Abstract
Purpose:
This study investigated the use of three-dimensional volumetric confocal imaging of the corneal subbasal nerve plexus and terminal epithelial nerves for quantitative analysis of nerve fiber loss in a diabetic mouse model in situ; and assessed changes in total corneal and epithelial thickness in diabetic mice using a remote controlled HRT confocal microscope in vivo.
Methods:
Type I diabetes was induced in male C57BL/6 mice using the Animal Models of Diabetics Complications Consortium high dose streptozotocin (150 mg/kg body weight) protocol. Mice were evaluated for corneal nerve and epithelial changes at 6 and 12 weeks. Age-matched vehicle treated mice were used as controls. Serum glucose levels and body weight were assessed at 6 and 12 weeks. A serum glucose level greater than 300 mg/dl was diagnostic of diabetes. Total corneal and epithelial thickness changes were assessed from three dimensional images using confocal microscopy through focusing. Corneas were then excised and stained with anti-beta-tubulin III and labeled with a FITC-conjugated secondary antibody. Nuclei were counter-stained with propidium iodide. Whole mount corneas were imaged on a laser scanning confocal microscope. Image stacks were reconstructed three-dimensionally and analyzed using MetaMorph and IMARIS software.
Results:
Total length of the subbasal nerve plexus was reduced at 6 and 12 weeks. This was associated with a reduction in basal cell density at both time points, while significant epithelial thinning was evident only after 12 weeks. In contrast, stromal thinning was detected in diabetic animals at 6 and 12 weeks and was associated with a significant reduction in body weight. Loss of terminal epithelial nerves was only weakly correlated with loss of the subbasal nerve plexus, but was significantly reduced as a function of increased age.
Conclusions:
Age and diabetes differentially mediate nerve loss in the corneal epithelium. Loss of the subbasal nerve plexus is associated with epithelial changes, which may contribute to epithelial thinning at prolonged disease durations. Further studies are necessary to investigate the impact of hyperglycemia on the corneal stroma.
Keywords: 482 cornea: epithelium •
498 diabetes