A novel ROCK inhibitor, Y-39983, promotes regeneration of crushed axons of retinal ganglion cells into the optic nerve of adult cats

https://doi.org/10.1016/j.expneurol.2007.02.002Get rights and content

Abstract

We investigated the effect of a novel ROCK inhibitor, Y-39983, on neurite regeneration in vitro and axonal regeneration in the crushed cat optic nerve in vivo. To determine the effective dose for neurite regeneration, retinal pieces were cultured with ROCK inhibitors, Y-39983 or Y-27632, a well-characterized ROCK inhibitor, and the number and length of TUJ-1-positive neurites were evaluated. The greatest number of neurites protruded at a dose of 3–10 μM Y-39983 and at a dose of 10–100 μM Y-27632, respectively. The neurite number at maximum effect of Y-39983 was greater than that of Y-27632. No significant difference was observed between values of neurite length with the inhibitors. Based on this finding, we examined the effect of Y-39983 on axonal regeneration in the crushed optic nerve in vivo. Immediately after crushing the left optic nerve, Y-39983 was injected into the vitreous and the crushed site. An injection of 10 μM Y-39983 induced the crushed axons to regenerate and pass over the crush site. In contrast, very few axons passed beyond the crush site in the optic nerve with phosphate-buffered saline injection. The second injection of 10 μM Y-39983 on day 7 doubled the number of regenerated axons, suggesting that new axons may have entered into the optic nerve after day 7 and that a continuous supply of the drug may make more axons to regenerate.

Introduction

Evidence for the axonal regeneration of retinal ganglion cells (RGCs) has been accumulated in adult mammals (So, 1988, Watanabe et al., 1997, Watanabe and Fukuda, 2002, Koeberle and Bähr, 2004). Axons of RGCs regenerate into a transplanted peripheral nerve (So and Aguayo, 1985, Keirstead et al., 1985, Watanabe et al., 1991, Watanabe et al., 1993, Dezawa and Adachi-Usami, 2000), but the number of regenerated fibers comprises only 10% of the total optic fibers (Vidal-Sanz et al., 1991, Watanabe et al., 1993, Watanabe and Fukuda, 2002). Moreover, terminals of regenerated fibers from the graft form synapses only within 0.5 mm in the superior colliculus (Vidal-Sanz et al., 1991, Carter et al., 1998), and no obvious evidence has been reported to support functional synaptic formation in the lateral geniculate nucleus (Carter and Jhaveri, 1997, Watanabe and Fukuda, 2002). Crushed axons of RGCs extend across the crush site in the optic nerve (OpN) of adult rats and cats when RGCs are activated by macrophages (Leon et al., 2000, Yin et al., 2003, Yin et al., 2006, Okada et al., 2005, Lorber et al., 2005) that secrete a 12-kDa protein, oncomodulin (Yin et al., 2006). Oncomodulin elicits robust rat OpN regeneration in collaboration with cAMP and mannose (Yin et al., 2006). These reports show that the ability of axonal regeneration of RGCs depends not only on the environment surrounding the injured axons but also on their own cellular properties.

The identified molecules that inhibit axonal regeneration in CNS, such as myelin-associated glycoprotein (MAG), Nogo and oligodendrocyte myelin glycoprotein (OMgp), exist in the oligodendrocyte membrane and glial scars at lesion sites. The molecules prevent axonal elongation by influencing intracellular signal transduction pathways (Yamashita and Tohyama, 2003, Sandvig et al., 2004, Mueller et al., 2005). In brief, MAG, Nogo or OMgp signals Rho GTPase through p75, their common receptor, to phosphorylate Lim kinase via Rho kinase (ROCK), which results in growth cone collapse. The proposed cascade suggests the potential for regenerating RGC axons by blocking the ROCK pathway. In fact, Rho inhibition with C3 transferase or ROCK inhibition with Y-27632 makes axons of CNS neurons regenerate in an inhibitory milieu (Lehmann et al., 1999, Dergham et al., 2002, Ellezam et al., 2002, Borisoff et al., 2003, Bertrand et al., 2005).

Here, we studied the effect of a novel ROCK inhibitor, Y-39983 (Nakajima et al., 2005), on the axonal regeneration of RGCs in adult cats. Y-39983 inhibits more potently ROCK by about 30 times than Y-27632, a common specific ROCK inhibitor (Uehata et al., 1997, Tokushige et al., in press), and promotes axonal regeneration of RGCs into a peripheral nerve segment transplanted to the cut OpN of adult rats (Takayama et al., 2004). To examine the most effective dose of the drug for axonal regeneration, we first cultured pieces of the cat retina. We also compared Y-39983 with Y-27632. Then, we injected Y-39983 into the vitreous and the crush site after microcrush of the OpN. Anterograde labeling of the optic fibers demonstrated the robust regeneration of crushed axons. Moreover, the second injection of Y-39983 resulted in an increase of regenerated fibers by 2- to 3.5-fold, implying that frequent administration of the drug may result in a larger number of regenerated fibers.

Section snippets

Animals and anesthesia

Animal treatments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, and the Institutional Guidelines for Laboratory Animal Care and Treatment. The number of animals used as well as their suffering was minimized. Adult cats of either gender, weighing 1.5 to 3.0 kg, were used in this study. The cats were sedated with an intramuscular injection of 50 mg ketamine hydrochloride (Sankyo, Tokyo, Japan), and injected subcutaneously with

Cell survival in culture with Y-39983

Activation of Rho/ROCK pathways mediates diverse cellular processes including apoptosis (Petrache et al., 2001, Coleman et al., 2001). Therefore, we examined whether Y-39983 induced protection against cell death that resulted in a greater number of regenerated axons. The LDH-cytotoxic test showed that the ratio of absorbance at 570 nm versus that at 450 nm in cultures containing 10 μM Y-39983 or 30 μM Y-39983 was similar to the ratio in the drug-free cultures (control, Fig. 2). This finding

Discussion

Molecules inhibiting regeneration of CNS axons exist in the oligodendrocyte membrane (Caroni and Schwab, 1988) and glial scars (Davies et al., 1997). These molecules have been identified as MAG (McKerracher et al., 1994, Mukhopadhyay et al., 1994), Nogo (Chen et al., 2000, Grandpre et al., 2000, Prinjha et al., 2000), OMgP (Wang et al., 2002), and can activate Rho-ROCK pathways to prevent axonal elongation (Schnell and Schwab, 1990, Jalink et al., 1994, Arimura et al., 2000, Fournier et al.,

Acknowledgments

We thank Dr. M. Takano for the technical advice on retinal 3D culture, Dr. T. Yamashita for the valuable comments on the manuscript, Dr. H. Tokushige for providing information about Y-39983, and Dr. A. Oohira for the support and encouragement during the study. This study was supported by Grants-in-Aid for Science Research from the Ministry of Education, Culture, Sports, Science and Technology to H.T. (16390497, 18390466), M.N. (16591746) and M.W. (16591780) and Grant-in-Aid from the Ministry of

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