Experimental Research Of A3R Activation Effects On Inflammation Reaction After Intracerebral Hemorrhage In Rat

Objective:To observe the effects of adenosine-3receptor(A3R) activation by receptor agonist (C1-IB-MECA) on inflammation cell (microglia,white blood cells), tumor necrosis factor-alpha (TNF-a),interleukin-10(IL-10) and MMP-9in brain damage after intracerebral hemorrhage(ICH) model established in rats; to explore the protection mechanism and effects of A3R receptor agonist (Cl-IB-MECA).Methods:1.The ICH model was established by autologous blood in caudate nucleus of rats.2.90SD rats were randomly divided into three groups:sham-operation group,ICH group and treatment group,ever group has30rats.Each group was observed at six time point, which were6h,24h,2d,3d,5d and7d after the operation. And at each time point five rats were observed.3.Delivery methods:A3R receptor agonist (Cl-IB-MECA) administered according to0.2mg/Kg for the treatment group165min before and180min after model established.ICH group was given NS at the same time,the sham group was fully consistent with ICH group,except injected autologous blood.4. We judged the ICH model after2h assessment, and neurologic defect grade were carried out at each time point.5. The were sacrificed to measure the brain water content with wet-dry weight method.6. The contents of TNF-a,IL-10,in the brain tissue were detected with radioimmunoassay method.7. We detected microglia and MMP-9by using immunohistochemistry and image analysis method.8. White blood cells’ changes of the cerebral hemorrhage tissue detected by HE staining. Result1. The sham group nerve dysfunction at each time,no significant difference(P>0.05);the ICH and treatment group obviously statistic significance with sham group(P<0.05);at each time point of the ICH and treatment group,the neurologic deficits at2d、3d was the lowest which was greatly different from other time point (P<0.05);between the ICH and treatment group at2d、3d was no significant difference(P>0.05);at each time point of the treatment group,the neurologic deficits at24h、2d、3d、5d and7d were greatly different from the sham group,except6h after postoperative(P<0.05).2.The brain water at each time point were no significant difference(P<0.05);the ICH and treatment group were obviously brain edema,greatly different from sham group at each time point(P<0.05); brain edema of the ICH and treatment group at2d、3d was greatly different from other time point (P<0.05);between the ICH and treatment group at2d、3d was no significant difference(P>0.05);at each time point of the treatment group, at24h、2d、3d、5d and7d were greatly different from the sham group,except6h after postoperative(P<0.05), it shown that treatment could reduce cerebral edema after ICH.3. TNF-α in brain tissue of the sham group was no obvious change,no significant difference(P>0.05); the ICH and treatment group were greatly different from sham group at each time point(P<0.05);TNF-α of the ICH group at2d、3d was greatly different from other time point (P<0.05);between the2d、3d was no significant difference(P>0.05);at each time point of the ICH and treatment group, at24h、 2d、3d、5d and7d were greatly different from the sham group,except6h after postoperative(P<0.05).4. IL-10content in brain tissue of the sham group was no obvious change,no significant difference(P>0.05);the ICH and treatment group were greatly different from sham group at each time point(P<0.05); between the ICH and treatment group at each time point was no significant difference after postoperative(P>0.05);5. There was no obvious expression of microglia in sham group,it greatly increased in brain tissue of ICH and treatment group at each time point;expression of microglia of the ICH and treatment group at2d、3d was greatly different from other time point (P<0.05);between the ICH and treatment group at each time point was significant difference after postoperative(P<0.05);6. There was no obvious expression of MMP-9in sham group,no significant difference(P>0.05);the MMP-9content of ICH group was greatly different between6h、24h、5d、7d and2d(P<0.05),but no difference between2d and3d(P>0.05);between the ICH and treatment group at each time point was significant difference(P<0.05);7. After HE coloring the brain cells structure is clear and complete,no obvious cell swelling and karyolysis pyknosis;the brain tissue of ICH group congestion and edema, a large number of white blood cells infiltration, nerve cell degeneration and necrosis after postoperative;the microglia obviously added with time lengthen,the cerebral edema reduced after3d.In ICH and treatment group the cerebral edema and leukocyte infiltration also lessened. Conclusion:1.The ICH model of rat was made by autologous blood that is simple and make easily,and the changes of pathophysiology of rat models are as almost same as the ICH of patients, thus the model can be used generally.2.TNF-α,IL-10, microglial cells and MMP-9around the brian hematoma markedly increased after ICH that showed they took part in the pathophysiological process of the secondary damage after ICH.3.IL-10significantly increased after cerebral hemorrhage, IL-10may be protect the neurons after cerebral hemorrhage.4. Cl-IB-MECA Can reduce the activation of microglial cells,reduce release of TNF-α、MMP-9and mitigate cerebral edema, thereby it can protect neurons.