School of Biomedical Sciences, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW, Australia.
Katharina Gaus Light Microscopy Facility, Mark Wainwright Analytical Centre, University of New South Wales, Sydney, NSW, Australia.
J Diabetes Investig. 2023 Apr;14(4):591-601. doi: 10.1111/jdi.13974. Epub 2023 Feb 2.
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.
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.
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 vs 4912.1 ± 1475.3/mm , 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.
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.
通过活体共聚焦显微镜对角膜上皮内基底神经参数进行量化，代表了一种有前途的方法，可以识别糖尿病周围神经病变的最早表现。然而，其诊断准确性受到神经元长度的限制，几乎没有考虑其他参数，包括这些神经的起源、角膜基质-上皮神经穿透部位。本研究旨在利用小鼠角膜神经的高分辨率图像，全面分析与 2 型糖尿病进展相关的形态变化。
对糖尿病前期和 2 型糖尿病小鼠及其各自对照的 βIII-微管蛋白免疫染色角膜进行扫描共聚焦显微镜成像，并自动分析神经参数。此外，手动确定穿透部位的数量和分布，并计算它们的轴突平均长度。
最早可检测到的变化包括神经密度（6.06±0.41%对 8.98±1.99%，P=0.03）和分支（2867.8±271.3/mm 对 4912.1±1475.3/mm ，P=0.03）以及穿透部位数量（258.80±20.87 对 422.60±63.76，P=0.0002）的显著增加，这些变化在 8 周龄时就已经出现。在 16 周时，角膜神经支配减少，尤其是在周边。在整个监测期间，穿透部位的数量相对于对照组仍然显著升高。同样，糖尿病前期小鼠的穿透部位数量增加（242.2±13.55 对 305.6±30.96，P=0.003），而神经没有明显变化。
我们的数据表明，糖尿病周围神经病变可能先于神经元生长而不是退化的阶段，并且外周角膜比中心更敏感地检测到神经支配的变化。
