Abstract
Purpose: :
The cornea is densely innervated with sensory fibers, which have a strong interaction with corneal epithelial cells, promoting cell differentiation, proliferation and wound healing. However little is known about role of glutamate-mediated signaling in this process. We have previously shown that Neuroprotectin D1 (NPD1) stimulates corneal nerve regeneration after injury and promotes wound healing (ARVO, 2010). Vesicular glutamate transporters (VGLUTs) are useful markers for functional glutaminergic systems. The purpose of this study was to assess the presence of VGLUT1 and VGLUT2 in trigeminal nerve fibers and in the cornea before and after corneal nerve damage and whether treatment with NPD1 influenced their expression.
Methods: :
An 8 mm corneal stromal dissection was performed in the left eyes of adult New Zealand rabbits. The treatment group received topical NPD1 (100ng) application three times a day for 6 weeks. The control group received vehicle drops (2μl of ethanol in 50μl of PBS). Rabbits were sacrificed at 8 weeks and corneas were processed for immunohistochemistry. Corneal wholemounts were stained with βIII tubulin and VGLUT1 or VGLUT2. The βIII tubulin-positive area at the subepithelial nerve plexus was calculated and compared to the total area. VGLUT-positive area at the epithelium level as well as at the subepithelial nerve plexus level was also calculated using an image analysis program.
Results: :
In agreement with our previous findings, corneal nerve area was increased four fold in the injured area in animals treated with NPD1 compared to vehicle treated group (6.2% vs 25.2%). VGLUT2 was more abundantly expressed than VGLUT1 in the corneal subepithelial nerve plexus prior to injury and only minimal staining for either VGLUT isoform was found in the basal corneal epithelial cells. However, six weeks after injury, VGLUT2 was strongly up-regulated in epithelial cells, with higher concentrations in the epithelium of the injured area than in the adjacent area (edge) (60.5% vs 25.8% of positive epithelial cells. p=0.0005). Animals treated with NPD1 showed the same pattern of localization of VGLUT2 compared to vehicle treated animals with a lesser amount of positive cells observed in general; but this difference was not statistically significant.
Conclusions: :
The presence of VGLUT2 within subepithelial corneal nerves under normal conditions suggests that glutamate-mediated signaling from trigeminal sensory nerve fibers occurs under control conditions. The selective induction of VGLUT2 in corneal epithelial cells after injury suggests a paracrine/autocrine role for glutamate in the control of epidermal renewal.
Keywords: cornea: epithelium • innervation: sensation • neuropeptides