Elsevier

Brain Research

Volume 898, Issue 2, 20 April 2001, Pages 281-287
Brain Research

Research report
The use-dependent sodium channel blocker mexiletine is neuroprotective against global ischemic injury

https://doi.org/10.1016/S0006-8993(01)02195-3Get rights and content

Abstract

Mechanisms responsible for anoxic/ischemic cell death in mammalian CNS grey and white matter involve an increase in intracellular Ca2+, however the routes of Ca2+ entry appear to differ. In white matter, pathological Ca2+ influx largely occurs as a result of reversal of Na+–Ca2+ exchange, due to increased intracellular Na+ and membrane depolarization. Na+ channel blockade has therefore been logically and successfully employed to protect white matter from ischemic injury. In grey matter ischemia, it has been traditionally presumed that activation of agonist (glutamate) operated and voltage dependent Ca2+ channels are the primary routes of Ca2+ entry. Less attention has been directed towards Na+–Ca2+ exchange and Na+ channel blockade as a protective strategy in grey matter. This study investigates mexiletine, a use-dependent sodium channel blocker known to provide significant ischemic neuroprotection to white matter, as a grey matter protectant. Pentobarbital (65 mg/kg) anesthetized, mechanically ventilated Sprague–Dawley rats were treated with mexiletine (80 mg/kg, i.p.). Then 25 min later the animals were subjected to 10 min of bilateral carotid occlusion plus controlled hypotension to 50 Torr by temporary partial exsanguination. Animals were sacrificed with perfusion fixation after 7 days. Ischemic and normal neurons were counted in standard H&E sections of hippocampal CA1 and the ratio of ischemic to total neurons calculated. Mexiletine pre-treatment reduced hippocampal damage by approximately half when compared to control animals receiving saline alone (45 vs. 88% damage, respectively; P<0.001). These results suggest that mexiletine (and perhaps other drugs of this class) can provide protection from ischemia to grey matter as well as white matter.

Introduction

The metabolic events associated with ischemic neuronal injury are complex. Numerous pathophysiological changes have been correlated with subsequent cell death. An excess release of glutamate and related excitatory substances, with a resultant increase in intracellular Ca2+, has traditionally been viewed as central in ischemic cell injury, particularly in grey matter [4], [5]. In white matter, a key role for Na+–Ca2+ exchange has been shown [13], [28] and the prevention of pathological Na+ influx by Na+ channel blockade has been found to be an effective strategy for protection of white matter in anoxia [13], [28]. Na+ channel blockade, and the contribution of pathological Na+ influx have received relatively less attention in relation to grey matter ischemia. Nonetheless, reports have appeared evaluating Na+ channel blocking agents as neuroprotectants. For example, lidocaine [6], [24], [22], [30], tetrodotoxin [2], [17], [19], [34] and phenytoin [1] are some of the Na+ channel blockers that have been evaluated. Often the effects demonstrated have been modest. Nonetheless, these results suggest that Na+ entry cannot be ignored as a contributing factor to grey matter damage resulting from an ischemic insult.

Sustained depolarizations that lead to non-inactivating Na+ currents are often seen during ischemia, and a massive TTX-sensitive Na+ accumulation occurs in in vitro brain slices during anoxia [15], [31]. The enhanced excitation during ischemia coincides with prolonged influxes of sodium during the depolarization process. These findings suggest that Na+ channels can contribute to the general excitability of the cell, prolonged Na+ influx, and a proceeding rise in cytotoxic levels of Ca2+. Under the pathological conditions of high Na+ influx, it is thought that the Na+–Ca2+ exchanger reverses and causes Ca2+ influx as Na+ is extruded [18]. In support of this, Stys et al. [29] have demonstrated in a model of white matter anoxia, that extracellular Ca2+ was necessary for damage to be incurred, and that ionic conditions favouring a reversal of the normal transmembrane Na+ gradient during the anoxic period resulted in greater injury. The reduction of Na+ permeability during ischemia could therefore potentially prevent a secondary Ca2+ influx into CNS neurons, and reduce ischemic damage.

While irreversible neuronal injury due to ischemia is thought to depend largely on Ca2+ influx, the mechanisms of Ca2+ entry may differ significantly between myelinated axons and neuronal cell bodies. During an anoxic challenge it is thought that the majority of Ca2+ flux in central, myelinated axons is mediated by the reversal of the Na+–Ca2+ exchanger, as described above [27], [29], although Ca2+ entry via axonal voltage-dependent Ca2+ channels cannot be ruled out as a contributing factor since Ca2+ channel blockers can significantly protect rat optic nerve from anoxic injury [8]. Nonetheless, in vitro experimental models of white matter anoxia have strongly focused on the link between Na+ entry to subsequent cell death by demonstrating the neuroprotective effects of sodium channel blockade using TTX [29], local anesthetics [28], antiarrhythmics [25], and certain anticonvulsants [7]. The traditional view of grey matter injury suggests the contribution of the Na+–Ca2+ exchanger in increasing intracellular Ca2+ is minimal in comparison. Yet Na+-mediated modes of injury may be present in grey matter. For example, Na+ has been implicated as a contributor to excitotoxicity from glutamate release via reversed Na+-glutamate co-transport [16], [32], [33]. Na+-dependent neuronal edema may also be present [4], [23]. In addition, the extent of Ca2+ entry by a reversal of the Na+–Ca2+ exchanger in global models of ischemia has not been determined. The net contributions of these Na+-dependent factors in cell body injury may be substantial and certainly merit consideration when designing potential therapeutic interventions.

Recently, we have demonstrated a direct correlation between Na+ channel blockade by the use-dependent sodium channel blocker, mexiletine, and ischemic protection in CNS white matter [26]. In addition, after intraperitoneal administration, in situ examination of CNS tissue suggested that mexiletine concentrations reached levels high enough to afford significant protection [26]. This particular characteristic may lend a particular advantage to the compound in instances of in vivo ischemia. Mexiletine is a primary amine with a pKa of 8.4, and exists in both neutral and protonated forms at physiological pH. The neutral form is able to cross the blood–brain barrier, where it is likely converted to the protonated form during the acidophilic conditions of ischemia. In addition, this protonated form has been proposed to be more potent at blocking open Na+ channels during an anoxic exposure [26]. These findings have characterized mexiletine as a use-dependent Na+ channel blocker that is capable of CNS penetration, and that can offer neuroprotection to white matter from ischemic injury. Given these characteristics of mexiletine, an assessment of the protective ability of mexiletine during a global ischemic insult affecting grey matter was warranted.

Section snippets

Materials and methods

A total of 23 male, Sprague–Dawley rats (Charles River, St. Constant, Québec), weighing 300–450 g, were randomly assigned to treatment groups. The experiments were approved by the animal care committee of the Ottawa Civic Hospital and the guidelines for animal care set out by the National Institutes of Health and the Canadian Council on Animal Care were strictly followed. Animals were denied access to food 6 h prior to surgery. After pretreatment with atropine (0.5 mg/kg, i.p.), general

Results

Following 10 min of global ischemia, control animals receiving saline (n=11) sustained 88±11% CA1 neuronal injury (Fig. 1). Pre-treatment with mexiletine (n=12) reduced this significantly by 50%, to 45±32% (P<0.001, Mann–Whitney U) (Fig. 1). Representative photomicrographs of hippocampal CA1 from saline treated animals are shown in Fig. 2A and from mexiletine treated animals in Fig. 2B.

The monitoring of arterial blood pressure demonstrated a dramatic hypotensive effect of mexiletine. In

Discussion

The use-dependent sodium channel blocker mexiletine significantly reduced neuronal damage in the CA1 sector of the dorsal hippocampus after global ischemia. This suggests that intra-ischemic Na+ influx is detrimental to cell survival, and contributes to the compromising metabolic cascade. These results are consistent with those of previous studies which have implemented various Na+ channel blockers as a method of averting cell death. For example, Prenen et al. [19] found that TTX could prevent

Acknowledgements

This research was supported by the Heart and Stroke Foundation of Ontario. Mexiletine–HCl was a generous gift from Boehringer-Ingelheim.

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