American Society for Peripheral Nerve

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VEGF-Receptor Inhibitor Impedes Macrophage Function At The End Target Muscle After Nerve Injury
Johnny Chuieng - Yi Lu, MD1,2; Katherine Bernadette Santosa, MD, MS3,4; Albina Jablonka-Shariff, PhD5; Alison K. Snyder-Warwick, MD3
1Washington University in St. Louis, St. Louis, MO, 2Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Chang Gung Medical College and Chang Gung University, Taoyuan, Taiwan, 3Washington University School of Medicine, St. Louis, MO, 4University of Michigan, Ann Arbor, MI, 5Washington University in St. Louis, Saint Louis, MO

Recovery in peripheral nerve injuries is limited by the critical time point beyond which muscle denervation is irreversible. A major component of functional recovery is muscle reinnervation, which depends on terminal Schwann cells (tSCs) at the neuromuscular junction (NMJ). The hallmark feature of the tSC response to injury is cytoplasmic process elongation that guides axon growth. In response to injury, macrophages secrete Vascular Endothelial Growth Factor (VEGF) which may induce angiogenesis for tSC migration. We hypothesize that VEGF inhibition impedes macrophage function at the NMJ, which in turn will impede reinnervation of the end target muscle.

Materials and Methods
Sciatic nerve transection with immediate repair was performed in C57BL/6 mice. To evaluate whether VEGF inhibition impedes end target muscle reinnervation, Carbozantinib, a VEGF Receptor-2 (VEGFR-2) inhibitor, was administered to mice via oral gavage for seven days following nerve injury to suppress VEGF secretion. For the control group, saline was given for the same duration. Mice were sacrificed on 14, 21 and 28 days after injury (n = 3 per time point), and the leg muscles were harvested. Immunohistochemical stains for VEGF, CD68 (macrophage marker), CD31 (endothelieal cell marker), and bungarotoxin (BTX, motor endplate marker) were used on extensor digitorum longus and tibialis anterior muscle sections. Functional innervation was determined by the immunohistochemical staining with NF-200 for axonal reinnervation into motor endplates marked by BTX in whole muscle mounts.

Results (Preliminary)
VEGF expression increased at postoperative day (POD) 6, and then peaked around POD 10. VEGF was found intracellularly within macrophages, and there were noticeable differences in expression between uninjured (1630.52 +/- 551.53 AU) and injured muscles (2303.42 +/- 33 AU). At 2 weeks after surgery, motor end plates were 41.2% fully innervated and 37.1% partially innervated in the control group, while the Carbozantinib group had only 7.5% fully innervated and 4.3% partially innervated motor end plates. A higher percentage of non-innervated motor end plates (78.2%) with fragmented debris was found in the muscles of the Carbozantinib group. The postoperative week 3 and 4 time points are currently under investigation.

VEGF inhibition on macrophages has a significant effect on the functional recovery of denervated muscle after nerve injury. The unrecognized angiogenetic potential of macrophages at the neuromuscular junction may possess a key role in the investigation of muscle reinnervation.

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