Type 2 diabetes influences intraepithelial corneal nerve parameters and corneal stromal‐epithelial nerve penetration sites

INTRODUCTION: The quantification of intraepithelial corneal basal nerve parameters by in vivo confocal microscopy represents a promising modality to identify the earliest manifestations of diabetic peripheral neuropathy. However, its diagnostic accuracy is hampered by its dependence on neuron length, with minimal consideration for other parameters, including the origin of these nerves, the corneal stromal‐epithelial nerve penetration sites. This study sought to utilize high‐resolution images of murine corneal nerves to analyze comprehensively the morphological changes associated with type 2 diabetes progression. MATERIALS AND METHODS: βIII‐Tubulin immunostained corneas from prediabetic and type 2 diabetic mice and their respective controls were imaged by scanning confocal microscopy and analyzed automatically for nerve parameters. Additionally, the number and distribution of penetration sites was manually ascertained and the average length of the axons exiting them was computed. RESULTS: The earliest detectable changes included a significant increase in nerve density (6.06 ± 0.41% vs 8.98 ± 1.99%, P = 0.03) and branching (2867.8 ± 271.3/mm(2) vs 4912.1 ± 1475.3/mm(2), P = 0.03), and in the number of penetration sites (258.80 ± 20.87 vs 422.60 ± 63.76, P = 0.0002) at 8 weeks of age. At 16 weeks, corneal innervation decreased, most notably in the periphery. The number of penetration sites remained significantly elevated relative to controls throughout the monitoring period. Similarly, prediabetic mice exhibited an increased number of penetration sites (242.2 ± 13.55 vs 305.6 ± 30.96, P = 0.003) without significant changes to the nerves. CONCLUSIONS: Our data suggest that diabetic peripheral neuropathy may be preceded by a phase of neuron growth rather than regression, and that the peripheral cornea is more sensitive than the center for detecting changes in innervation.