Objective: To establish a murine model of hindlimb ischemia and denervation and evaluate the effects of neural factors on angiogenesis and skeletal muscle fiber remodeling.
Methods: A total of 54 mice were randomly allocated into three groups of control (n = 6), hindlimb ischemia (n = 24), hindlimb ischemia and denervation (n = 24). The model of hindlimb ischemia was established by ligating femoral artery. And the model of hindlimb ischemia and denervation was established by transecting sciatic nerve on the basis of hindlimb ischemia modeling. Gastrocnemius of ischemic hindlimbs were harvested under the condition of peritoneal cavity anesthesia at Days 7, 14, 21 and 28 postoperation. And then the mice were sacrificed. The gastrocnemius of three groups were tested by hematoxylin-eosin staining, CD31 and PCNA immunohistochemical staining and myosin ATPase staining. The protein expressions of nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) were detected by enzyme-linked immunosorbent assay.
Results: At Day 7 postoperation, hindlimbs of two intervention groups showed ischemic manifestations as compared with controls. And semiquantitative assessment of ischemic damage showed the proportion level I-IV of hindlimb ischemia and denervation group was significantly higher than that of hindlimb ischemia group (33.4% vs. 16.6%, χ(2) = 7.94, P = 0.047). At Day 28 postoperation, the values of endothelial cell proliferation index [(2.4 ± 0.4)% vs. (5.7 ± 1.0)%, P = 0.000], capillary density [(0.44 ± 0.05) vs. (0.61 ± 0.07), P = 0.000], NGF protein expression [(6.71 ± 0.94) vs. (8.36 ± 0.77) pg/ml, P = 0.008] and VEGF protein expression [(11.32 ± 1.56) vs. (15.13 ± 2.93) pg/ml, P = 0.018] of hindlimb ischemia and denervation group significantly decreased as compared with hindlimb ischemia group. At Day 28 postoperation, the proportion of type II muscle fiber in gastrocnemius was significantly higher than that of hindlimb ischemia group [(84.9 ± 3.8)% vs. (78.8 ± 1.8)%, P = 0.008].
Conclusion: Neural factors play important roles in promoting angiogenesis and maintaining muscle fiber types in ischemic limbs. But its molecular regulatory mechanism requires further studies.