In the present study co administration of TM AT

In the present study, co-administration of TM (AT1 receptor blocker) and PD (AT2 receptor blocker) were made in ischemic mice to explore the significance of AT2 receptors in cerebral ischemia while blocking central AT1 receptors. Also to prove that AT2 receptors should be free after ischemic injury in order to bind and accomplish the agonist activity of released endogenous Ang II peptide which may be beneficial in cerebral ischemia. Also, the role of brain angiotensin system (BAS) i.e., AT receptors in cerebral ischemia was evaluated by administering AT antagonists in global ischemic mice model. The alteration in the brain Ang II content and its receptors (AT1 & AT2) expressions were studied. Further, glutamate and its transporter system, especially excitatory amino clemastine transporter- 2 (EAAT-2) expressions in the brain, were measured and correlated to understand the possible link between excitotoxicity with respect to BAS. Cerebral blood perfusion, inflammatory cytokines level, inflammatory marker’s expression such as nuclear factor kappa-B (NF-κB), Glycogen synthase kinase-3β (GSK-3β), synaptophysin, and glial fibrillary acidic protein (GFAP) immunohistochemistry staining were performed to assess the possible role of angiotensin receptors in pathogenesis of cerebral ischemia.
Materials and methods
Results
Discussion Induction of ischemia has increased the Ang II peptide level and elevated AT1 & AT2 receptor expression [21]. In the present we have shown increased GSK-3β and NF-κB activity and decreased EAAT-2 level, indicating the activation of inflammatory responses in ischemic condition. Increased Ang II peptide level during ischemia would have decreased the flux mean due to vasoconstriction [22]. Further, Ang II peptide directly modulates the permeability of BBB through activation of AT1 receptors and it could contribute to the pathophysiology of cerebrovascular disorders [23]. Treatment of AT1 blocker TM antagonized the IR effect, while PD failed to do so. Interestingly, PD decreased the TM effect but the reason for antagonistic response is not clear. However, earlier report supports that AT2 receptors null mice has decreased the neuroprotective and cognitive enhancement activity of AT1 receptor blockers suggesting that AT2 stimulation is very important during AT1 blockade in neurodegenerative conditions [24]. Ang II is the major endogenous peptide in BAS, which acts with equal affinity at central AT1 and AT2 membrane bound receptors [25]. The activation of inflammatory cascades by Ang II during ischemia may be attributed through AT1 receptor mediated responses and not through AT2 because TM has antagonized the elevated inflammatory markers and not PD. Experimental evidences have indicated that the stimulation of AT1 receptor by Ang II enhances the activation of NADPH-oxidase [7] and stimulates the NF-κB inflammatory pathway [26]. Central administration of Ang II peptide activates the GSK-3β and tau phosphorylation in brain [27]. AT1 receptors may exacerbate the NF-κB mediated cytokines release in the brain [6,28]. Treatment with TM alters the excitotoxicity through reduced brain cytokines and NF-κB expression in ischemic brain [19,29]. On the other hand, treatment with AT2 blocker PD did not protect the brain from ischemic insult and increased the pro-inflammatory cytokines levels and NF-κB expression along with decreased EAAT-2 expression. This response indicates that AT2 receptor could have physiological role and agonistic activity at AT2 might be beneficial [10]. Earlier report showed increased ischemic area and decreased CBF with AT2 receptor deficit mice [30]. In the present study, AT1 blocking effect has been attenuated with co-administration of PD and similar observations were made in middle cerebral artery occluded rats [31,32], which supports our present observation. In addition, neuroprotective effect of AT1 blockers was decreased but not completely abolished in AT2 gene deleted mice [30]. Earlier report indicate that administration of AT2 receptors agonist CGP42112 protected the primary cortical neural cells of C57Bl6 mice exposed to glucose deprivation for 24 h and in cerebral ischemic mice model [9]. Treatment of CGP42112 has shown dose dependent neuroprotection and neurogenesis in traumatic brain injury [10] and in spontaneously hypertensive rats [31] and AT2 receptor antagonist PD compound failed to produce any response. In middle cerebral artery occluded model, pre-administration of novel AT2 receptor agonist compound 21 protected the neuronal damage and this effect was attributed due to microglial activation, evoking the release of brain derived neurotrophic factor [32]. Consequently, the study with AT2 receptor agonists might help to elucidate the mechanism behind AT2 role in ischemic protection. Since several studies have suggested that activation of AT2 receptor will decrease the brain injury in ischemia [9,33], central AT2 receptor could be a potential target for stroke treatment. Hence, at this juncture, it can be stated that AT1 neuroprotective effect is interlinked with AT2 receptor in ischemic condition.