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.

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.

YFP(+) BMCs in thy1-YFP mice have immunophenotypic features of MDSCs. They secrete NGF and promote neuroregeneration. Their immunosuppressive and neurotrophic actions are preserved after corneal infiltration. These findings increase our understanding of the beneficial roles played by leukocyte trafficking in the cornea and may lead to therapeutic strategies that use NGF-secreting myeloid cells to repair diseased or injured neurons.

Purpose To characterize the involvement of Semaphorin 7A (Sema7a) in corneal neovascularization (NV). Methods We generated anti-Sema7A antibodies to detect protein expression. Corneal fibroblast cells were cultured, stimulated with bFGF, immunostained with anti-Sema7A antibodies, and visualized by confocal microscopy. bFGF pellets were implanted in mouse corneal micropockets for 3–10 days, and corneal sections were immunostained with anti-Sema7A antibodies. Mouse corneas were injected with naked Sema7A DNA and vector control for 3, 7, and 10 days. Mouse corneas were imaged by slit lamp microscopy, and areas of corneal NV were calculated using the ImageJ program. Mouse corneal sections were also immunostained with anti-macrophage marker (F4/80) and anti-vascular endothelial growth factor (VEGF)-A antibodies. Results Our data showed enhanced Sema7A expression levels in bFGF-stimulated cultured corneal fibroblasts. bFGF corneal implantation also demonstrated enhanced Sema7A expression. Corneas injected with a Sema7A expression vector showed evidence of significant corneal NV compared to controls on day 10 (1.8 mm2 vs. 0.11 mm2; p<0.02). Additionally, immunolocalization of Sema7A DNA-injected corneas (at day 7) revealed macrophage recruitment and enhanced VEGF-A levels. Conclusions We demonstrated that Sema7A was expressed in vascularized corneas and showed pro-angiogenic properties in our corneal model. Understanding the mechanism of Sema7A in angiogenesis may provide a therapeutic target for the treatment of corneal angiogenesis-related disorders.