The Dynamic Expression Of OX40L In Brain Tissues Of MCAR Rats

Objectives: The mechanism of ischemic cerebrovascular disease is complicated after reperfusion injury,such as the toxic effect of acidexcitatory amino acids, inflammation and apoptosis. It is a hallmark that excessive immunological reaction in necrosis and ischemic region that results in inflammatory injury, where it has been implicated in tissue damage as well as in repair. However,anti-inflammation therapies have not reach the expected therapeutic effect.Looking for new inhibitors of inflammation may bring progress in treatment of cerebral ischemic reperfusion. To date, we[1-6] studied a serie of the dynamic expression of inflammation factors on cerebral infarction, such as platelet-derived growth factor-BB ( PDGF-BB ) , aquaporin-4 (AQP-4), IL–6, TNF-α, COX-2. At the same time, we also sieved traditional Chinese drugs to inhibit inflammation reation, such as naoxintong, curcumine, atorvastatin, salviol, ammothamnine. All the studies were published periodicals[7]. The study about OX40L is one of them.Costimulatory molecules are drawing more and more attention from scholars. Take OX40 ligand (OX40L,also known as tumor necrosis factor superfamily member 4,TNFSF4) for example. OX40L belongs to tumor necrosis factor superfamily and which expresses on the surface of immune cells, including B cells,monocytes,and dendritic cells (DCs), microglia, T cells, as well as nonimmune cells such as vascular endothelial cells, epithelial cells and in the tissues of hearts, skeletal muscles, testis, lungs. The ligand possess a variety of biological activities, for example the promotion of DCs differentiation and cellular adhesive attraction and the regulation of inflammation and inmmune responses. These exciting findings expand our knowledge of the complex immunological reaction. In recent years, there are many experimental studies conducted on OX40L, most of them are focused on artherosclerosis and acute coronary syndrome. There are rare reports on the effects of OX40L on ischemic cerebrovascular diseases. Therefore,the study on the dynamic expression of OX40L perhaps elucidates the mechanisms of reperfusion injury and suggests a treatmemt for ischemic cerebrovascular diseases.This study tested the dynamic expression of OX40L in ischemic rats brain tissues after reperfusion using immunohistochemistry technology and western blot. What's more, we studied brain infarction volume to investigate the dynamic expression of OX40L in brain tissues of MCAR rats.Methods:1 MCAR animal model was established by integrating Koizumi [8] method with our experience.The advantage of the model [9] includs non-invasive operation and reperfusion after ischemia.And there also are some disadvantage [9]: 1.the weight of experimental animal should be 250 - 300 g; 2.a monofilament was introduced to influence the blood circulation of hypothalamus thermotaxic center so that there is fervescence at postischemia, which is bad for results; 3.The operation needs higher skills; 4. the model is embolic apoplexy, different with stroke in crowds. We closely observed and controled some aspects to depress the death rate: 1.maintain ambient temperature 26℃, or rats is hard to analepsia, even to die; 2.slowly pull out the bolt, or the blood flow in reperfusion is so big to aggratate injury; 3.dysjunct blood thoroughly, don't ligate nerve that parallels external carotid artery (ECA) , ganglion sympatheticum and trachea;⒋operations shoud be quick to avoid thrombosis;⒌make up for body fluid;⒍guarantee Animal nutrition, especially post-operation, make animal feeds farina;⒎avoid stimulate excessive to decrease stringent state;⒏avoid duplicate anesthesia to influence the scores of model and the protection of the drug;⒐give a mark halfhour after analepsia.2 Male Sprague-Dawley rats were randomly assigned to the following 2 groups: MCAR and sham operation group. MCAR was divided into 5 subgroups randomly: 6 h, 24 h, 48 h, 72 h and 96 h after operation.The rats were sacrificed at 6 h ,24 h ,48 h , 72 h and 96h after operation.3 the ischemic brain tissues were used for testing brain infarction volume, immunohistochemistry and at 48h used for TTC staining (n = 3) respectively.Results:1 The infarcted volume: The brain tissues of sham group were stained red by TTC and there were no infarct, and that of MCAR group were stained white. The difference was statistically significant (P < 0.05) .2 The dynamic expression of immunopositive cells of OX40L: There is no definite immunopositive cells of OX40L of the MCAR group.3 The dynamic expression of OX40L by western blot is negative. Conclusion:The expression of OX40L was doubtful after ischemical reperfusion. That suggests there is no inevitable relationship between OX40L and ischemical reperfusion. There needs further studies.