Objective To investigate the effect of heme oxygenase-1 (HO-1) on hypoxia-reoxygenation (H/R) injury in cultured neonatal rat cardiomyocytes. Methods Primary cultured neonatal rat cardiomyocytes were randomly divided into4 groups: Ⅰ control group (group C), Ⅱ H/R group, Ⅲ hemin group and Ⅳ hemin+zinc protoporphyrin (ZnPP) group. In group H/R, the cardiomyocytes were exposed to 2 h of hypoxia followed by 6 h of reoxygenation. Hemin was added to the culture medium with a final concentration of 20 μmol/L 24 h before hypoxia and immediately before hypoxia in group hemin. Hemin and ZnPP were added to the culture medium with final concentrations of 20 μmol/L 24 h before hypoxia and immediately before hypoxia in group Henin + ZnPP. The cells were incubated in 35 mm culture dishes (2 ml in each dish) and 50 ml culture flasks (3 ml in each flask)(45 dishes and 3 flasks in each group). The HO-1 expression, cell survival rate, LDH activity, malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were detected after the end of reoxygenation. Results Compared with group C, the LDH activity, MDA level and HO-1 expression were significantly increased, while the cell survival rate and SOD activity decreased in the other three groups ( P ＜ 0.05). Compared with group H/R,the LDH activity and MDA level were significantly decreased, while the HO-1 expression, cell survival rate and SOD activity increased in group hemin ( P ＜ 0.05 ), but no significant change was found in group hemin + ZnPP (P ＞0.05). H/R injury was obvious in group H/R and hemin + ZnPP, but was significantly attenuated in group hemin. Conclusion HO-1 can protect cardiomyocytes against H/R injury in neonatal rats.

Objective To study the cardioprotection effects of heme oxygenase-1(HO-1)on cardiopulmonary resuscitation(CPR).Method Male Sprague-Dawley rats were asphyxiated for 9 minutes and resuscitated.Rats wefe randomly divided into 4 groups,namely,sham asphysiation group,CPR group,Hemin group and Heroin +ZnPP group(zinc protoporyphyrin IX).Resuscitated groups were further divided into two subgroups according to various intervals:6 hours and 24 hours after resuscitation.Hemodynamic was observed.Serum creatine phosphokinase-MB(CPK-MB)and lactate dehydrogenase were determined.HO-1 in heart tissue homogenates was assayed.Ultrastructure of rats hearts was examined.Statical evaluation was performed with analysis of variance.Results The mean blood pressure(MBP)in resuscitated groups was significantly reduced after resuscitation,hadn't any difference between supgroups.The scores of dp/dt 40 and-dp/dt were significantly decreased in CPR group and Hemin+ZnPP group after resuscitation(all P＜0.05),but dP/dt40 in Heroin group did nol differ significantly after resuscitation.and-dp/dt decreased only 0.5 hours and one hour after resuscitation and returned to baseline values two hours after resuscitation.The scores of dp/dt 40and-dp/dt in Heroin group at different intervals after resuscitation were significantly higher than those in CPR group and Hemin+ZnPP group(all P＜0.05).Serum CPK-MB and LDH in CPR group and Hemin+ZnPP group at 6 hours and 24 hours after resuscitation were significantly higher than those in Hemin group(all P＜0.05).The cardiac tissue ultrastructure of rats in Hemin group was more intact than that of CPB group and Hemin+ZnPP goup.HO-1 levels in heart tissue homogenates of Hemin group at 6 hours and 24 hours after resuscitation were significantly higher than those in CPR group and Heroin+ZnPP group(all P＜0.05).Conclusions HO-1 expression induced by Heroin can effectively improve post-resuscitation myocardial dysfunction,alleviate cardiac injury,keep the ultrassructure integrity of cardiac myocytes.It may be a new approach to treat myocardial dysfunction after resuscitation.

Objective To study the protective effect of δ-opioid receptor agonist DADLE Oil hypoxia-in-duced injury of rat cortical neuronfl.Method Cortical neuron,q cultured for 8,lays were assessed by MAP2 im-munofluorescenee and randonly divided into hypoxia group(n=6),and DADLE preconditioning plus hypoxia group(n=6),normoxia group(n=6),and DADLE preconditioning plus normoxia group(n=6).The cells in hyoo O'oup Were cultured in a hyooxic incubator containing I%02,5%c02,and 94%N2 at 37℃.The cens in DADLE preconditioning groups Wel'preconditioned with 10 ttmol/L DADLE before culture.The cells in normoxia group were cultured in an incubator COntaining95%air and 5%c02 at 37℃.The survival rate ofneu.rOllS cultured for 72 hours was analyzed with hochest-33258 staining,and lactate dehydrogenase(LDH)release Wills detected.Glucose in the culture medium was detected to assess glucose/energy metabolism.The data of LDH re.1ease and ducose level were analyzed by A.NOVA.and cell survival rates wG:qanalyzed by Chi-square Tests.Re.vtats When cortical neurons were cultured for 8 davs,95%the cells were cortical neurous as assayed by MAP2 immunofluorescence·Cell survival rate was significantly higher in DADLE preconditioning Cus hypoxia group than in hypoxia group[(71.88±1.77)%vs.(43.58±3.07)%(P<0.05)].LDH release was significantly lower in DADLE preconditioning plus hypoxh group than in hypoxgroup[(3824.27±294.86)vs.(4516.59±605.02),P<0.05].Glucose level in the culture medium was significantly higher in DADLE preconditioning plus hypoxia group than in hypoxia group[(11.92-4-2.05)mmol/L vs.(9.88±0.71)mmol/L,P<0.051.Conclusions 6-opioid receptor agonist DADLE Can protect rat cortical neurons from hypoxia injury, which might be the mechanism of reducing glucose/energy metabolism in cortical neurons during the period of hypoxia.

Objective To explore the therapeutic potential and mechanism of stem cells mobilized by granulocyte colony-stimulating factor (G-CSF) and AMD3100 to repair global cerebral ischemia injuries in a rat model of cardiac arrest (CA) and cardiopulmonary resuscitation (CPR).Methods Cardiac arrest was induced by asphyxia.Fifty-six SD rats were randomly assigned into four groups:G-CSF group,G-CSF + AMD3100 group,CPR control group and sham operated group.The animals were sacrificed at 3d and 6d after CPR respectively.The neurological status and morphological changes of damaged cerebrum,the apoptosis of nerve cells and vascular endothelial growth factor (VEGF) expressed in brain tissue and capillary density in hippocampus and temporal lobe cortex were measured and analyzed by means of neurological deficit score (NDS),adhesive tape removal test (TRT),ELISA,MRI and immunofluorescence.Results NDS in G-CSF + AMD3100 group (61.4 ± 10.7) was significantly higher than that in CPR control group (49.9 ± 10.4) at 3 d after CPR (P ＜0.05).And less time consumption for TRT found in G-CSF + AMD3100 group (85.5 ±28.9) s rather than was in CPR control group (148.1 ± 23.8) s and G-CSF group (118.5 ± 30.4) s (P ＜ 0.05).The severity of cerebral injury assessed by MRI was significantly milder at both 3 d and 6 d in the two stem cell mobilization groups.The apoptosis rate of nerve cells in G-CSF + AMD3100 group (0.23 ± 0.06) was significantly lower than that in G-CSF group (0.34 ±0.08) at 3 d after CPR,and that in both stem cell mobilization groups was lower than that in CPR control group (0.44 ± 0.09) (P ＜ 0.05).At 3 d and 6 d after CPR,the levels of VEGF in brain tissue were (106.2 ±23.3) pg/mL and (79.9 ± 18.4) pg/mL in G-CSF + AMD3100 group,and were (50.6 ± 13.7) pg/mL and (73.9 ± 16.6) pg/mL in G-CSF group,which were both significantly higher than that in CPR control group (23.1 ± 10.2) pg/mL and (36.2 ± 12.8) pg/mL (P ＜0.05).At 3 d after CPR,the cerebral capillary density (351.8 ±67.9) branches in every high power field (A/HPF) was significantly higher in G-CSF + AMD3100 group than that (301.4 ± 77.3) A/HPF in G-CSF group and (250.4 ± 48.0) A/HPF in CPR control group (P ＜ 0.05).The cerebral capillary density in G-CSF group elevated to (348.4 ±76.7) A/HPF at 6 d after CPR which was significantly higher than that at 3 d (P ＜0.05),and there was no difference between that at 3 d and 6 d in G-CSF + AMD3100 group.Conclusions The mobilization stem cells improve the impaired neurological function.The increased expression of VEGF in brain tissue,the neo-vascularization promoted by the mobilized stem cells and the inhibition of nerve cell apoptosis may be associated with the protective effects of the stem cell mobilization.

Objective To compare the difference in cardiac injuries between asphyxia and ventricular fibrillation modes in different periods after cardiac arrest (CA).Methods The model was established in Cardiopulmonary Resuscitation Lab,Sun Yat-sen University.A total of 35 male SD rats were used to produce the asphyxia or ventricular fibrillation (VF) cardiac arrest models randomly.Both of the two modes were induced 8 minutes cardiac arrest.The myocardial HE stains,mitochondrial respiratory control ratio (RCR),and echocardiography were observed at 4 h,24 h and 72 h after ROSC (restoration of spontaneous circulation).The results were expressed as (-x ± s),t test was performed to compare between two groups,and one way analysis of variance was used to compare multiple groups.P ＜ 0.05 was considered as significant difference.Results HE stains showed damages were more serious in the VF mode than in asphyxia mode at 4 h,and both of them had a disorderly-arranged myocardium at 72 h.RCR in VF mode became worse at 4 h,and RCR resumed at 24 h in both modes without significant difference compared with the sham operated rats.The echocardiography showed VF mode had a lower left ventricular ejection fraction (LVEF) than asphyxia mode at 4 h (29.68％ vs.42.16％,P =0.03),and there was no difference in LVEF between VF mode and the sham operated rats at 24 h,however no difference in LVEF between the asphyxia and sham operated rats at 72 h.Both of them had a thicker left ventricular anterior wall than the sham operated rats at 72 h (2.41 mm vs.1.72 mm,P=0.013; 2.61 mmvs.1.72 mm,P=0.007),and there was no significant difference between them.Conclusions The ventricular fibrillation mode has a more severe injuries in early period,but it recovers sooner than asphyxia one.Both of two groups get compensatory left ventricular hypertrophy in later period of ROSC.

[Objective] To explore the variety of matrix metalloproteinase 9 (MMP9) and blood brain barrier (BBB) in cardiopulmonary resuscitation rats.[Methods] Eighty rats were randomly divided into 2 groups:the sham-operated group (n ＝ 40) and the resuscitation group (n ＝ 40).The two groups were anaesthetized and endotracheally intubated,the resuscitation group was also induced to cardiac arrest by aphysia.Then the rats were put to death and samples were taken at immediate,3 h,9 h,24 h,and 48 h.After that,the expression of MMP9,MMP9 mRNA,water content and Evans blue content in brain tissue were detected.Ultramicrostructure of brain tissue was observed with electron microscope.[Results] Compared to the sham-operated group,at 3 h,9 h,24 h and 48 h,the expression of MMP9 of resuscitation group was significantly changed.MMP9mRNA significantly increased.Water content statistically increased and so was Evans blue content.The change of ultramicrostructure in the resuscitation group at 3 h,9 h,24 h,and 48 h was obvious.[Conclusion] The expression of MMP9 and MMP9mRNA obviously increased in the cerebral ischemia model with CPR rats,and got to peak at 24 h.Water content and Evans blue content in brain tissue obviously increased in the cerebral ischemia model with CPR rats,BBB was destroyed,and the peak was 24 h.The injury of ultramicrostructure of brain tissue with electron microscope was obvious,and the peak was 24 h.

Objective To establish the oxygen glucose deprivation (OGD)/reoxygenation experimental model of hippocatnpal neurons of rat in vitro, and to try to identify the length of time for producing optimum injury in this model. Method The primary hippocampal neurons of newborn Sprague-Dawley rats were cultured for 7 days and randomly (random number) divided into a control group and OGD groups. The OGD groups were assigned into 1 h, 2 h, 4 h, 6h, 8 h and 10 h subgroups in accordance with different lengths of time for oxygen glucose deprivation. The neurons of OGD groups were placed into a tri-gas incubator containing 0.5% oxygen and the culture medium was substituted with the glucose-free Earle' s balanced salt solution, simulating cerebral ischemia injury in vivo. The morphology of neurons was observed after reoxygenation for 24 hours. The MIT assay was used to determine the rate of survived cells derived from the value of optical density (OD) of cells. The lactate dehydro-genase (LDH) content in culture medium was detected to evaluate the neuron injury. The apoptotic rate of neurons was measured by using flow cytometry. Dunnett-test and Spearman correlation analysis were used to analyze the data with SPSS version 16.0 soft ware package. Results The morphological damage of neurons in OGD groups aggravated gradually, optical density and cell survival rate decreased (rs= -0.961 and rs = -0.966, P <0.01), and the amount of LDH increased (rs = 0.990, P <0.01) with longer duration of exposure to oxygen glucose deprivation, and the rate of neuron apoptosis increased obviously which was significantly statistical difference in com-parison with the control group (P < 0.05). Under the setting of oxygen glucose deprivation for 6 hours, the apop-tosis rate of neurons approximated to 50% . Conclusions The oxygen glucose deprivation/reoxygenation model of rat's hippocampal neurons in vitro was established successfully. From the findings of morphological changes and apoptosis rate of neurons, the oxygen glucose deprivation for 6 hours may be the suitable length of time for inducing neuron injury in this model.

Objective To compare the changes of physiological parameters after cardiac arrest caused by asphyxia with that of cardiac arrest induced by ventricular fibrillation in rats and assess the values of the parameters on predicting ROSC and 24 h survival rate. Method Two groups of Sprague-Dwaley rats, which randomly (ramdom number) included 30 animals in each group, were investigated. Cardiac arrest were induced by asphyxia (AS group) or ventricular fibrillation(VF group). PETCO2, aortic pressure, left ventricular pressure and ECG of limb lead Ⅱ were recorded continuously, dP/dt4o was calculated with the windaq software. The parameters were compared between the two groups at baseline, precordial compression(PC) 10 s, PC 1 min, PC 3 min, ROSC 1 h and ROSC 2 h. The relations were explored between the parameters and ROSC/24 h survival rate. Results PETCO2,aortic pressure, left ventricular pressure and ECG have distinctive changes in the two groups. In group VF, PETCO2 of ROSC rats at BL, PC 1 min and PC 3 min were higher than those of Non-ROSC rats (P ＜ 0.05); PETCO2of 24 h survival rats at ROSC 1 h and ROSC 2 h were higher than those of 24 h death rats (P ＜ 0.05), which were not observed in the group AS. dP/dt40 and - dP/dt40 at ROSC 1 h and ROSC 2 h in group VF were higher than those in group AS (P ＜ 0.05). Conclusions Physiological parameters after cardiac arrest caused by asphyxia or that of cardiac arrest induced by ventricular fibrillation in rats have unique features respectively. PETCO2 in cardiac arrest caused by ventricular fibrillation may predict ROSC and 24 h survival rate. Researchers have to select the appropriate cardiac arrest model according their research purposes and clinical requirments.

Objective To investigate the effects of bone marrow mesenchymal stem cells (MSCs)treatment on TSG-6 in a rat model of cardiopulmonary resuscitation (CPR).Methods Sprague Dawley (SD) rats were randomly (random number) divided into sham group,phosphate buffer solution (PBS)-treated group and MSCs-treated group.Animals were subjected to asphyxial cardiac arrest followed by CPR.In PBS-treated group or MSCs-treated group,animals were injected intravenously with PBS or MSCs at 2h after resuscitation.Neurological deficit scores (NDS) were assessed at 1,3 and 7 d after CPR.Serum S-100B was assayed using enzyme linked immunosorbent assay (ELISA).Immunofluorescence was performed to detect donor MSCs and the expression of TSG-6 in brain.TSG-6 and proinflammatory cytokines in brain were assayed using real time reverse transcription-polymerase chain reaction (RT-PCR).Western blot analysis was performed to measure the levels of neutrophil elastase (NE) in brain.Multiple comparisons were made by analysis of variance.Results At 3d and 7d,MSCs-treated group demonstrated higher NDS than PBS-treated group (P ＜ 0.01),and serum S-100B levels significantly reduced in MSCs-treated group compared with PBS-treated group (P ＜ 0.01).DAPI-labeled MSCs migrated into the ischemic brain and some DAPI + cells colocalized with TSG-6.Compared with PBS-treated group,MSCs treatment significantly up-regulated the expression of TSG-6 and reduced the expression of NE and proinflammatory cytokines in brain at 3 d and 7 d after CPR (P ＜ 0.05).Conclusion Systemically administered MSCs suppressed inflammatory responses in brain after CPR and improved neurological function in rats possibly via induction of TSG-6.

Objective To study the effects of mild hypothermia on cardiomyocyte contractility improvement after ischemia-repeffusion injury and on the preservation of well-functioning mitochondrial respiratory capability.Methods A total of 50 newborn SD rats 1 ～ 2 days after delivery were sacrificed and their hearts taken to preserved in 4 ℃ cold D-hanks buffer solution with 0.12％ pancreatic proteinase and collagenase and then processed with 37 ℃ water bath to collect the cardiomyocytes cultured in DMEM medium with 10％ FBS for 5 days.The cardiomyocytes of rats were subjected to ischemia/reperfusion,in vitro,by oxygen and glucose deprivation(OGD)/oxygen and glucose restoration(OGR).The cardiomyocytes of rats after ischemia/reperfusion were divided into three groups:control group,hypothemia group and normothermia group.Contractile frequency and velosity were determined before OGD and 0 h,0.5h,1 h,1.5 h and 2 h after OGR.Ultrastructure changes of cardiomyocytes and mitochondrion were observed under transmission electron microscope(TEM)0 h and 2 h after OGR as well as assessment ot respiratory rate and respiratory control rate(RCR)with Clark oxygen electrode in each group.All data were analyzed with statistical software of SPSS 13.0.Results Contractile function of cardiomyocytes in hypothermia group and normothermia group declined to nadir at 0 h after OGR(P =0.000)and the contractile function of cardiomyocytes in hypothermia group was improved one hour later,compared with the normothermia group(P =0.000).Obvious swelling of mitochondrion was observed under TEM in normothermia group with little alteration after OGR.The RCR assessments indicated respiratory function in normothermia group was impaired after OGR(P =0.000)and this may be responsible for contractility dysfunction.Conclusions Mild hypothemia used after ischaemia can optimize the contractility of cardiomyocytes after a normothermia OGR,and the well-functioning respiratory capability of mitochondrion may be preserved in this process.