Effects Of Hydrogen-rich Saline On Expression Of P38MAPK During Cerebral Is Chemia-reperfusion In Rats

Background and objectiveApoptosis plays an important role in cerebral ischemia diseases through the activation of apoptosis cells and pro-apoptosis cytokines. Saturated hydrogen saline are considered to actively accelerate apoptosis resolution. In the present study, we investigated the effect of saturated hydrogen saline on apoptosis responses induced by ischemia/reperfusion injury. To investigated whether saturated hydrogen saline reduces apoptosis responses following cerebral ischemia/reperfusion injury. MethodsAdult male Sprague-Dawley rat were used for t MCAO model. Animals were randomly assigned into experimental groups. Cerebral ischemia was induced by occluding the origin of the middle cerebral artery(MCA) for 2h using the intraluminal thread model. Saturated hydrogen saline or vehicle was given into lateral cerebroventricle through microsyringe after the reperfusion.The rats(n=10 per group) were evaluated for motor-deficits by an observer blinded to the identity of the groups at 24 h after reperfusion. The motor-deficit was scored on a four-point neurological scale as previous reported, no observable neurological deficit(normal); 1,failure to extend left forepaw on lifting the whole body by tail(mild);2,circling to the contralateral side but normal posture at rest(moderate);3,leaning to the contralateral side at rest(severe);4,no spontaneous motor activity.Rats were sacrificed 24 hours after MCAO, and brains were removed, frozen, and six coronal serial sections were cut 2-mm intervals from the frontal pole using a mouse Brain Matrix(Roboz surgical instrument). Coronal sections were stained with 2% triphenyl-2,3,4-tetrazolium-chloride(TTC) for 25 min at 37℃. Sections were scanned, digitalized and infarct areas were measured blindly using the image pro-plus software. The wet minus dry weight method was used for determining water content of the ischemia brain at 24 hours after t MCAO. Transmission of the p38 MAPK.the activation of apoptosis text by TUNEL.p38 MAPK and p-p38 MAPK was observed by Western blot analysis and immunostaining. Results 1.Saturated hydrogen saline ameliorated neurological dysfunctions.Neurologic deficits, evaluated at 24 h after reperfusion, were significantly increased in I/R group compared with saturated hydrogen saline group(neurological scores 2.12±0.58 versus 1.19±0.63,n=6,respectively,Tab.3). No rats appeared with neurological impairment symptoms in the sham group. 2.Saturated hydrogen saline extenuated pathological damage in the cortex Hematoxylin and eosin staining was showed in Fig.2. In the sham group no neuronal damage observed and no inflammatory cells infiltrated. Neurons were eumorphism with normal cellular architecture. The nuclei were in the cell center and clear stained. In I/R group, the brain tissue characterized by a majority of degenerated and necrotic cells, which contained pyknotic nuclei, cavitation with neuronal loss and disorders. In saturated hydrogen saline group, there was moderate neuronal damage in the ischemic area. 3.Saturated hydrogen saline reduced brain infarction and water content of the brainRepresentative coronal brain sections from the experiment groups were showed in fig3. Infarct volume at 24 h after reperfusion decreased significantly in saturated hydrogen saline group compared with I/R group. The infarct size was quantified by the ratio of corrected infracted volume to whole brain volume. The edema of the ipsilateral brain in Saturated hydrogen saline group was significant reduced than I/R group. 4.Saturated hydrogen saline inhibited neural apoptosis.The apoptotic rate of the I/R group was significantly increased after I/R. Saturated hydrogen saline treatment dramatically reduced the apoptotic rate. In the sham group, there are seldom apoptotic cells in the neural. 5.Saturated hydrogen saline inhibited p38-mapk activation.As shown in Fig.5, p38 MAPK expressed in total protein extracts and nuclear protein extracts of ischemic cortex was measured by western blotting. Western blot analysis showed than the expression of p38 MAPK in total protein was more prominently at 24 hours after t MCAO in rat. In Saturated hydrogen saline group, the expression of p38 MAPK was profoundly attenuated, but significantly increased in I/R group. ConclusionSaturated hydrogen saline significantly suppressed apoptosis response after ischemia/reperfusion injury. It attenuated ischemia/reperfusion induced acute apoptosis reaction in the ischemic region.