Neutrophils Invade Atrophied Skeletal Muscles Without Causing Muscle Dysfunction Unless They Are Stimulated With LPS.

Abstract

Important muscle atrophy and loss of force are observed following unloading and early reloading periods, with subsequent muscle recovery within 14 to 28 d. The reloading period is associated with neutrophil (PMN) and macrophage infiltration. PMNs phagocyte necrotic debris and release various cytokines and oxidative factors. Although PMNs can induce secondary damage in different pathologies, their roles in muscle dysfunction and regrowth following unloading and reloading periods have never been fully investigated. PURPOSE: To determine the contribution of PMNs to muscle injury, dysfunction and regrowth in an unloading and reloading model. METHODS: Mice were submitted to 10 d of hindlimb unloading followed by reloading periods of 1, 3 or 14 d. They were intraperitoneally injected with 50 μL (0.025 mg) of rat anti-PMN (anti-Ly6G/Ly6C) or placebo 1 d before and after reloading. This dose was sufficient to deplete 95 % of circulating PMNs. Ambulatory mice were used as control. Soleus muscles were incubated in a physiological solution for measurements of contractile properties. In a second set of experiments, solei were incubated in vitro with lipopolysaccharide (LPS, 100 μg/mL) to activate PMNs. Contractile properties were obtained every hour for 6 h and muscles were subsequently incubated in 1 % procion orange at 4 °C to assess muscle damage. Comparisons between groups were performed with a one-way ANOVA followed by a Fisher's a posteriori test. RESULTS: Our findings show that PMN depletion neither affects the loss of muscle force nor the time of recovery. Interestingly, reloaded soleus muscles that contain a high PMN density showed a 20 % greater loss of force when compared to reloaded solei depleted in PMNs following LPS incubation. These significant differences appear after 4 h of incubation in LPS (p<0.05). Procion orange staining also confirms that presence of PMNs in atrophied muscles induces a 3-fold increase in muscle membrane damage following LPS stimulation (26 % vs 9 %, p<0.05). CONCLUSIONS: Together, these results show that secondary damage following PMN infiltration is strongly limited during muscle atrophy and regrowth. However, when activated by other stimuli, such as LPS, PMNs can potentially damage skeletal muscle. Supported by the Canadian Space Agency and Canadian Institutes of Health Research.