Endothelial cell damage by temporary arterial occlusion with surgical clips

Transcription

1 Endothelial cell damage by temporary arterial occlusion with surgical clips Study of the clip site by scanning and transmission electron microscopy S. DAVID GERTZ, PH.D., MARSHALL L. RENNELS, PH.D., MICHAEL S. FORBES, PH.D., JUNICHIRO KAWAMURA, M.D., TOSHIAKI SUNAGA, M.D., AND ERLAND NELSON, M.D., PH.D. Departments of Neurology and Anatomy, University of Maryland School of Medicine, Baltimore, Maryland ~" The effects of temporary vascular occlusion with surgical clips on the underlying endothelial lining were studied with scanning (SEM) and transmission (TEM) electron microscopy. Twenty-five rabbits were anesthetized and both common carotid arteries exposed. A Heifetz clip was used to occlude the right carotid artery for 5, 15, and 30 minutes, and 2 hours in five animals each. The clips were removed and the vessels immediately perfused with glutaraldehyde. In the five remaining animals, the right carotid arteries were occluded for 30 minutes followed by removal of the clip and resumption of blood flow for 30 minutes prior to fixation. Combined SEM and TEM examination of the endothelium of compressed segments revealed "craters" and "balloons," blebs and vacuoles, swollen mitochondria, dilated granular endoplasmic reticulum, and subendothelial edema. There were also areas of endothelial cell flattening, discontinuity, and desquamation exposing the subendothelial tissues. Following restoration of flow, platelets and fibrin were found adherent to altered endothelial cells and to exposed subendothelial tissues. Endothelial craters and balloons were also found distal and, significantly less frequently, proximal to the site of occlusion. It is suggested that antiplatelet aggregating agents may prove beneficial for the prevention of thrombus formation at the site of the clip as well as craters and balloons distal to the clip following procedures requiring temporary vascular occlusion. KEY WORDS 9 endothelium 9 carotid artery 9 thrombosis 9 scanning electron microscopy 9 transmission electron microscopy 9 platelets 9 vascular occlusion 9 endothelial damage T HE use of removable clips for temporary vascular occlusion is common practice in many surgical procedures. However, several scanning and transmission electron microscopic studies of vascular endothelium have emphasized the vulnerability of this tissue to a variety of injurious stimuli. 4,Sa,8,12,1s The present study was under- taken to examine the effect of varying periods of total temporary vascular occlusion on the endothelial lining underlying the clip. Materials and Methods Twenty-five New Zealand white rabbits were lightly anesthetized with Nembutal, 30 to 40 mg/kg administered intravenously, and 514 J, Neurosurg. / Volume 45 / November, 1976

2 Endothelial damage by temporary clips both common carotid arteries surgically exposed. A Heifetz clip e was used to occlude the right carotid artery at the level of the laryngotracheal junction for 5 minutes, 15 minutes, 30 minutes, or 2 hours in five animals each. The clips were then removed and all animals immediately sacrificed by intracardiac perfusion with 110 mm Hg of 2.5% glutaraldehyde in 0.1 M Sorensen's phosphate buffer (ph 7.4, 4 ~ C). In five additional animals, the right carotid artery was occluded for 30 minutes followed by removal of the clip permitting resumption of blood flow for 30 minutes prior to glutaraldehyde perfusion. Arterial segments 3 cm in length were excised from the area of the right carotid artery, which had been compressed by the surgical clip, as well as from the contralateral sham-operated control carotid artery. All tissues were then immersed in fresh fixative overnight. Specimens for scanning electron microscopy (SEM) were rinsed in 0.15 M phosphate buffer for 1 hour and postfixed in 1% osmium tetroxide in veronal acetate buffer for 45 minutes. Tissues were dehydrated in ethanol, immersed in increasing concentrations of isoamyl acetate, and dried in a Polaron critical point drying apparatus.* Specimens were coated with gold palladium and examined in a Cambridge $4-10 scanning electron mi~roscope.t Specimens for transmission electron microscopy (TEM) were rinsed in buffer and postfixed for 90 minutes. The tissues were dehydrated in ethanol and propylene oxide and embedded in Epon 812 epoxy resin. Thin sections were stained with uranyl acetate and lead citrate land examined in a Philips EM- 200 transmission electron microscope.~: FIG. 1. Scanning electron micrograph of the endothelial surface of a normal rabbit common carotid artery shows cell borders (B), marginal fold (F), and nuclear protrusions (N) common carotid artery confirmed previous morphological descriptions of arterial endothelial surfaces ',5,8,1~ (Figs. 1 and 2). Endothelial cells varied from fusiform to rhomboid in shape with ovoid protrusions reflecting the position of underlying nuclei. Cell borders, marginal folds, and surface striations could be identified. The carotid arteries that had been occluded by the surgical clip appeared normal macroscopically; however, SEM examination of the endothelial surface of the compressed segments revealed a broad spectrum of alterations. Variations in the severity of the observed lesions are probably due to differences in compression varying with the concavity of the clip blades. In some areas the Results The SEM and TEM features of the luminal surface of the normal, unoccluded rabbit *Polaron critical point drying apparatus manufactured by Polaron, Inc., 1760 Costnee Circle, Warrington, Pennsylvania tcambridge $4-10 scanning electron microscope manufactured by Image Analyzing Computers, Inc., Kent-Cambridge subsidiary, 40 Robert Pitt Drive, Monsey, New York ~Philips EM-200 transmission electron microscope manufactured by Philips Electronic Instruments, 750 South Fulton Avenue, Mt. Vernon, New York FIG. 2. Transmission electron micrograph of a normal rabbit common carotid artery shows normal endothelial cell lining (EC), endothelial nucleus (N), internal elastic lamina (EL), medial smooth muscle (SM), and vascular lumen (L) J. Neurosurg. / Volume 45 / November,

3 S. D. Gertz, et al. FIG. 3. Scanning electron micrographs of the endothelial surface of an arterial segment compressed by a surgical clip. Upper Left." After compression for 30 minutes, an endothelial bridge (Br), balloon (BAL), and leukocyte (Lt) can be seen Upper Right: After compression for 15 minutes craters (C) are visible Lower Left: After compression for 15 minutes, endothelial cell flattening is seen, most marked in the region of the nucleus (arrows) Lower Right: After compression for 30 minutes there is cellular disruption and desquamation with exposure of subendothelial connective tissue (arrows). E = erythrocytes endothelial surface appeared relatively normal except for the presence of bridge-like distortions 12a5 (Fig. 3 upper left). Although these structures occasionally appeared to form obliqud connections between endothelial cells, their course was usually difficult to determine. These structures have also been observed on the endothelial surface of normal specimens where there was considerable contraction of the vessel wall. Crater-like defects 5,8a~ and balloon-like protrusions 5,8 were found at the clip site and distal and proximal to it (Fig. 3 upper). In many areas the endothelial cells appeared compressed. This was most marked over endothelial nuclei (Fig. 3 lower left), which normally form ovoid elevations along the luminal surface. Marked transverse folding of the endothelial membrane was also found associated with fragmentation of cells and disruption of endothelial continuity. This varied from partial endothelial detachment to total desquamation exposing the subendothelial connective tissue (Fig. 3 lower right). Occasionally, platelets and fibrin were adherent to damaged endothelial cells or subjacent intimal tissue. With TEM, swollen mitochondria and dilated rough endoplasmic reticulum were frequently seen in the endothelium of arterial segments compressed by the surgical clip (Fig. 4 left). In addition, numerous blebs, 1'1~ ranging from 0.5 to 2.0 ~z, were observed protruding into the vascular lumen (Fig. 4 left). Interendothelial junctions were frequently disrupted (Fig. 4), and focal areas of endothelial cell desquamation with cleavage between endothelial cells and the underlying internal elastic lamina were also seen. All of these alterations were detected in arterial segments that had been compressed for as little as 5 minutes. No further increase 516 J. Neurosurg. / Volume 45 / November, 1976

4 Endothelial damage by temporary clips FIG. 4. Transmission electron micrographs of the endothelium of an arterial segment compressed by the surgical clip for 15 minutes. Left: Swollen mitochondria (Mi), bleb (BI), and disrupted intercellular junction (J) can be seen. L = vascular lumen, EL = internal elastic lamina, and M = media. 10,070. Right: Marked endothelial compression with transverse folding (small arrows) and endothelial detachment (large arrow) with exposure of internal elastic lamina (EL). Vascular lumen (L), media (M) in frequency or severity of these alterations was apparent following periods of compression of up to 2 hours. Similar alterations were observed in segments that had been compressed for 30 minutes followed by removal of the clip and resumption of blood flow for 30 minutes. However, the adherence of platelets and fibrin to altered endothelial cells and to exposed subendothelial tissues (as seen with both SEM and TEM) was markedly increased following resumption of blood flow for 30 minutes (Fig. 5). Discussion Combined SEM and TEM examination of the endothelium of arterial segments, that had been compressed by a surgical clip for 5 minutes to 2 hours followed immediately by glutaraldehyde perfusion, revealed craters and balloons, blebs and vacuoles, swollen mitochondria, dilated granular endoplasmic reticulum, and subendothelial edema. There were also areas of endothelial cell flattening, discontinuity, and desquamation exposing the subendothelial tissues. Endothelial craters and balloons were often found distal and, significantly less frequently, proximal to the site of occlusion (see Cover). Craters have also been reported to occur in monkey carotid arteries following double arterial ligation, TM in rabbit arteries following cholesterol feeding and epinephrine injection, 1' at or near the ostia of intercostal arteries of the aorta in egg yolk-fed miniature swine, xl and spontaneously in the vicinity of branch orifices of rabbit s and swine aortas, ix It has been suggested that the craters and balloons seen with SEM may represent endothelial blebs and vacuoles previously reported with TEM. 5's,x2 It is probable that these alterations represent a non-specific reaction of endothelium to a variety of injurious stimuli. The frequency of occurrence of craters and balloons in arteries subjected to ischemia was markedly reduced by pretreatment with heparin or aspirin in doses considered to have significant antiplatelet aggregating activity. 5 However, these drugs would not be expected to influence the mechanical disruption of the endothelium at the site of the clip? '~ This marked disruption and fragmentation of the endothelial lining might be sufficient to cause dramatic alteration of intimal permeability. When blood flow was resumed for 30 minutes after removal of the clip, platelets and fibrin were found attached to altered endothelial cells and to the exposed subendothelial tissue. This is consistent with the known affinity of platelets for subendothelial tissues. 2 It is therefore suggested that the use of antiplatelet aggregating agents might prove beneficial for the prevention of thrombus formation at the site of the clip and subsequent embolism. Furthermore, the prevention of the formation of craters and balloons distal to the site of the clip would be important in microcirculatory beds where such J. Neurosurg. / Volume 45 / November, ]7

5 S. D. Gertz, et al. FIG. 5. Upper." Scanning electron micrograph of the endothelial surface of an arterial segment compressed by the surgical clip for 30 minutes followed by restoration of flow for 30 minutes. Left." Platelets (P) are shown adherent to altered endothelial cells. E = erythrocyte Right." Platelets are adherent to exposed subendothelial tissue (arrows). X Lower." Transmission electron micrograph of the endothelium of an arterial segment compressed by the surgical clip for 30 minutes followed by restoration of flow for 30 minutes shows adherence of platelets (P) to disrupted endothelium (large arrows) and to subendothelial tissue (small arrow). L = vascular lumen, E = erythrocyte, EL = internal elastic lamina, and M = media. 12, J. Neurosurg. / Volume 45 / November, 1976

6 Endothelial damage by temporary clips alterations would be large enough to cause partial or complete obstruction of blood flow and ischemia of the parenchyma following surgical procedures requiring temporary vascular occlusion. Acknowledgments The authors gratefully acknowledge the technical assistance of Miss Barbara A. Plantholt and Ms. Camilla Maruszewski-Joy. References 1. Chiang J, Kowada M, Ames A 3rd, et al: Cerebral ischemia. III. Vascular changes. Am J Pathol 52: , Deykin D: Emerging concepts of platelet function. N Engl J Med 290: , Ehrlich J, Stivala SS: Chemistry and pharmacology of heparin. J Pharm Sci 62: , Gertz SD, Rennels ML, Forbes MS, et al: Preparation of vascular endothelium for scanning electron microscopy: a comparison of the effects of perfusion and immersion fixation. J Microse (Oxf) 105: , Gertz SD, Rennels ML, Nelson E: Endothelial cell ischemic injury. Protective effect of heparin or aspirin assessed by scanning electron microscopy. Stroke 6: , Heifetz MD: A new intracranial aneurysm clip. Technical suggestion. J Neurosurg 30:753, Hoff HF: Vascular injury: a review, in Koller F, Brinkhous KM, Biggs R, et al (eds): Vascular Factors and Thrombosis. Stuttgart: Schattauer Verlag, 1970, pp Kawamura J, Gertz SD, Sunaga T, et ah Scanning electron microscopic observations on the luminal surface of the rabbit common carotid artery subjected to ischemia by arterial occlusion. Stroke 5: , Nakano J, Koss MC: Pathophysiologic roles of prostaglandins and the action of aspirin-like drugs. South Med J 66: , Nelson E: Endothelial ischemia as studied by correlated scanning and transmission electron microscopy and by fluorescent antibody staining, in Shimamoto T, Numano F (eds): Atherogenesis II. Amsterclam: Excerpta Medica, 1973, pp Nelson E, Gertz SD, Rennels ML, et al: Endothelial lesions in the aorta of egg yolk-fed miniature swine: scanning and transmission electron microscopic observations. Circulation 52 (Suppi 2):103, 1975 (Abstract 404) 12. Nelson E, Sunaga T, Shimamoto T, et ah Ischemic carotid endothelium: scanning electron microscopic studies. Arch Pathol 99: , Shimamoto T, Numano F: Atherogenesis II. Amsterdam: Excerpta Medica, Shimamoto T, Sunaga T: The contraction and blebbing of endothelial cells accompanied by acute infiltration of plasma substances into the vessel wall and their prevention, in Shimamoto T, Numano F (eds): Atherogenesis II. Amsterdam: Excerpta Medica, 1973, pp Sunaga T, Shimamoto T, Nelson E: Correlated scanning and transmission electron microscopy of arterial endothelium, in Johari O, Corvin I (eds): Scanning Electron Microscopy/1973. Chicago: ITT Research Institute, 1973, pp This work was supported in part by U.S. Public Health Service Grants NS and NS Present address for Dr. Gertz: Department of Anatomy and Embryology, Hebrew University, Hadasseh Medical School, P.O. Box 1172, Jerusalem, Israel. Address reprint requests to: Erland Nelson, M.D., Department of Neurology, University of Maryland School of Medicine, Baltimore, Maryland J. Neurosurg. / Volume 45 / November,