Roles Of Cysteinyl Ieukotriene Receptor2in Ischemic Brain Injury And Microglial Actiyation

BackgroundsCerebral ischemia-induced neuronal injury and inflammation are the main features of post-ischemic lesions. Microglia are the primary inflammatory cells in the brain, and their activation is implicated in progress of post-ischemic inflammation and the recovery of chronic neurological function. Cysteinyl leukotrienes (CysLTs) are potent inflammatory mediators, and play important roles in cerebral ischemia, traumatic brain injury and other diseases in the central nervous system.CysLTs act their specific receptors, the CysLT receptors. Two main subtypes (CysLT1and CysLT2receptors) have been cloned and characterized. CysLT1and CysLT2receptors are widely expressed in the body. CysLT1receptor is weakly expressed in human brain and CysLT2receptor is relatively higher expressed in the brain. Pharmacological studies have shown that CysLT1receptor mediates acute in vivo neuronal injury and chronic astroglial proliferation after cerebral ischemia; it regulates post-ischemic inflammation by increasing the permeability of blood-brain barrier and mediates expression of intercellular adhesion molecule1(ICAM-1). After focal cerebral ischemia, CysLT2receptor mRNA is up-regulated in rat brain; however, whether CysLT2receptor mediates ischemic brain injury and microglial activation after ischemia is still unknown.AimsTo clarify the roles of CysLT2receptor in ischemia-induced neuronal injury and microglial activation, in the present study we performed the following investigations:(1) After preparation of a polyclonal antibody specifically against rat CysLT2receptor, we examined the spatiotemporal properties of CysLT2receptor expression in the brain and the cellular localization after focal cerebral ischemia in rats.(2) By RNA interference tequenich, we investigated the role of CysLT2receptor in oxygen-glucose deprivation (OGD)-induced neuronal injury and microglial activation in mixed cultures of rat cortical cells.(3) We also investigated CysLT2receptor-mediated micoglial activation and phagocytosis in primary rat microglia.MethodsThe polyclonal antibody against CysLT2receptor was prepared in rabbits immunized with KLH-coupled CysLT2receptor peptide. Focal cerebral ischemia was induced by middle cerebral arterial occlusion (MCAo). The spatiotemporal properties of CysLT2receptor protein expression were determined by immunohistochemistry and Western blot analysis, and the cellular distribution of the receptor was assessed by double immunofluorescence.In the mixed culture of rat cortical cells and rat primary microglia, ischemic injury was induced by OGD, or the changes were induced by the agonist LTD4at various concentrations. The gene expression of CysLT2receptor was silenced by a targeted lentivirus shRNA. Then, the effect of RNA interference on OGD-induced neuronal injury and microglia activation was observed in mixed cultures of rat cortical cells. Also, the effects of CysLT2receptor shRNA and CysLT receptor antagonists on changes in microglial activation and phagocytosis in vitro were determined.ResultsPart â…  Spatiotemporal properties of expression and cellular localization of CysLT2receptor in rat brain after focal cerebral ischemiaThe specific polyclonal antibody against CysLT2receptor was successfully prepared and identified, and used in the following experiments. Western blot analysis showed that CysLT2receptor protein expression was significantly up-regulated in the ischemic core both6-24h and14days after reperfusion in rats with MCAo. In the boundary zone, no significant change was found within7days, but significantly increased14-28days after reperfusion. In the contralateral cortex, the expression did not change during reperfusion after ischemia. In accordance with Western blot results, the number of CysLT2receptor positive cells was significantly increased within and around the ischemic area as detected by immunohistochemistry. Double immunofluorescence results showed that CysLT2receptor was mainly expressed in astrocytes around the ventricles and in the cortex. After MCAo, in the ischemic core, CysLT2receptor was primarily localized in neurons and few in astrocytes and microglia at24h, but it was restricted to microglia at14days after reperfusion. Whereas, in the boundary zone, it was primarily localized in proliferated astrocytes, few in microglia, but none in neurons14days after reperfusion. These findings suggest that CysLT2receptor might mediate acute neuronal damage, later microglia activation and astrogliosis. Part â…¡ Effect of CysLT2receptor interference on OGD-induced acute neural injury and microglia activation in primary mixed cellsIn the mixed cultures of rat cortical cells, RT-PCR and immunofluorescence results confirmed that the lentivirs shRNA inhibited CysLT2receptor expression. OGD/R (OGD1h, recovery24h) or LTD4induced LDH release, and cell necrosis and apoptosis as determined by PI/Hoechst33342staining. After treatment with CysLT2shRNA and the non-selective CysLT receptor antagonist BAY u9773, OGD-or LTD4-induced LDH release was inhibited, and the number of necrotic cells, not apoptotic cells, was decreased. Immunofluorescence showed that CysLT2shRNA inhibited morphological changes of microglial activation. Double immunofluoresence showed that the main type of dead cells was neuron, and few was astrocytes and microglia. CysLT2receptor shRNA significantly decreased neuronal death. Thus, CysLT2receptor might mediate neuronal injury and microglial activation.Part â…¢ Regulatory roles of CysLT2receptor in microglial activation and phagocytosisIn the primary cultures of rat microglia, immunofluoresence showed that CysLTi and CysLT2receptors were weakly expressed under normal conditions, and significantly up-regulated after1-h OGD and24-h recovery. CysLT2shRNA inhibited the protein expression of CysLT2receptor, but significantly up-regulated CysLT1receptor expression. Microglial activation and phagocytosis induced by OGD or LTD4was inhibited by CysLT2shRNA and the non-selective CysLT receptor antagonist BAY u9773; however, microglial phagocytosis was accelerated by the selective CysLT1receptor antagonist pranlukast. N-methyl leukotriene C4, a selective CysLT2receptor agonist, activated microglial phagocytosis, and this was inhibited by BAY u9773but not by pranlukast. These findings suggest that the CysLT2receptor might mediate microglial activation and phagocytosis, the CysLT1receptor might mediate inhibition of microglial phagocytosis, and an interaction might occur between CysLTi and CysLT2receptors in microglial activation.Conclusions:1. After focal cerebral ischemia in rats, the spatiotemporal patterns of CysLT2receptor expression in the brain show that CysLT2receptor mediates acute neuronal injury and later microglia activation, and may also participate in the process of later astroglial proliferation and glial scar formation.2. In the mixed cultures of rat cortical cells, the results of CysLT2receptor shRNA and antagonist show that CysLT2receptor mediates OGD-induced neuronal injury and microglial activation.3. In the primary cultures of rat microglia, OGD up-regulated both CysLT1and CysLT2receptors. The results of the shRNA and antagonist demonstrate that the CysLT2receptor mediates OGD-induced microglial activation and phagocytosis. Results of antagonist indicate that the CysLT1receptor may mediate inhibition of microglial phagocytosis. Interference of CysLT2receptor expression up-regulates CysLTi receptor expression, suggesting a possible interaction between the two subtypes in regulation of microglial function.