Analysis of corneal axons remodeling in vivo during development and aging using long-term imaging in mice

The cornea serves as a critical interface between the external environment and the inner eye tissues, such as the retina. Its rich sensory innervation is essential to mediate protective reflexes and support corneal homeostasis. Despite its importance, our understanding of corneal nerve development, maturation, and remodeling, particularly in vivo, remains limited. Here, we performed long-term live imaging of corneal nerves with spinning-disk confocal microscopy. We used CGRP:GFP transgenic mice of either sex, in which the corneal nociceptive axons are labeled. We were able to track the same corneal region from the third postnatal week until almost 2 years of age. We found that the most superficial nerve plexus and terminals are continuously remodeled, with individual axons displaying high dynamics. In contrast, deep stromal nerves remain remarkably stable over time. In addition, we observed a decrease in corneal axonal coverage during aging. Our study introduces the use of spinning-disk confocal imaging as a high-resolution, minimally invasive tool to study corneal tissue in living mice over extended time periods. It provides new insights into the plastic behavior of corneal nerves. To our knowledge, this is the longest longitudinal study of axonal morphology and plasticity in any system and animal species. Significance statement Corneal sensory nerves are essential for ocular surface health, yet their long-term remodeling and degeneration remain poorly characterized in vivo. Using longitudinal imaging in CGRP:GFP mice, we tracked corneal nerve dynamics during development and aging, revealing superficial axon plasticity, stromal stability, and age-related nerve loss. These findings provide a baseline for understanding alterations in corneal innervation in conditions including refractive surgery, dry eye disease, neurotrophic keratopathy, and sensory neuropathies. We present a minimally invasive platform for tracking peripheral axons across the lifespan in live animals, establishing the cornea as a powerful model to investigate mechanisms of axonal plasticity and aging in the peripheral nervous system.