Elsevier

Brain Research

Volume 959, Issue 1, 3 January 2003, Pages 29-38
Brain Research

Research report
A possible role of RhoA/Rho-kinase in experimental spinal cord injury in rat

https://doi.org/10.1016/S0006-8993(02)03717-4Get rights and content

Abstract

Secondary injury following traumatic spinal cord injury is induced by the activation of a number of cellular and molecular changes. RhoA, a small GTPase, regulates the organization of the actin cytoskeleton, gene expression, cell proliferation, and has been implicated in the regenerative process. This study was undertaken to investigate the involvement of the RhoA signaling pathway in the secondary injury that follows traumatic spinal cord injury in rats. RhoA mRNA and protein expressions were enhanced significantly in the injured spinal cord 1 week after surgery (P<0.05, ANOVA). C3 exozyme (RhoA inhibitor), Y-27632 (selective Rho kinase inhibitor), and Fasudil (non-selective protein kinase inhibitor) were administered after spinal cord injury, and the subjects were evaluated for 5 weeks as per BBB locomotor score. Poor rat response interrupted the C3 experiment. Y-27632 slightly, but significantly (P<0.05, ANOVA), delayed the recovery. Fasudil significantly improved the BBB score (P<0.05, ANOVA). In conclusion, spinal cord injury activates the RhoA/Rho-kinase α, β associated pathway. However, their role in secondary injury or in the improvement of functional recovery remains unclear. Fasudil might exert a cytoprotective effect by mechanisms other than inhibiting Rho-kinase α, β.

Introduction

Secondary injury following traumatic spinal cord injury is induced by activating a number of cellular and molecular changes [9], [11]. These secondary pathophysiological events result in a host of biochemical changes, including impaired spinal cord blood flow, intracellular Ca2+ overload, excess release of excitatory amino acids, free radical formation, apoptosis, immune activation, and altered gene expression [9], [5], [32]. Derangement of the neuronal cytoskeleton is another important pathological consequence of traumatic central nervous systemic injury [10].

The small GTPase RhoA belongs to the Rho subfamily of GTPases within the Ras superfamily. The best characterized members of this subfamily, which include RhoA-E, RhoG, Rac1-2, Cdc42, and TC10, regulate the organization of the actin cytoskeleton, gene expression, and cell proliferation [8], [14], [26]. A specific extracellular signal activates Rho family proteins and directs the organization of a specific type of actin cytoskeleton to induce the characteristic morphological change(s), respectively [25]. Although the effects of Rho GTPases on the organization of the actin cytoskeleton are perhaps still best characterized in fibroblasts, compelling evidence now demonstrates a similar role for these proteins in all eukaryotic cells including neuronal cells [8], [21]. Rho family members are implicated in the regenerative process, regulating growth cone formation, and elongating neurites.

The possible role of RhoA in secondary injury following spinal cord injury was investigated in this study. Based on the study by Hara et al. [9] that reported that Fasudil hydrochloride showed effectiveness in promoting neurological recovery after traumatic spinal cord injury, we hypothesize that RhoA and its downstream Rho-kinase α, β associated pathway participate in the secondary injury of the injured spinal cord. The spatiotemporal expression of RhoA mRNA and protein expressions, and the effects of RhoA (C3), Rho-kinase (Y-27632), and protein kinase (Fasudil) inhibitors on motor recovery were evaluated.

Section snippets

Materials and methods

All animal experiments were performed in accordance with the guidelines of the National Institutes of Health (NIH) and were approved by the University of Mississippi Medical Center Institutional Standing Committee on Animals.

RhoA mRNA expression in the spinal cord

The competitive RT-PCR assay was used to measure RhoA mRNA collected from the spinal cords of the control group and the rats sacrificed at 1 h, 3 h, 1 day, 3 days, 1 week, and 3 weeks after injury. The expression level of RhoA mRNA (molecules/μg total mRNA) was very low in the uninjured spinal cords and decreased slightly at 1 h after injury. The level of RhoA mRNA expression increased slightly in the injured cords during the period 1–3 days after injury, and values of expression levels peaked

Discussion

In the present study, we have demonstrated the following: (1) the weight-drop method we used created a spinal cord injury model that clinically and histologically resembles the weight-drop models reported by other investigators. (2) RhoA mRNA was highly expressed in contused spinal cord samples taken 1 week post-injury when compared to the samples collected from the control group. (3) The RhoA protein expression directly and significantly increased in contused spinal cords, especially with

Acknowledgements

This work was partially supported by a grant from the American Heart Association National Grant-in-Aid, and a grant from the American Heart Association Bugher Foundation Award for the Investigation of Stroke to J.H.Z.

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      Interestingly, RhoA inhibition with C3 exoenzyme has been shown to reduce cell death following SCI in rodents demonstrating the potential neuroprotective benefits of RhoA inhibition (Dubreuil et al., 2003). Cell penetration with C3 exoenzyme is very low, and as a result has yielded varying results in SCI models (Dergham et al., 2002; Dubreuil et al., 2003; Fournier et al., 2003; Sung et al., 2003). Ba-210 (trademarked as Cethrin) is a recombinant engineered variant of C3 exoenzyme that can readily cross the dura of the spinal cord and has shown promising effects in rodent SCI models, however, its role in blocking CSPGs effects have not been tested (Lord-Fontaine et al., 2008).

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