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M. Watanabe, M. Ichikawa, H. Sagawa, Y. Tokita; Effects of Two Rho Inhibitors on Axonal Regeneration Into the Crushed Cat Optic Nerve. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5513.
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Inhibition of Rho pathways results in axonal regeneration in the crushed cat optic nerve (OpN) (Sagawa et al., 2007). To know whether promotion of extension of neurites and/or glial processes is necessary for axonal regeneration, we investigated effects by Fasudil, which is used clinically to relieve vaso-spasm in patients after subarachnoid hemorrhage, and by Y-27632 on process extension in culture and axonal regeneration in the OpN.
Retinal Culture To determine the optimal dose, retinal pieces were cultured with Fasudil or Y-27632 and the number and length of extending processes were measured at day 14. Processes in some plates were immunostained with anti-TUJ-1 antibodies for neurites and anti-GFAP antibodies for glial processes. OpN Regeneration Fasudil or Y-27632 was injected into the vitreous and the crush site after the OpN was microcrushed applying 0.2 N tension, 60 s. Regenerated fibers were anterogradely labeled with WGA-HRP injected into the vitreous at day 12. At day 14 the animals were perfused with fixative. The dissected OpN was sectioned at 30 µm in a cryostat and the sections were reacted with HRP reaction. Numbers and length of HRP-positive fibers were measured in whole series of sections.
Neurites in culture: Many processes protruded at doses of 10 - 100 µM for Y-27632 and 10 - 30 µM for Fasudil but very few neurites in control culture containing no drugs. Most processes were neurites in 10 µM Y-27632, 55% of them were neurites and 45% were glial processes in 100 µM Y-27632, and all processes were glial in Fasudil. The number and length of neurites were greatest in 100 µM Y-27632. Regeneration in crushed OpN: Injection of 10 µM and 100 µM Y-27632 induced extension of axons beyond the crush site. The numbers of regenerated axons at 0.5 mm were 3-fold in 10 µM Y-27632, at which mainly neurites extended in culture, and 5-fold in 100 µM Y-27632, at which neurites and glial processes extended in culture, than in PBS injection. No axons extended beyond the crush site with Fasudil injection. We measured the length of 10 longest axons from the crush site in each OpN. The average length of these axons was 1.3-fold longer in 100 µM Y-27632 than those in PBS or 10 µM Y-27632 injection. Injection of 300 µM Y-27632 decreased the mean number of regenerated axons into 22% of 100 µM Y-27632 at 0.5 mm.
The results suggest that neurite extension is essential for OpN regeneration and glial process extension may be beneficial to elongate neurite extension.
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