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Intraoperative Trauma to Human Saphenous Veins: Scanning Electron Microscopic Comparison of Preparation Techniques
Intraoperative Trauma to Human Saphenous Veins: Scanning Electron Microscopic Comparison of Preparation Techniques Steven R. Gundry, M.D., Michael Jones, M.D., Tokuhiro Ishihara, M.D., and Victor J. Ferrans, M.D., Ph.D.
ABSTRACT To determine optimal preparation techniques for human saphenous veins, a scanning electron microscopic comparison was made of the effects of variations in solutions, temperatures, and distention pressures on human vein morphology. Segments of saphenous veins obtained from 20 patients undergoing myocardial revascularization were divided into five groups of 5 veins each. Group 1 segments (controls) were immediately distended and perfusion-fixed with glutaraldehyde at 30 mm Hg to simulate in vivo saphenous vein hydrostatic pressure. Segments comprising Groups 2 and 3 were placed in heparinized normal saline solution at 28°C for one hour and then distended to either 100 mm Hg (Group 2) or 300 mm Hg (Group 3). Segments making up Groups 4 and 5 were immersed in heparinized blood for one hour at either 28°C (Group 4) or 4°C (Group 5), and then distended to 100 mm Hg. Except for controls, which were fixed as described, all other groups of vein specimens were perfusion-fixed at 100 mm Hg to simulate in vivo arterial pressure. Control veins showed focal intimal fractures with minimal endothelial cell loss due to harvesting manipulation. Groups 2 and 3 had marked endothelial loss, which led to exposure of basement membrane, collagen fibrils, and smooth muscle cells and was associated with intimal and medial edema. Veins prepared with blood appeared similar to controls, showing focal endothelial cell loss, but preservation of intimal and medial structure. Changes were less marked in Group 5 than in Group 4. We conclude that human saphenous veins are best preserved by
immersion in cold blood and distention to a pressure of 100 mm Hg or less.
Aortocoronary bypass grafts using autogenous saphenous veins are subject to early and late occlusion. Problems related to operative technique, distal coronary artery runoff, hyperlipidemia, graft ischemia, and vein preparation techniques have been implicated as causes of bypass graft failure [2, 5, 9, 13, 25, 261. Although investigations in animals suggest that vein immersion in saline solution and vein distention above physiological pressures cause vein endothelial cell and mural damage [ l , 6, 13, 20, 22, 251, no systematic evaluations of these problems have been performed using human saphenous veins [2, 3, 6, 7, 13, 22, 281. To determine preferable techniques for preserving vein integrity, we compared human graft preparation techniques, evaluating the veins by scanning electron microscopy.
Materials and Methods Portions of saphenous veins from 20 patients undergoing coronary artery bypass operations were divided in 25 segments, each measuring 4 to 5 cm in length. These segments were divided into five groups of 5 veins each. Segments from Group 1 (control) were immediately fixed by perfusion with cold, buffered (pH 7.2) 3% glutaraldehyde at 30 mm Hg distention pressure, using a constant pressure perfusion device (Fig 1) to approximate the in vivo hydrostatic venous pressure in the standing position. Group 2 veins were immersed in heparinized From the Clinic of Surgery and Pathology Branch, National normal saline solution (0.9% sodium chloride) Heart, Lung, and Blood Institute, Bethesda, MD. Presented at the Sixteenth Annual Meeting of The Society of at 28°C for one hour and then distended to a Thoracic Surgeons, Jan 21-23, 1980, Atlanta, GA. pressure of 100 mm Hg with normal saline Address reprint requests to Dr. Gundry, Clinic of Surgery, solution. Group 3 veins were immersed in National Heart, Lung, and Blood Institute, Building 10, heparinized normal saline solution at 28°C for Room 6N252, National Institutes of Health, Bethesda, one hour, followed by distention to a pressure MD 20205. 40
0003-49751801070040-08$01.25 @ 1980 by The Society of Thoracic Surgeons
41 Gundry et al: Intraoperative Trauma to Saphenous Veins
Pressure
Saphenous Vein
Adjustable Clamp
damage; 1= less than 10%; 2 = 10 to 25%; 3 = 25 to 50%; and 4 = more than 50%. We utilized the sum of these grades for each of the pathological alterations in each of the five groups of vein segments as a numerical representation of of 300 mm Hg. Group 4 veins were immersed in the total amount of vein trauma produced by heparinized arterial blood at 28°C for one hour each of the five techniques for preparation of and afterward distended to a pressure of 100 vein grafts (Table). mm Hg with autologous human blood. Group 5 veins were immersed in heparinized arterial Results blood at 4°C for one hour and then distended All vein segments in Group 1 (control) demonwith blood to a pressure of 100 mm Hg. After strated occasional endothelial cell separation pressure distention, all groups of veins, except and endothelial cell loss with minimal exposure for controls, were fixed with cold, buffered (pH of fibrillar collagen or basement membrane (Fig 7.2) 3% glutaraldehyde at a perfusion pressure 2). These specimens showed fractures, with of 100 mm Hg, simulating human in vivo arte- clefts extending through the intima and into the rial pressure. media. We presume that these fractures occur After fixation, the vein segments were di- from manipulation during operation because vided in half longitudinally. From each, two 0.5 veins in all groups showed similar intimal cm sections were prepared for scanning elec- fractures. Group 1 veins had no separation of tron microscopy by critical-point drying and the intima from the media and no edema (Fig coating with gold-palladium. Each half of the 3A). The total pathological damage score for vein segment was examined for pathological Group 1 was 4, the lowest score for all groups damage both en face and in cross section in a studied. JEOL 35C scanning electron microscope. The All veins in Group 2 (28°C saline solution, 100 types of pathological damage observed in- mm Hg) had marked, grade 4, endothelial cell cluded endothelial cell separation, endothelial separation and grade 3 endothelial cell loss, excell loss, exposed basement membrane, ex- posing large amounts of basement membrane posed fibrillar collagen, intimal edema, medial and fibrillar collagen (Fig 4A). Intimal fractures edema, and intimal and transmural vein frac- occurred more frequently than in control veins tures. These types of pathological damage were and separation of the intima from the media graded on a scale of 0 to 4 on the basis of per- occurred in 3 specimens (Fig 3B). Edema was cent involvement of the vein segment: 0 = no present in all specimens in this group. The total Fig I . Constant pressure perfusion device, which allows perfusion and fixation of a saphenous vein in various solutions at constant distention pressure.
42 The Annals of Thoracic Surgery Vol30
No 1 July 1980
Damage Scores in Five Groups of Vein Segmentsa Group (5 veins each) Group 1 Control 30 mm Hg Group 2 NSS storage 28°C 1 hr 100 mm Hg Group 3 NSS storage 28°C 1 hr 300 mm Hg Group 4 WB storage 28°C 1 hr 100 mm Hg Group 5 WB storage 4°C 1 hr .lo0 mm Hg
Endothelial Cell Separation
Endothelial Cell Loss
Exposed Basement Membrane
Exposed Collagep
Intimal Edema
Medial Edema
Intimal Fractures
Total Damage Score
1
1
0
0
0
0
2
4
4
3
2
2
2
2
3
18
4
2
4
4
3
24
2
1
1
1
2
11
2
1
0
0
2
7
"Damagescore: 0 = no change; 1 = occasionally observed; 2 = 10 to 25% of vein involved; 3 = 30 to 50% of vein involved; 4 = more than 50% of vein involved. NSS = normal saline solution; WB = whole blood.
Fig 2 . Endothelial surface of Group 1 vein (control) shows good preservation of endothelial cells with intact intercellular connections. Bulging nuclei and surface microvilli are seen clearly. ( ~ 1 , 3 0 0before 40% reduction.)
pathological damage score for Group 2 vein segments was 18. Veins in Group 3 (28°C saline solution, 300 mm Hg) showed grade 4 endothelial cell separation and endothelial cell loss, exposing large areas of basement membrane (Fig 4B). Intimal
fractures were more pronounced than in control veins. The wall thickness was decreased, with separation of the intima from the media (Fig 3C). Edema was present between smooth muscle fibers. This group showed more damage than any of the others, having a damage score of 24. Veins in Group 4 (28°C blood, 100 mm Hg) demonstrated endothelial cell separation and endothelial cell loss involving 10 to 25% of the intimal surface. The remaining intima appeared identical to that of control veins. Grade 2 areas of exposed basement membrane occurred while fibrillar collagen was exposed only occasionally (Fig 5A). There was minimal intimal or medial edema. The total damage score for Group 4 was 11. Veins in Group 5 (4°C blood, 100 mm Hg) had mild endothelial cell separation with endothelial cell loss similar to that of control veins. Fibrillar collagen and basement membrane were only occasionally exposed (Fig 5B). Fractures appeared in numbers similar to control veins. There was no intimal or medial edema (Fig 3D). The total damage score for Group 5 was 7, similar to that of Group 1.
43 Gundry et al: Intraoperative Trauma to Saphenous Veins
Fig 3. Low-power views of scanning electron micrographs of vein walls. Endothelial surface (E) is shown on the left and the adventitial surface (A) on the right. ( A ) Cross section of Group 1 vein (control) shows endothelial surface and adventitial surface. Edema in the wall is absent. (X150 before 10% reduction.) (B) Cross section of Group 2 vein (saline immersion) shows separation of intima (arrowheads) from media. Areas of muscle fiber separation in media represent edema. (X130 before lOo/o reduction.) (C) Cross section of Group 3 vein (saline immersion with 300 mm Hg distention) shows fine separation of intima from media and marked fiber separation in media, representing severe edema. ( X 9 4 before 10% reduction.) ( D ) Cross section of vein immersed in cold blood (Group 5) is representative of blood-immersed veins. The appearance is compact, similar to that of a control vein, with no intimal-medial separation and minimal separation of medial fibers. (X540 before 10% reduction.)
44 The Annals of Thoracic Surgery Vol30
No 1 July 1980
Fig 4 . ( A ) Endothelial surface of saline-immersed vein (Group 2 ) shows an area of total endothelial cell loss, with exposed basement membrane (BM) and fibrillar 0 40% reduction.) ( B ) Encollagen (C).( ~ 7 2 before dothelial surface of saline-immersed vein distended with 300 mm Hg pressure (Group 3 ) shows typical rolled-up appearance of separated endothelial cells (E), beneath which exposed basement membrane and collagen are seen. ( ~ 1 , 6 0 0before 40% reduction.)
Fig 5. (A) Endothelial surface of veins immersed in blood (Group 4 ) shows occasional areas of endothelial cell separation with rolled-up appearance of a few cells (arrowheads). Note the generally intact endothelial cell layer and normal nuclei. ( ~ 1 , 0 0 0before 40% reduction.) (B)Endothelial surface of vein immersed in cold blood (Group 5) shows a single rolled-up endothelial cell (arrowhead) with good preservation of the remainder of the surface layer. Note prominent nuclei and microvilli. ( ~ 9 4 before 0 40% reduction.)
Comment Coronary artery vein graft patency rates drop dramatically during the first year following implantation, with reported patency rates of 50 to 85% at one year [12, 14, 15, 19, 291. Animal studies suggest that 60% of failures occur within the first week, usually from thrombosis [5]. Following the high rate of attrition during the first year, occlusions occur at rates of less than 2% per year [8,24]. These markedly different rates of early and late graft failure suggest that they result from two separate processesearly thrombosis and late luminal stenosis or occlusion. Examination of postmortem and surgical
specimens of vein grafts demonstrates that 70% of occlusions within the first year are secondary to mural thrombus formation overlying areas of endothelial cell loss [28]. Jones [16], Ferrans [13], Barboriak [3], Reiche [22], and their associates observed adherent fibrin and thrombus over denuded endothelium in vein grafts, while Bulkley and Hutchins [7] demonstrated circumferential intimal thrombus in 73% of human vein grafts obtained one hour to one month postoperatively. Studies of vein grafts in dogs have shown large areas of sloughed endothelium, leaving basement membrane, fibrillar collagen, and elastin to which microthrom.bi of platelets, red blood cells, and fi-
45 Gundry et al: Intraoperative Trauma to Saphenous Veins
brin are adherent [5,21,27,30]. Thus, denuding of intimal endothelium predisposes to mural thrombus formation, which, in turn, is a cause of early graft occlusion. Late graft failures appear to arise from focal or diffuse luminal stenoses secondary to subendothelial fibromuscular hyperplasia or atheromatous plaques [2, 7, 13, 16, 281. Medial necrosis, hydraulic alterations, arterialization of the vein wall, and venous valve stenosis have been implicated in the pathogenesis of fibromuscular hyperplasia [4-6, 14, 16, 171. Although the fate of the original endothelium in implanted vein grafts is not known with certainty, after several months of implantation, the endothelium of vein grafts appears to be regenerated, presumably from endothelial cells remaining in the wall. Nevertheless, late endothelial deficits do occur [2, 3, 6, 281. Additionally, intima underlying regenerated endothelium is substantially thicker and more likely to accumulate lipid than is intima under undamaged endothelium [20]. Studies by Duguid [lo, 111, Jones and co-workers [161, and Ross and colleagues [23] have indicated that the development of late fibromuscular hyperplasia may be secondary to the formation and organization of early intimal thrombi and plateletfibrin aggregates. A thrombotic origin of intima1 hyperplasia is supported further by the finding that drugs that inhibit platelet aggregation and activation also reduce intimal thickening in animal coronary bypass grafts [191. Thus, despite endothelial regeneration, the initial endothelial damage at operation may predispose to the late development of fibromuscular intimal hyperplasia, late graft stenosis, and late graft occlusion. Therefore, the preservation of the structural integrity of the vein appears to be important. The results of our investigations indicate that the endothelium of human veins immersed in blood is better preserved than the endothelium of veins immersed in saline solution for identical time periods, at identical temperatures, and subjected to identical distention pressures. Immersion in cold blood provides additional protection for human veins, as suggested by animal experiments [l]. Furthermore, distention above physiological pressures with saline
solution damages the vein intimal and medial components [l, 18, 251. Vein wall edema is minimal or absent following blood immersion, whereas edema is consistently present following immersion in saline solution, especially when associated with high distention pressures. In conclusion, human saphenous vein endothelium is damaged by intraoperative manipulation and particularly by immersion in saline solution and by distention to pressures above 100 mm Hg. Human saphenous veins are best preserved by gentle handling, immersion in cold, heparinized blood and avoidance of distention above physiological pressures. Preservation of the endothelium and prevention of mural edema may reduce early thrombus formation and the late development of subendothelial fibromuscular hyperplasia. References 1. Abbott WM, Wieland S, Austen WG: Structural changes during preparation of autogenous venous grafts. Surgery 76:1031, 1974 2. Barboriak JJ, Batayias GE, Pintar K, et al: Late lesions in aorta-coronary artery vein grafts. J Thorac Cardiovasc Surg 73:596, 1977 3. Barboriak JJ, Von Horn DL, Pintar K, et al: Scanning electron microscope study of human veins and aorta-coronary artery vein grafts. J Thorac Cardiovasc Surg 71:673, 1976 4. Bosher LP, Deck JD, Thubrikar M, et al: Role of the venous valve in late segmental occlusion of vein grafts. J Surg Res 26:437, 1979 5. Brody WR, Angell WW, Kosek JC: Histologic fate of venous coronary artery bypass grafts in dogs. Am J Pathol 66:111, 1972 6. Brody WR, Kosek JC, Angell WW: Changes in vein grafts following aorto-coronary bypass induced by pressure and ischemia. J Thorac Cardiovasc Surg 642347, 1972 7. Bulkley BH, Hutchins GM: "Accelerated atherosclerosis": a morphologic study of 97 saphenous vein coronary artery bypass grafts. Circulation 55:163, 1977 8. Campeau L, Lesperance J, Corbara F, et al: Late changes in aorto-coronary saphenous vein bypass grafts (5 to 7 years after surgery). Circulation 55, 56:Suppl3:111-132, 1977 9. Cooley DA: Revascularization of the ischemic myocardium. J Thorac Cardiovasc Surg 78:301, 1979 10. Duguid JB: Thrombosis as a factor in the pathogenesis of coronary atherosclerosis. J Pathol Bacteriol 58:207, 1946
46 The Annals of Thoracic Surgery Vol30 No 1 July 1980
11. Duguid JB: Thrombosis as a factor in the pathogenesis of aortic atherosclerosis. J Pathol Bacteriol 60:57, 1948 12. Effler DB, Favaloro RG, Groves LK, et al: The simple approach to direct coronary artery surgery. J Thorac Cardiovasc Surg 62:503, 1971 13. Ferrans VJ, Jones M, Roberts WC: The pathology of saphenous vein aorto-coronary artery bypass grafts, in Proceedings of the International Symposium on Selected Topics in Cardiac Surgery. Edited by PG Cevese. Padova, Italy (in press) 14. Grondin CM, Meere C, Castonguay Mi, et al: Blood flow through aorto-to-coronary artery bypass grafts and early postoperative patency: a study of 100 patients. Ann Thorac Surg 12:574, 1971 15. Itscoitz SB, Redwood DR, Stinson EB, et al: Saphenous vein bypass grafts: long-term patency and effect on the native coronary circulation. Am J Cardiol 36:739, 1975 16. Jones M, Conkle DM, Ferrans VJ, et al: Lesions observed in arterial autogenous vein grafts, light and electron microscopic evaluation. Circulation 47, 48:S~ppl3:III-198, 1973 17. Kennedy JH, Wieting DW, Hwang NHC, et al: Hydraulic and morphologic study of fibrous intimal hyperplasia in autogenous saphenous vein bypass grafts. J Thorac Cardiovasc Surg 675305, 1974 18. MacGregor DC, Agarwal VK, Silver MD: Changes produced in the wall of the saphenous vein of dogs by distending media and pressures. Surg Forum 23:135, 1972 19. Metke MI’, Lie JT, Fuster V, et al: Reduction of intimal thickening in canine coronary bypass vein grafts with dipyridamole and aspirin. Am J Cardiol 43:1144, 1979 20. Minick CR, Stemerman MB, Insull W: Role of endothelium and hypercholesterolemia in intima1 thickening and lipid accumulation. Am J Pathol 95:131, 1979 21. Nunn DB, Chun 8 , Whelan TJ, et al: Autogenous veins as arterial substitutes: a study of their histologic fate with special attention to endothelium. Ann Surg 160:14, 1964 22. Reichle FA, Stewart GJ, Essa N: A transmission and scanning electron microscopic study of luminal surfaces: Dacron and autogenous vein bypasses in man and dog. Surgery 74:945, 1973 23. Ross E, Gomset J, Harker L: Response to injury and atherogenesis. Am J Pathol 86:675, 1977 24. Seides SF, Borer JS, Kent KM, et al: Long-term anatomic fate of coronary artery bypass grafts and functional status of patients five years after operation. N Engl J Med 298:1213, 1978 25. Stanley JC, Sottiurai V, Fry RE, et al: Comparative evaluation of vein graft preparation media: electron and light microscopic studies. J Surg Res 18:235, 1975
26. Stiles QR: Technique of saphenous vein aortocoronary bypass grafting. J Thorac Cardiovasc Surg 78:305, 1979 27. Ts’ao CH, Glagov S: Platelet adhesion to subendothelial components in experimental aortic injury: role of fine fibrils and basement membrane. Br J Exp Pathol 51:423, 1970 28. Unni KK, Kottke BA, Titus JL, et al: Pathologic changes in aorto-coronary saphenous vein grafts. Am J Cardiol 34:526, 1974 29. Walker JA , Friedberg HD, Flemma RJ, et al: Determinants of angiographic patency of aortocoronary vein bypass grafts. Circulation 45: Suppl 1:1-86, 1972 30. Wyatt AP, Taylor GW: Vein graft changes in the endothelium of autogenous free vein grafts used as arterial replacements. Br J Surg 53:943, 1966
Discussion DR. GORDON N . OLINGER (Milwaukee, WI): I will accent Dr. Gundry’s concern regarding trauma to veins by relating some biochemical data pertinent to the effect of trauma on delayed vein graft atherogenesis. I also will comment on the pressure limits for vein distention Dr. Gundry has advocated. My data were derived from a nonhuman primate model developed at the laboratory at the Medical College of Wisconsin. A forearm cephalic vein was inserted as a reversed graft from the common femoral to the superficial femoral artery, bypassing an occlusive ligature. Before insertion, the distal half of the graft was distended with heparinized blood to 350 mm Hg and the proximal half to 700 mm Hg. The particular group of animals used was normolipemic, and grafts were harvested at three months. I believe this model is reasonably analogous to use of human saphenous vein as an artery replacement. Monkeys’ cephalic veins are muscular vessels like human saphenous veins, and the pressure required to overcome spasm in these veins is similar to that observed intraoperatively in human veins, that is, somewhere from 200 to as high as 500 mm Hg. This is more pressure than Dr. Gundry found suitable. I wonder if storage for one hour prior to distentionreally a clinical impracticality in our practicefacilitated lower pressures in this study. My colleagues and I found by electron microscopy examination that endothelium of monkey veins distended at 300 mm Hg, seen prior to implantation, is generally well preserved. Higher pressure distention, on the other hand, denudes endothelium, exposing basement membrane and collagen to formed blood elements. Now 700 mm Hg may seem outrageously high, but such intraluminal pressures or greater ones are common when human saphenous veins are distended by the unfortunately popular technique of occluding one end and applying pressure to the other with a fluid-filled syringe.
47 Gundry et al: Intraoperative Trauma to Saphenous Veins
When these grafts were harvested, biochemical analysis defined an increased concentration of cholesterol in grafts compared with normal vein left in situ, and a concentration of cholesterol in the higher pressure segments that was significantly greater than that in the lower pressure segments. The same relationships held for beta-apolipoprotein. Thus, there are both histological and biochemical data to support the concept that intraoperative endothelial damage may be a precursor to vein graft atheromatous degeneration. Injury from hypooncotic agents can and should be avoided. However, if the surgeon wants to work with veins free from spasm, some intraluminal distending pressure will have to be applied. We found a range from 300 to 400 mm Hg to be necessary usually. Our observations suggest such pressures will permit preservation of endothelial integrity not only in monkey veins but, in contradistinction to Dr. Gundry’s studies, in human saphenous veins as well. At this time we are distending veins in the operating room with a simple pressure-limiting balloon device inserted between the syringe and vein that permits vein distention at less than 400 mm Hg. We hope it will help mitigate at least one of the factors in vein graft atherogenesis.
DR. GERALD M . LAWRIE (Houston, TX): I present some original data, compiled by Dr. Jack Titus and Dr. Saenz at Baylor, from 60 grafts recovered from the
time of operation in humans to about two weeks after operation. In a graft recovered 6 hours after operation, there is necrosis in the intima, some changes in the endothelium, and some edema. One day after operation, almost all the veins are lined by fibrin to which platelets are adhering and there are platelet thrombi. This may explain the dichotomy between the theoretical advantages of aspirin or Persantine (dipyridamole) and the practical effects. The process is well advanced long before any of these agents can be started. Three days after operation, there is quite extensive necrosis developing progressively in the intima with pyknotic nuclei. Fibrin is overlying the endothelium, which has undergone patchy loss and has some edema. Nine days after operation, the fibrin lining is present, the entire vein wall is infiltrated with inflammatory cells, and the entire thickness of the vein wall has undergone patchy necrosis. This has occurred independent of the extent of intimal integrity. Despite these early findings in long-term, follow-up pathological studies, findings that I think have not been documented before, we found excellent late histology. So although I think the early techniques used to prepare the veins are important, more work must be done to document what happens in the first two weeks. Our study leads us to believe that the veins universally undergo patchy, generalized necrosis regardless of the technique used to prepare them.
ABSTRACT To determine optimal preparation techniques for human saphenous veins, a scanning electron microscopic comparison was made of the effects of variations in solutions, temperatures, and distention pressures on human vein morphology. Segments of saphenous veins obtained from 20 patients undergoing myocardial revascularization were divided into five groups of 5 veins each. Group 1 segments (controls) were immediately distended and perfusion-fixed with glutaraldehyde at 30 mm Hg to simulate in vivo saphenous vein hydrostatic pressure. Segments comprising Groups 2 and 3 were placed in heparinized normal saline solution at 28°C for one hour and then distended to either 100 mm Hg (Group 2) or 300 mm Hg (Group 3). Segments making up Groups 4 and 5 were immersed in heparinized blood for one hour at either 28°C (Group 4) or 4°C (Group 5), and then distended to 100 mm Hg. Except for controls, which were fixed as described, all other groups of vein specimens were perfusion-fixed at 100 mm Hg to simulate in vivo arterial pressure. Control veins showed focal intimal fractures with minimal endothelial cell loss due to harvesting manipulation. Groups 2 and 3 had marked endothelial loss, which led to exposure of basement membrane, collagen fibrils, and smooth muscle cells and was associated with intimal and medial edema. Veins prepared with blood appeared similar to controls, showing focal endothelial cell loss, but preservation of intimal and medial structure. Changes were less marked in Group 5 than in Group 4. We conclude that human saphenous veins are best preserved by
immersion in cold blood and distention to a pressure of 100 mm Hg or less.
Aortocoronary bypass grafts using autogenous saphenous veins are subject to early and late occlusion. Problems related to operative technique, distal coronary artery runoff, hyperlipidemia, graft ischemia, and vein preparation techniques have been implicated as causes of bypass graft failure [2, 5, 9, 13, 25, 261. Although investigations in animals suggest that vein immersion in saline solution and vein distention above physiological pressures cause vein endothelial cell and mural damage [ l , 6, 13, 20, 22, 251, no systematic evaluations of these problems have been performed using human saphenous veins [2, 3, 6, 7, 13, 22, 281. To determine preferable techniques for preserving vein integrity, we compared human graft preparation techniques, evaluating the veins by scanning electron microscopy.
Materials and Methods Portions of saphenous veins from 20 patients undergoing coronary artery bypass operations were divided in 25 segments, each measuring 4 to 5 cm in length. These segments were divided into five groups of 5 veins each. Segments from Group 1 (control) were immediately fixed by perfusion with cold, buffered (pH 7.2) 3% glutaraldehyde at 30 mm Hg distention pressure, using a constant pressure perfusion device (Fig 1) to approximate the in vivo hydrostatic venous pressure in the standing position. Group 2 veins were immersed in heparinized From the Clinic of Surgery and Pathology Branch, National normal saline solution (0.9% sodium chloride) Heart, Lung, and Blood Institute, Bethesda, MD. Presented at the Sixteenth Annual Meeting of The Society of at 28°C for one hour and then distended to a Thoracic Surgeons, Jan 21-23, 1980, Atlanta, GA. pressure of 100 mm Hg with normal saline Address reprint requests to Dr. Gundry, Clinic of Surgery, solution. Group 3 veins were immersed in National Heart, Lung, and Blood Institute, Building 10, heparinized normal saline solution at 28°C for Room 6N252, National Institutes of Health, Bethesda, one hour, followed by distention to a pressure MD 20205. 40
0003-49751801070040-08$01.25 @ 1980 by The Society of Thoracic Surgeons
41 Gundry et al: Intraoperative Trauma to Saphenous Veins
Pressure
Saphenous Vein
Adjustable Clamp
damage; 1= less than 10%; 2 = 10 to 25%; 3 = 25 to 50%; and 4 = more than 50%. We utilized the sum of these grades for each of the pathological alterations in each of the five groups of vein segments as a numerical representation of of 300 mm Hg. Group 4 veins were immersed in the total amount of vein trauma produced by heparinized arterial blood at 28°C for one hour each of the five techniques for preparation of and afterward distended to a pressure of 100 vein grafts (Table). mm Hg with autologous human blood. Group 5 veins were immersed in heparinized arterial Results blood at 4°C for one hour and then distended All vein segments in Group 1 (control) demonwith blood to a pressure of 100 mm Hg. After strated occasional endothelial cell separation pressure distention, all groups of veins, except and endothelial cell loss with minimal exposure for controls, were fixed with cold, buffered (pH of fibrillar collagen or basement membrane (Fig 7.2) 3% glutaraldehyde at a perfusion pressure 2). These specimens showed fractures, with of 100 mm Hg, simulating human in vivo arte- clefts extending through the intima and into the rial pressure. media. We presume that these fractures occur After fixation, the vein segments were di- from manipulation during operation because vided in half longitudinally. From each, two 0.5 veins in all groups showed similar intimal cm sections were prepared for scanning elec- fractures. Group 1 veins had no separation of tron microscopy by critical-point drying and the intima from the media and no edema (Fig coating with gold-palladium. Each half of the 3A). The total pathological damage score for vein segment was examined for pathological Group 1 was 4, the lowest score for all groups damage both en face and in cross section in a studied. JEOL 35C scanning electron microscope. The All veins in Group 2 (28°C saline solution, 100 types of pathological damage observed in- mm Hg) had marked, grade 4, endothelial cell cluded endothelial cell separation, endothelial separation and grade 3 endothelial cell loss, excell loss, exposed basement membrane, ex- posing large amounts of basement membrane posed fibrillar collagen, intimal edema, medial and fibrillar collagen (Fig 4A). Intimal fractures edema, and intimal and transmural vein frac- occurred more frequently than in control veins tures. These types of pathological damage were and separation of the intima from the media graded on a scale of 0 to 4 on the basis of per- occurred in 3 specimens (Fig 3B). Edema was cent involvement of the vein segment: 0 = no present in all specimens in this group. The total Fig I . Constant pressure perfusion device, which allows perfusion and fixation of a saphenous vein in various solutions at constant distention pressure.
42 The Annals of Thoracic Surgery Vol30
No 1 July 1980
Damage Scores in Five Groups of Vein Segmentsa Group (5 veins each) Group 1 Control 30 mm Hg Group 2 NSS storage 28°C 1 hr 100 mm Hg Group 3 NSS storage 28°C 1 hr 300 mm Hg Group 4 WB storage 28°C 1 hr 100 mm Hg Group 5 WB storage 4°C 1 hr .lo0 mm Hg
Endothelial Cell Separation
Endothelial Cell Loss
Exposed Basement Membrane
Exposed Collagep
Intimal Edema
Medial Edema
Intimal Fractures
Total Damage Score
1
1
0
0
0
0
2
4
4
3
2
2
2
2
3
18
4
2
4
4
3
24
2
1
1
1
2
11
2
1
0
0
2
7
"Damagescore: 0 = no change; 1 = occasionally observed; 2 = 10 to 25% of vein involved; 3 = 30 to 50% of vein involved; 4 = more than 50% of vein involved. NSS = normal saline solution; WB = whole blood.
Fig 2 . Endothelial surface of Group 1 vein (control) shows good preservation of endothelial cells with intact intercellular connections. Bulging nuclei and surface microvilli are seen clearly. ( ~ 1 , 3 0 0before 40% reduction.)
pathological damage score for Group 2 vein segments was 18. Veins in Group 3 (28°C saline solution, 300 mm Hg) showed grade 4 endothelial cell separation and endothelial cell loss, exposing large areas of basement membrane (Fig 4B). Intimal
fractures were more pronounced than in control veins. The wall thickness was decreased, with separation of the intima from the media (Fig 3C). Edema was present between smooth muscle fibers. This group showed more damage than any of the others, having a damage score of 24. Veins in Group 4 (28°C blood, 100 mm Hg) demonstrated endothelial cell separation and endothelial cell loss involving 10 to 25% of the intimal surface. The remaining intima appeared identical to that of control veins. Grade 2 areas of exposed basement membrane occurred while fibrillar collagen was exposed only occasionally (Fig 5A). There was minimal intimal or medial edema. The total damage score for Group 4 was 11. Veins in Group 5 (4°C blood, 100 mm Hg) had mild endothelial cell separation with endothelial cell loss similar to that of control veins. Fibrillar collagen and basement membrane were only occasionally exposed (Fig 5B). Fractures appeared in numbers similar to control veins. There was no intimal or medial edema (Fig 3D). The total damage score for Group 5 was 7, similar to that of Group 1.
43 Gundry et al: Intraoperative Trauma to Saphenous Veins
Fig 3. Low-power views of scanning electron micrographs of vein walls. Endothelial surface (E) is shown on the left and the adventitial surface (A) on the right. ( A ) Cross section of Group 1 vein (control) shows endothelial surface and adventitial surface. Edema in the wall is absent. (X150 before 10% reduction.) (B) Cross section of Group 2 vein (saline immersion) shows separation of intima (arrowheads) from media. Areas of muscle fiber separation in media represent edema. (X130 before lOo/o reduction.) (C) Cross section of Group 3 vein (saline immersion with 300 mm Hg distention) shows fine separation of intima from media and marked fiber separation in media, representing severe edema. ( X 9 4 before 10% reduction.) ( D ) Cross section of vein immersed in cold blood (Group 5) is representative of blood-immersed veins. The appearance is compact, similar to that of a control vein, with no intimal-medial separation and minimal separation of medial fibers. (X540 before 10% reduction.)
44 The Annals of Thoracic Surgery Vol30
No 1 July 1980
Fig 4 . ( A ) Endothelial surface of saline-immersed vein (Group 2 ) shows an area of total endothelial cell loss, with exposed basement membrane (BM) and fibrillar 0 40% reduction.) ( B ) Encollagen (C).( ~ 7 2 before dothelial surface of saline-immersed vein distended with 300 mm Hg pressure (Group 3 ) shows typical rolled-up appearance of separated endothelial cells (E), beneath which exposed basement membrane and collagen are seen. ( ~ 1 , 6 0 0before 40% reduction.)
Fig 5. (A) Endothelial surface of veins immersed in blood (Group 4 ) shows occasional areas of endothelial cell separation with rolled-up appearance of a few cells (arrowheads). Note the generally intact endothelial cell layer and normal nuclei. ( ~ 1 , 0 0 0before 40% reduction.) (B)Endothelial surface of vein immersed in cold blood (Group 5) shows a single rolled-up endothelial cell (arrowhead) with good preservation of the remainder of the surface layer. Note prominent nuclei and microvilli. ( ~ 9 4 before 0 40% reduction.)
Comment Coronary artery vein graft patency rates drop dramatically during the first year following implantation, with reported patency rates of 50 to 85% at one year [12, 14, 15, 19, 291. Animal studies suggest that 60% of failures occur within the first week, usually from thrombosis [5]. Following the high rate of attrition during the first year, occlusions occur at rates of less than 2% per year [8,24]. These markedly different rates of early and late graft failure suggest that they result from two separate processesearly thrombosis and late luminal stenosis or occlusion. Examination of postmortem and surgical
specimens of vein grafts demonstrates that 70% of occlusions within the first year are secondary to mural thrombus formation overlying areas of endothelial cell loss [28]. Jones [16], Ferrans [13], Barboriak [3], Reiche [22], and their associates observed adherent fibrin and thrombus over denuded endothelium in vein grafts, while Bulkley and Hutchins [7] demonstrated circumferential intimal thrombus in 73% of human vein grafts obtained one hour to one month postoperatively. Studies of vein grafts in dogs have shown large areas of sloughed endothelium, leaving basement membrane, fibrillar collagen, and elastin to which microthrom.bi of platelets, red blood cells, and fi-
45 Gundry et al: Intraoperative Trauma to Saphenous Veins
brin are adherent [5,21,27,30]. Thus, denuding of intimal endothelium predisposes to mural thrombus formation, which, in turn, is a cause of early graft occlusion. Late graft failures appear to arise from focal or diffuse luminal stenoses secondary to subendothelial fibromuscular hyperplasia or atheromatous plaques [2, 7, 13, 16, 281. Medial necrosis, hydraulic alterations, arterialization of the vein wall, and venous valve stenosis have been implicated in the pathogenesis of fibromuscular hyperplasia [4-6, 14, 16, 171. Although the fate of the original endothelium in implanted vein grafts is not known with certainty, after several months of implantation, the endothelium of vein grafts appears to be regenerated, presumably from endothelial cells remaining in the wall. Nevertheless, late endothelial deficits do occur [2, 3, 6, 281. Additionally, intima underlying regenerated endothelium is substantially thicker and more likely to accumulate lipid than is intima under undamaged endothelium [20]. Studies by Duguid [lo, 111, Jones and co-workers [161, and Ross and colleagues [23] have indicated that the development of late fibromuscular hyperplasia may be secondary to the formation and organization of early intimal thrombi and plateletfibrin aggregates. A thrombotic origin of intima1 hyperplasia is supported further by the finding that drugs that inhibit platelet aggregation and activation also reduce intimal thickening in animal coronary bypass grafts [191. Thus, despite endothelial regeneration, the initial endothelial damage at operation may predispose to the late development of fibromuscular intimal hyperplasia, late graft stenosis, and late graft occlusion. Therefore, the preservation of the structural integrity of the vein appears to be important. The results of our investigations indicate that the endothelium of human veins immersed in blood is better preserved than the endothelium of veins immersed in saline solution for identical time periods, at identical temperatures, and subjected to identical distention pressures. Immersion in cold blood provides additional protection for human veins, as suggested by animal experiments [l]. Furthermore, distention above physiological pressures with saline
solution damages the vein intimal and medial components [l, 18, 251. Vein wall edema is minimal or absent following blood immersion, whereas edema is consistently present following immersion in saline solution, especially when associated with high distention pressures. In conclusion, human saphenous vein endothelium is damaged by intraoperative manipulation and particularly by immersion in saline solution and by distention to pressures above 100 mm Hg. Human saphenous veins are best preserved by gentle handling, immersion in cold, heparinized blood and avoidance of distention above physiological pressures. Preservation of the endothelium and prevention of mural edema may reduce early thrombus formation and the late development of subendothelial fibromuscular hyperplasia. References 1. Abbott WM, Wieland S, Austen WG: Structural changes during preparation of autogenous venous grafts. Surgery 76:1031, 1974 2. Barboriak JJ, Batayias GE, Pintar K, et al: Late lesions in aorta-coronary artery vein grafts. J Thorac Cardiovasc Surg 73:596, 1977 3. Barboriak JJ, Von Horn DL, Pintar K, et al: Scanning electron microscope study of human veins and aorta-coronary artery vein grafts. J Thorac Cardiovasc Surg 71:673, 1976 4. Bosher LP, Deck JD, Thubrikar M, et al: Role of the venous valve in late segmental occlusion of vein grafts. J Surg Res 26:437, 1979 5. Brody WR, Angell WW, Kosek JC: Histologic fate of venous coronary artery bypass grafts in dogs. Am J Pathol 66:111, 1972 6. Brody WR, Kosek JC, Angell WW: Changes in vein grafts following aorto-coronary bypass induced by pressure and ischemia. J Thorac Cardiovasc Surg 642347, 1972 7. Bulkley BH, Hutchins GM: "Accelerated atherosclerosis": a morphologic study of 97 saphenous vein coronary artery bypass grafts. Circulation 55:163, 1977 8. Campeau L, Lesperance J, Corbara F, et al: Late changes in aorto-coronary saphenous vein bypass grafts (5 to 7 years after surgery). Circulation 55, 56:Suppl3:111-132, 1977 9. Cooley DA: Revascularization of the ischemic myocardium. J Thorac Cardiovasc Surg 78:301, 1979 10. Duguid JB: Thrombosis as a factor in the pathogenesis of coronary atherosclerosis. J Pathol Bacteriol 58:207, 1946
46 The Annals of Thoracic Surgery Vol30 No 1 July 1980
11. Duguid JB: Thrombosis as a factor in the pathogenesis of aortic atherosclerosis. J Pathol Bacteriol 60:57, 1948 12. Effler DB, Favaloro RG, Groves LK, et al: The simple approach to direct coronary artery surgery. J Thorac Cardiovasc Surg 62:503, 1971 13. Ferrans VJ, Jones M, Roberts WC: The pathology of saphenous vein aorto-coronary artery bypass grafts, in Proceedings of the International Symposium on Selected Topics in Cardiac Surgery. Edited by PG Cevese. Padova, Italy (in press) 14. Grondin CM, Meere C, Castonguay Mi, et al: Blood flow through aorto-to-coronary artery bypass grafts and early postoperative patency: a study of 100 patients. Ann Thorac Surg 12:574, 1971 15. Itscoitz SB, Redwood DR, Stinson EB, et al: Saphenous vein bypass grafts: long-term patency and effect on the native coronary circulation. Am J Cardiol 36:739, 1975 16. Jones M, Conkle DM, Ferrans VJ, et al: Lesions observed in arterial autogenous vein grafts, light and electron microscopic evaluation. Circulation 47, 48:S~ppl3:III-198, 1973 17. Kennedy JH, Wieting DW, Hwang NHC, et al: Hydraulic and morphologic study of fibrous intimal hyperplasia in autogenous saphenous vein bypass grafts. J Thorac Cardiovasc Surg 675305, 1974 18. MacGregor DC, Agarwal VK, Silver MD: Changes produced in the wall of the saphenous vein of dogs by distending media and pressures. Surg Forum 23:135, 1972 19. Metke MI’, Lie JT, Fuster V, et al: Reduction of intimal thickening in canine coronary bypass vein grafts with dipyridamole and aspirin. Am J Cardiol 43:1144, 1979 20. Minick CR, Stemerman MB, Insull W: Role of endothelium and hypercholesterolemia in intima1 thickening and lipid accumulation. Am J Pathol 95:131, 1979 21. Nunn DB, Chun 8 , Whelan TJ, et al: Autogenous veins as arterial substitutes: a study of their histologic fate with special attention to endothelium. Ann Surg 160:14, 1964 22. Reichle FA, Stewart GJ, Essa N: A transmission and scanning electron microscopic study of luminal surfaces: Dacron and autogenous vein bypasses in man and dog. Surgery 74:945, 1973 23. Ross E, Gomset J, Harker L: Response to injury and atherogenesis. Am J Pathol 86:675, 1977 24. Seides SF, Borer JS, Kent KM, et al: Long-term anatomic fate of coronary artery bypass grafts and functional status of patients five years after operation. N Engl J Med 298:1213, 1978 25. Stanley JC, Sottiurai V, Fry RE, et al: Comparative evaluation of vein graft preparation media: electron and light microscopic studies. J Surg Res 18:235, 1975
26. Stiles QR: Technique of saphenous vein aortocoronary bypass grafting. J Thorac Cardiovasc Surg 78:305, 1979 27. Ts’ao CH, Glagov S: Platelet adhesion to subendothelial components in experimental aortic injury: role of fine fibrils and basement membrane. Br J Exp Pathol 51:423, 1970 28. Unni KK, Kottke BA, Titus JL, et al: Pathologic changes in aorto-coronary saphenous vein grafts. Am J Cardiol 34:526, 1974 29. Walker JA , Friedberg HD, Flemma RJ, et al: Determinants of angiographic patency of aortocoronary vein bypass grafts. Circulation 45: Suppl 1:1-86, 1972 30. Wyatt AP, Taylor GW: Vein graft changes in the endothelium of autogenous free vein grafts used as arterial replacements. Br J Surg 53:943, 1966
Discussion DR. GORDON N . OLINGER (Milwaukee, WI): I will accent Dr. Gundry’s concern regarding trauma to veins by relating some biochemical data pertinent to the effect of trauma on delayed vein graft atherogenesis. I also will comment on the pressure limits for vein distention Dr. Gundry has advocated. My data were derived from a nonhuman primate model developed at the laboratory at the Medical College of Wisconsin. A forearm cephalic vein was inserted as a reversed graft from the common femoral to the superficial femoral artery, bypassing an occlusive ligature. Before insertion, the distal half of the graft was distended with heparinized blood to 350 mm Hg and the proximal half to 700 mm Hg. The particular group of animals used was normolipemic, and grafts were harvested at three months. I believe this model is reasonably analogous to use of human saphenous vein as an artery replacement. Monkeys’ cephalic veins are muscular vessels like human saphenous veins, and the pressure required to overcome spasm in these veins is similar to that observed intraoperatively in human veins, that is, somewhere from 200 to as high as 500 mm Hg. This is more pressure than Dr. Gundry found suitable. I wonder if storage for one hour prior to distentionreally a clinical impracticality in our practicefacilitated lower pressures in this study. My colleagues and I found by electron microscopy examination that endothelium of monkey veins distended at 300 mm Hg, seen prior to implantation, is generally well preserved. Higher pressure distention, on the other hand, denudes endothelium, exposing basement membrane and collagen to formed blood elements. Now 700 mm Hg may seem outrageously high, but such intraluminal pressures or greater ones are common when human saphenous veins are distended by the unfortunately popular technique of occluding one end and applying pressure to the other with a fluid-filled syringe.
47 Gundry et al: Intraoperative Trauma to Saphenous Veins
When these grafts were harvested, biochemical analysis defined an increased concentration of cholesterol in grafts compared with normal vein left in situ, and a concentration of cholesterol in the higher pressure segments that was significantly greater than that in the lower pressure segments. The same relationships held for beta-apolipoprotein. Thus, there are both histological and biochemical data to support the concept that intraoperative endothelial damage may be a precursor to vein graft atheromatous degeneration. Injury from hypooncotic agents can and should be avoided. However, if the surgeon wants to work with veins free from spasm, some intraluminal distending pressure will have to be applied. We found a range from 300 to 400 mm Hg to be necessary usually. Our observations suggest such pressures will permit preservation of endothelial integrity not only in monkey veins but, in contradistinction to Dr. Gundry’s studies, in human saphenous veins as well. At this time we are distending veins in the operating room with a simple pressure-limiting balloon device inserted between the syringe and vein that permits vein distention at less than 400 mm Hg. We hope it will help mitigate at least one of the factors in vein graft atherogenesis.
DR. GERALD M . LAWRIE (Houston, TX): I present some original data, compiled by Dr. Jack Titus and Dr. Saenz at Baylor, from 60 grafts recovered from the
time of operation in humans to about two weeks after operation. In a graft recovered 6 hours after operation, there is necrosis in the intima, some changes in the endothelium, and some edema. One day after operation, almost all the veins are lined by fibrin to which platelets are adhering and there are platelet thrombi. This may explain the dichotomy between the theoretical advantages of aspirin or Persantine (dipyridamole) and the practical effects. The process is well advanced long before any of these agents can be started. Three days after operation, there is quite extensive necrosis developing progressively in the intima with pyknotic nuclei. Fibrin is overlying the endothelium, which has undergone patchy loss and has some edema. Nine days after operation, the fibrin lining is present, the entire vein wall is infiltrated with inflammatory cells, and the entire thickness of the vein wall has undergone patchy necrosis. This has occurred independent of the extent of intimal integrity. Despite these early findings in long-term, follow-up pathological studies, findings that I think have not been documented before, we found excellent late histology. So although I think the early techniques used to prepare the veins are important, more work must be done to document what happens in the first two weeks. Our study leads us to believe that the veins universally undergo patchy, generalized necrosis regardless of the technique used to prepare them.