Objective To observe the expression characteristics of green fluorescent protein(GFP)-positive myeloid cells(mainly monocytes,macrophages,and neutrophils)and their pattern of response to diphtheria toxin(DT)in Lyz2 internal ribosome entry site(IRES)DT receptor/enhanced GFP(DTREGFP)mice.Methods Transgenic Lyz2 IRES DTREGFP mice(6～8 weeks old)and C57BL/6J mice with the same genetic background were selected randomly and injected intraperitoneally with DT(25 ng/g)for 3 consecutive days.Bone marrow,peripheral blood,and spleen cells were separated and made into single-cell suspensions with GFP and CD11b as markers.Changes in the proportion of GFP+cells to total myeloid cells(CD11b+)was detected in these tissues at different time points before and after drug administration,using flow cytometry.Results Peripheral blood CD11b+GFP+cells accounted for(24.62±5.655)％of CD11b+cells before DT injection,and this proportion gradually decreased after injection and reached a minimum of(7.982±2.729)％on the third day.The proportions in spleen and bone marrow were(13.73±2.994)％and(65.23±4.261)％before DT injection,respectively,decreased most significantly on the first day after injection to(3.468±0.5862)％and(50.98±7.957)％,respectively,and then gradually recovered.Conclusions DT effectively reduced the proportions of monocytes,macrophages,and neutrophils in Lyz2 IRES DTREGFP mice,but the pattern of change varied among tissues.

Objective To observe the expression characteristics of green fluorescent protein(GFP)-positive myeloid cells(mainly monocytes,macrophages,and neutrophils)and their pattern of response to diphtheria toxin(DT)in Lyz2 internal ribosome entry site(IRES)DT receptor/enhanced GFP(DTREGFP)mice.Methods Transgenic Lyz2 IRES DTREGFP mice(6～8 weeks old)and C57BL/6J mice with the same genetic background were selected randomly and injected intraperitoneally with DT(25 ng/g)for 3 consecutive days.Bone marrow,peripheral blood,and spleen cells were separated and made into single-cell suspensions with GFP and CD11b as markers.Changes in the proportion of GFP+cells to total myeloid cells(CD11b+)was detected in these tissues at different time points before and after drug administration,using flow cytometry.Results Peripheral blood CD11b+GFP+cells accounted for(24.62±5.655)％of CD11b+cells before DT injection,and this proportion gradually decreased after injection and reached a minimum of(7.982±2.729)％on the third day.The proportions in spleen and bone marrow were(13.73±2.994)％and(65.23±4.261)％before DT injection,respectively,decreased most significantly on the first day after injection to(3.468±0.5862)％and(50.98±7.957)％,respectively,and then gradually recovered.Conclusions DT effectively reduced the proportions of monocytes,macrophages,and neutrophils in Lyz2 IRES DTREGFP mice,but the pattern of change varied among tissues.

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 mitochondrial permeability transition pore (mPTP) of hippocampal neurons in process of hydrogen-rich saline attenuating global cerebral ischemia-reperfusion (I/R) injury in rats.Methods Seventy-two male Sprague Dawley rats,weighing 250-300 g,were randomly divided into six groups ( n =12 each):sham operation group (group S),cerebral ischemia-reperfusion group (group IR),normal saline group (group NS),hydrogen-rich saline group (group H),atractyloside group (group A) and hydrogen-rich saline + atractyloside group (group HA).Global cerebral I/R injury was produced by four-vessel occlusion method.Bilateral vertebral arteries were cauterized.Then bilateral common carotid arteries were occluded for 15min and followed by reperfusion.In groups H and HA,hydrogen-rich saline 5 ml/kg was injected intraperitoneally immediately after reperfusion,while equal volume of normal saline was injected in the other four groups.The rats in groups A and HA received intracerebroventricular injection of atractyloside 15 μl 10 min before reperfusion,while groups NS and H received intracerebroventricular injection of equal volume of normal saline.After the neurological behavior was evaluated at 24 h of reperfusion,8 rats in each group were sacrificed and the hippocampi were immediately isolated and homogenized followed by density gradient centrifugation.The opening degree of mPTP was assayed with spectrophotometry and the mitochondrial membrane potential (MMP) was detected with Rhodamine 123 method.Four rats in each group were killed at 72 h of reperfusion and the brains were removed for microscopic examination of the area CA1 of the hippocampus and determination of the number of normal pyramidal neurons.Results Compared with group S,the neurological behavior was compromised,MMP was decreased and mPTP opening degree was enhanced in the other five groups ( P ＜ 0.05).The neurological behavior was better,MMP was increased and mPTP opening degree was decreased in groups H and HA as compared with group IR ( P ＜ 0.05).Compared with group H,the neurological behavior was compromised,MMP was decreased and mPTP opening degree was enhanced in group HA ( P ＜ 0.05).Compared with group IR,the number of normal pyramidal neurons at 72 h of reperfusion in the CA1 region of the hippocampus was higher in group HA ( P ＜0.05).The injury of the CA1 region of the hippocampus at 72 h of reperfusion was attenuated in group H as compared with groups IR,NS,A and HA.Conclusion Hydrogen-rich saline can attenuate global cerebral I/R injury throngh inhibiting the mPTP opening and reducing the dissipation of MMP,thus maintaining the mitochondrial function.

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.

17B~estradiol. Displacement experiments showed that when RU38486 concentration reached 100~280-fold that of [3H]corticosterone, it began to inhibit [3H]corticosterone binding, while low concentration of RU38486 had no inhibitory effect.