Effect Of Adenosine A_2A Receptor Deficiency On Ischemic Brain Injury Induced By Transient Middle Cerebral Artery Occlusion In Mice

Background Stroke is not only one of the principal diseases which lead to the death of human being, but also the chief cause of disability resulted from diseases.Cerebral ischemic stroke is the main type of it. Although the scholars home and abroad have carried out many studies and researches, no surely effective neuroprotective agents have been used in the clinical treatment of this disease at present. Therefore to explore a new approach to neural protection is in great need. Adenosine is an important neuromodulator of the central nervous system. Up to now four kinds of subgroups of receptor of it have been cloned: A1,A2A,A2B and A3. Adenosine can participate in the regulation of the important physiological function in the brain. The unusual activity of it is related to the occurrence and development of some diseases of nervous system, and the interference may prevent or deteriorate the process of diseases. According to the animal experiments in the past, A2A receptor antagonist can prevent the neuron from injury induced by ischemia, but the exact functional sectors and mechanism are still not clear and need the further discussion and exploration.It is well known that after the acute cerebral ischemia, the ischemic brain damage may develop through many stages, mainly including: acute-stage injury, reperfusion injury and cerebral edema. After minutes of cerebral ischemia, with the cell energy being exhausted, both cell acidosis and the functional and structural damage of cell membrane occur, and then the nerve injury of early stage comes into being. Cerebral ischemia causes the glutamate to be let out overly in the brain. Glutamate makes the calcium ion internal flow by activating the receptor of NMDA and help the intra-cellular storage calcium let out, leading to the increase of the intra-cellular free calcium. Thus the lactonase and the proteinase in the cell are activated, and the cytoskeletal structure of the cell are destroyed, which result into a series of cascade reaction. Former studies and researches have proved that when the receptor of A2A located in front of the glutamate neuron synapse is activated, the outlet of glutamate is promoted. Therefore inference is made that the activation of the A2A receptor may lead to the calcium overload because of the promotion of the glutamate outlet. After reperfusion, with the restoration of cerebral blood flow,a lot of oxygen free radical is produced and inflammatory mediators increases, which cause the structural damage of cell membrane and the split of DNA. And many mechanisms work together and lead to reperfusion injury, blood-brain barrier destruction and cerebral edema. Cerebral edema, an important pathological link of reperfusion injury afer cerebral ischemia, may lead to and aggravate the delayed neuronal damage.It is found through experiments that Aquaporin4 (AQP4) were expressed high after the ischemic stroke, which is identical to the development of cerebral edema. Researches have proved that when the A2A receptor located in the vascular endothelial cell and the smooth muscle cell is activated, the cerebral vessels can be dilated. Hence another inference can be made that after cerebral ischemia the activation of the A2A receptor in the phase of reperfusion is likely to aggratate the cerebral edema out of the dilation of the cerebral vessels.Although the animal experiments in the past have discovered that adenosine A2A receptor deficiency can alleviate the ischemic brain damage, it is difficult to clearly figure out the probable sector and mechanism where it works because the time and point of observation on pathological injury of tissue are confined only after the reperfusion and at the same time there is no report on the influence of it on the cerebral edema. So it is necessary to further explore the impact of adenosine A2A receptor deficiency on the pathological injury of different phase and different type after cerebral ischemia.Objective: The study forms the model of middle cerebral artery occlusion(MCAO) by adenosine A2A receptor knockout in mice, to tentatively explore the sector of protective effect where adenosine A2A receptor deficiency works on the cerebral injury in connection with acute ischemic brain damage with different phases and different pathological types. It is also expected to be an important clue contributing to account for the protective mechanism of adenosine A2A receptor deficiency.Methods:1. Adenosine A2A receptor knockout (A2ARKO) mice and their wild-type littermates (A2ARWT) were divided into 2h cerebral ischemia group, cerebral ischemia with 22h reperfusion group and cerebral ischemia with 46h reperfusion group respectively. 2. Transient(2h) cerebral ischemia was induced by the middle cerebral artery occlusion (MCAO) in mice with suture method.3. Cerebral infarction volume was measured by image analysis of brain sections stained with cresyl violet (CV).4. Brain water content was evaluated with the dry-wet weighing method.5. The expression of calbindin D-28k (CB) and aquaporin-4 (AQP4) in ischemic brain was determined with immunohistochemical method.Results:1. The cerebral infarction volume in 2h cerebral ischemia group, cerebral ischemia with 22h reperfusion group and cerebral ischemia with 46h reperfusion group of A2ARKO mice were less than that in the corresponding groups of A2ARWT mice(P<0.01).2. Under the microscope positive cells of Calbindin D-28k can be seen in the cytoplasm of neuron. Those positive cells are mainly distributed over hippocampus and cortex. The cells at the peripheral area of infarction were counted and then the comparison were made among the groups.2h cerebral ischemia group was compared with sham operated group and it was found that the positive cells in A2ARWT mice are obviously less(P<0.01) and no clear differnce in A2ARKO mice(P>0.05). Then another comparison was made between the group of cerebral ischemia with 22h and 46h reperfusion and sham operated group , the positive cells in both A2ARKO mice and A2ARWT mice are found to be less(P<0.05,P<0.01). The comparison of each point time after cerebral ischemia,the positive cells of Calbindin D-28k in A2ARKO mice are apparently more than those in A2ARWT mice. There is no significant difference between 2h cerebral ischemia and 22h reperfusion in A2ARKO mice and A2ARWT mice,yet compared with the point of 2h cerebral ischemia and 22h reperfusion there have much less Calbindin D-28k immunoreactive material at the point of 46h reperfusion(P<0.01).3. Under the microscope AQP4 immunoreactive positive materials lie under the cell membrane,mainly expressed in astrocytes,also found in vascular endothelial cells and ependymal cells,and there are more positive cells in the surrounding area of infarction. The cells at the peripheral area of infarction were counted and then the comparison were made among the groups. At each time point after cerebral ischemia, compared with sham-operated group,A2ARWT mice and A2ARKO mice had more AQP4 immunoreactive positive cells (P <0.01). AQP4 immunoreactive positive cells in 2h cerebral ischemia group, cerebral ischemia with 22h reperfusion group and cerebral ischemia with 46h reperfusion group of A2ARKO mice were less than that in the corresponding groups of A2ARWT mice(P<0.05,P<0.01,P<0.01). Compared with 2h cerebral ischemia group , cerebral ischemia with 22h reperfusion group and 46h reperfusion group of A2ARKO mice had more AQP4 immunoreactive positive cells(P<0.01);furthermore, made the comparison with 22h reperfusion time point,AQP4 immunoreactive positive cells increased significantly at the 46h reperfusion time point(P<0.05). Compared among all time points after cerebral ischemia,there are significant deviation in A2ARWT mice (P <0.01). With the extension of ischemic time,AQP4 immunoreactive positive cells gradually increased.4. At the time of cerebral ischemia(2h)with 46h reperfusion, brain water contentsof A2ARKO mice and A2ARWT mice at the side of infarction were significantly great than the non-infarcted side (P <0.01).Compared with A2ARWT mice,A2ARKO mice had less brain water contents at the side of infarction,non-infarcted side was no significant difference (P> 0.05).5. The cerebral infarction volume in both A2ARKO mice and A2ARWT mice presents the negative correlation to the number of Calbindin D-28k immunoreactive positive cells(r=-0.764,p<0.01; r=-0.506,p<0.05), and the positive correlation to the number of AQP4 immunoreactive positive cells (r=0.837,p<0.01; r=0.809,p<0.01).Conclusion:1. Adenosine A2A receptor deficiency exerts the protection against ischemic brain injury both in the acute phase and reperfusion phase2. Adenosine A2A receptor deficiency can attenuate brain edema caused by cerebral ischemia3. The protection of Adenosine A2A receptor deficiency to ischemic brain injury may be due to the inhibition of intracellular calcium overload and AQP4 expression.