In Vivo Serial Imaging of Regenerating Corneal Nerves after Surgical Transection in Transgenic Thy1-YFP mice

Abstract

To determine the effect of lamellar transection surgery on the nerve fiber density (NFD) and pattern of nerve regeneration in the cornea of thy1-YFP transgenic mice. Wide-field stereo fluorescence microscopy was used to obtain serial images of nerves in live thy1-YFP mice, which express a fluorescent protein in their axons. NFD (mm/mm(2)) was calculated from maximum intensity projection images as the total length of fibers within the area of the contour in which nerves were traced. Whole-mount confocal microscopy was performed to analyze the arrangement of nerves and the types of regenerating fibers. NFD in normal corneas was 35.3 ± 1.8 mm/mm(2). Stereo fluorescence microscopy revealed the presence of a subbasal hairpin nerve layer and an intrastromal nerve trunk layer. After surgery, regenerative sprouting was observed from transected distal ends of intrastromal nerve trunks. NFD also increased, with this increase being maximal between 4 and 6 weeks after surgery. NFD approximated baseline values at 6 weeks and did not change any further at 8 weeks. Regenerated nerves did not readopt 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. Immunofluorescence 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). Although corneal NFD recovers to normal levels by 8 weeks after nerve transection, the arrangement of regenerated nerves is abnormal.