Macrophage-Derived Vascular Endothelial Growth Factor-A Is Integral to Neuromuscular Junction Reinnervation after Nerve Injury

Functional recovery in the end target muscle is a determinant of outcome after peripheral nerve injury. The neuromuscular junction (NMJ) provides the interface between nerve and muscle and includes non-myelinating terminal Schwann cells (tSCs). After nerve injury, tSCs extend cytoplasmic processes between NMJs to guide axon growth and NMJ reinnervation. The mechanisms related to NMJ reinnervation are not known. We used multiple mouse models to investigate the mechanisms of NMJ reinnervation in both sexes, specifically whether macrophage-derived vascular endothelial growth factor-A (VEGF-A) is crucial to establishing NMJ reinnervation at the end target muscle. Both macrophage number and VEGF-A expression increased in end target muscles after nerve injury and repair. In mice with impaired recruitment of macrophages and monocytes (CCR2-/- mice), the absence of CD68+ cells (macrophages) in the muscle resulted in diminished muscle function. Using a Vegf-receptor 2 (VEGFR2/KDR/Flk-1) inhibitor (cabozantinib; CBZ) via oral gavage in wild-type (WT) mice resulted in reduced tSC cytoplasmic process extension and decreased NMJ reinnervation compared with saline controls. Mice with VEGF-A conditionally knocked out in macrophages (VEGF-A^fl/fl; LysM^Cre mice) demonstrated a more prolonged detrimental effect on NMJ reinnervation and worse functional muscle recovery. Together, these results show that contributions of the immune system are integral for NMJ reinnervation and functional muscle recovery after nerve injury.SIGNIFICANCE STATEMENT This work demonstrates beneficial contributions of a macrophage-mediated response for neuromuscular junction (NMJ) reinnervation following nerve injury and repair. Macrophage recruitment occurred at the NMJ, distant from the nerve injury site, to support functional recovery at the muscle. We have shown hindered terminal Schwann cell (tSC) injury response and NMJ recovery with inhibition of: (1) macrophage recruitment after injury; (2) vascular endothelial growth factor receptor 2 (VEGFR2/KDR/Flk-1) signaling; and (3) VEGF secretion from macrophages. We conclude that macrophage-derived VEGF is a key component of NMJ recovery after injury. Determining the mechanisms active at the end target muscle after motor nerve injury reveals new therapeutic targets that may translate to improve motor recovery following nerve injury.