Experimental Study On The Effects Of Exogenous Thrombin On Angiogenesis In Rabbit Ischemic Hindlimb

Objective:(1) To observe the effects of intramuscular injection of exogenous thrombin on angiogenesis in rabbit ischemic hindlimb.(2) To investigate the mechanism of thrombin-induced angiogenesis in ischemic hindlimb.Methods: After hindlimb ischemia models were induced by removing the left femoral artery and deligating its branch vessels in 40 New Zealand rabbits, they were divided into two groups randomly. Rabbits were received intramuscular injection of thrombin solution (500IU/L, 2ml) in ischemic zone in the group THR and equal dose of normal saline in the control group NS. Four time points (Day 3, 7, 14 and 21 after operation) were established, animals in each group were divide into each time point randomly (n=5). The number of collateral vessels in ischemic hindlimbs were closely observed and evaluated by angiography as well as the number of capillary vessels in the left adductor by histopathologic analysis(MVD). And the VEGF protein expression were observed and evaluated by immunohistochemistry(IOD). All experimental datas were processed by SPSS13.0.Results:(1) Clinical observation: Animals in each group were all alive till their time point dates. Some ischemia symptoms such as claudication and hair falling of the left hindlimbs were observed in each group.(2) Angiography of hindlimb: The photographs of angiography showed that femoral artery and its branch vessels are invisible in all the ischemic hindlimbs. Only few thinner collateral vessels can be seen on the day 3 in each group. The number of collateral vessels increased slightly on the day 7 and increased obviously on the day 14 as well as on the day 21 in each group. There was no statistically significant difference in the number of collateral vessels between the two groups on all the corresponding time points (P>0.05).(3) Histopathological observation: There were atrophia and deformation of muscle cells, abundant capillary vessels in the widened diastema between muscle cells and no necrosis in the tissue slices of adductor muscle in each group. The MVD increased on the day 3, reached the peak level on the day 7 and decreased gradually in the following days in each group. The MVD of group THR was higher than that of group NS group, there was statistically significant difference in the value of MVD between the two groups on all the corresponding time points(P<0.05).(4) Immunohistochemistrical observation: There were VEGF-expressed endothelial cells in the tissue slices of adductor muscle in each group. The IOD of VEGF protein increased on the day 3, reached the peak amplitude on the day 7 and decreased gradually in the following days in each group. The IOD of group THR was higher than that of group NS. The IOD of VEGF was statistically significant difference between the two groups on the four corresponding time points(P<0.05).(5) Correlation analysis:There was no statistically significant correlation between IOD of VEGF and the number of collateral vessels(P.>0.05).There was statistically significant correlation between IOD of VEGF and MVD of capillary vessels(P<0.05).Conclusions:(1) There was significant correlation between the expression of VEGF and angiogenesis, there was no significant correlation between the expression of VEGF and the formation of collateral vessels in the early stage of rabbit hindlimb ischemia.(2) Intramuscular injection of exogenous thrombin(1000IU) induced angiogenesis in rabbit ischemic hindlimb and little effect on the formation of collateral vessels.(3) The mechanism of thrombin-induced angiogenesis could have been due to upregulate the expression of VEGF in rabbit ischemic hindlimb.