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H. Matsui, H. Terasaki, M. Nakamura, Y. Tokita, M. Watanabe; Rho/ROCK Inhibitor Enables Cat Retinal Ganglion Cells to Regenerate Axons Into the Crushed Optic Nerve . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1578.
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© ARVO (1962-2015); The Authors (2016-present)
A novel Rho/ROCK inhibitor, Y39983, promotes axonal regeneration of retinal ganglion cells (RGCs) of adult rats (Takayama et al., 2004 ARVO). We examined whether Y39983 can make injured RGC axons regenerate into the crushed optic nerve (OpN) of cats.
Retinal culture: The eye was enucleated from anesthetized adult cats. The retina was dissected aseptically. Small pieces of the retina were embedded in collagen gel, and cultured in Dubeccos MEM containing Y39983 for 14 days. After fixation with paraformaldehyde, the neurites were stained with anti–TUJ–1 antibody. Number and length of TUJ–1 positive processes were obtained. Microcrush of optic nerve (OpN): After an intravitreal injection of drug or saline, the left OpN of anesthetized cats was tied up with 6–0 surgery thread. The OpN was crushed with 0.2 N tention for 60 s (Okada et al., Exp. Neurol. 196:153). Anterograde labeling of regenerated axons: Twelve days after crush, 0.5 mg of WGA–HRP in 30 micro L saline was injected into the vitreous. On day 14, the cats were deeply anesthetized, perfused with 1% glutaraldehyde–1% paraformaldehyde in 0.1 M phosphate buffer. The OpN was dissected and embedded in gelatin. Sections were cut in a cryostat, reacted for HRP with TMB reaction.
Retinal culture: To obtain the optimum concentration for axonal regeneration, we examined effect of Y39983 on neurite outgrowth of cultured retinal pieces. On day 14 the number of TUJ–1 positive processes was maximum at the concentrations of 3 and 10 micro M in central, intermediate and peripheral retinas. Similarly, length of neurites in retinal pieces was longest at 3 and 10 micro M. No neurites protruded in culture containing 33 micro M or 100 micro M Y39983. Axonal regeneration in crushed OpN: Taking account of drug leak from the vitreous (mean volume, 2.7 ml), we injected Y39983 at 10 and 100 micro M. An injection of 10 micro M Y39983 increased regenerated axons longer than 0.5 mm from the crush site. Second injection of 10 micro M Y39983 at day 7 increased the number 2 fold at 0.5 mm, 3.5 fold at 2 mm than in one injection of 10 micro M Y39983. Single or double injections of 100 micro M 39983 increased the number regenerated axons.
Rho/ROCK inhibitor, Y39983, enabled injured axons of RGCs of adult cats to regenerate into the crushed OpN.
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