The Protective Effect Of Adenosine A_2A Receptor Deficiency In Cerebral Ischemia/Reperfusion Injury And Approaching To Its Mechanism

Ischemic apoplexy, a serious disorder, is of high morbility and fatality. According to epidemiological analysis, most of the survivors have remain permanent disability. Nowadays, recovery of the blood fluid at the ealier phage and protection of the ischemic neuron are the main strategies in in the treatment of cerebral infarction (CI ) . However, as the reperfusion of the blood fluid is limited by the time window (<3 h ), the patients always lossed the optimal opportumity for therapy, which could aggravate the injury induced by the reperfusion beyond the time window. Up to now, limited efficiecy measures have been applied in clinic to protect the ischemic neuron from injury. The brain protecting drugs such as calcium ion antagonist and glutamate receptor antagonist were failed in clinic trails. Therefore, it is urgent to explore a new strategy for curing ischemic apoplexy.Adenosine is a ubiquitous homeostatic substance released from most cells, including neurones and glia. Once in the extracellular space, adenosine modifies cell functioning by operating the receptors. The adenosine is also serves as a neuromodulator under physiological conditions in central nervous system (CNS ), such as Parkinson disease, Alzheimer disease, Dyssomnia, epilepsy, cognitive disorder, depression, anxiety and so on. Extracellular adenosine acts through multiple Gprotein coupled receptors (adenosine receptor subtypes A1,A2A, A2B and A3) to exert a variety of physiological effects. Adenosine levels rise markedly in response to ischemia, which has long been known to act predominantly as a neuroprotectant under this condition.Recent studies have demonstrated that the inactivation of adenosine A2A receptor (A2AR) protects against brain injury caused by cerebral ischemia in various animal models. In these previous studies, pathohistological lesions were assessed after reperfusion or after 24 h persistent focal cerebral ischemia. Therefore, it is still unclear whether A2AR blockade protects against ischemic neuronal injury during acute ischemia, reperfusion or both stages.The underlying neurochemical mechanism remains to be determined. A2AR activation may promote glutamate release in the brain under both normoxic and ischemic conditions, facilitate the inflammatory activities of inflammatory cells. Therefore, we infer that the beneficial effects of A2AR inactivation on ischemic brain injury attributed to reduced glutamate outflow and inhibitory inflammatory activities after ischemia and reperfusion.However, few direct evidence has been obtained to determine the effect of the A2AR inactivation on glutamate outflow and glia reaction in cerebral ischemia. In this study, we critically evaluated the effect of A2AR inactivation on ischemic brain injury in both the ischemic and reperfusion stages using an A2AR knock-out mouse (A2AR-KO) model. In addition, we observed the glia reaction at the delay injury stage after reperfusion, in order to reveal the underly mechanisim of the the neuroprotective effect of the A2A adenosine receptor deficiency.Method: The PCR protocol used to genotype the mice, A2AR gene knockout mice(A2AR-KO) and wild-type littermates(A2AR /WT). Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery (MCA) with an 8-0 nylon monofilament with a silicon-coated tip to establish the MCAo model according to Huang, et al. Evaluating the neurological deficits at ischemic phase and reperfusion phase respectively. Measuring the infarction volumes of the A2AR/KO mice and A2AR/WT mice by Cresyl violet(CV)staining and image analysis system, in order to investigate the impact on the infarction volumes at ischemic stage and reperfusion stage by the A2AR/KO. Collecting the extracellular fluid of the two groups by microdialysis before the ischemia and during the ischemia/reperfusion, then determining the glutmate, aspartate, gamma-aminobutyric acid (GABA) and glycine content by high performance liquid chromatography -fluorescene detection (HPLC-FL). Immunohistochemical staining was used to observe the glia cells quantity and morphologic changes influenced by the inactivation of the A2AR.Result: 1. The mice MCAo model was established stably. The successful rate of execution was 90% and the neurological deficit scoring of 1-3 grades was 91.1% of all these MCAo-mice. After the 2,3,5-Triphenyltetrazolium Chloride(TTC)and CV staining, it was proved that the infarction degrees and areas after the MCAo were stability with neurological deficit scoring of 1-3 grades.2. Establishing a reliable HPLC-FL detecting technique, which could separat and determin multi-amino acids including glutmate, aspartate, GABA, glycine and so on within 18 min. The modified HPLC-FL was a more quick , reliable and sensitive method to measure the amino acid neurotransmitters in brain.3. The extracellular concentration of glutmate, aspartate, GABA and glycine increased in ischemia phase and reperfusion phase respectively, which became a"double-peaks"excessive release.4. Adenosine A2AR deficiency reduced ischemia-induced amino acid neurotransmitters excessive release in striata. A2AR deficiency ameliorates neurological deficit (P<0.01 compared with the A2A/WT), and attenuates cerebral infarct volume (P<0.01 compared with the A2A/WT) after transient MCAo.5. Adenosine A2AR deficiency reduced the excessive release of these amino acid neurotransmitters once more induced by reperfusion. A2AR deficiency ameliorates neurological deficit (P<0.05 compared with the A2A/WT), and attenuates cerebral infarct volume (P<0.01 compared with the A2A/WT) after 22h reperfusion.6. Adenosine A2AR deficiency inhibited the glial cells responding after ischemia/reperfusion. inhibiting the hyperplasy of the microglial cells and astrocytes, the number of positive CD11b and GFAP significantly reduced in A2AR/KO group (P<0.01 compared with the A2A/WT), (P<0.01 compared with the A2A/WT), and inhibiting the morphological change responding to activation.Conclusion:1. The modified HPLC technique is a ideal measuring method could detect multi-amino acid neurotransmitters in the samples in one time, which improve the sensitivenes and reproducibility, save the time, cut down the cost. 2. Adenosine A2AR deficiency inhibits ischemia- and reperfusion-induced excessive release of glutamate and other amino acid neurotransmitters in striata, protects against ischemic cerebral injury in both ischemia and reperfusion phases, ameliorating the neurological deficit and attenuating the cerebral infarct volumes at last.3. Blockading the adenosine A2AR could inhibit the glial cells responding induced by ischemia/reperfusion, depressing the hyperplasy degree of the the microglial cells and astrocytes. Inhibition of the inflammatory cells could be an other important reason to attenuating ischemic cerebral injury by the A2AR inactivation.