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The use-dependent sodium channel blocker mexiletine is neuroprotective against global ischemic injury
Brain Research 898 (2001) 281–287 www.elsevier.com / locate / bres
Research report
The use-dependent sodium channel blocker mexiletine is neuroprotective against global ischemic injury Kimberley E. Hewitt, Peter K. Stys*, Howard J. Lesiuk Loeb Health Research Institute, Division of Neuroscience, Ottawa Hospital – Civic Campus, University of Ottawa, 725 Parkdale Avenue, Ottawa, Ont., Canada K1 Y 4 K9 Accepted 23 January 2001
Abstract Mechanisms responsible for anoxic / ischemic cell death in mammalian CNS grey and white matter involve an increase in intracellular Ca 21 , however the routes of Ca 21 entry appear to differ. In white matter, pathological Ca 21 influx largely occurs as a result of reversal of Na 1 –Ca 21 exchange, due to increased intracellular Na 1 and membrane depolarization. Na 1 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 Ca 21 channels are the primary routes of Ca 21 entry. Less attention has been directed towards Na 1 –Ca 21 exchange and Na 1 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. 2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Stroke; Neuroprotection; Sodium channel; Mexiletine; Hippocampus
1. 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 Ca 21 , has traditionally been viewed as central in ischemic cell injury, particularly in grey matter [4,5]. In white matter, a key role for Na 1 –Ca 21 exchange has been shown [13,28] and the prevention of pathological Na 1 influx by Na 1 channel blockade has been found to be an effective strategy for *Corresponding author. Tel.: 11-613-761-5444; fax: 11-613-7615330. E-mail address: [email protected] (P.K. Stys).
protection of white matter in anoxia [13,28]. Na 1 channel blockade, and the contribution of pathological Na 1 influx have received relatively less attention in relation to grey matter ischemia. Nonetheless, reports have appeared evaluating Na 1 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 1 channel blockers that have been evaluated. Often the effects demonstrated have been modest. Nonetheless, these results suggest that Na 1 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 1 currents are often seen during ischemia, and a massive TTX-sensitive Na 1 accumulation occurs in in vitro brain slices during anoxia [15,31]. The enhanced
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excitation during ischemia coincides with prolonged influxes of sodium during the depolarization process. These findings suggest that Na 1 channels can contribute to the general excitability of the cell, prolonged Na 1 influx, and a proceeding rise in cytotoxic levels of Ca 21 . Under the pathological conditions of high Na 1 influx, it is thought that the Na 1 –Ca 21 exchanger reverses and causes Ca 21 influx as Na 1 is extruded [18]. In support of this, Stys et al. [29] have demonstrated in a model of white matter anoxia, that extracellular Ca 21 was necessary for damage to be incurred, and that ionic conditions favouring a reversal of the normal transmembrane Na 1 gradient during the anoxic period resulted in greater injury. The reduction of Na 1 permeability during ischemia could therefore potentially prevent a secondary Ca 21 influx into CNS neurons, and reduce ischemic damage. While irreversible neuronal injury due to ischemia is thought to depend largely on Ca 21 influx, the mechanisms of Ca 21 entry may differ significantly between myelinated axons and neuronal cell bodies. During an anoxic challenge it is thought that the majority of Ca 21 flux in central, myelinated axons is mediated by the reversal of the Na 1 – Ca 21 exchanger, as described above [27,29], although Ca 21 entry via axonal voltage-dependent Ca 21 channels cannot be ruled out as a contributing factor since Ca 21 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 1 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 1 –Ca 21 exchanger in increasing intracellular Ca 21 is minimal in comparison. Yet Na 1 -mediated modes of injury may be present in grey matter. For example, Na 1 has been implicated as a contributor to excitotoxicity from glutamate release via reversed Na 1 glutamate co-transport [16,32,33]. Na 1 -dependent neuronal edema may also be present [4,23]. In addition, the extent of Ca 21 entry by a reversal of the Na 1 –Ca 21 exchanger in global models of ischemia has not been determined. The net contributions of these Na 1 -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 1 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 1 channels during an anoxic exposure [26]. These findings have characterized mexiletine as a use-dependent Na 1 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.
2. Materials and methods A total of 23 male, Sprague–Dawley rats (Charles ´ River, St. Constant, Quebec), 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 anesthesia was induced with sodium pentobarbital (65 mg / kg, i.p.). Supplemental doses of sodium pentobarbital (15–30 mg / kg, i.p.) were administered, if needed, to maintain adequate anesthesia. Once anesthesia was induced, the trachea was intubated with a [16 Teflon IV catheter under direct vision with a laryngoscope. Mechanical ventilation (using a Model 665 small animal ventilator, Harvard apparatus, South Natick, MA) was adjusted according to arterial blood gas (ABG) determinations to maintain normocapnia ( pCO 2 535–45 Torr) and normoxia ( pO 2 590–120 Torr), throughout the operative procedure. A thermocouple probe was placed at the tympanic membrane into the middle ear to allow continuous monitoring and adjustment of head temperature. Temperature was maintained at 37.560.28C using a heat lamp as necessary for the duration of the surgery. A [24 Teflon IV catheter, charged with heparinized (10 U / ml) normal saline, was inserted through the tail artery into the descending aorta for: continuous blood pressure monitoring, aspiration of blood to induce controlled hypotension (see below), and arterial blood sampling. For blood pressure monitoring, the cannula was connected to a pressure transducer, and blood pressure was sampled 40 times a second by an analog to digital converter, and stored on a computer disk. Once blood gases and temperature were within the desired ranges, animals were given either mexiletine (80 mg / kg, i.p.; n512) 25 min prior to ischemia or an equal volume of physiological saline (1 ml / kg, i.p.; n511). The
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common carotid arteries were exposed bilaterally through a midline, ventral, cervical incision. Forebrain ischemia was induced through bilateral carotid artery occlusion with temporary cerebral aneurysm clips and coincident controlled hypotension (mean BP55062 Torr) induced by temporary, partial exsanguination. This was achieved by the aortic cannula, with blood withdrawn into a syringe containing 0.5 ml of heparinized (10 U / ml) saline. After 10 min of ischemia, the clips were removed and the blood was reinfused over 30 s. Repeat ABG was then obtained. The cervical incision was sutured, the arterial cannula was removed, and the tail incision closed. Ventilatory support was continued until the animal was breathing well on its own. During this recovery phase, animals were maintained under a heating lamp coupled to a rectal probe, capable of maintaining body temperature within 60.28C of the preset value. Temperature was maintained post-operatively until the animal was completely awake and thermoregulating normally. A number of animals were studied for up to 8 h post-surgery and no late hypothermic events were noted. Food and water were allowed ad libitum following surgery. Animals were allowed to recover for 7 days before being sacrificed under deep barbiturate anesthesia with transcardial perfusion fixation and immediate harvesting of the brains. A series of 7-mm coronal serial sections at the level of the dorsal hippocampus were taken from paraformaldehyde fixed brains embedded in paraffin, and stained with hematoxylin and eosin. The ratio of dead cells to the total cells present was determined for each animal, and expressed as a percentage. The statistical analysis was carried out using a Mann– Whitney U test for non-parametric data. Data are presented as means, and errors are reported as standard deviations.
3. Results Following 10 min of global ischemia, control animals receiving saline (n511) sustained 88611% CA1 neuronal injury (Fig. 1). Pre-treatment with mexiletine (n512) reduced this significantly by 50%, to 45632% (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 addition to its neuroprotective properties, pre-treatment of animals with mexiletine caused a marked reduction in mean arterial blood pressure prior to occlusion. Fig. 3 illustrates the pattern of induced hypotension following mexiletine administration, and the response following 10 min of global ischemia with hypotension (50 mmHg) for each group. Animals receiving mexiletine had an average reduction in blood pressure of 60 mmHg 20 min after drug administra-
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Fig. 1. Bar graph illustrating the degree of neuronal cell death following global ischemia in the hippocampal CA1 region of rats pretreated with either saline or mexiletine (80 mg / kg, i.p.). Heights of the bars represent the number of compromised cells, expressed as a percentage of the total counted. Filled circles (d) represent individual scores from each animal. A 10-min global ischemic insult resulted in the death of 88611% of hippocampal CA1 pyramidal cells, when animals were pretreated with saline. Pretreatment with mexiletine 25 min before ischemia reduced this by half, resulting in only 45632% cell mortality. Bars represent means, and error bars are standard deviations. The asterisk indicates a statistically significant difference from the saline treated group (P,0.001; Mann– Whitney U test for non-parametric data).
tion. Control rats showed relatively no change prior to exsanguination. Post-ischemically, control rats showed a slight elevation in blood pressure in comparison to preischemic baseline measurements (Fig. 3). Rats receiving mexiletine did show an increase in blood pressure following ischemia, however they did not reach their pre-injection baseline level, and remained hypotensive during the post-operative recording period. No differences in body temperature were observed between the treatment groups. However, mexiletine-treated rats were thermoregulated for longer periods of time postsurgically due to an extended recovery time from anesthetic. This was likely due to the sedating effects of mexiletine at this dose, and is consistent with the sideeffects observed in a previous experiment [26]. No other difficulties were observed following mexiletine administration at this dose.
4. Discussion The use-dependent sodium channel blocker mexiletine significantly reduced neuronal damage in the CA1 sector of the dorsal hippocampus after global ischemia. This
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Fig. 2. Photomicrographs of representative coronal sections through the dorsal hippocampus CA1 region of rats treated with saline (A) or mexiletine (B). Saline-treated rats incurred severe CA1 damage when assessed at 7 days post-ischemia. A significant loss of CA1 pyramidal cells was evident as exemplified in the photomicrograph (A). By contrast, mexiletine administration (80 mg / kg, 25 min pre-occlusion) resulted in a marked improvement in CA1 pyramidal cell survival (B). The number of remaining viable neurons was greatly increased, and a continuous band of pyramidal cells can be seen. Scale bar represents 50 mm.
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Fig. 3. Illustration of the hypotensive effects mexiletine. Initial blood pressure readings did not differ between the two groups. However, following mexiletine administration, blood pressure fell dramatically, and by 20 min post-administration, blood pressure had dropped by an average of 60 mmHg. During ischemia, exsanguination maintained hypotension at 50 mmHg. Post-ischemically, blood pressure returned to a slightly higher level than pre-ischemic values in saline treated rats. While those rats receiving mexiletine did show a rise in post-ischemic pressure above that recorded immediately before exsanguination, mean arterial blood pressure did not return to the initial levels observed prior to drug administration. Filled circles (d) represent the mean pressure values for saline-treated animals, while open circles (s) represent mexiletine-treated rats. Error bars represent the standard deviation from the mean.
suggests that intra-ischemic Na 1 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 1 channel blockers as a method of averting cell death. For example, Prenen et al. [19] found that TTX could prevent increases in Na 1 concentration (as indicated by measuring cation concentrations in brain tissue by flame emission spectrometry) with a coincident increase in cell survival. Burke and Taylor [2] found that TTX administration also prevented glutamate release during ischemia. More recently, Yamasaki et al. [34] and Lysko et al. [17] reported that TTX via intracranial injection afforded protection from ischemic cell death in both rats and gerbils, respectively. While intracranial administration of TTX does not represent an attractive therapeutic approach, these findings strongly support a role for Na 1 influx as a contributing factor in ischemic neuronal death. Administration of other Na 1 channel blockers such as phenytoin [1] and lidocaine [22,24,30] have also resulted in improvements in cell outcome after ischemia. More recently, a use-dependent blocker of voltage-dependent sodium channels, BW619C89, was also shown to be successful in reducing infarct volume after MCA occlusion in the rat [11]. Use-
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dependent compounds such as mexiletine and BW619C89 may offer more advantages therapeutically since they would be most effective during instances of high open channel activity, while leaving normal signalling relatively unaffected. Together, these findings suggest that Na 1 influx resulting from ischemia contributes to the pathological events leading to cell death, and that a reduction in Na 1 permeability via Na 1 channel blockade can effectively reduce cellular damage. While it is clear that Na 1 channel blockade may be advantageous in reducing ischemia-induced cell death, the role of Na 1 in producing grey matter injury following an ischemic insult is not as well characterized mechanistically as it is for white matter injury. As discussed, in models of white matter anoxia, it has been shown that energy failure is followed by axonal depolarization, and Na 1 influx via non-inactivating Na 1 channels, causing a reversal of the Na 1 –Ca 21 exchanger [29]. This leads to an increase in intracellular Ca 21 , and the pathological activation of Ca 21 dependent effector systems. An interruption of this cascade by Na 1 channel blockers in white matter anoxia is neuroprotective [7,21,25,28,29]. The results of this experiment, and those discussed above using other Na 1 channel blockers, suggest that this relationship between Na 1 influx and anoxia / ischemia-induced injury may indeed be similar in neuronal cell bodies. In support of this, an increase in [Na 1 ] i has been observed following anoxia in dissociated hippocampal neurons, and in cultured cortical neurons [9], and removal of [Na 1 ] o can attenuate anoxia-induced injury [10]. The present study has demonstrated that mexiletine is capable of significantly ameliorating cellular injury in a global ischemia model, putatively by reducing Na 1 permeability. This inhibition could ultimately impact a number of factors which contribute to cellular decline. Edema and cell swelling may be direct consequences of Na 1 influx, resulting in cellular damage independently of Ca 21 mediated events [9]. Na 1 -mediated anoxia-induced depolarization can also activate voltage-gated Ca 21 channels [12] causing a coincident increase in intracellular Ca 21 . Therefore, an increase in intracellular Na 1 can impact the cell in Ca 21 -dependent and independent ways, both rendering it susceptible to anoxic / ischemic damage. In addition, Na 1 / glutamate co-transport may be affected by increased intracellular Na 1 , resulting in the release and / or diminished uptake of glutamate during an anoxic / ischemic event [16,32,33]. Further mechanistic studies, such as the determination of glutamate release, or Ca 21 entry following mexiletine administration in instances of ischemia, will be necessary to more precisely define the specific effects of the drug. Mexiletine administration also caused significant hypotension in rats beginning soon after drug delivery. These hypotensive effects are most likely to be detrimental to outcome. This suggests that other compounds without such properties and with greater selectivity for neuronal Na 1 channels versus those in the myocardium and vascular tree
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may be even more effective in preventing cell death. Indeed, the differences in molecular structure of neuronal versus cardiac and muscle Na 1 channels [14,20] suggests that developing more selective compounds may be feasible. The possibility remains that the hypotensive effects of mexiletine may be contributing to the positive effects of the drug, although it seems unlikely. Peri-ischemic hypotension correlates with a poor prognosis in the clinical setting and with increased cellular injury in ischemia models (for example, see Zhu and Auer [35]). In addition, induced hypertension during ischemia reduces infarct size [3] suggesting that the maintenance of blood pressure at or above normal levels results in less damage following ischemia. An examination of other drugs in this class that do not result in hypotension may prove to be advantageous when treating ischemic injury. Nonetheless, mexiletine provided marked neuroprotection despite its coincident hypotensive effects, suggesting Na 1 channel blockade can provide robust neuroprotection following a global ischemic insult. In conclusion, we suggest that mexiletine is an effective neuroprotectant in this model of transient forebrain ischemia. We further suggest that Na 1 fluxes contribute to cellular injury in grey matter as well as white matter ischemia models. Na 1 channel blockade appears to be an effective protective strategy for both grey and white matter damage. Further evaluation of mexiletine or other related use-dependent Na 1 channel blockers in a post-treatment paradigm is indicated.
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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Research report
The use-dependent sodium channel blocker mexiletine is neuroprotective against global ischemic injury Kimberley E. Hewitt, Peter K. Stys*, Howard J. Lesiuk Loeb Health Research Institute, Division of Neuroscience, Ottawa Hospital – Civic Campus, University of Ottawa, 725 Parkdale Avenue, Ottawa, Ont., Canada K1 Y 4 K9 Accepted 23 January 2001
Abstract Mechanisms responsible for anoxic / ischemic cell death in mammalian CNS grey and white matter involve an increase in intracellular Ca 21 , however the routes of Ca 21 entry appear to differ. In white matter, pathological Ca 21 influx largely occurs as a result of reversal of Na 1 –Ca 21 exchange, due to increased intracellular Na 1 and membrane depolarization. Na 1 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 Ca 21 channels are the primary routes of Ca 21 entry. Less attention has been directed towards Na 1 –Ca 21 exchange and Na 1 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. 2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Stroke; Neuroprotection; Sodium channel; Mexiletine; Hippocampus
1. 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 Ca 21 , has traditionally been viewed as central in ischemic cell injury, particularly in grey matter [4,5]. In white matter, a key role for Na 1 –Ca 21 exchange has been shown [13,28] and the prevention of pathological Na 1 influx by Na 1 channel blockade has been found to be an effective strategy for *Corresponding author. Tel.: 11-613-761-5444; fax: 11-613-7615330. E-mail address: [email protected] (P.K. Stys).
protection of white matter in anoxia [13,28]. Na 1 channel blockade, and the contribution of pathological Na 1 influx have received relatively less attention in relation to grey matter ischemia. Nonetheless, reports have appeared evaluating Na 1 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 1 channel blockers that have been evaluated. Often the effects demonstrated have been modest. Nonetheless, these results suggest that Na 1 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 1 currents are often seen during ischemia, and a massive TTX-sensitive Na 1 accumulation occurs in in vitro brain slices during anoxia [15,31]. The enhanced
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02195-3
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excitation during ischemia coincides with prolonged influxes of sodium during the depolarization process. These findings suggest that Na 1 channels can contribute to the general excitability of the cell, prolonged Na 1 influx, and a proceeding rise in cytotoxic levels of Ca 21 . Under the pathological conditions of high Na 1 influx, it is thought that the Na 1 –Ca 21 exchanger reverses and causes Ca 21 influx as Na 1 is extruded [18]. In support of this, Stys et al. [29] have demonstrated in a model of white matter anoxia, that extracellular Ca 21 was necessary for damage to be incurred, and that ionic conditions favouring a reversal of the normal transmembrane Na 1 gradient during the anoxic period resulted in greater injury. The reduction of Na 1 permeability during ischemia could therefore potentially prevent a secondary Ca 21 influx into CNS neurons, and reduce ischemic damage. While irreversible neuronal injury due to ischemia is thought to depend largely on Ca 21 influx, the mechanisms of Ca 21 entry may differ significantly between myelinated axons and neuronal cell bodies. During an anoxic challenge it is thought that the majority of Ca 21 flux in central, myelinated axons is mediated by the reversal of the Na 1 – Ca 21 exchanger, as described above [27,29], although Ca 21 entry via axonal voltage-dependent Ca 21 channels cannot be ruled out as a contributing factor since Ca 21 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 1 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 1 –Ca 21 exchanger in increasing intracellular Ca 21 is minimal in comparison. Yet Na 1 -mediated modes of injury may be present in grey matter. For example, Na 1 has been implicated as a contributor to excitotoxicity from glutamate release via reversed Na 1 glutamate co-transport [16,32,33]. Na 1 -dependent neuronal edema may also be present [4,23]. In addition, the extent of Ca 21 entry by a reversal of the Na 1 –Ca 21 exchanger in global models of ischemia has not been determined. The net contributions of these Na 1 -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 1 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 1 channels during an anoxic exposure [26]. These findings have characterized mexiletine as a use-dependent Na 1 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.
2. Materials and methods A total of 23 male, Sprague–Dawley rats (Charles ´ River, St. Constant, Quebec), 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 anesthesia was induced with sodium pentobarbital (65 mg / kg, i.p.). Supplemental doses of sodium pentobarbital (15–30 mg / kg, i.p.) were administered, if needed, to maintain adequate anesthesia. Once anesthesia was induced, the trachea was intubated with a [16 Teflon IV catheter under direct vision with a laryngoscope. Mechanical ventilation (using a Model 665 small animal ventilator, Harvard apparatus, South Natick, MA) was adjusted according to arterial blood gas (ABG) determinations to maintain normocapnia ( pCO 2 535–45 Torr) and normoxia ( pO 2 590–120 Torr), throughout the operative procedure. A thermocouple probe was placed at the tympanic membrane into the middle ear to allow continuous monitoring and adjustment of head temperature. Temperature was maintained at 37.560.28C using a heat lamp as necessary for the duration of the surgery. A [24 Teflon IV catheter, charged with heparinized (10 U / ml) normal saline, was inserted through the tail artery into the descending aorta for: continuous blood pressure monitoring, aspiration of blood to induce controlled hypotension (see below), and arterial blood sampling. For blood pressure monitoring, the cannula was connected to a pressure transducer, and blood pressure was sampled 40 times a second by an analog to digital converter, and stored on a computer disk. Once blood gases and temperature were within the desired ranges, animals were given either mexiletine (80 mg / kg, i.p.; n512) 25 min prior to ischemia or an equal volume of physiological saline (1 ml / kg, i.p.; n511). The
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common carotid arteries were exposed bilaterally through a midline, ventral, cervical incision. Forebrain ischemia was induced through bilateral carotid artery occlusion with temporary cerebral aneurysm clips and coincident controlled hypotension (mean BP55062 Torr) induced by temporary, partial exsanguination. This was achieved by the aortic cannula, with blood withdrawn into a syringe containing 0.5 ml of heparinized (10 U / ml) saline. After 10 min of ischemia, the clips were removed and the blood was reinfused over 30 s. Repeat ABG was then obtained. The cervical incision was sutured, the arterial cannula was removed, and the tail incision closed. Ventilatory support was continued until the animal was breathing well on its own. During this recovery phase, animals were maintained under a heating lamp coupled to a rectal probe, capable of maintaining body temperature within 60.28C of the preset value. Temperature was maintained post-operatively until the animal was completely awake and thermoregulating normally. A number of animals were studied for up to 8 h post-surgery and no late hypothermic events were noted. Food and water were allowed ad libitum following surgery. Animals were allowed to recover for 7 days before being sacrificed under deep barbiturate anesthesia with transcardial perfusion fixation and immediate harvesting of the brains. A series of 7-mm coronal serial sections at the level of the dorsal hippocampus were taken from paraformaldehyde fixed brains embedded in paraffin, and stained with hematoxylin and eosin. The ratio of dead cells to the total cells present was determined for each animal, and expressed as a percentage. The statistical analysis was carried out using a Mann– Whitney U test for non-parametric data. Data are presented as means, and errors are reported as standard deviations.
3. Results Following 10 min of global ischemia, control animals receiving saline (n511) sustained 88611% CA1 neuronal injury (Fig. 1). Pre-treatment with mexiletine (n512) reduced this significantly by 50%, to 45632% (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 addition to its neuroprotective properties, pre-treatment of animals with mexiletine caused a marked reduction in mean arterial blood pressure prior to occlusion. Fig. 3 illustrates the pattern of induced hypotension following mexiletine administration, and the response following 10 min of global ischemia with hypotension (50 mmHg) for each group. Animals receiving mexiletine had an average reduction in blood pressure of 60 mmHg 20 min after drug administra-
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Fig. 1. Bar graph illustrating the degree of neuronal cell death following global ischemia in the hippocampal CA1 region of rats pretreated with either saline or mexiletine (80 mg / kg, i.p.). Heights of the bars represent the number of compromised cells, expressed as a percentage of the total counted. Filled circles (d) represent individual scores from each animal. A 10-min global ischemic insult resulted in the death of 88611% of hippocampal CA1 pyramidal cells, when animals were pretreated with saline. Pretreatment with mexiletine 25 min before ischemia reduced this by half, resulting in only 45632% cell mortality. Bars represent means, and error bars are standard deviations. The asterisk indicates a statistically significant difference from the saline treated group (P,0.001; Mann– Whitney U test for non-parametric data).
tion. Control rats showed relatively no change prior to exsanguination. Post-ischemically, control rats showed a slight elevation in blood pressure in comparison to preischemic baseline measurements (Fig. 3). Rats receiving mexiletine did show an increase in blood pressure following ischemia, however they did not reach their pre-injection baseline level, and remained hypotensive during the post-operative recording period. No differences in body temperature were observed between the treatment groups. However, mexiletine-treated rats were thermoregulated for longer periods of time postsurgically due to an extended recovery time from anesthetic. This was likely due to the sedating effects of mexiletine at this dose, and is consistent with the sideeffects observed in a previous experiment [26]. No other difficulties were observed following mexiletine administration at this dose.
4. Discussion The use-dependent sodium channel blocker mexiletine significantly reduced neuronal damage in the CA1 sector of the dorsal hippocampus after global ischemia. This
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Fig. 2. Photomicrographs of representative coronal sections through the dorsal hippocampus CA1 region of rats treated with saline (A) or mexiletine (B). Saline-treated rats incurred severe CA1 damage when assessed at 7 days post-ischemia. A significant loss of CA1 pyramidal cells was evident as exemplified in the photomicrograph (A). By contrast, mexiletine administration (80 mg / kg, 25 min pre-occlusion) resulted in a marked improvement in CA1 pyramidal cell survival (B). The number of remaining viable neurons was greatly increased, and a continuous band of pyramidal cells can be seen. Scale bar represents 50 mm.
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Fig. 3. Illustration of the hypotensive effects mexiletine. Initial blood pressure readings did not differ between the two groups. However, following mexiletine administration, blood pressure fell dramatically, and by 20 min post-administration, blood pressure had dropped by an average of 60 mmHg. During ischemia, exsanguination maintained hypotension at 50 mmHg. Post-ischemically, blood pressure returned to a slightly higher level than pre-ischemic values in saline treated rats. While those rats receiving mexiletine did show a rise in post-ischemic pressure above that recorded immediately before exsanguination, mean arterial blood pressure did not return to the initial levels observed prior to drug administration. Filled circles (d) represent the mean pressure values for saline-treated animals, while open circles (s) represent mexiletine-treated rats. Error bars represent the standard deviation from the mean.
suggests that intra-ischemic Na 1 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 1 channel blockers as a method of averting cell death. For example, Prenen et al. [19] found that TTX could prevent increases in Na 1 concentration (as indicated by measuring cation concentrations in brain tissue by flame emission spectrometry) with a coincident increase in cell survival. Burke and Taylor [2] found that TTX administration also prevented glutamate release during ischemia. More recently, Yamasaki et al. [34] and Lysko et al. [17] reported that TTX via intracranial injection afforded protection from ischemic cell death in both rats and gerbils, respectively. While intracranial administration of TTX does not represent an attractive therapeutic approach, these findings strongly support a role for Na 1 influx as a contributing factor in ischemic neuronal death. Administration of other Na 1 channel blockers such as phenytoin [1] and lidocaine [22,24,30] have also resulted in improvements in cell outcome after ischemia. More recently, a use-dependent blocker of voltage-dependent sodium channels, BW619C89, was also shown to be successful in reducing infarct volume after MCA occlusion in the rat [11]. Use-
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dependent compounds such as mexiletine and BW619C89 may offer more advantages therapeutically since they would be most effective during instances of high open channel activity, while leaving normal signalling relatively unaffected. Together, these findings suggest that Na 1 influx resulting from ischemia contributes to the pathological events leading to cell death, and that a reduction in Na 1 permeability via Na 1 channel blockade can effectively reduce cellular damage. While it is clear that Na 1 channel blockade may be advantageous in reducing ischemia-induced cell death, the role of Na 1 in producing grey matter injury following an ischemic insult is not as well characterized mechanistically as it is for white matter injury. As discussed, in models of white matter anoxia, it has been shown that energy failure is followed by axonal depolarization, and Na 1 influx via non-inactivating Na 1 channels, causing a reversal of the Na 1 –Ca 21 exchanger [29]. This leads to an increase in intracellular Ca 21 , and the pathological activation of Ca 21 dependent effector systems. An interruption of this cascade by Na 1 channel blockers in white matter anoxia is neuroprotective [7,21,25,28,29]. The results of this experiment, and those discussed above using other Na 1 channel blockers, suggest that this relationship between Na 1 influx and anoxia / ischemia-induced injury may indeed be similar in neuronal cell bodies. In support of this, an increase in [Na 1 ] i has been observed following anoxia in dissociated hippocampal neurons, and in cultured cortical neurons [9], and removal of [Na 1 ] o can attenuate anoxia-induced injury [10]. The present study has demonstrated that mexiletine is capable of significantly ameliorating cellular injury in a global ischemia model, putatively by reducing Na 1 permeability. This inhibition could ultimately impact a number of factors which contribute to cellular decline. Edema and cell swelling may be direct consequences of Na 1 influx, resulting in cellular damage independently of Ca 21 mediated events [9]. Na 1 -mediated anoxia-induced depolarization can also activate voltage-gated Ca 21 channels [12] causing a coincident increase in intracellular Ca 21 . Therefore, an increase in intracellular Na 1 can impact the cell in Ca 21 -dependent and independent ways, both rendering it susceptible to anoxic / ischemic damage. In addition, Na 1 / glutamate co-transport may be affected by increased intracellular Na 1 , resulting in the release and / or diminished uptake of glutamate during an anoxic / ischemic event [16,32,33]. Further mechanistic studies, such as the determination of glutamate release, or Ca 21 entry following mexiletine administration in instances of ischemia, will be necessary to more precisely define the specific effects of the drug. Mexiletine administration also caused significant hypotension in rats beginning soon after drug delivery. These hypotensive effects are most likely to be detrimental to outcome. This suggests that other compounds without such properties and with greater selectivity for neuronal Na 1 channels versus those in the myocardium and vascular tree
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may be even more effective in preventing cell death. Indeed, the differences in molecular structure of neuronal versus cardiac and muscle Na 1 channels [14,20] suggests that developing more selective compounds may be feasible. The possibility remains that the hypotensive effects of mexiletine may be contributing to the positive effects of the drug, although it seems unlikely. Peri-ischemic hypotension correlates with a poor prognosis in the clinical setting and with increased cellular injury in ischemia models (for example, see Zhu and Auer [35]). In addition, induced hypertension during ischemia reduces infarct size [3] suggesting that the maintenance of blood pressure at or above normal levels results in less damage following ischemia. An examination of other drugs in this class that do not result in hypotension may prove to be advantageous when treating ischemic injury. Nonetheless, mexiletine provided marked neuroprotection despite its coincident hypotensive effects, suggesting Na 1 channel blockade can provide robust neuroprotection following a global ischemic insult. In conclusion, we suggest that mexiletine is an effective neuroprotectant in this model of transient forebrain ischemia. We further suggest that Na 1 fluxes contribute to cellular injury in grey matter as well as white matter ischemia models. Na 1 channel blockade appears to be an effective protective strategy for both grey and white matter damage. Further evaluation of mexiletine or other related use-dependent Na 1 channel blockers in a post-treatment paradigm is indicated.
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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