Macrophage Recruitment and Activation to Skeletal Muscle after Acute Nerve Injury
Katherine B. Santosa, M.D., Albina Jablonka-Shariff, Ph.D., Anja G. Fuchs, Ph.D., Alexandra M. Keane, B.A., Isaiah R. Turbull, M.D., Ph.D., Alison K. Snyder-Warwick, M.D..
Washington University, Saint Louis, MO, USA.
PURPOSE: The skeletal muscle has a unique immunologic profile that is particularly dynamic following muscle injury. Immune cells such as macrophages, which are normally few in number during physiologic conditions, are recruited to the muscle following injury to assist in inflammatory and reparative processes. In response to their muscle microenvironment, macrophages are able to change their phenotypes and functions during these events. Although macrophage recruitment and activation have been demonstrated in direct muscle injury models, this dynamic process has not been described in muscle after nerve injury. The goal of this study was to determine if acute nerve injury resulted in the recruitment of macrophages to the distal muscle target, and to characterize the phenotype of these activated macrophages.
METHODS: We utilized two strategies to determine if macrophages are recruited to muscle after sciatic nerve transection without repair. First, we evaluated the extensor digitorum longus (EDL) muscles of 15 adult wildtype C57BL/6 mice (n=3 per time point) at days 1, 3, 5, 7, and 14 after sciatic nerve injury. The uninjured EDL muscles served as the experimental controls. These muscles were harvested for immunostaining with CD68 (monocytes/macrophages) and DAPI (nuclear) staining. Next, using the same injury and mouse model, flow cytometry was utilized to evaluate total cells present in EDL muscle after sciatic nerve injury. Animals were sacrificed at days 1 and 5 after nerve injury, and all muscles of the hindlimb innervated by the sciatic nerve were harvested from the right injured and left uninjured legs. Cells were analyzed following muscle digestion.
RESULTS: At all timepoints after nerve injury, there were significantly more CD68+ cells recruited to denervated EDL muscles than to uninjured controls in our immunocytochemistry analysis. On flow cytometry, there was a higher number of CD45+ hematopoietic cells isolated from denervated muscle than uninjured controls. Moreover, data demonstrate significantly more Ly6C+F480- monocytes and CD206+MerTK+ macrophages recruited to the muscle following acute nerve injury. At postoperative day 5, CD206+MerTK+ macrophages had decreased CD11c expression, suggesting activation of these immune cells.
CONCLUSIONS: Our studies demonstrate the novel finding that acute nerve injury induces macrophage recruitment to the distal target muscle. Moreover, recruited macrophages have an altered phenotype, which may suggest a functional transformation of these important inflammatory and regenerative immune cells. Further studies are ongoing to determine the functional impact of this macrophage phenotypic change on reinnervation of the muscle following acute nerve injury. Knowledge of this process may provide new therapeutic targets to improve functional recovery following nerve injury.
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