Illuminating Corneal Nerve Injury: Analysis of Neuronal Phenotypes Following Acute and Chronic Corneal Nerve Injury

The cornea is uniquely avascular, transparent, and densely innervated with a highly complex and rapidly adapting sensory system. Combined with the tear film coating its surface, it is the first line of protection for the visual system. Clear vision provides tremendous survival advantage and thus uses long evolved and highly conserved mechanisms within the corneal protective barrier to preserve its function. Corneal nerves have a critical role in maintaining corneal homeostasis, initiating blinking, and tearing circuits to block potentially damaging environmental insults, and for engaging rapid-response regeneration programs. Many of these critical molecular mechanisms have been broadly characterized over the past century. However, with new advances in transgenic models and precision medicine, current studies focus on specific molecular factors that can be targeted in translational research and treatments. Sry-Box Transcription Factor 11 (SOX11) is a known upstream regulator of the injury response regeneration associated gene network and is highly expressed in primary afferent neurons following injury. Currently, little is known about the role of SOX11 in nerve regeneration following corneal injury. The aim of this study was to examine corneal injury-induced pathology in direct and indirect corneal injury models, which include trephine-only (TO), corneal abrasion (CA), and lacrimal gland excision (LGE). Both wild-type C57B6/J, Nav1.8Cre-tdTomato and Nav1.8Cre-Sox11-tdTomato mice were used to characterize corneal pathology following these injuries. LGE-induced dry eye reduces the aqueous component of tears, resulting in persistent corneal epithelial cell damage and retraction of corneal afferent nerve terminals. TO ligates axon terminals to the subbasal plexus in the corneal epithelium. CA first uses TO injury directly followed by mechanically removing the corneal epithelium and axon terminals from the central cornea. Before and after injury, assays were performed to evaluate tearing, mechanical sensitivity, ocular discomfort, and the corneal epithelium. Tissue was collected at terminal timepoints, and whole-mount corneas were imaged, and afferent terminals were analyzed. Before injury, corneal cell bodies were labeled using retrograde tracer, and somal phenotype was evaluated using immunohistochemistry and in situ hybridization. Additionally, rt-qPCR was performed on both corneas and trigeminal ganglia in some experiments. The results show that while corneal innervation density decreased between 1-2 weeks following LGE in control animals, nerves regenerated to near normal levels by four weeks, albeit in a disorganized manner. In Nav1.8Cre-tdTomato-Sox11f/f (Sox11fl/fl) animals, innervation density was significantly reduced at the 4-week time point compared to control animals. As determined with fluorescein staining, corneal epithelial cell damage was similar between Sox11fl/fl and control animals over the 4 -weeks after LGE. Directly following CA-induced injury, both control and Sox11fl/fl animals showed significant decreases in innervation density at 24 hours. By 48 hours after injury, Sox11fl/fl animals showed a small yet significant increase in nerve growth. Both control and Sox11fl/fl animals demonstrated comparable reductions in corneal epithelial cell damage 48 hours after injury. Taken together, these results provide support for the critical role of SOX11 in nerve regeneration and healing following corneal injury