Effects of Experimental Desiccating Stress on Corneal Nerve Density and Barrier Function

Abstract

Purpose: : To evaluate the effects of experimental desiccating stress on corneal nerve density and the relationship between changes in nerve density and corneal barrier disruption.

Methods: : Experimental dry eye (EDE) was created in C57BL/6 mice aged 6-8 weeks for 2, 5, and 10 days (D). At these time points, whole corneas from those and unstressed mice were removed. Oregon green dextran (OGD) permeability and tubulin beta-3 chain (tubb3) immunostaining were assessed by laser scanning confocal microscopy. Pictures were taken using a Z-stack of 10 µm for OGD and 50 µm for tbb3. The depth and intensity of corneal OGD staining and density of tubulin staining were measured using the NIS Elements software. Epithelial and stromal tbb3 intensities in the central and peripheral corneas were quantified separately; the density of tubb3 was calculated by dividing the tbb3 intensities (pixels) by the area of the image (mm2). Statistical analyses were performed with GraphPad Prism software. Statistical differences were evaluated by one-way ANOVA analysis with post-hoc analysis calculated by the Dunn’s Multiple Comparison test. Values of p ≤0.05 were considered significant.

Results: : Corneal OGD permeability and innervation significantly changed in EDE. The intensity of OGD staining was significantly greater than baseline at 2 and 5 D (p<0.0001), and OGD depth was significantly greater than baseline at 5 D (p<0.0001). Tbb3 density in the central cornea significantly increased after 5D and 10D (p<0.0001) with respect to baseline and between 2 and 5 D (p<0.0001) and between 2 and 10 D (p<0.0001), in both the epithelium and stroma (p<0.0001 and p<0.001, respectively). Tbb3 density in the peripheral cornea significantly increased after 10D of EDE comparing to baseline (p<0.0001) and between 2 and 10 D (p<0.001) in the epithelium, and between baseline and all time points in the stroma (p<0.0001).

Conclusions: : Acute disruption of apical corneal epithelial barrier function in dry eye is accompanied by increased nerve density in the corneal epithelium and stroma. It is possible that these findings contribute to the alterations in corneal sensitivity that can be seen in acute and chronic dry eye.

Keywords: cornea: tears/tear film/dry eye • pathobiology • innervation: sensation