ObjectiveTo explore the effects of different doses of PcTx1,a specific blocker of acid-sensing ion channel 1a,on global cerebral ischemia/repedfusion (I/R) injury in rats,MethodsSixty adult male Sprague Dawley rats (weighing 250-300 g) were randomly divided into 6 grups ( n =10 each):sham operation group (group S),I/R group,different doses of PcTx1 ( 10 ng/ml,group P1 ; 25 ng/ml,group P2 ; 50 ng/ml,group P3 ;and 500 ng/ml,group P4 ) groups.Global cerebral ischemia was induced by the modified procedure of Pulsinelli 4-vessel occlusion.In groups P1,P2,P3 and P4,different doses of PcTx1 ( 10,25,50 and 500 ng/ml),6 μl each,were respectively injected into the lateral cerebral ventricle at the initiation of reperfusion,while equal volume of double distilled water was injected instead in group I/R.Six rats in each group were sacrificed at 24 h of reperfusion,and the brains were immediately removed,Thereafter,the contents of malondialdehyde (MDA),reduced glutathione (GSH) and ritric oxide (NO),the activities of constitutive NO synthase (eNOS) snd inducible NO synthase (iNOS) were detected in hippocampus.Four rats in each group were sacrificed at 72 h of reperfusion,and hematoxylin and eosin staining was used to observe the pathomorphological changes of the hippocampal neurons.ResultsCompared with group S,the other groups showed decreases in the contents of GSH,while increases in the contents of MDA and NO and the activities of cNOS and iNOS ( P ＜ 0.05 or 0.01 ).The contents of GSH increased,while the contents of MDA and NO and the activities of cNOS and iNOS decreased in groups P2,P3 and P4 compared with group I/R ( P ＜ 0.05 or 0.01).Compared with group P1,the contents of GSH increased,the contents of MDA and the activities of cNOS decreased in groups P2,P3 and P4,and the contents of NO and the activities of iNOS decreased in groups P3 and P4 ( P ＜ 0.05 or 0.01 ).Compared with group P2,the activities of iNOS decreased in groups P3 and P4(P ＜ 0.05 or 0.01).The damage to neurons in hippocampal CAI was severe in groups I/R and P1,but it was attenuated in groups P3 and P4.ConclusionPcTx1 25,50 and 500 ng/ml (6 μl)injected into lateral cerebral ventricle can attenuate global cerebral I/R injury in rats,and the dose 50 ng/ml (6 μl) is more suitable.

Objective To investigate the role of acid-sensing ion channel 1a(ASIC1a) in global cerebral ischemia-reperfusion injury in rats.Methods Forty male SD rats weighing 250-300 g were randomly divided into 4 groups (n =10 each): sham operation group (group S),cerebral ischemia-reperfusion group (group I/R),solvent control group (group SC) and group PcTX1 (a ASIC1 a blocker,group P).Global cerebral ischemia-reperfusion was induced by four-vessel occlusion.PcTX1(500 ng/ml)6 μl or solvent 6 μl was injected into the crerbral ventricular at the begining of reperfusion in groups P and SC respectively.The rats were sacrificed at 24 h of reperfusion,and then the hippocampi were removed for determination of Caspase-3,Bcl-2 and Bax protein expression and microscopic examination.Results Compared with group S,the expression of Caspase-3,Bcl-2 and Bax protein was up-regulated in groups I/R,SC and P (P ＜ 0.05).Compared with group I/R,the expression of Caspase-3 and Bax was down-regulated,and the expression of Bcl-2 was up-regulated in group P ( P ＜ 0.05).There was no significant difference in Caspase-3,Bcl-2 and Bax protein expression between groups I/R and SC (P ＞ 0.05).The histopathologic damage was ameliorated in group P as compared with group I/R.Conclusion ASIC1a can induce global cerebral ischemia-reperfusion injury in rats by up-regulating Caspase-3 and Bax expression,and down-regulating Bcl-2 expression and inducing apoptosis.

Objective To evaluate the effects of hydrogen-rich saline on the expression of miR-210 and miR-21 in hippocampus during global cerebral ischemia-reperfusion (I/R) in rats.Methods Seventy-two healthy male Sprague-Dawley rats,aged 9-10 weeks,weighing 250-300 g,were randomly divided into 3 groups (n =24 each):sham operation group (group S),group I/R,and hydrogen-rich saline group (group H).Global cerebral I/R was produced by 4-vessel occlusion method.In group H,0.6 mmol/L hydrogen-rich saline 5 ml/kg was injected intraperitoneally at 0 and 6 h of reperfusion,while the equal volume of normal saline was injected instead of hydrogen-rich saline in the other two groups.Rats were sacrificed at 24 and 72 h of reperfusion,and then the bilateral hippocampi were removed for detection of the expression of miR-210 and miR-21 using RT-PCR.The global brain tissues were also obtained and stained with HE for examination of the changes in pyramidal cells in the CA1 region of hippocampus.Results Compared with group S,the expression of miR-210 and miR-21 was significantly up-regulated,and the number of pyramidal cells was decreased in group I/R (P ＜ 0.05).Compared with group I/R,the expression of miR-210 and miR-21 was significantly down-regulated,and the number of pyramidal cells was increased in group H (P ＜ 0.05).The pathological changes were significantly ameliorated in group H.Conciusion The mechanism by which hydrogen-rich saline attenuates global cerebral I/R injury is related to downregulation of the expression of miR-210 and miR-21 in rat hippocampus.

Objective To evaluate the effect of hydrogen-rich saline on the regulatory T cells (Tregs ) in the peripheral blood during global cerebral ischemia-reperfusion (I/R ) in rats .Methods Seventy-seven male Sprague-Dawley rats ,aged 2-3 months ,weighing 260-300 g ,were randomly divided into 3 groups using a random number table:sham operation group (group S , n=11) ,group I/R (n=33) ,and hydrogen-rich saline group (group H , n=33 ) .Global cerebral I/R was produced by 4-vessel occlusion method .The bilateral carotid arteries were blocked for 15 min followed by reperfusion in I/R and H groups .In group H ,0.6 mmol/L hydrogen-rich saline 5 ml/kg was injected intraperitoneally at 0 and 6 h of reperfusion ,while the equal volume of normal saline was injected instead in the other two groups .Before ischemia (T0 ) in group S and at 6 ,24 and 72 h of reperfusion (T1-3 ) in I/R and H groups ,7 rats were chosen ,the blood samples from the peripheral vein were collected for determination of the number of Tregs . Then the animals were sacrificed and the spleen was removed for measurement of transforming growth factor-β1 (TGF-β1) content .The left 4 rats of each group were sacrificed at T0 and T1-3 and the brains were obtained for examination of the pyramidal cell morphology in the hippocampal CA 1 region and for determination of the number of pyramidal cells in brain tissues .Results Compared with group S , the number of pyramidal cells in the hippocampal CA1 region ,the number of Tregs in the peripheral blood and content of TGF-β1 in the spleen were significantly decreased at T1-3 in group I/R ( P<0.05) .Compared with group I/R ,the number of pyramidal cells in the hippocampal CA 1 region and the number of Tregs in the peripheral blood at T2-3 ,and the content of TGF-β1 in the spleen at T1-3 were significantly increased ( P<0.05) ,and the pathological changes of pyramidal cells were attenuated in group H .Conclusion The mechanism by which hydrogen-rich saline attenuates global cerebral I/R injury may be related to the increased number of Tregs in peripheral blood and promoted secretion of TGF-β1 in rats .