allows platelets to adhere to the wall, did not take place in this little isolated area near the injury . The fact that this area is exposed to the same tissue-activated platelets and the same blood flow patterns as the surrounding platelet-covered area indicates, perhaps, that there is still normal homeostasis . That is, the interfacial process during homeostasis are still occurring in this region . In similar experiments, the effect of more severe injury on the surface charge of the vessel wall was determined using streaming potential techniques . When injury was induced (again by hemostat clamping) the amount of charge of the blood vessel wall became less negative as the extent of injury was increased . This experiment, therefore, is consistent with the hypothesis that negative surface charge and vascular homeostasis are directly related and that injury and/or vascular disease destroy the processes necessary to maintain this charge 14-19



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Fig . 4 :

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Normal endothelial folds two centimeters distal

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to injury (750X) .

It is proposed to continue these studies in essentially two directions . The first will be to measure the effects of anticoagulant, vasoactive and antiatherosclerotic drugs, on the healing of vessels which have been injured and/or endarterectomized . Secondly, it would seem meaningful to work on an improved procedure (and/or device) for vascular clamping to minimize injury to the blood vessel wall during surgery . ACKNOWLEDGEMENTS The authors would like to thank Drs . F . Attinger and A . Navarro, Department of Biomedical Engineering, University of Virginia, for surgical assistance in this study . REFERENCES 1 .

VROMAN, L ., ADAMS, A .L ., and KLINGS, M . Interactions among human

blood proteins at surfaces . Federation Proceedings, 30, 1494, 1971 .



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SALZMAN, E .W .

705

Role of platelets in blood surface interactions . Ibid,

30, 1503, 1971 . 3.

BARNETT, M .K ., and ZISMAN, W .A .

Surface chemical aspects of clotting .

Ibid, 30, 1633, 1971 . 4.

CHISOLM, G .M ., GAINER, J .L ., STONER, G .E ., GAINER, J .V .,JR .

Plasma_

proteins, oxygentransportand atherosclerosis, Atheroscler ., In Press . 5.

CHISOLM, G .M .

Plasmaproteins,oxygentransportandatherosclerosis .

Ph .D . Dissertation, University of Virginia, 1972 . 6.

SHIMAMOTO, T ., YAMASHITA, Y ., NUMANO, F ., SUNAGA, T .

Scanning and

transmission EM observations of endothelial cells in the normal condition and in the initial stages of atherosclerosis .

Acta Pathol . Japonica, 21,

(1), 93, 1971 . 7.

SUNAGE, T ., YAMASHITA, Y ., NUMANO, F ., SHIMAMOTO, T .

Scanning

electronmicroscopy, P . 241, I .I .T . Research Institute, Chicago, Ill ., 1970 . 8.

SHIMAMOTO, T .

Atherogenesis .

PERRIM, A ., (EDS .), Excerpta Medical 9.

WEBER, G ., TOSI, P .

SHIMAMOTO, T ., NUMANO, F ., HALES, C .N ., Foundation, P . 5, Amsterdam,

1969 .

Observations with the SEM on the development

of cholesterol induced aortic atherosclerosis in the guinea pig .

Virchows

Arch . Abt . A . Path . Anat ., 353, 325 . 1971 . 10 .

GARBARSCH, C ., CHRISTENSEN, B .C .

boundaries after staining with AgN0 3 . 11 .

RIEDE, U .N ., VILLIGER, W .

surface of the aorta . 12 .

Angiologica, 7, 365, 1970 .

Topographical features of the endothelium

Virchows Arch . B . Zellpath ., 5, 294, 1970 .

SMITH, U ., RYAN, J .W ., MICHIE, D .D ., SMITH, D .S .

jestions as revealed by SEM . 13 .

SEM of aortic endothelial cell

Endothelium pro-

Science, 173, 925, 1971 .

BOATMAN, J .B ., CARTER, S .D ., DORSEY, B .O ., WHITE, R .R . SEM study

of the ulstrastructure surface of the atherogenic aorta .

Intersociety

Session No . 5, Paper No . 52, 56th Annual Meeting of Fed . Amer . Soc . Exp . Biol ., Atlantic City, New Jersey, (April, 1972) . 14 .

SAWYER, P .N .

living cells . 15 .

Ann . of N .Y . Acad . of Sci ., 146, 49, 1968 .

SAWYER, P .N ., and SRINIVASAN, S .

vascular thrombosis . 16 .

The effect of various Me+ interfaces on blood and other

Amer . J . of Surg ., 113, 42, 1967 .

SAWYER, P .N ., ZUFI, D ., WESOLOWSKI, S .A ., BURROWES, C .B .

surface charge as a factor in maintenance aorta . 17 .

Studies on the biophysics of intra-

Electrical

of patency of endarterectomized

Surg ., 59, 1019, 1966 . SAWYER, P .N ., OGONIAK, J .G ., BODDY, P .J .

Interactions between human

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erythrocytes and metal surfaces . 18 .

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Surgery, 61, 448, 1967 .

SRINIVASAN, S ., and SAWYER, P .N .

The effect of thrombogenic and anti-

thrombogenic drugs on the surface charge characteristics of the blood vessel wall and blood cells . Bull . N .Y . Acad . Med ., 44, 9, 1968 . 19 .

SAWYER, P .N ., SETO, S ., and SRINIVASAN, S .

Electrokinetic characteris-

tics of normal and atherosclerotic human aortas, Surgery, 68, 822, 1968 .