Macrophages are Integral to Neuromuscular Junction Reinnervation after Nerve Injury
Katherine Bernadette Santosa, M.D.1; Alexandra Marie Keane, B.A.1; Bianca Vannucci, BA1; Albina Jablonka-Shariff, Ph.D.2; Alison K. Snyder-Warwick, MD3; (1)Washington University, Saint Louis, MO, (2)Division of Plastic and Reconstructive Surgery, Washington University, Saint Louis, MO, (3)Plastic Surgery, Washington University School of Medicine, St. Louis, MO
Purpose: Macrophages are well-known for their role in Wallerian degeneration following a nerve injury. Recent studies, however, have suggested additional macrophage roles such as response to hypoxia and angiogenesis propagation, both of which are necessary for nerve regeneration across an injury site. We hypothesize that similar mechanisms occur at the neuromuscular junction (NMJ), and we have previously shown that macrophages are not only recruited to the site of nerve injury, but also to the NMJ and end-target muscle after nerve injury. By utilizing Ccr2-/- mice, which have markedly impaired recruitment of macrophages to sites of inflammation and injury, we sought to determine the importance of recruited macrophages to the NMJ on reinnervation following peripheral nerve injury.
Methods: We performed right sciatic nerve transections with immediate repairs on adult wildtype (C57BL/6) and Ccr2-/-mice. At two and three weeks following nerve injury, mice from both groups were sacrificed for collection and evaluation of extensor hallucis longus (EHL) and extensor digitorum longus (EDL) muscles. Using whole mount confocal microscopy, we characterized the morphology of the NMJ and quantified reinnervation by imaging axons (NF200), Schwann cells (SCs, S100), and motor endplates (?-bungarotoxin).
Results: At homeostasis, there were no morphological differences in the appearance of the NMJs of wildtype versus Ccr2-/- mice. At two weeks after injury, however, we observed more denervated endplates in Ccr2-/- mice than in wildtype mice (68.1% vs. 45.4%, p=0.016). Similarly, at three weeks after nerve injury, more denervated endplates remained in Ccr2-/- mice as compared to age-matched wildtype controls (26.2% vs. 7.3%, p=0.0043).
Conclusions: Successful reinnervation of the NMJ following nerve injury is complex and multifaceted. Our data demonstrate that Ccr2-/- mice, which have impaired monocyte-derived macrophage recruitment, have delayed reinnervation as compared to age-matched wildtype controls. These results suggest that macrophages are important for reinnervation of the NMJ following peripheral nerve injury. Further investigations to determine the functional consequences of the differences in reinnervation between the two groups are ongoing.
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