The Study Of Adventitia Applied Slow-releasing Rapamycin Inhibiting Intima Hyperplasia Of Vascular Grafts In Rabbits

Objective:Establish a model of rabbit vascular grafts on infrarenal abdominal aorta and administrate rapamycin through vascular adventitia; observe the pathological changes of the vessel grafts and the expression of a-actin, vWf, PCNA, p27kip1, and study its relationship with the proliferation of vascular smooth muscle cells; investigate the mechanism of rapamycin inhibiting restenosis and the feasibility of administration through vascular adventitia.Methods:Thirty-six healthy male New Zealand rabbits weighted 2.5-3.Okg were randomly divided into 6 groups:Group A, B, C, D, E, and F, six rabbits each. Two models were then established:antigen removed allogeneic abdominal aorta transplantation and PTFE artificial vascular graft on abdominal aorta. Vascular anastomosis was performed before the groups of rabbits were subjected to different experimental conditions. Group A:antigen removed allogeneic abdominal aorta transplantation, no treatment for the grafts; Group B:antigen removed allogeneic abdominal aorta transplantation, and 0.5ml 20% pluronic F-127 gel was locally applied to the adventitia of the grafts and anastomoses; Group C:antigen removed allogeneic abdominal aorta transplantation, and 0.5ml 20% pluronic F-127 gel containing 0.5mg rapamycin was locally applied to the adventitia of the grafts and anastomoses; Group D:PTFE artificial vascular graft on abdominal aorta, the grafts accepted no any management; Group E:PTFE artificial vascular graft on abdominal aorta, and 0.5ml 20% pluronic F-127 gel was locally applied to the adventitia of the grafts and anastomoses; Group F:PTFE artificial vascular graft on abdominal aorta, and 0.5ml 20% pluronic F-127 gel containing 0.5mg rapamycin was locally applied to the adventitia of the grafts and anastomoses. The grafts were acquired four weeks after the experiment. Histomorphologic methods were used to detect the intima hyperplasia of the specimen. Electronic imaging system was used to measure the thickness of intima and media of the grafts followed by calculating the degree of intima hyperplasia (thickness of intima/thickness of media),α-actin, vWf, PCNA and p27kip1 were investigated by using immunohistochemistry. Data were analyzed by using SPSS 13.0 statistics software and then were compiled in the form of (x±s).Results:1. The models of antigen removed allogeneic abdominal aorta transplantation and PTFE artificial vascular graft on abdominal aorta were successfully constructed.2. One month after the operation, the intima of Group A and B thickened obviously (P<0.05) in comparison to C. Similarly, the intima of D and E also thickened obviously (P<0.05) in comparison to F.3. Weigert's elastic stain: four weeks after antigen removed allogeneic abdominal aorta transplantation, the elasticity fibers were well stained and preserved.4. Result of a-actin immunohistochemistry:the hyperplastic intima was marked obviously, which is consistent with the smooth muscle cells of blood vessels.5. Result of vWf immunohistochemistry:the surface layer of the thickened intima was marked positive, which was proved to be a monolayer endothelial cell.6. Result of PCNA and p27kip1 immunohistochemistry:both of them were stained in brown in cell nucleus, and expressed in the thickened intima of every group to study the difference.7. Compared to A, B and D, E the expression of a-actin and PCNA in C and F were remarkably inhibited (P<0.05), while the expression of p27kip1 was remarkably enhanced (P< 0.05). No significant deviation was found among A, B and C, D (P>0.05).Conclusion:1. Formaldehyde can remove the vessels antigenic determinants and preserve the elastin and collagen extracellular matrix.2. The main reason of restenosis after vessel transplantation is the hyperplasia of intima, which is bioactive for vascular smooth muscle cells. Inhibiting the hyperplasia of vascular smooth muscle cells is the key factor to keep vessels patency intact for long time.3. Smearing rapamycin mixed with pluronic F-127 gel on graft adventitia can effectively inhibit the hyperplasia of vascular smooth muscle cells.4. The mechanism of rapamycin inhibiting restenosis may be related to increase the expression of p27kip1 and inhibition cell generation cycle.5. The administration route through vascular adventitia is feasible and effective.