Study On The Programmed Cryopreservation Of Rat Aorta

Vessel transplantation is one of the most useful methods for the treatment of many vascular diseases. Aortic replacement, as studied experimentally by Carrel at the turn of last century, was introduced for clinical use in the early years of vascular surgery. Generally speaking, homograft is the most suitable grafts for the vessel transplantation, which have no graft rejections. Because of their limited resource, homografts are only used in the infected fields or important areas. Allograft is another kind of adequate grafts. Cryopreservation is a useful technique for the long term storage of various tissues. In recent years, there have been great interests in the area of the vascular cryopreservation.Vascular cryopreservation refers to the storage of a vessel at ultra-low-temperature such that it can be revived and restored to the same living state as before it was stored. During the cryopreservation process, cells shrink as they are cooled below the freezing point of the solution and remain shrunken during storage, then return to their isosmotic volume once more upon thawing. Interactions between cells and extracellular ice crystals can lead to cell injury during freezing and thawing, presenting an obstacle to the development of cryopreservation techniques for long term stabilization and storage of biological materials. The most effective methods to minimize these potentially lethal effects are to control the transient cooling and wanning rates during the cryopreservation, and to add a cryoprotective compound to the culture prior to its freezing for storage. The endothelial function of the allograft is very important because it decides the prognosis directly.The study on the vascular cryopreservation has been started at 1980s. Ku D D tested the relaxant responses in the canine and human coronary arteries. Schoeffter tested the contractile response with the prostaglandin in the swine coronary artery. Ellis did some experiments on the cryopreservation of the human pulmonary arteries.Many investigators had reported the function or the viability of the cryopreserved grafts in different manners. However their conclusions were different. Bodnar and Bellon reported marked endothelial damages in cryopreserved blood vessels. But Pukacki and Pascual showed that cryopreserved porcine femoral arteries could be preserved in approximately 50% contraction and approximately 90% relaxation of fresh arteries. In the late 90s of last century, there were some reports on the new bovine aorta cryopreservation in China; however, there have no reports or papers on the studies of programmed cryopreservation of the rat aorta.Objectives: To design an adequate technique with which to cryopreserve rat aorta and to assess the influence of cryopreservation in vascular function and endothelial apoptosis. Methods:1) Sixty-five rat aortas were randomly distributed in thirteen groups. In group A(control), five arteries were studied after harvest; In group B1-B6, thirty arteries were suspended in RPMI 1640 plus fetal calf serum plus dimethylsulfoxide, cryopreserved at 1 °C per minute and kept frozen for one hour, one day, one week, two week, one month or three months respectively; In groupCi-C6, thirty arteries were suspended in modified Krebs-Henseleit plus dimethylsulfoxide plus sucrose, cryopreserved at 0.7 °C per minute and kept frozen for one hour, one day, one week, two week, one month or three months respectively. After being thawed, arteries were examined for vasoconstrictions, vasodilatations(organ bath studies) and vessel structure (microscopy).2) Twenty rat aortas were randomly distributed in four groups. In group O (control), five arteries were studied after harvest; In group A, B and C, fifteen arteries were suspended in Celsior, UW and modified Krebs-Henseleit plus dimethylsulfoxide respectively and kept frozen for three months. After being thawed, cryopreservation damages in Celsior, UW and KH solutions were assessed through measurement of isometric contractions and relaxations in an organ bath.3) Forty rat aortas were randomly distributed in eight groups. In group B1-B4,8twenty arteries were suspended in RPMI 1640 plus fetal calf serum plus dimethylsulfoxide, cryopreserved at 1 °C per minute and kept frozen for one day, one week, one month or three months respectively; In group C1-C4, twenty arteries were suspended in modified Krebs-Henseleit plus dimethylsulfoxide plus sucrose, cryopreserved at 0.7 °C per minute and kept frozen for one day, one week, one month or three months respectively. Endothelial apoptosis were observed by TdT mediated dUTP-biotion nick end labeling method and the immunohistochemistry method. Simultaneously, levels of cellular Bax gene protein expression were measured by counting the number of the positive cells.Results:1 ^ With one hours of cryopreservation, the maximal contractile responses to all the contracting agonists tested were preserved as follows in group Bi vs Cj: KC1: 0.387g±0.058g vs 1.244g±0.152g;PE: 0.777g±0.177g vs 1.244g±0.151g; ET-l:0.136g±0.049g vs 0.536g±0.055g and the maximal relaxant responses to SNP were preserved as follows in group Bi and Ci: 0.114g±0.050g vs 0.934g±0.129g,in which each value of group C\ was significantly greater than that of Group BI However the relaxation to Ach was diminished in all the groups (0.057g±0.048g vs 0.080g±0.041g). Time course of retention at -80 °C showed gradual decrescence in both contractile and relaxant responses and group C descended more slowly than group B.2^ The maximal contractile and the relaxant responses were better preserved in group A than in group B(P<0.05) within three months of cryopreservation. The contractile and the relaxant responses were similar in Group B and Group C after the cryopreservation.3 > Apoptosis were obviously found in the rat aorta endothelium. And there were significant differences in the numbers of apoptotic group Bx and Cx.Conclusions:1) A modified cryopreservation technique can better preserve vasoconstriction and endothelial independent vasodilation of the rat aorta than the conventional method, but the endothelial dependent vasodilation is badly preserved in allgroups after the cryopreservation. Time course of retention at -80 °C showed gradual decrescence in both contractile and relaxant responses and experimental group descended more slowly than conventional group.2) Celsior solution was superior to UW solution and KH solution in the protection of rat aorta endothelial function against injury from the programmed cryopreservation.3) Apoptosis of rat aorta endothelium is markedly involved during the cryopreservation which may be related with cellular Bax gene protein.ProspectPeople study the vascular cryopreservation for only about 10 years, and have got lots of exciting results. Now, there are already some vascular banks established providing the adequate grafts for the clinical uses. Besides the treatment of vascular diseases, Carbognani tried the tracheal transplantation using a cryo-preserved aortic allograft. It has been proved that it is superior to the silica gel tract, which provides a new application. And there are still many questions deserved to be study, such as the function of the cryo-grafts after the transplantation, the problem of the recessive process. And we also should study more about the re-growth of the endothelium after the transplantation.