| Objective: The vascular injuries are common in othopaedic emergency. Autografts of vein are usually used to repair the defect of artery. When the veingraft is transplanted to the artery, it will be changed a lot in many respects. There are plenty of reasons contribute to those changes, for instance, ischemia, the injury during transplantation and the changes of the hemodynamics. The procedure contains the exfoliation of endothelial cell, the deposit of phlegmasia cells, the proliferation and migration of smooth muscle cell and the deposit of the extracellular matrix in the intima. With the intimal and medium hyperplasia, the lumen of vessel become restenosis gradually. According to some clinical data, the ratio of postoperative restenosis is 30%-50% after 5 years. The currently studis are concentrated on the mechanism of restenosis and the method to prevent and treat restenosis of the vein graft. Fasudil, a kind of Rho Kinase inhibitor, could depress the permeability and abscission of endothelial cell, the proliferation and migration of smooth muscle cell and the generation of inflammatory factor. Rabbits were used in our study and the femoral vein to femoral artery transplantation model was established, and some were treated with fasudil. The vein grafts were stained with HE to observe the intimal hyperplasia, and were stained with proliferating cell nuclear antigen (PCNA) to study the proliferation of the cells. Electron microscope was used to observe the ultrastructure of the vein graft. The terminal deoxynucleotidyl transferase- mediated dUTP-biotin nick end labeling (TUNEL) method was used to detect in cell situ apoptosis. Based on these results, the mechanism of restenosis after transplantation and the effect of fasdil would be analyzed.Methods: Twenty-four healthy rabbits (2.3-2.5 kilogram weight) were used and divided into two groups which were control (A) group and the fasudil (B) group. The femoral artery was cut and replaced with the harvested autologous ipsilateral femoral vein. The group B was treated with fasudil 30 mg/kg intravenous from three days before the operation until harvest, and the group A was given 0.9% NaCl intravenous instead. After the operation, rabbits were given haparin sodium 500u/kg intramuscular injection for anticoagulation, penicillin 800ku to prevent infection for 3 days.At two and four weeks after operation, every time 6 rabbits were harvested in both groups. The intimal hyperplasia of vein grafts was assessed. The grafts were stained with HE to observe the thickness of the intima and were stained with proliferating cell nuclear antigen (PCNA) to study the proliferation of the wall cells. Electron microscope was used to observe the ultrastructure of the cells. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method was used to detect in situ apoptosis. These graphic results were analyzed with computer image-analysis system. All the experimental data was analyzed by SPSS 11.0.Results1 Macroscopic observation: All the experimental animals survived when the experiment completed. The patency rate of the vein grafts was 100% and there was no rupture of the vein grafts until they were harvested. There was no infection in the wound.2 Morphology: Two weeks after the transplantation, HE staining showed that the intima of the Group A is thick (44.83±3.53μm), but the intima of the Group B (30.33±3.23μm) changed a little. Four weeks after the transplantation, the intimal hyperplasia of the Group A (66.16±8.45μm) was obvious. On the other hand, the Group B (43.11±4.92μm) got less intimal hyperplasia ( P<0.05).Ultrastructure observed by electron microscope. In group A, two weeks after operation, the size of the smooth muscle cells was larger than normal. The number of secreting smooth muscle cell increased. Four weeks later, there are lots of smooth muscle cell in the intima, and more intercellular substance and collagen fibers in the media. On the contrary, in group B, two weeks after the transplantation, there was no significate proliferation. Four weeks later, the secreting smooth muscle cells increased but were less than group A. 3 Immunohistochemistry of PCNA: Two weeks after the transplantation, there were more PCNA positive cells in the intima in Group A (20.08±3.56% ) than Group B (14.28±2.76% ). Four weeks later, PCNA positive cells in the intima in both group were reduced. The percent of PCNA positive cells in the intima of the Group A was more than Group B (P<0.05).4 The percentage of TUNEL positive cells in Group B(3.55±0.36% and 1.22±0.18% ) is more than Group A (1.11±0.31% and 0.55±0.11%) (P<0.05) in the two and four weeks after operation.Conclusions: These novel findings clearly demonstrate that fasudil can suppress intimal hyperplasia of the vein graft by suppression of cell proliferation and apoptosis in the neointima in vivo. Furthermore, these results strongly support the clinical use of fasudil to prevent intimal hyperplasia of vein graft. |
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