| Objective: 1.To study the therapeutical effect of angiogenesis and collateral circulation formation in a rabbit model of hindlimb ischemia with recombinant human basic fibroblast factor (rh-bFGF);2.applied several methods of inspection ( such as calf blood pressure ratio, digital subtraction angiography and pathologic) to investigate a valuable method of a single intramuscular injection treating chronic arterial ischemic diseases in lower limbs;3.evaluated the synergistic effect of heparin and bFGF by compared the therapeutical effect with applied single bFGF or bFGF mixed heparin.Material and methods: We used 24 rabbits to establish the animal modelof lower limb ischemia by removed femoral artery of left and deligated its branch vessels . Randomized at four groups. A group without any therapy was control group, put to death on 10 days after operation;B group without any therapy was control group, put to death on 30 days after operation;C group that intramuscular injected bFGF (rh- bFGFlO μg+5ml mMTris/each) was the experiment group;D group that intramuscular injected bFGF mixed heparin (rh- bFGF10 μg+5ml mMTris + heparin 1000U/each) was also the experiment group. 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. The calf blood pressure ratio and digital subtraction angiography was observed at physiological and anatomic levels after treatment. The tissue samples of injected spots were analyzed and observed for the changes of pathologic structure with microscope.Results: 1. clinical observation: Animals of each groups were all alive in theperiod of experiment, and showed some kinds of hindlimb ischemic symptoms after model was made, such as: claudication, hairs of hindlimb becoming little, hindlimb showed redness, and so on.The ischemic symptoms were still existence in B group at the end. And the most serious symptom of hindlimb ischemia was B group. We found that two animals happened ischemic ulcer of digitipedis in B group(33.3%) , and limping became serious in animals of B groups. However, The animals of experiment groups(C group, D group) have no anabrosis and digitipedis, and their tresis vulnus healing well, and the animals of experiment groups relief from lameness after drug was given.2. The calf blood pressure ratio: The calf blood pressure could not find in 10 days after model was made. At the end of treatment, significant improvements in collateral formation and hind limb perfusion were observed in C group and D group compared with the B group (calf blood pressure ratio: 0.62+0.053, 0.71+0.032, 0.32 + 0.046, respectively). The experiment groups(C group , D group ) were all significant higher than the control group(B group) (P<0.01);In addition, D group was also significant higher than C group (P<0.05) .3. Digital subtraction angiography of hindlimb: DSA of A group (10 d) showed that femoral artery and its branch vessels can not be find in the operated limb. There are some vasculogenesis and angiogenesis of the B group, but A group and B group were not obvious difference. However there are many neovasculars and collateral vasculars in the experimental groups. Such vasculars creeping in mid thigh and distribute like reticulodromous;and internal iliac artery and femoral artery of operated hindlimb became pachy because of compensation. We could not find that the normal vascular became narrow-minded, and there are no vascular malformation in the animals of treatment groups.4. Micro vessel density and micro vessel density/ muscle fiber ratio: Althoughgroup A and group B were put to death at different time, microvessel density and microvessel density/ muscle fiber ratio of the two control groups have no significant difference (P>0.05) . But microvessel density and microvessel density/ muscle fiber ratio of the experiment groups(C group, D group) were all significant higher than the control group(B group) (P<0.01) . D group was also significant higher than C group (P<0.05) .5. Pathological observation: we can see that the animales ischemic hindlimb of the control group exhibited serious ischemia changes. There are little vascular between skeletal muscle and no hyperplastic endothelia in group A. Ischemic exhibition of group B was the most serious, and many skeletal muscle were ischemic muscular atrophy and denaturalization. In addition, the vacular was also sparseness. But there are a lot of angiogenesis and collateral circulation formation in the experimental groups. The ischemia symptom of the experimental groups were catabatic. We could not find malformed capillary vessel and tumorous vessel administration of the experiment groups. Besides, skeletal muscle cell and other cell can not abnormal proliferation, polarization, tumorous development and so on was not found.Conclusion: 1. rh-bFGF induced therapeutic angiogenesis and collateralcirculation formation in a rabbit model of chronic hind limb ischemia. Thus, bFGF injected may be a new therapeutic strategy for the treatment of peripheral vascular diseases. 2. A single intramuscular injection in many spots of limbs in short time with bFGF or bFGF mixed heparin as one of the best methods of therapy limb of ischemia disease was suggested. 3. heparin can cooperated of the bFGF in therapeutical effect of angiogenesis and collateral circulation formation.
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