Early Differential Time Kinetics between Corneal Nerve Fiber Recovery and Innate Immune Response Following Murine Corneal Nerve Injury

초록

Purpose : While the timeline and underlying mechanisms for peripheral nerve regeneration after injury are well known, the specifics of early corneal nerve regeneration and the associated mechanisms of innate immunity remain unclear. In this study, following a corneal circular nerve cut (CNC), we analyzed the corneal nerve regeneration timeline over three weeks and investigated the kinetics of innate immunity at a very early stage. Methods : The corneal subbasal nerve plexus (SBNP) and stromal nerves were severed using a 2.0 mm circular punch in Balb/c mice. To visualize corneal nerves, whole mount βIII-tubulin staining was performed immediately after injury and at 1, 3, 7, 14, and 21 days post-injury (N=10 eyes per group). Corneal nerves were imaged using confocal microscopy, and SBNP fiber length was measured with NeuronJ software. QRT-PCR was employed to analyze monocyte, macrophage, and myeloid cell markers, as well as innate immunity-related inflammatory cytokines over 3 weeks, including within 12 hours post-injury. Results : After CNC, the SBNP fiber length significantly decreased at both the center and cutting edge 1 day post-injury. By day 21, nerve fibers at the cutting edge had fully regenerated, while those at the center recovered to about 44%. Inflammatory cytokines, including IL1B and IL6, and innate immune cells, especially myeloid cells and M1 macrophages, spiked within 6 hours. Interestingly, M2 macrophages showed early differential marker kinetics. MRC1 (CD206) was activated within 12 hours, while ARG1 and CD163 were suppressed. Notably, ARG1 expression increased on days 7 and 14 post-CNC. Conclusions : After corneal CNC in murine eyes, corneal nerve fibers gradually regenerated over a period of 3 weeks, with the center recovering more slowly compared to the cutting edge. In contrast, modulation of myeloid cell and monocyte/macrophage marker expressions in the cornea was pronounced within just 12 hours post-injury. These findings highlight a distinct temporal difference between nerve regeneration and innate immune response in the cornea following corneal circular nerve injury.