AM-36, a novel neuroprotective agent, profoundly reduces reactive oxygen species formation and dopamine release in the striatum of conscious rats after endothelin-1-induced middle cerebral artery occlusion
Introduction
Reactive oxygen species (ROS) generation is considered to be an important contributor to neuronal damage in cerebral ischemia (Chan, 2001, Kuroda and Siesjo, 1997). Obtaining evidence for the involvement of ROS in the pathophysiology of cerebral ischemia has been difficult due to problems of directly measuring these species because of their extremely reactive nature and rapid degradation. However, methods relying on hydroxylation of compounds such as salicylate or 4-hydroxybenzoate by ROS to form stable detectable adducts (2,3 dihydroxybenzoic acid or 3,4-dihydroxybenzoate, respectively), have been utilised as an index of brain ROS formation (Floyd et al., 1984, Ste-Marie et al., 2000). Studies employing these techniques together with in vivo microdialysis, have detected increases in ROS in the hippocampus or striatum following transient global cerebral ischemia and reperfusion in rats (Globus et al., 1995, Piantadosi and Zhang, 1996, Ste-Marie et al., 2000, Yamamoto et al., 1997). Increases in ROS have also been demonstrated in the cortex (Gido et al., 2000, Morimoto et al., 1996, Solenski et al., 1997, Yang et al., 1996), and striatum (Lancelot et al., 1995, Li et al., 1997) in models of focal cerebral ischemia and reperfusion, suggesting a role for ROS in ischemia-induced neuronal damage. However, few studies have employed middle cerebral artery (MCA) occlusion models in rats (Bogaert et al., 2000, Li et al., 1997, Morimoto et al., 1996) and it is these models which are most pertinent to stroke in humans since the MCA is most commonly occluded (Garcia, 1984).
Antioxidants have been shown to attenuate ROS formation and neuronal damage in the reperfusion period in a global ischemia model (Yamamoto et al., 1997) however, there is limited data demonstrating a direct effect of antioxidants at reducing measured ROS formation during focal ischemia. Li et al. (1997) demonstrated that high doses of melatonin given prior to ischemia, inhibited production of hydroxyl radicals in the striatum of rats following MCA occlusion. However, these authors did not investigate subsequent neuroprotective effects of melatonin on ischemia-induced brain damage. Intracerebral administration of high concentrations of L-NAME, has been found to attenuate ROS generation in the cortex following a three-vessel occlusion model of focal ischemia (Solenski and Kwan, 2000). High systemic doses of the spin trapping agent PBN (alpha-phenyl-tert-butyl nitrone) were ineffective against hydroxyl radical formation in the cortex when administered before occlusion or immediately after reperfusion (Gido et al., 2000). To our knowledge, no previous studies have examined changes in ROS in conscious rats following focal ischemia by occlusion of the MCA, nor has attenuation of ROS generation by proven neuroprotective doses of an antioxidant compound been demonstrated post-ischemia.
There is ample evidence for an increase in extracellular glutamate in the brain following both global and focal ischemia (Beneviste et al., 1984, Hillered et al., 1989, Morimoto et al., 1996). However, increases in other neurotransmitters have also been detected, including dopamine (DA) (Bogaert et al., 2000, Phebus and Clemens, 1989). DA has been shown to contribute to subsequent neuronal damage following cerebral ischemia (Buisson et al., 1992, Clemens and Phebus, 1988, Globus et al., 1987). The susceptibility of the striatum to ischemia has been associated with DA oxidation (Allen et al., 1994, Chiueh et al., 1993, Ste-Marie et al., 2000) and increased vulnerability of the nigrostriatal pathway to oxidative stress (Chiueh et al., 1992). The striatum is generally considered to be the core of the ischemic lesion in rats and humans after MCA occlusion (Fisher and Garcia, 1996, Tyson et al., 1984) and previously has proved relatively refractory to neuroprotection. We have recently shown that AM-36 (1-(2-(4-chlorophenyl)-2-hydroxy)ethyl-4-(3,5-bis(1,1-dimethyl)-4-hydroxyphenyl)methylpiperazine), a novel neuroprotective agent with antioxidant and sodium (Na+) channel blocking activity (Callaway, 2001, Jarrott et al., 1997, Jarrott et al., 1999), can reduce ischemic damage in the striatum (Callaway et al., 1999). Blockade of Na+ channels has previously been shown to inhibit ischemia-induced glutamate and DA release in vitro (Taylor et al., 1995, Toner and Stamford, 1997) and in vivo (Lysko et al., 1994). Previous experiments in our laboratory, demonstrated inhibition by AM-36 of veratridine stimulated glutamate release from the cortex of anaesthetised rats in a concentration dependent manner (unpublished observations).
The purpose of the present study was to obtain in vivo evidence of antioxidant activity by determining whether AM-36 could inhibit salicylate hydroxylation in the dorsolateral striatum following MCA occlusion using the endothelin-1 (ET-1) method in conscious rats. Additionally, we sought evidence that AM-36 may inhibit the release of DA. The ET-1 model is less invasive than other models, incorporates gradual reperfusion, avoids damage to the endothelium by occlusion of the artery with filaments (Macrae, 1992) and has the advantage that rats are conscious during the stroke (Sharkey et al., 1993). The use of anaesthesia during stroke may confound experimental findings since barbiturates have been reported to be neuroprotective, may also potentially affect ROS production and inhibit neurotransmitter release (Bhardwaj et al., 1990). Inhalational anaesthetics such as isoflurane have also been reported to be neuroprotective (Warner et al., 1993).
The present studies report potent inhibition of ischemia-induced increases in salicylate hydroxylation and extracellular DA efflux by AM-36. These findings support the contribution of free radical scavenging and inhibition of transmitter release as the mechanism of action of AM-36 in protecting against neuronal damage and functional deficits in this rat model of stroke. Part of this work was presented at the 31st annual meeting of the Society for Neuroscience and at the 22nd annual meeting of the Australian Society for Neuroscience (Callaway and Jarrott, 2001b).
Section snippets
Animals
All procedures used in this study were carried out in accordance with the Prevention of Cruelty to Animals Act 1986 under the guidelines of the NH and MRC code of Practice for the Care and Use of Animals for Experimental Purposes in Australia. Male Long Evans rats (Monash University Animal Services, Australia) weighing 280–320 g were used throughout this study. These animals were housed in a room with controlled temperature (22±1 °C) and humidity (50±5%) under a 12:12 h light–dark cycle. Rats
Microdialysis
Basal striatal levels in nM (mean±SEM) in freely moving rats were 5.9±1.2 for 2,3 DHBA, 16.5±4.5 for DA, 15.3±7.2 for DOPAC and 21.4±7.8 for HVA (n=14). Since measured levels of 2,5 DHBA have been suggested to reflect microsomal P450 enzymatic hydroxylation of salicylate rather than in situ salicylate hydroxylation (Halliwell et al., 1991) the levels of 2,5 DHBA are not presented.
Fig. 1, Fig. 2 show the time course of 2,3 DHBA, DA, DOPAC and HVA outflow for 1 h prior, and 5 h following
Discussion
Only two studies have investigated changes in ROS generation in the striatum following focal cerebral ischemia (Lancelot et al., 1995, Li et al., 1997) and only the latter study employed an MCA occlusion model. The free radical scavenger, MCI-186, and the nitric oxide synthase inhibitor, L-NAME, have been shown to reduce 2,3 DHBA (the stable adduct of salicylate) formation in the cortex and hippocampus in models of transient cerebral ischemia in the rat (Yamamoto et al., 1997, Zhang et al., 1995
Concluding remarks
In conclusion, the present study has demonstrated in vivo evidence for inhibition of the generation of ROS following MCA occlusion-induced ischemia in conscious rats by AM-36. The mechanism of action is likely to be through antioxidant activity of this compound, but may also include inhibition of DA efflux possibly through blockade of neuronal Na+ channels. Correlations between 2,3 DHBA, DA and striatal damage, support inhibition of oxidative stress as a mechanism of neuroprotection in the
Acknowledgements
The authors gratefully acknowledge AMRAD Corporation Limited, Melbourne, Australia, for their contribution to the discovery and development of AM-36. This work was supported by the National Health and Medical Research Council, Australia.
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