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Sonja Mertsch, Inga Neumann, Gerd Geerling, Stefan Schrader; Evaluation of the effects of Rho Kinase Inhibitor on corneal nerve regeneration in vivo using confocal microscopy. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4148.
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Injury or damage of the corneal nerve plexus (CNP) is one of the main causes of neurotrophic keratopathy as well as neurogenic induced dry eye syndrome. Spontaneous regeneration of the corneal nerves is often incomplete and the underlying mechanisms are mostly unknown. Therefore, stimulation of corneal nerve regeneration might be a promising curative approach to treat these diseases. The aim of this study was to evaluate the effect of Rho kinase inhibitor on corneal nerve regeneration in a novel in vivo model using confocal microscopy.
The CNP of the right eyes of 8 – 10 week old C57BL/6 mice (n=24) was cut through with a 1.2 mm trephine. Eyes were treated twice a day with eye drops containing either 100µM Y27632/NaCl/50%Glycerin or NaCl/50%Glycerin as control. At D7, D14, D21 and D28 measurements of corneal nerves using confocal microscope HRTIII with RCM was performed. Nerve length was evaluated using CCMetrix software in 25 HPF/mouse. After decapitation at D14 and D28, corneas were removed, fixed and whole mount fluorescence staining using βIIItubulin was done. Trigeminal ganglia (TG) were removed and qrtPCR was performed using ROCK1 (R1) and 2 (R2), LIMK, cdc42, GAP43 and NGF comparing TG of treated, operated side (op-G) with control-treated, operated side (op-Co-G) or with native side (Co-G).
Fiber length measurement using HRT data revealed a highly significant increase of mean fiber length after 4 weeks from 3.37 ± 0.33 mm control group to 7.73±0.38 mm in the Y27632 treated group (n=6 per group). QRT-PCR analysis revealed slight changes in expression in the TG comparing the ganglion of the treated eye side with a non-treated operated eye side or with native non-treated eye side. ROCK2 showed a significant reduction of expression level at D14 of 89.1% ±0.87% (p=0.007) compared to native control (Co-G). NGF showed an increase at D14 to 113.5%±4.51% with a reduction to 96.7%±3.78% at D28 compared to Co-G (p=0.049). Comparing op-G with op-Co-G, no significant changes in the expression of primers were found except a trend for GAP43 D28 (176.8%±8.63%, p=0.088).
The data showed that the inhibition of Rho kinase leads to a significant enhanced neuronal fiber length in the damaged cornea compared to control. This might be a step towards a new therapeutic concept to treat impairment of corneal nerves in diseases such as neurotrophic keratopathy.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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