| Background: With an aging population, the ischemic cerebrovascular disease showedan increasing incidence, seriously threated to human health and brang a heavy burden to thesociety and families because of its high morbidity and mortality. Although people of theneuroscience community in many countries invested a lot of energy to the study of suchdiseases, but there is none of them can pass the clinical trials to make real benefit to protectthe nerve in patients with cerebral infarction, including oxygen free radical scavengers,calcium antagonists, glutamate receptor inhibitors and so all. Therefore, exloring the newneuroprotective treatment strategies of ischemic brain injury is very necessary and urgent.Adenosine (Adenosine, ADO) as a neurotransmitter and neuromodulator, widelydistributed in the organization of the nervous system, cardiovascular system, kidneys,gastrointestinal tract, regulating a variety of important physiological functions. ADO is acrucial role in the central nervous system, and involved in the pathophysiological process of avariety of neurological diseases through its specific receptor A1R,A2AR, A2BR, and A3R.The distribution and abundance of A1R and A2AR in the brain were significantly more thanthe other two receptors, and they also have stronger affinities with the ADO. Previous studieshave found that ADO as an important neurotransmitter, its neuroprotective function wasachieved through activating its inhibitory receptors A1. They also proved that the activationof A1receptor have neuroprotection and this process was involved the in inhibition ofglutamate-mediated excitotoxicity. In normal circumstances, the physiological concentrationsof ADO were only40nmol/L-400nmol/L. Animal studies have found that adenosine levels ofextracellular fluid in the brain after cerebral ischemia were significantly higher (200timesmore than normal). The increated adenosine acted on its receptors to play its regulatory role.At the same time, glutamate, as the representative excitatory amino acid, its concentrationsalso increased rapidly. This amino acids started a series of pathophysiological process aftercerebral ischemia and eventually lead to cell death. Our early experiments found that theadenosine A2A receptor knockout can significant reduce extracellular glutamate concentration in the district of ischemia/reperfusion brain, improve neurological function, and reduceinfarct volume. But the exact mechanism has not been elucidated. It can be seen, a sharpincrease of ADO in the state of cerebral ischemia did not play a neuroprotective role by theactivation of A1receptors. On the contrary, the activation of A2AR and inhibition of A1Rmay be an important reason leading to cerebral ischemic injury after cerebralischemia/reperfusion. Therefore, we assume that there is a mutual antagonism betweenA2AR and A1R. In the conditions of focal cerebral ischemia, the expression of adenosineA2A receptor increased and the functions of the A1R were inhibited. The ischemicneuroprotective effects of blocking A2AR were related with the activation or protection ofthe A1receptor function and inhibition of glutamate-mediated excitotoxicity cascade reaction.Previous studies have also found that in the central nervous system, glutamate transporters(glutamate transporter, the GLT) is responsible for the intracellular translocation of glutamate.As a subtype of the glutamate transporters, GLT-1is widely distributed in the glial cells ofthe forebrain, and take the90%of the glutamate transporter task. The pre-experiment foundthat neuroprotection of using A2AR antagonists in rat MCAO model was related to theenhanced glutamate-transport function of the GLT-1on glial cells in the ischemic region.Therefore, this project plan to explore the effects of interaction between A1and A2on theischemia/reperfusion injury in mice brain from the perspective of the GLT-1expression, andthe molecular mechanisms of regulating the release of glutamate.In this experiment, we used brain arterial ischemia2hours/reperfusion22hours modelin the adenosine A2A receptor gene knock mice to study the effect of A2AR knockout on theexpression of the A1R, and of activating or inhibiting the function of the A1R on theneuroprotection when A2AR was deleted on the overall level. The expression of GLT-1wereobserved after ischemia/reperfusion in attempt to reveal the possible mechanism of theadenosine receptors on the neuroprotection after ischemia. We aimed at providing theexperimental basis for developing new ways of ischemic neuroprotective treatment.Methods: We studied the Adenosine A2AR gene knock out mice and used PCR toidentify the genotype of mice and screening adenosine A2A receptor knockout the mice(A2AR/KO) and the wild-type mice (A2AR/WT) for the MCAo model. We detected changesof A1R expression in ischemia/reperfusion (corpus striatum) by immunofluorescence Franceand Western-blot; Adenosine A1receptor antagonist DPCPX and the agonist CPA were given before MCAo,and the region of neurological deficit and infarct volume of each groupmice was observed after2hours of ischemia/22hours of reperfusion; GLT-1expression wereassayed2hours after MCAo ischemia/22hours after reperfusion by immunofluorescence andWestern-blot in ischemia/reperfusion area (corpus striatum) of each group rats.Results：1. Adenosine A2A receptor missing increased the A1receptor expression afterischemia/reperfusion（1）Western Blot：Adenosine A2A receptor gene knockout mice (A2AR/KO) and wildtype mice (A2AR/WT) were made into2hours of MCAo ischemia and ischemia2hours/22hours reperfusion model, the sham operation group KO mice and the corpus striatum WTmice showed no significant difference of A1experssion; after2hours of ischemia A2AR/WTand A2AR/KO group, the expression of A1was no significant difference; after ischemia2hours/22hours reperfusion,WT mice A1expression was decreased, while KO mice A1expression was significantly increased.（2）Immunofluorescence：Adenosine A2A receptor knockout mice (A2AR/KO) andwild type mice (A2AR/WT) made into cerebral artery ischemia2hours/22hours reperfusionmodel, the sham operation group KO mice and WT mice had no significant difference on thenumber of positive cells of A1R in striatum District; after two hours of ischemia, WT miceA1R-positive cells decreased, while KO mice A1R positive cells decrease is not obvious;after ischemia2hours/reperfusion22hours WT mice striatum A1R-positive cells wassignificantly decreased, while KO mice the A1R positive cells increased significantly,basically the same results with WB results.2. After blocking A1receptors, cerebral ischemia/reperfusion injury protective effectproduce by the A2AR gene knockout is weakened, activation of A1R, this protective effectwas enhanced（1）Neurological function defect score: after ischemia2hours/22hours reperfusion, thenon-drug intervention group, the A1antagonist and KO mice neurological function scoreswere significantly lower than the same group of WT mice (P <0.05, P <0.01); using A1Rantagonist, regardless of KO mice or WT mice, the neurological score increased, wassignificantly higher than the non-intervention group (P <0.05, P <0.01); A1agonist group KOmice or WT mice, neurological function scores were lower, significantly lower than the non- drug intervention group (P <0.05, P <0.01).（2）Cerebral infarct volume calculation: Ischemia2hours/22hours reperfusion, thenon-drug intervention group, the A1antagonist and A1agonist group KO mice the infarctvolume was significantly lower than the same group of WT mice (P <0.01); after using A1receptor antagonist, regardless of KO mice or WT mice, the volume of cerebral infarctionwere significantly greater than the non-intervention group (P <0.01); after using A1receptoragonist, the KO mice or WT mice, the volume of cerebral infarction were significantly lessthan the non-drug intervention group (P <0.01)3. The influence of A2AR gene knockout to the expression of GLT-1after cerebralischemia in different A1active state（1）Western Blot detecte the expression of GLT-1：sham operation group, WT and KOmice striatum in GLT-1expression was no significant difference. after Ischemia2hours/22hours reperfusion the GLT-1expression reduced in WT mice compared with before ischemia,while KO mice GLT-1expression is no significant decrease, the expression was significantlymore than WT mice; after using A1antagonist KO mice the GLT-1expression in decline,significantly lower than non-drug intervention group; while after using A1agonist KO micestriatum in the GLT-1expression was increased, in the WT group, the expression of GLT-1not significantly difference in every intervention group.（2）Immunofluorescence assay the expression of the GLT-1：Before ischemia, WT andKO mice the GLT-1expression was no significant difference, after2hours ischemia/22hours reperfusion, in non-drug intervention group WT and KO mice striatum the GLT-1positive cell count decrease compared to the sham group,WT mice reduction is more obvious;After using A1R antagonist, WT mice and KO mice striatum the GLT-1positive cells wassignificantly less than non-drug intervention group; after using A1R agonists KO mice GLT-1positive cells was significantly increased than non-drug intervention group and the A1Rantagonist group.Conclusions：1. Under the conditions of ischemia/reperfusion injury, adenosine A2A receptor deletioncan enhance expression of adenosine A1, its activation may protect the A1receptor function2.By blocking adenosine receptors A1, generated by the lack of adenosine A2ARneuroprotective effect was weakened, and activation of adenosine receptors A1adenosine due to A2A receptor missing the neuroprotective effect was enhanced. Thus, we suggest: ischemicneuroprotective effects produced by blocking the adenosine A2AR are related to A1Rexpression increase and functional activation. after blocking adenosine A2A receptors andactivating A1receptors can work together to enhance this protective effect of ischemic nerve3. Adenosine A2AR deficiency can enhance the GLT-1expression in theischemia/reperfusion district. When blocking adenosine receptors A1function, this enhancedexpression trend is weakened; When adenosine receptors A1function is activated, thisenhanced expression trend is strengthening. Combined with previous experiments of thisgroup found that: after the adenosine A2A receptor gene knockout can significantly reduceextracellular glutamate concentration in cerebral ischemia/reperfusion district, and improveneurological function, thus we speculate: ischemic neuroprotective function due to adenosineA2A receptor absence may activate the A1R functionality and increase the expression ofGLT-1, Increase the capacity of the glutamate transporter into cell, thereby reducing theaccumulation of a large number of glutamate-mediated excitotoxicity injury4. This study lay a theoretical foundation for the development of new ways inneuroprotection of ischemic brain injury, the role of target and combined treatment program,for combined using the adenosine A2A receptor suppressing drugs and A1receptor activationdrugs into clinical research, and give to the timing of drugs a theoretical basis. |
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