Corneal Reinnervation Following Surgical Transection

Abstract

Purpose: : To determine nerve fiber density (NFD), pattern of regenerated nerves, and the expression of neurotrophins (NTs), regeneration associated genes (RAGs), and nerve guidance proteins (NGs ) in the cornea of thy1-YFP transgenic mice after lamellar flap surgery.

Methods: : Lamellar corneal flaps with multiple hinges were created in thy1-YFP neurofluorescent mice. Serial in vivo wide-field stereofluorescent microscopy was performed to determine changes in nerve fiber density (NFD).Mice were sacrificed at weeks 2, 4, and 8. Whole-mount confocal microscopy was performed to analyze the arrangement of nerves and the type of regenerating fibers. Quantitative PCR was performed to determine the expression of NTs, RAGs, and NGs. Whole-mount confocal immunofluorescence and Western analyses were performed for localization and abundance of robustly expressed genes.

Results: : NFD in normal corneas was 35.3 ± 1.8 mm/mm2. Stereofluorescent microscopy revealed the presence of a subbasal hairpin nerve layer and an intrastromal nerve trunk layer. Following surgery, regenerative sprouting was observed from transected distal ends of intrastromal nerve trunks. NFD also increased, with this increase being maximal between 4 to 6 weeks after surgery. NFD approximated baseline values at 6 weeks and did not change any further at 8 weeks. Regenerated nerves did not re-adopt the normal corneal nerve arrangement. A dense interlacing network of regenerated nerves was present in the corneal bed. Branches from this network traversed the flap to innervate the epithelium. Immunofluorescent staining revealed that regenerating fronds contained peptidergic nociceptive fibers (positive for calcitonin gene-related peptide and substance P) and myelinated non-nociceptive fibers (positive for neurofilament 200). 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.

Conclusions: : Although corneal NFD recovers to normal levels by 8 weeks after nerve transection, the arrangement of regenerated nerves is abnormal. The neurotrophin Bdnf and regeneration-associated gene Sprr1a are robustly and significantly expressed during corneal nerve regeneration in vivo.

Keywords: cornea: basic science