Purpose

Impairment of corneal nerves can lead to neurotrophic keratopathy accompanied with severe ocular surface damage, which due to limited treatment options, can result in severe visual deterioration. This study evaluates a possible new treatment by enhancing the corneal nerve regeneration using a Rho Kinase inhibitor (Y27632). ROCK is known to play an important role in regulating cell morphology, adhesion and motility but little is known about its role in corneal nerve regeneration.

Methods

Effects of ROCK inhibition on murine peripheral nerves was assessed in single cell- and wound healing assays as well as a 3D in vitro model. Furthermore, Sholl analysis evaluating neuronal branching and life-death assays evaluating toxicity of the inhibitor were performed. An in vivo mouse model was established, with monitoring weekly corneal nerve regrowth using confocal microscopy. Additionally, corneal nerve fiber length was evaluated by immunofluorescence staining. Underlying pathways were examined by qrtPCR.

Results

ROCK inhibition leads to a significant enhancement of fiber growth in vitro. Sholl analysis revealed a higher degree of branching of treated fibers. Cytotoxicity assay showed no influence of Y27632 on cellular survival. In vivo measurement revealed significant enhanced regeneration after injury in the treated group. QrtPCR of trigeminal ganglia confirmed ROCK knock-down as well as altered pathways.

Conclusion

The inhibition of ROCK after corneal nerve injury resulted in an enhanced regrowth of fibers in vitro and in vivo. This might be a step towards a new therapeutic concept for the treatment of impaired corneal nerves in diseases such as neurotrophic keratopathy.

中文翻译：

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Rho激酶抑制在体内外对角膜神经再生的影响

目的

角膜神经受损可导致伴有严重眼表损伤的神经营养性角膜病变，由于治疗选择有限，可导致视力严重恶化。本研究通过使用 Rho 激酶抑制剂 (Y27632) 增强角膜神经再生来评估可能的新治疗方法。已知 ROCK 在调节细胞形态、粘附和运动方面发挥重要作用，但对其在角膜神经再生中的作用知之甚少。

方法

在单细胞和伤口愈合试验以及 3D 体外模型中评估了 ROCK 抑制对小鼠外周神经的影响。此外，还进行了评估神经元分支的 Sholl 分析和评估抑制剂毒性的生死分析。建立了体内小鼠模型，使用共聚焦显微镜监测每周角膜神经再生。此外，通过免疫荧光染色评估角膜神经纤维长度。通过qrtPCR检查潜在途径。

结果

ROCK 抑制导致体外纤维生长的显着增强。Sholl 分析显示处理过的纤维具有更高的支化度。细胞毒性试验显示 Y27632 对细胞存活没有影响。体内测量显示治疗组损伤后再生显着增强。三叉神经节的 QrtPCR 证实了 ROCK 敲低以及通路的改变。

结论

角膜神经损伤后 ROCK 的抑制导致体外和体内纤维的再生增强。这可能是朝着治疗神经营养性角膜病变等疾病中受损角膜神经的新治疗概念迈出的一步。