The Effects Of Macrophages On Skeletal Muscle Regeneration After Acute Contusion In Mice

Abstract

Macrophages are known to play key roles in skeletal muscle regeneration. However, interaction between macrophages and myogenic cells and the molecular mechanisms remain to be clarified. PURPOSE: Our objective was to evaluate the effects of macrophages on skeletal muscle regeneration after acute contusion. METHODS: One hundred and twenty-eight 8-10 week male C57BL6 mice, weight 18.8 to 22.3 g, were randomized to acute-contused group (C) and macrophage-depleted group (MpD), then sacrificed at 0, 12 h, 1, 3, 5, 7, 14 and 21 days (n=8 mice/group at each time point) after acute contusion. The model of skeletal muscle acute contusion was made by a ball (16.2g, 1.6cm in diameter) dropped from 120 cm above the gastrocnemius muscle. The model of macrophage depletion was established through intraperitoneal inject 2 mg clodronateliposomes 3 days before acute contusion, then 0.5 mg on day 0, 3, 6, 9, 12 and 15 after injury. The protein levels of MyoD, Myogenin, uPA, COX-2 were detected using Western Blotting and immunohistochemistry. Masson trichrome staining was used to identify the fibrosis and scar formation. RESULTS: The MyoD expression reached a peak on day 1 in C and on day 3 in MpD (1.04 ± 0.35 vs. 0.85± 0.22, p<0.01); the Myogenin expression reached a peak on day 3 in C and on day 5 in MpD (0.98 ± 0.21 vs. 0.71 ± 0.13, p<0.05). In MpD, the COX-2 protein levels up-regulated on day 1, reached the peak on day 5, and dropped to the lowest point on day 21, were higher than that in the control group at each time point (p<0.05); similar time-dependent expression was observed in the uPA protein, but there was no difference between MpD and C at each time point (p>0.05). The fibrotic area was significantly larger in the MpD21d group than that in the C21d group (51.8 % vs.19.5 %, p<0.01). CONCLUSION: Macrophages directly related to the chronological change of myogenic cells. Macrophages depletion impaired skeletal muscle regeneration, leading to fibrosis. Supported by the National Natural Science Foundation of China (No. 31271273, 31300975, 31471135) and the Science Foundation of Liaoning Provincial Education Department of China(No.L2014465).