Abstract
Purpose :
Ion channels receptors confers high sensitivity and multimodality function to corneal nerves. We aim to characterize the corneal and trigeminal ganglia (TG) expression of the TRPM8-EGFP receptor in intact and injured corneas and determine its correlation with the anatomic nerve regeneration.
Methods :
A transgenic mouse line that contains an enhanced green fluorescent protein (EGFP) in the promoter of the transient receptor potential cation channel subfamily M member 8 gene (TRMP8-EGFP) was obtained from Jackson Labs (Strain #:020650). The analysis of TRPM8-EGFP expression were performed in corneas, TGs and isolated TG neurons from male and female mice with intact or corneas subjected to 2mm central epithelium debridement. Corneal epithelium recovery was evaluated using fluorescein staining and slit lamp imaging. Mice were sacrificed after 3, 7, and 21 days and tissues collected for analysis (n=3 per condition). Dissected corneas were fixed and mounted on 1% agarose for light sheet microscopy (LSM). Corneas, TG cryosections and isolated TG neurons were immunostained with either anti B-3 tubulin or anti TRMP8 antibodies, to validate the endogenous expression of TRMP8. Samples were mounted on vectashield and imaging was performed using an AxioObserver fluorescent microscope.
Results :
The transgenic mice line exhibited normal growth and no phenotypic variation was observed. LSM showed TRPM8-EGFP expression in subbasal and stromal nerves throughout the cornea. Colocalization with beta-3-tubulin and TRPM8 antibody staining was observed. TGs and TG neurons exhibited TRPM8-EGFP expression in the soma and neurites. After injury variable TRMP8-GFP expression was observed in the subbasal corneal nerve plexus and it is being currently analyzed for its correlation with anatomic recovery using Neurolucida software.
Conclusions :
The TRMP8-EGFP transgenic mice line could be useful to investigate the expression and function of TRMP8 receptor in normal and injured corneas. The TRMP8 receptors are responsive to cooling, hyperosmolarity, and wetness of the cornea. Therefore, further investigation of TRPM8 expression in studies related to impaired corneal sensation may provide insights into how this receptor relate to cornea nerve repair.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.