Unravelling the causes of dry eye disease: the role of distal degeneration on TRPM8 corneal nerve endings

Abstract

Purpose : The causes and pathogenesis of Dry Eye Disease (DED) remain unknown, while evidence for the neurosensory chapter of the disease is accumulating. Here we investigated the role of TRPM8 in age and diabetic-degenerating corneal sensory terminals and its relevance to pain, tear regulation and inflammation in DED.

Methods : We used transgenic TRPM8^BAC-EGFP and TRPM8^-/- mice from 3 to 24 months of age to study the degenerative cellular events during mouse ageing and diabetic disease and their relation to DED. Streptozotocin was used to induce dysregulation of glucose metabolism. All procedures were performed according to the ARVO Statement for the Use of Animals in Research and the corresponding Spanish and EU guidelines.
Immunofluorescence of whole-mounted corneas and trigeminal ganglia and q-PCR analysis were performed to evaluate the expression of TRPM8, TRPV1, Piezo2, NF200, peripherin, TrKA, CGRP, VGlut2, GABA and markers of mitochondrial function HO-1, cytochrome C oxidase, Nrf2 and Keap1.
Behavioural analysis of pain responses was used to assess variation in stimulus perception. TBUT and phenol red thread tests were used to analyse tear film production and stability.

Results : Aging and diabetic mice showed reduced sensitivity to corneal stimulation and altered tear production. Trigeminal ganglion neurons showed increased expression of CGRP, TrKA and VGlut2 transcripts, while decreased expression of TRPM8 and reduced number of TRPM8-positive corneal fibres were observed. Oxidative stress markers were increased in TRPM8-positive cells from ageing and diabetic mice, suggesting a role for oxidative stress in the onset of nerve terminal degeneration. This was slowed by the antioxidant N-acetylcysteine and TRPM8 agonists.
Two distinct populations of TRPM8-containing neurons were identified innervating the cornea, showing different characteristic markers and undergoing changes in protein expression with age or in diabetic DED.

Conclusions : Our results indicate a direct role for TRPM8 in maintaining corneal homeostasis and suggest TRPM8 as a potential target for neuroprotection of corneal sensory innervation.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.