Effects Of Neurological Factors On Angiogenesis And Skeletal Muscle Remodeling In Ischemic Limbs

Objective (1) To establish a hindlimb ischemia and denervation model in mice, and then evaluate the effects of neural factors on VEGF protein expression, compensatory angiogenesis and skeletal muscle fiber remodeling. (2) To conduct the NGF gene and VEGF gene transfection in mice hindlimb ischemia model, and then evaluate effects of NGF on angiogenesis and skeletal muscle fiber remodeling in ischemic hindlimb, and study the relationship of NGF and VEGF on angiogenesis.Methods (1) Fifty-four mice were randomly allocated to control group (n=6), hindlimb ischemia group (n=24), hindlimb ischemia and denervation group (n=24). The hindlimb ischemia model was established by ligating the femoral artery. The hindlimb ischemia and denervation model was established by transecting the sciatic nerve on the basis of hindlimb ischemia model. Gastrocnemius of ischemic hindlimbs were harvested and weighted under the condition of peritoneal cavity anesthesia on the 7th,14th,21th, and 28th after operation, and then the mice were sacrificed. The gastrocnemius specimens were tested by HE staining, CD31 and PCNA IHC staining, myosin ATPase staining. The expression of NGF protein and VEGF protein were detected by ELISA. (2) Twenty-four mice were randomly allocated to normal control group (n=6), blank control group (n=6), VEGF gene transfection group (n=6), and NGF gene transfection group (n=6). The left hindlimb ischemia model was established as previous discribed. VEGF-ATF plasmid (125μg) and NGF plasmid (125μg) was injected into the mouse ischemic gastrocnemius of VEGF group and NGF group, separately. Left hindlimb function and ischemic damage were assessed at post-operative 28d (21 days after gene transfection), and then gastrocnemius of left hindlimb were harvested and weighted as previous described. Finally, the mice were sacrificed. The gastrocnemius specimens of four groups were tested as we previous described.Results (1) On the 7th day after surgery, hindlimbs of the two intervention groups showed ischemic manifestations compared with the control hindlimbs. In addition, Semiquantitative assessment of ischemic damage showed the proportion level 1-4 of hindlimb ischemia and denervation group was significantly higher than that of hindlimb ischemia group (P<0.05). Compared with normal control group, the relative muscle weight were significantly lower in the two intervention groups (P<0.05). On the 28th day after surgery, the endothelial cell proliferation index, the capillary density, the NGF protein expression, and the VEGF protein expression of the hindlimb ischemia and denervation group were significantly decreased compared with the hindlimb ischemia group (P<0.05). On the 28th day after surgery, the proportion of type II muscle fiber in gastrocnemius was significantly higher than that of hindlimb ischemia group (P<0.05). (2) On the 28th day after surgery, the functional assessment score and skeletal muscle atrophy degree of the VEGF group and NGF group were significantly lower than that of the normal control group and blank control group (P<0.05). Compared with normal control group, the relative muscle weight of blank control group, VEGF group and NGF group were significantly decreased (P<0.05). Nevertheless, the relative muscle weight of NGF group and VEGF group were still significantly higher than that of blank control group (P<0.05). The endothelial cell proliferation index and the capillary density of the VEGF group and NGF group were significantly increased compared with the normal control group and blank control group (P<0.05). The NGF and VEGF protein expression of NGF group showed a significant rise when compared with the blank control group (P<0.05). Similarly, the VEGF protein expression of VEGF group was significantly higher than that of blank control group (P<0.05), but there was no significant difference of the NGF protein expression between the VEGF group and blank control group (P>0.05). The type I skeletal muscle fiber proportion in gastrocnemius of the NGF group and VEGF group were significantly higher than that of blank control group (P< 0.05).Conclusion (1) Neural factors play an important role in maintaining VEGF protein expression and compensatory angiogenesis, reducing the skeletal muscle ischemia, and maintaining muscle fiber types in ischemic limbs. (2) NGF gene transfection in ischemic limbs, not only can induce NGF protein expression, but also can induce the expression of VEGF protein, and then promote new blood vessels to form. Both NGF protein and VEGF protein can induce type I skeletal muscle fibers formation in ischemic hindlimb, thus improve ischemic limb motor function.