Vegf-receptor Inhibitor Impedes Macrophage Function At The End Target Muscle After Nerve Injury
Johnny Chuieng - Yi Lu, M.D.1, Albina Jablonka-Shariff, PhD2, Katherine Santosa, M.D., M.S.2,3, Alison Snyder-Warwick, M.D.2.
1Chang Gung Memorial Hospital, Linkou, Taiwan, 2Washington University at St.Louis, St. Louis, MO, USA, 3University of Michigan, Ann Arbor, MI, USA.
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 to support tSC process extension. We hypothesize that VEGF inhibition impedes tSC function at the NMJ, which in turn will impede reinnervation of the end target muscle.
Sciatic nerve transection with immediate repair was performed in C57BL/6 mice. To evaluate whether VEGF inhibition impedes end target muscle reinnervation, Cabozantinib, 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, 28 and 42 days after injury (n = 3 per time point), and the leg muscles were harvested. Immunohistochemical stains for NF200 (axon marker), S100 (Schwann cell marker), VEGF, CD68 (macrophage marker), and bungarotoxin (BTX, motor endplate marker) were used on extensor digitorum longus and tibialis anterior muscle sections. Functional innervation was determined by two methods: 1) immunohistochemical staining with NF-200 for axonal reinnervation into motor endplates marked by BTX in whole muscle mounts and 2) electrophysiological stimulation of the sciatic nerve to test tibialis anterior CMAP amplitude.
After nerve injury, extracellular and intracellular VEGF expression increased significantly at postoperative day (POD) 3 compared to non-injured muscle, and then peaked at POD 12. At two weeks (POD14) after injury, motor end plates were 41.2% fully innervated and 37.1% partially innervated in the control group, while the Cabozantinib group had only 7.5% fully innervated and 4.3% partially innervated motor end plates. VEGF expression at POD 14 was greatly different between the two groups (11.48 percent pixels vs 3.49 percent pixels, p <0.001). By POD 21, however, 97% of the motor end plates in the Control group and 93.2% in the Cabozantinib group had reached full innervation. Axonal sprouting between motor end plates was more prevalent in the control group (46.9% vs 18.5%). VEGF expression decreased in both groups by POD 21 and POD 28, while CD68 positive cells co-stained with VEGF remained significantly higher in the control group at POD 28. Electrophysiological stimulation at 10 Hz showed significant differences at POD 21 (t test, mean difference 2.151 mV, p = 0.047) but no differences at POD 42 (t test -0.822, p = 421).
Drug induced VEGF inhibition had a marked but temporary effect on the functional recovery of denervated muscle after nerve injury. Although the differences between the two experimental groups became nonsignificant over time, the effects seen at the neuromuscular junction of the target muscles highlighted the unrecognized role of VEGF in macrophages on the functional recovery after nerve injury.
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