Role of Galectins in Corneal Nerve Regeneration

Abstract

Purpose: Corneal nerve damage results in corneal hypoesthesia, dry eye, neurotrophic ulceration, corneal melting or pain. Corneal nerve regeneration is, in part, mediated by nerve growth factor (NGF) that binds to glycosylated tyrosine kinase receptor TrkA. The purpose of this study was to determine the role of galectins, a family of carbohydrate-binding proteins, in corneal nerve regeneration.

Methods: Ophthalmic branch of trigeminal ganglia (OTG) were dissected from E9 days chick embryos and incubated with DMEM (control), DMEM/NGF, NGF and pan-galectin inhibitor (asialofetuin) or with NGF/bovine serum albumin (BSA) for 24 hours. Cultures were fixed and immunostained with anti-β-III tubulin antibody to visualize axons. Wild-type (WT) and MGAT5 and Galectin-3 (GAL3) KO underwent excimer laser (Visx Star S4) transepithelial ablation to create 2-mm corneal wounds. After 1 and 3 weeks, corneas were excised, and immunostained with anti-βIII-tubulin antibody and compared to normal corneas. WT, MGAT5 and GAL3 KO normal and inflamed (by thermal cautery) corneas were excised and stained for a pan-leukocyte maker CD45 and density of nerve and CD45+ cells were quantified. Pull down experiments using GAL3-conjugated agarose beads were performed to determine if TrkA binds to GAL3.

Results: Robust neurite outgrowth was seen in OTG cultures incubated with NGF, as compared to cultures in media alone (p<0.05). NGF-induced neurite outgrowth was significantly (p<0.05) reduced by asialofetuin, but not by BSA. Pull down experiments confirmed that TrkA receptor is a GAL3-binding protein. WT and galectin KO mice demonstrated similar nerve density at baseline. However, MGAT5 KO mice demonstrated incomplete corneal regeneration in the central cornea (45±7 mm/mm2; p<0.05) and delayed regeneration in corneal periphery, as compared to controls (115±20), WT (90±34) and GAL3KO (79±24) mice 3 weeks after surgery. The density of CD45+ cell was not statistically significant at baseline or after inflammation in the corneal center between WT and KO groups (P>0.05), but was higher in the periphery of MGAT5KO mice.

Conclusions: The data suggest that one or more members of the galectin family modulate the function of NGF receptor. Impaired nerve regeneration after laser surgery in KO mice lacking galectin ligands suggests that galectins are a critical determinant of corneal nerve regeneration.

Keywords: 565 innervation: sensation • 765 wound healing • 480 cornea: basic science