Experimental Study On The Dose Of Basic Fibroblast Growth Factor To Treat Low Limb Ischemia

Objective:To screen the optimal dose of recombinant human basic fibroblast factor (rh-bFGF)and evaluate the effect of different doses of rh-bFGF in 30 rabbit models of hindlimb ischemia through observation in general,the number of collateral branch, microvessel density and microvessel density/myolin ratio.To evaluate the safety of different doses of rh-bFGF by testing the liver and kidney function,digital subtraction angiography and pathological observation.To provide theoretial basis for clinically practising rh-bFGF to treat low limb ischemia.Material and methods:30 rabbits were used to establish the animal model of lower limb ischemia by removed femoral artery of left and deligated its branch vessels. Randomized at five groups(6 each group),A group was intramuscular injected 5ml Tris/each;B,C,D and E groups are treating groups.The four groups were respectively intramuscular injected bFGF 2.5μg,5μg,10μg and 20μg+5ml Tris/each. The animals of each experiment group were once intramuscular injected in five spots of its thigh in 10 days after operation,and were put to death in 30 days after operation. Before injection,10 days after injection and 20 days after injection,the venous blood was collected to detect the liver and kidney function.The number of collateral branch was counted by digital subtraction angiography before the rabbits were put to death. The sample of medial vastus muscle was analyzed and observed for the changes of pathologic structure with microscope.Results:1.observation in general:Animals of each groups were all alive in the period of experiment,and showed claudecation in distinct degrees in 10 days after the models were made.The claudication in A group and B group became more serious gradually,But it improved gradually in C,D and E groups.The cut healed up well and no ulcer and gangrene in each group.Abscess was found in one of A group.2.Digital subtraction angiography of lower limb:DSA showed that the left femoral artery broke off.A few slight collateral vessels were distributed over the middle of thigh in A group.In B group and C group,several collateral vessels originated from internal iliac artery and deep femoral artery,the distant artery didn't develop.However,many collateral vascular creeped in D group and E group,and the distant artery of operated hindlimb developed because of collateral circulation,the speed of blood was a little slower than the uninjured side.There are no malformed capillaries and tumorous vessels in each group.The number of collateral casculars in D group and E group were all higher than A group,B group and C group(P＜0.05);but D group and E group were not statistically significant(P＞0.05).Among A,B and C group,the numbers were not statistically significant(P＞0.05).3.Microvessel density and microvessel density/muscle fiber ratio:microvessel density and microvessel density/muscle fiber ratio among A group,B group and C group was no statistical difference(P＞0.05).But it in D group and E group was higher than A group(P＜0.05).It was also higher than B group and C group(P＜0.05). But D group and E group have no significant difference(P＞0.05).4.Pathological observation:In A group,many skeletal muscle fibers were atrophy,space between muscle fibers turn long,and there were little blood vessels in mesenchymal.In B group and C group,the muscular atrophy was slight,there were a few blood vessels.But there were a lot of angiogenesis and collateral vessels in D group and E group.Some red cells could be seen in the vessels and no signifivant muscular fibers atrophy.Besides,skeletal muscle cell and other cell can not abnormal proliferation.Tumorous development was not found.5.Liver and kidney function test:Before injection,10 days and 20 days after injection,the venous blood was collected to test ALT,AST,BUN,Cr.There were no statistical difference of the result in different time among each group(P＞0.05).Conclusion:1.bFGF could promote therapeutic angiogenesis and collateral circulation formation effectively in a rabbit model of hind limb ischemia.Its curative effect strengthen with the dose of bFGF added.The curative effect was the best in 10μg,but it didn't increase in 20μg.10μg was the best dose for bFGF to treat lower limb ischemia.2.Applying experimental dose of bFGF to treat lower limb ischemia was safe,it has no affact on liver and kidney function and can not lead to abnormal hyperplasia and tumorous growth of skeletal muscle cell and other cells.