Complement inhibitor prevents disruption of sodium channel clusters in a rabbit model of Guillain–Barré syndrome

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Abstract

Complement-mediated disruption of voltage-gated sodium channels at the nodes of Ranvier acts in the development of acute motor axonal neuropathy. Nafamostat mesilate, a synthetic serine protease inhibitor, used in clinical practice for more than 20 years, has anti-complement activity. Acute motor axonal neuropathy rabbits obtained by GM1 ganglioside sensitization were or were not given nafamostat mesilate intravenously. Complement deposition and sodium channel disruption in the spinal anterior roots were significantly less frequent in the treated rabbits than in the controls. Nafamostat mesilate inhibited complement deposition and prevented sodium channel disruption. This provided the rationale for a clinical trial.

Introduction

Clinical data indicates that complement activation followed by membrane attack complex (MAC) formation is an important mechanism for neuronal and glial injury in Guillain–Barré syndrome (GBS). SC5b-9 (a marker for complement activation) has been detected in GBS sera, and C9neo antigen (a component of MAC) found in segments of myelinated nerve fibers of a GBS patient (Koski et al., 1987). In GBS patients, deposits of complement components along myelinated fibers, C9neo antigen at sites of active myelin breakdown, and MAC on Schwann cell membranes have been reported (Putzu et al., 2000, Wanschitz et al., 2003). GBS is divided into 2 subtypes, acute inflammatory demyelinating polyneuropathy and acute motor axonal neuropathy (AMAN). In reports, many fibers showed a rim of C3dg (a biologically active, degradation product of C3b) and MAC along the outer surface of Schwann cells in patients with acute inflammatory demyelinating polyneuropathy (Hafer-Macko et al., 1996b); whereas, in AMAN patients the nodal and internodal axolemma of myelinated motor fibers was immunostained intensely for C3d and MAC (Hafer-Macko et al., 1996a). Inhibition of complement activation therefore might attenuate the disease process of both GBS subtypes. In a murine model of Fisher syndrome, a variant of GBS, complement inhibitors prevent anti-ganglioside antibody-mediated neuropathy (Halstead et al., 2005, Halstead et al., 2008).

Modeling of AMAN was established in rabbits sensitized with GM1 ganglioside (Susuki et al., 2003, Yuki et al., 2001). Anti-GM1 IgG antibodies cause complement-mediated disruption of clusters of voltage-gated Na+ (Nav) channels at the nodes of Ranvier in peripheral motor nerve fibers of the disease model (Susuki et al., 2007). Because nodal Nav channels are responsible for nerve conduction, disruption of their clusters is likely to cause limb weakness at onset in AMAN rabbits. Several serine proteases activate classical and alternative pathways of the complement system, and a synthetic serine protease inhibitor, nafamostat mesilate (NM; 6-amidino-2-naphtyl-p-guanidino-benzoate dimethanesulfonate), used clinically in Japan for more than 20 years with no serious adverse effects, has anti-complement activity (Fujii and Hitomi, 1981, Nafamostat, 2008). To test whether NM is a suitable candidate for treatment trials on GBS patients, we compared the clinical signs, activated C3 fragment depositions and disrupted Nav channel clusters of NM-treated and non-treated AMAN rabbits.

Section snippets

Induction of the disease model

This research was approved by the Animal Care and Use Committee, Dokkyo Medical University (approval no. 0458). Japanese white rabbits were injected subcutaneously to the back with 1 mg of GM1 (Sigma, St. Louis, MO), keyhole lympet hemocyanin (Sigma) and complete Freund's adjuvant (Sigma) at 3-week intervals until disease onset or 3 weeks after the 7th inoculation (Susuki et al., 2003). Table 1 shows the clinical scale modified from that of a previous report (Nishimoto et al., 2004). Each

Results

Osmotic pumps were implanted in 9 AMAN rabbits. There was no significant difference in any of the baseline characteristics of the NM-treated (n = 4) and non-treated (n = 5) groups (Table 2). Total C3 depositions and large or long forms were significantly less in NM-treated rabbits as compared with non-treated ones (Fig. 1). As reported elsewhere (Susuki et al., 2007), a large or long C3-deposit form was associated with disrupted Nav channel clusters. Disruption or the absence of Nav channel

Discussion

Autoantibodies of the IgG class bind to GM1 at the nodes, initiate complement activation and form C3 products and MAC, then disrupt Nav channel clusters in the spinal anterior roots of the AMAN model (Susuki et al., 2007). Anti-rabbit MAC antibodies were unavailable for the present study. Because MAC formation is associated with activated C3 fragment deposition as well as with Nav channel cluster disruption (Susuki et al., 2007), we evaluated complement activation by deposited C3 fragments and

Acknowledgments

This work was supported by a grant for Hi-Tech Research from Dokkyo Medical University (K.H.). We thank Prof. T. Kinoshita (Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University) for suggesting NM as a candidate for GBS treatment; Messrs. H. Hirata and Y. Machida for animal assistance (Animal Research Center, Dokkyo Medical University); Ms. F. Yokotsuka (Institute for Medical Science, Dokkyo Medical University) for microscopy assistance; and Drs. S.

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