PURPOSE. The aim of this study was to determine and characterize the effect of topical application of benzalkonium chloride (BAK) on corneal nerves in vivo and in vitro.METHODS. Thy1-YFP+ neurofluorescent mouse eyes were treated topically with vehicle or BAK (0.01% or 0.1%). Widefield stereofluorescence microscopy was performed to sequentially image the treated corneas in vivo every week for 4 weeks, and changes in stromal nerve fiber density (NFD) and aqueous tear production were determined. Whole-mount immunofluorescence staining of corneas was performed with antibodies to axonopathy marker SMI-32. Western immunoblot analyses were performed on trigeminal ganglion and corneal lysates to determine abundance of proteins associated with neurotoxicity and regeneration. Compartmental culture of trigeminal ganglion neurons was performed in Campenot devices to determine whether BAK affects neurite outgrowth.RESULTS. BAK-treated corneas exhibited significantly reduced NFD and aqueous tear production, and increased inflammatory cell infiltration and fluorescein staining at 1 week (P < 0.05). These changes were most significant after 0.1% BAK treatment. The extent of inflammatory cell infiltration in the cornea showed a significant negative correlation with NFD. Sequential in vivo imaging of corneas showed two forms of BAK-induced neurotoxicity: reversible neurotoxicity characterized by axonopathy and recovery, and irreversible neurotoxicity characterized by nerve degeneration and regeneration. Increased abundance of beta III tubulin in corneal lysates confirmed regeneration. A dose-related significant reduction in neurites occurred after BAK addition to compartmental cultures of dissociated trigeminal ganglion cells. Although both BAK doses (0.0001% and 0.001%) reduced nerve fiber length, the reduction was significantly more with the higher dose (P < 0.001).CONCLUSION. Topical application of BAK to the eye causes corneal neurotoxicity, inflammation, and reduced aqueous tear production. (Invest Ophthalmol Vis Sci.

Extracellular DNA production and clearance mechanisms are dysregulated in DED. Nuclease deficiency in tear fluid allows eDNA and NETs to accumulate in precorneal tear film, and results in ocular surface inflammation. These findings point to novel therapeutic interventions in severe DED based on clearance of eDNA, NETs, and other molecular components from the ocular surface.

Sema7a is expressed constitutively in the cornea, and potently stimulates nerve regeneration and inflammatory cell influx. Therefore, this immune semaphorin links nerve regeneration and inflammatory processes in the cornea.

Purpose To determine the in vivo expression of neurotrophins (NTs) and nerve regeneration-associated genes (RAGs) after surgically creating a hinged lamellar corneal flap in thy1-YFP mice. Methods Lamellar corneal flaps with multiple hinges were created in thy1-YFP mice. Mice were sacrificed weeks 2, 4, and 8. Quantitative PCR was performed to determine the expression of NTs and RAGs in the corneas following lamellar transection. Nerve growth factor (Ngf), Brain-derived neurotrophic factor (Bdnf), Glial cell-derived neurotrophic factor (Gdnf), Neurotrophin-3 (Ntf3), Neurotrophin 5 (Ntf5), Small proline-rich repeat protein 1A (Sprr1a), Growth-associated protein 43 (Gap43) and Beta III tubulin (Tubb3) gene expressions were analyzed. Whole-mount confocal immunofluorescence and Western analyses were performed for localization and abundance of robustly expressed genes. Results Sprouts of fine YFP positive fronds emanating from transected (injured) nerve bundles were seen in the flap area at 2 weeks onwards. Bdnf and Sprr1a were robustly and significantly expressed at 2 weeks postoperatively (> 2 folds increase in expression and p < 0.05). Bdnf localized to thy1-YFP+ cells in operated corneas. Sprr1a localized to corneal epithelial cell membranes. At 8 weeks, none of the NTs and RAGs had increased expression. Bdnf (ρ = 0.73, p = 0.001) and Sprr1a (ρ = 0.76, p = 0.001) showed a significant positive correlation with Tubb3. Conclusion The neurotrophin Bdnf and regeneration-associated gene Sprr1a are robustly and significantly expressed during corneal nerve regeneration in vivo.