Long-term Patency of Vein Grafts Preserved in Liquid Nitrogen in Dimethyl Sulfoxide THOMAS R. WEBER, M.D., S. MARTIN LINDENAUER, M.D., THOMAS L. DENT, M.D., EDWARD ALLEN, PH.D., CLAUDIO A. SALLES, M.D., LEE WEATHERBEE, M.D.

Autogenous canine jugular veins were stored in 15% dimethyl sulfoxide (DMSO) in liquid nitrogen vapor for one to 28 days and then implanted in the carotid artery as autografts. The patency rate at one year was 62.5-87.5%. The patency rate of fresh jugular vein autografts placed in the carotid artery for one year was 75%. Similar autografts stored in liquid nitrogen vapor for one to 28 days without the cryopreservative DMSO exhibited a zero to 12.5% patency rate at one year. Scanning electron microscope studies revealed preservation of the endothelium in DMSO protected veins and a damaged or sloughed endothelium in veins frozen without DMSO cryopreservation.

THE autogenous vein segment is the only graft material that has been consistently successful in terms of long term patency in small vessel arterial reconstruction. The autogenous saphenous vein is the most often used vessel for both distal arterial reconstruction10 and aortocoronary bypass.7 Unfortunately, the saphenous vein may not be useable in many patients due to prior venous disease, surgical stripping, or previous arterial graft procedures. Because synthetic prostheses have had limited success in small vessel applications,2 there is a great need for a ready source of biologic tissue for use as grafts in these situations. Homologous vein grafts have been used with increasing frequency for peripheral vascular reconstruction with reasonable long term patency rates.36'" Immunologic studies have shown the vein to be a weak antigenic stimulus, so rejection of these grafts is probably less important.8 However, the lack of a ready supply of fresh homologous veins for transplantation has proven to be a serious drawback, since fresh veins are the only

From the Department of Surgery, Section of General Surgery, University of Michigan Medical Center and the Vascular Research Laboratory, Veterans Administration Hospital, Ann Arbor, Michigan 48105

tissue that have been shown to be consistently successful in grafting procedures.3'6'8 Preliminary studies in this laboratory1'12 have shown that 15% dimethyl sulfoxide (DMSO) will preserve smooth muscle contraction in canine veins frozen and stored in liquid nitrogen for up to 28 days. These studies also indicated that preserved canine vein segments were identical to fresh veins in terms of short term patency when implanted as autografts in the carotid artery. The present study is a continuation of these preliminary experiments, in which long term patency of preserved vein grafts is assessed.

Materials and Methods Adult mongrel dogs of both sexes, weighing between 15 and 20 kg, were anesthetized with intravenous sodium thiamylol. In 10 dogs, the jugular veins were exposed using aseptic surgical technique. An 8 cm segment of one vein was excised and immersed in 15% (V/V) dimethyl sulfoxide (DMSO) at room temperature for one hour. The vein segment was then sealed in a small plastic bag with 10-15 ml of the DMSO solution. Care was taken to exclude all air from the bag prior to sealing. The bag was then immersed in liquid nitrogen until frozen, usually between 30-45 sec (freezing rate 5°/sec), and the veins stored in liquid nitrogen Submitted for publication March 8, 1976. Reprint requests: S. Martin Lindenauer, M.D., 2215 Fuller vapor (- 120°) for 24 h. The opposite jugular vein was imRoad, Ann Arbor, Michigan 48105. mersed in a physiologic salt solution (PSS) for one hour, 709

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WEBER AND OTHERS TABLE 1. Patency of Fresh Veins and Frozen Veins Preserved in DMSO or PSS When Used as Arterial Autografts

Group

No. Patent 1 Mo.

No. Patent 6 Mos.

Fresh DMSO frozen 1 day DMSO frozen 7 days DMSO frozen 28 days PSS frozen I day PSS frozen 28 days

6/8 (75%) 8/10 (80%o) 7/8 (87.5%) 5/8 (62.5%) 2/8 (25%) 0/8 (0)

6/8 8/10 7/8 5/8 1/8 0/8

(75%) (80%)

(87.5%) (62.5%) (12.5%) (0)

No. Patent 1 Yr. 6/8 (75%) 8/10 (80Wo) 7/8 (87.5%) 5/8 (62.5%)

1/8 (12.5%) 0/8 (0)

sealed in a plastic bag in PSS and then frozen and stored in liquid nitrogen for 24 h. Both veins were thawed by immersion of the plastic bag in a 450 water bath. Thawing was completed usually within 30-45 sec (thawing rate 5°/sec). The veins were then implanted as autografts in an end-to-end fashion in the carotid arteries, using a continuous 6-0 Tevdek suture. In a second group of 8 dogs, one jugular vein was frozen and stored in 15% DMSO in a manner identical to the first group, except the veins were stored for 28 days. The contralateral vein was likewise frozen and stored for 28 days in PSS. After thawing, the veins were implanted as autografts in the carotid arteries. A third group of 8 dogs underwent unilateral jugular vein excision, and these veins were frozen and stored in 15% DMSO in liquid nitrogen for 7 days. The veins were thawed and implanted as autografts in the carotid arteries. At the time of implantation, the contralateral jugular vein was excised and implanted immediately in the other carotid artery. All grafts were evaluated one month postoperatively

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by surgical exploration at which time thrombosed grafts were removed. Grafts found to be patent were biopsied in their midportion, with continuity restored by end-toend anastomosis. Thrombosed grafts and biopsy specimens were examined by light microscopy using hematoxylin and eosin, Verhoeff's elastic tissue stain, and Masson's trichrome. Scanning electron microscope studies were performed on selected specimens using the techniques of Stewart et al.9 with minor modifications to accomodate the electron microscope in use at our institution. The vein grafts were further evaluated by arteriography 6 months postoperatively, using standard techniques. All vein grafts patent one year postoperatively were removed, and selected specimens were examined by the light microscope, and scanning electron microscope. Results

Graft Studies All dogs survived each operative procedure, and all dogs with patent grafts survived for the one year postoperative observation period. Wound infection occurred rarely, and responded well to systemic antibiotics and local wound care. The results of the implantation studies are summarized in Table 1. Fresh vein grafts had a 75% patency rate one month postoperatively, and this same proportion remained patent throughout the one year observation period. The 25% graft thrombosis in this group was probably due to faults in operative technique. Vein grafts frozen and stored in 15% DMSO in liquid nitro-

FIGS. la and b. Scanning electron photomicrograph x450. (left) Fresh canine jugular vein. (right) Canine jugular vein after freezing in 15% DMSO in liquid nitrogen for 28 days. Note similarity in appearance of both specimens.

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FIGS. 2a and b. Scanning electron photomicrograph x450. (left) Fresh canine jugular vein. (right) Canine jugular vein after freezing in PSS in liquid nitrogen. Note disruption of endothelial architecture with fissures in 2b compared to appearance of fresh veins (2a) and DMSO protected vein (lb).

with the scanning electron microscope (SEM). Figure 1 is a photograph of: a) SEM of the endothelium of a fresh canine jugular vein, and b) one which was frozen in 15% DMSO in liquid nitrogen for 28 days. Note that there is little difference in the protruding nuclei and overall architecture of the two specimens. In contrast, Fig. 2 compares: a) a fresh vein segment, Microscope Studies and b) one frozen in liquid nitrogen in PSS, without Light microscopy showed no differences among the the cryoprotectant DMSO. There is severe disruption various groups. However, significant differences were of the architecture in the endothelium of the unprofor one, 7 and 28 days all showed patency rates similar to fresh veins. In contrast, veins frozen and stored without the cryoprotectant, i.e. in PSS only, were poor arterial substitutes. Only one of 16 grafts in these two groups remained patent for more than one month postoperatively.

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FIGS. 3a and b. Scanning electron photomicrograph x450. (left) Canine jugular vein preserved in 15% DMSO in liquid nitrogen and then implanted in carotid artery for one year. Note normal architecture under thin fibrin layer. (right) Canine jugular vein frozen without cryoprotectant and then implanted in carotid artery. Note ab- _ sence of endothelium and thrombus in upper right _ comner.

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tected vein segment, with actual gaps in the endothelium readily apparent. In Fig. 3, the fate of the endothelium in veins preserved in PSS and DMSO and then implanted in the carotid artery is seen. The DMSO preserved vein graft, which was removed one year after implantation (Fig. 3a) has a normal architecture with a layer of fibrin overlying normal endothelial nuclei. However, the vein frozen without the cryoprotectant and then implanted (Fig. 3b) is devoid of endothelium, with a highly thrombogenic collagen layer exposed. In addition, a thrombus is seen attached to the collagen surface. The "washing away" after implantation of the endothelium of veins frozen without DMSO may explain the consistent thrombosis these segments undergo when used as arterial grafts.

In contrast, veins frozen and thawed without the cryoprotectant showed a high rate of thrombosis one month postoperatively, and only one of the 16 vein grafts in these groups remained patent for one year. The scanning electron photomicrographs show severe disruption of endothelial architecture, which appears to have sloughed after insertion in the arterial circulation, exposing the flowing blood to a highly thrombogenic collagen surface. It would appear that methods of vascular preservation must retain the endothelium if patency is to be maintained. Dimethyl sulfoxide allows preservation of the endothelium of veins frozen in liquid nitrogen. In addition, the integrity of the other vein wall elements, i.e. smooth muscle and collagen is also maintained which probably Discussion accounts for the absence of aneurysmal dilatation after Homologous veins have been used sporadically for one year in the arterial circulation. Veins preserved in arterial reconstruction, with reasonable patency.3'6"'l 15% DMSO can be used for arterial reconstruction with However, the lack of a reliable method of long term the potential for long term patency. storage of veins has limited the application of this technique. Homologous vein grafts would be most beneficial References in small vessel procedures and low flow situations where synthetic prostheses have proven to be poor vascular 1. Dent, T. L., Weber, T. R., Lindenauer, S. M., et al.: Cryo-

substitutes.2 Dimethyl sulfoxide (DMSO) has been used to preserve tissues at liquid nitrogen temperatures (- 1900) for long periods of time,5 but has not been used extensively for preservation of venous tissue. Since it is a highly penetrating compound it seems particularly well suited for preservation of multi-layered tissues such as veins. Previous studies in this laboratory have shown that 15% DMSO is superior to other cryoprotective agents for the preservation of vein smooth muscle contraction, and therefore DMSO was chosen as a cryoprotectant in this investigation. The present study demonstrates clearly that autologous veins preserved in 15% DMSO for up to 28 days are identical in function to fresh veins when used as arterial substitutes. The early patency rates of 62.5%-87.5% for both fresh and preserved vein grafts continued for one year, with no aneurysmal degeneration of the graft wall. In addition, there was no difference in the ultrastructure of fresh and DMSO preserved vein grafts, both immediately after thawing and after implantation as an arterial conduit. The scanning electron photomicrographs show an intact endothelium in the veins frozen and thawed in DMSO, which probably accounts for the high patency rates in these vessels.

preservation of Vein Grafts. Surg. Forum, 25:241, 1974. 2. DeWeese, J. A., Terry, R., Barner, H. B., and Rob, C. R.: Autogenous Venous Femoropopliteal Bypass Grafts. Surgery,

59:28, 1966. 3. Jackson, D. R.: Living Homologous Saphenous Vein in Geriatric Femoropopliteal Bypass Grafting: Report of a Case. Vasc. Surg., 5:6, 1971. 4. Litvan, G. G.: Mechanism of Cryoinjury in Biological Systems. Cryobiology, 9:182, 1972. 5. Meryman, H. T.: Preservation of Living Cells. Fed. Proc., 22:81, 1963. 6. Ochsner, J. L., DeCamp, P. T., and Leonard, G. L.: Experience with Fresh Venous Allografts as an Arterial Substitute. Ann. Surg., 173:933, 1971. 7. Reul, G., Cooley, D., Wukash, D., et al.: Long Term Survival Following Coronary Artery Bypass: Analysis of 4522 Consecutive Cases. Arch. Surg., 110: 1419, 1975. 8. Schwartz, S. I., Kutner, F. R., Neistadt, A., et al.: Antigenicity of Homografted Veins. Surgery, 61:471, 1967. 9. Stewart, G. J., Ritchie, W. G. M., and Lynch, P. R.: A Scanning and Transmission Electron Microscopic Study of Canine Jugular Veins. In Scanning Electron Microscopy 1973, Proceedings of IIT Research Institute Chicago, Illinois, April 1974; pp. 474-479. 10. Szilagyi, D. E., Elliott, J. P., Hageman, J. H., et al.: Biologic Fate of Autogenous Vein Implants as Arterial Substitutes. Ann. Surg., 178:3:232, 1971. 11. Tice, D. A. and Santeri, E.: Use of Saphenous Vein Homografts for Arterial Reconstruction: A Preliminary Report. Surgery, 67:493, 1970. 12. Weber, T. R., Dent, T. L., Lindenauer, S. M., et al.: Viable Vein Graft Preservation. J. Surg. Res., 18:247, 1975.

Long-term patency of vein grafts preserved in liquid nitrogen in dimethyl sulfoxide.

Autogenous canine jugular veins were stored in 15% dimethyl sulfoxide (DMSO) in liquid nitrogen vapor for one to 28 days and then implanted in the car...
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