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Jiucheng He, Thang Luong PHAM, Azucena H Kakazu, Haydee E P Bazan; Remodeling of substance P sensory nerves and TRPM8 cold receptors after corneal experimental surgery. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4726. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate changes in corneal nerves positive to SP and to TRPM8 as well as gene expression in the trigeminal ganglia (TG) following experimental corneal surgery to unveil the peripheral nerve mechanism of surgery-induced dry eye-like pain (DELP).
Surgery was performed on the right eye of adult mice of both sexes by removing the central epithelial and anterior stromal nerves. Mice were euthanized at different times up to 15 weeks. Immunostaining was performed with TRPM8, SP or PGP9.5 antibodies, and relative epithelial nerve densities were calculated by computer-assisted analysis. The origin of TRPM8 and SP TG neurons were analyzed by retrograde tracing and immunofluorescence. The gene expression in TG were studied by Fluidigm real-time PCR analysis.
SP-positive epithelial nerves were more abundant than TRPM8 in mouse corneas and they have a different expression in TG neurons. After injury, corneal epithelial nerve regeneration occurs in two distinct stages. The early regeneration consisted of the remaining epithelial bundles around the wound, which extended through the edge of the wound and ran centripetally to cover the wound area and reached the highest density on day 3 and then rapidly degraded. From day 5, the epithelial nerves originate from the underlying stromal nerve branches, which formed the whirl-like structure at week 4 and reached to the highest density at week 6, about 80% of normal level, but still lower than normal levels by 15 weeks. The recovery of SP- and TRPM8-positive nerve fibers followed the same pattern as the total nerves. TRPM8-positive terminals increased slowly, and they reached only half of normal values by 3 months after injury. Contrarily, corneal sensitivity, gradually increased from the third day and reached normal values on day 12, and there was an increase in tear production after surgery. Real-time PCR showed that corneal injury induced significant changes in TG gene expression, decreasing trpm8 and tac1 genes.
This is the first study to show a map of the entire epithelial TRPM8 nerves in the mouse cornea. Our findings that abnormal SP expression, and low amounts of TRPM8 terminals as well as hypersensitive nerve response occur long after the injury and changes in the expression of selective genes in the TG during after corneal injury, suggest a contribution to the pathogenesis of corneal surgery-induced DELP.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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