Analysis of Excitotoxicity and Acidotoxicity mediated by NMDAR and ASIC following Cerebral Ischemia/Reperfusion Injury

Abstract

The intracellular calcium overload is considered a major cause for activating various signaling pathways leading to neuronal death following cerebral ischemia. Earlier this overload was thought mainly due to glutamate mediated excitotoxicity, until recently acidosis activated ASIC1a also showed to significantly promote intracellular calcium influx during cerebral ischemia. Thus an effort was made to analyze the neuroprotective effectiveness attained by targeting these pathways in rat MCAO model of focal cerebral ischemia. At the outset, the standardization of rat MCAO model was carried out. Eventually, one hour ischemia and subsequent 24 hr reperfusion successfully produced marked damage in MCAO rat brain, as evidenced by significant neurological deficit, formation of an extensive cerebral infarct area and rise in levels of oxidative stress markers i.e. nitrite and MDA. Besides this, an increase in spatiotemporal protein expression of NR1, NR2B and ASIC1a occurred following I/R injury, which highlights their significance in the pathology of transient focal cerebral ischemia. Ex-vivo studies with synaptoneurosomes revealed the potential of NMDA receptors and ASIC1a in increasing the intracellular calcium levels, following activation. This strengthen the concept of acidosis mediated neuronal damage following cerebral ischemia and further, attenuation of acid mediated increase in intracellular calcium by flurbiprofen confirms its ASIC1a inhibitory activity. The excitotoxicity was modulated using Ifenprodil, a specific NR2B –NMDA receptor antagonist in MCAO rats. The ifenprodil pre and post treatment at early hours of ischemia was found to be neuroprotective as evidenced by significant reduction in cerebral infarct volume, neurological deficit, and oxidative stress marker levels in brain i.e. nitrite and MDA. The histological studies further revealed the protective effect of ifenprodil in cortical structures of MCAO rats. The ASIC’s were interestingly found to be inhibited by certain NSAID’s like ibuprofen and flurbiprofen. Thus acidotoxicity in MCAO rats was modulated by flurbiprofen and neuroprotective efficacy analyzed. In our studies flurbiprofen treatment, thirty minutes prior to ischemia and four hour post reperfusion, afforded protection from ischemic injury as evidenced by significant reduction in cerebral infarct volume and neurobehavioral deficit scores. Further, an early calcium dependant rise in levels of nitrite and MDA in ischemic brain regions were also found to be significantly reduced following flurbiprofen treatment. Moreover, flurbiprofen also reduced the proteolysis caused by ischemic activation of calcium dependant protease calpain. These studies thus provide firsthand information that flurbiprofen inhibits ASIC1a mediated acidotoxicity apart from its anti-inflammatory property and could be used to target multiple pathways in treatment of cerebral stoke. The pathology of cerebral stroke is complex and multifactorial hence multiple drug therapy had long been advocated by STAIR committee for stroke successful treatment. Thus, we further analyzed the effect of ifenprodil and its combination at lower doses with flurbiprofen on rat model of focal cerebral ischemia. We found that the combination produced significant neuroprotective effect as produced by ifenprodil at higher doses, which was evidenced by reduction in cerebral infarct volume, neurological deficit and MDA levels. Further, histopathological studies revealed that, the combination not only attenuated the cell damage in striatal regions of ischemic brain but also significantly inhibited apoptotic cell death, which was more pronounced than monotherapy with ifenprodil or flurbiprofen. Hence, the combination therapy appears to be more efficacious in offering neuroprotection on one hand and also lower the risks associated by mono-therapy with ifenprodil at higher doses. Thus it can be concluded that following ischemia, excitotoxicity as well as acidotoxicity both play significant role in pathology of cerebral injury. Moreover, modulation of these pathways may provide a novel approach for development of new entities for cerebral stroke therapy.