The Impact of Aging and Sex on Corneal Nerve Density and Function

Purpose: To quantify the role of aging and sex in corneal nerve morphology and function and to identify cellular and molecular mechanisms involved in corneal nerve aging in male and female mice. Methods: This study included young (8-week-old) and old (52-week-old) C57BL/6N male and female mice. Corneal nerve function was measured using eye wiping and Cochet-Bonnet tests. Corneal nerve density was analyzed after β3-tubulin staining in the sub-basal nerve plexus, the vertical nerve projections, and the superficial nerve terminals. Neuronal and neuroinflammatory markers were quantified in the trigeminal ganglia using immunofluorescence and real-time PCR. Results: Aging significantly reduces nerve density and the corneal nociceptive response (P < 0.0001). Mechanical sensitivity was decreased in male mice (P < 0.0001), but not in females. Female mice showed milder nerve fiber degeneration compared to males (P = 0.0453). Moreover, we identified an age-dependent decrease of substance P-positive neurons in the ophthalmic branch of the trigeminal ganglion (P = 0.0003), which was milder in females (P = 0.0005). Gene expression analysis in the trigeminal ganglion revealed a higher expression of neuroprotective and pro-regenerative markers in female mice. Immunofluorescence analysis revealed an age-dependent increase in the uptake of leukocytes in the trigeminal ganglion (P = 0.0068). M2 anti-inflammatory macrophage numbers were preserved in aging females versus males (P = 0.0008). Conclusions: Aging was associated with sensory corneal neuropathy, altered sensory abnormalities, and neuroinflammation. Interestingly, female mice appeared to be protected from nerve degeneration compared to males.