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Objective:To evaluate the effect of propofol anesthesia on autophagy in hippocampal neurons of newborn rats.Methods:Thirty-nine healthy Sprague-Dawley rats, aged 7 days, weighing 10-12 g, were divided into 3 groups ( n=13 each) using a random number table method: control group (group C), fat emulsion group (group F) and propofol group (group P). Normal saline 8 ml/kg was intraperitoneally injected for 5 consecutive days in group C. Medium-/long-chain fatty emulsion injection 8 ml/kg was intraperitoneally injected for 5 consecutive days in group F. Medium-/long-chain propofol injection 80 mg/kg was intraperitoneally injected for 5 consecutive days in group P. Five rats were sacrificed on 1st day after the end of propofol anesthesia, and hippocampal tissues were taken for determination of the expression of microtubule-associated protein 1 light chain 3B (LC3B) and Beclin-1 (by Western blot). The remaining rats in each group underwent the Morris water maze test on 19th day after the end of propofol anesthesia (30 days after birth), and the escape latency, percentage of time of staying at the target quadrant and the number of crossing the original platform were recorded. Results:Compared with group C, no significant change was found in the expression of hippocampal LC3B and Beclin-1, escape latency, percentage of time of staying at the target quadrant, and the number of crossing the original platform in group F ( P>0.05), and the expression of hippocampal LC3B and Beclin-1 was significantly up-regulated, the escape latency was prolonged, percentage of time of staying at the target quadrant was decreased, and the number of crossing the original platform was decreased in group P ( P<0.05 or 0.01). Conclusion:The mechanism by which propofol anesthesia causes long-term cognitive dysfunction may be related to promoting autophagy in hippocampal neurons of newborn rats.
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Objective:To evaluate the effects of exogenous biliverdin (BV) on the expression of Litaf in PC12 cells subjected to oxygen-glucose deprivation and restoration (OGD/R).Methods:PC12 cells were seeded in a 96-well cell culture plate at a density of 1×10 4 cells/well for 3 days and were divided into 3 groups ( n=18 each) by a random number table method: control group (group C), OGD/R group, and biliverdin group (BV group). Group C was incubated in a 37 ℃ incubator (95% air+ 5%CO 2) for 6 h. To establish the OGD/R model, cells were incubated with sugar-free medium in a 37 ℃ incubator (95% air+ 5%CO 2) for 2 h, and the medium was then replaced with normal medium and cells were continuously incubated in a 37 ℃ incubator (95% N 2+ 5% CO 2). In BV group, 2 μg/ml biliverdin was added immediately after oxygen-glucose restoration.Cells in 6 wells in each group were selected at 6 h of restoration for determination of the expression of Litaf protein and mRNA (by real-time polymerase chain reaction) and tumor necrosis factor-alpha (TNF-α) concentration (by enzyme-linked immunosorbent assay). Results:Compared with group C, the expression of Litaf protein and mRNA was significantly up-regulated, and TNF-α concentration in supernatant was increased in group OGD/R ( P<0.05). Compared with group OGD/R, the expression of Litaf protein and mRNA was significantly down-regulated, and TNF-α concentration in supernatant was decreased in group BV ( P<0.05). Conclusion:The mechanism by which exogenous biliverdin reduces OGD/R damage to PC12 cells is related to inhibiting up-regulated expression of Litaf and alleviating the inflammatory responses.
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Objective To evaluate the clinical effects of ultrasound-guided internal jugular vein catheterization in long axis plane,short axis plane and oblique axis plane,in order to identify the opti-mal axis plane for this procedure.Methods One hundred and eighty patients (male 94 cases,female 86 cases,aged 34-82 years)requiring ultrasound-guided internal jugular vein catheterization were in-cluded in this study.They were randomly divided into three groups (n =60 each),long axis group, short axis group and oblique axis group,with 60 cases in each group.The details of catheterization in-cluding the time accessing into vein,the time finishing cannulation,needle redirecting times,number of skin points of puncture,puncture successful rate and complications in the three groups were recor-ded.Results Compared with long axis plane and short axis plane,the oblique axis plane was associat-ed with decreased time for venous access and cannulation.The oblique axis plane also needed less changes of needle direction.The complication of arterial puncture in the oblique axis plane group was significantly lower than long axis plane group and short axis plane group(P <0.05).The number of skin puncture points were similar between the three groups.Conclusion The oblique plane can provide a safe and more effective route to perform the IJV catheterization with minimal risk for carotid artery puncture,which demonstrates the practical superiority over the classic short axis plane and long axis plane for critically ill patients.
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Objective To investigate the gene expression change of TNF-α,IL-6 and IL-1β at different time points in brain tissues of rats with cerebral ischemia reperfusion injury.Methods A total of 24 adult male SD rats were randomly divided into four groups:sham group and 3 groups with brain ischemia reperfusion of 3h,6h and 12h.Real-Time PCR was used to analyze the gene expression ofTNF-α,IL-6 and IL-1 β at 3h,6h,and 12h after reperfusion.Results In the sham group,the mRNA expression levels of TNF-α,IL-6 and IL-1 β in were low,but increased immediately after brain ischemia injury and decreased gradually thereafter.The gene expression of TNF-α mRNA at 3h after reperfusion was significantly increased and reached the peak (P ＜0.01) then significantly decreased at 12h after reperfusion.The gene expression of IL-6 mRNA was notably increased at 3h after reperfusion and peaked at 6h (P＜0.01),and significantly decreased at 12h compared with 6h (P＜0.01).The gene expression of IL-1 β mRNA at 3h after reperfusion was significantly increased,peaked at 6h (P＜0.01) and significantly decreased at 12h (P ＜0.01).Conclusion The gene expression levels of TNF-α,IL-6 and IL-1β mRNA increased significantly in the early stage of reperfusion and decreased gradually after reaching the peak,which suggested that the gene expression change of TNF-α,IL-6 and IL-1β was involved in the mechanism of cerebral ischemia reperfusion injury.
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Aim To investigate the effect of transduct-ed-hemeoxygenase-1 ( HO-1 ) protein on brain ischemi-a-reperfusion ( I/R ) rat hippocampal neurons injury. Methods 11 R ( arginine residues )-fused HO-1 pro-tein was established and 50 male Mongolian gerbils were randomly divided into 5 groups ( n=10 ):I/R ( control group ) , I/R + 5 mg · kg-1 saline group ( group S ) , I/R + 5 mg · kg-1 11 R group ( group R), I/R + 5mg·kg-1 11R-HO-1 group (group H1) and I/R + 25 mg · kg-1 11 R-HO-1 group ( group H2). For I/R experiments, ischemia was induced for 5 min by occluding the common carotid arteries bilater-ally with aneurysm clips under Ketamine anesthesia. The experiment was conducted after the neurons were intraperitoneally injected with 5mg·kg-1 saline,11R , 11R-HO-1,or 25mg · kg-1 11R-HO-1 for 3 h. The rats were killed after 24h of reperfusion. Hippocampus was removed immediately for determination of cAMP level, neuronal apoptotic rate, and expression of HO-1 and Caspase-3 protein, mitochondria was observed un-der electron microscope. Results Among group C, group S and group R,there were no differences in the expressions of HO-1, Caspase-3 protein, cAMP level , neuronal apoptotic rate and mitochondria damage ( P>0. 05). Compared with group C, group S and group R, the expression of HO-1 protein was up-regulated, the expression of Caspase-3 protein was down-regula-ted, cAMP level increased, the apoptotic rate was sig-nificantly decreased and mitochondria damage de-creased in group H1 ( P < 0. 01 ) . Compared with group H1 , the expression of HO-1 protein was up-regu-lated, the expression of Caspase-3 protein was down-regulated, cAMP level increased, the apoptotic rate was significantly decreased and mitochondria damage decreased in group H2 ( P <0. 01 ) . Conclusion Transducted-HO-1 protein can attenuate brain ischemi-a-reperfusion rat hippocampal neurons injury.
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Objective To investigate the effect of transduction of heme oxygenase-1 (HO-1) protein on oxygen-glucose deprivation and restoration (OGD/R)-induced injury to hippocampal neurons in rats.Methods Plasmid 11R-HO-1 was constructed using plasmid pET-21a(+)-p53-11R (plasmid 11R) and 11R-HO-1 fusion protein was identified and collected.Hippocampal neurons obtained from newborn Wistar rats (＜ 48 h) were cultured for 7 days in vitro and then the neurons were randomly divided into 5 groups (n =171 each) using a random number table:OGD/R group,normal saline group (group NS),plasmid 11R group (11R group),300 nmol/L 11R-HO-1 group (H1 group),and 1 500 nmol/L 11R-HO-1 group (H2 group).In NS,11R,H1 and H2 groups,the neurons were incubated for 2 h with 300 nmol/L normal saline,300 nmol/L plasmid 11R,300 nmol/L 11R-HO-1 fusion protein,and 1 500 nmol/L 11R-HO-1 fusion protein,respectively,and then OGD/R was performed.The neurons were incubated in deoxygenated glucose-free DMEM medium and sealed under 5 ％ CO2-95 ％ N2 in an anaerobic chamber equilibrated to 37 ℃ for 45 min.OGD was terminated by replacement of the medium with high glucose DMEM medium and by returning the cultures to a standard incubator maintained at 37 ℃ in 5 ％ CO2-95 ％ air and the neurons were then incubated for 24 h.Immediately after OGD/R was established,the cell survival rate (by MTT assay),apoptosis rate (using TUNEL),and expression of HO-1 and caspase-3 protein (by using Western blot) were measured.Results Compared with group OGD/R,the cell survival rate was significantly increased,the apoptosis rate was decreased,the caspase-3 expression was down-regulated,HO-1 protein expression was up-regulated in H1 and H2 groups (P ＜ 0.05),and no significant change was found in the parameters mentioned above in NS and 11R groups (P ＞ 0.05).Compared with group H1,the cell survival rate was significantly increased,the apoptosis rate was decreased,the caspase-3 expression was down-regulated,and HO-1 protein expression was up-regulated in group H2 (P ＜ 0.05).Conclusion Transduction of HO-1 protein can reduce OGD/R-induced injury to hippocampal neurons of rats.
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ObjectiveTo investigate the effect of heme oxygenase-1 (HO-1) on cyclin-dependent kinase 5 (CDK5)-ataxia telangiectasia mutated (ATM)-P53 signal transduction pathway in rat hippocampal neurons subjected to oxygen-glucose deprivation (OGD) injury.MethodsHippocampal neurons of newborn Wistar rats ( ＜ 48 h) were cultured for 7 days in vitro.The primary cultured neurons were randomly divided into 4 groups with 10 wells in each group:control group (group C),OGD (group D),OGD + hemin (HO-1 inducer) group (group D + H ) and OGD + hemin + zinc protoporphyrin ( HO-1 inhibitor) group ( group D + H + T).For OGD experiments,cultures were washed three times in a glucose-free balanced salt solution (BSS).They were then placed in deoxygenated glucose-free medium and sealed under 95％ N2-5％ CO2 in an anaerobic chamber equilibrated to 37°C and 100％ humidity for 45 min.OGD was terminated by replacement of stored medium and by returning the cultures to a standard incubator maintained at 37 ℃ in 95％ air-5％ CO2.The OGD model was established after the neurons were preconditioned with hemin 10 μmol/L for 24 h in group D + H.The OGD model was established after the neurons were preconditioned with hemin 10 μmol/L and zinc protoporphyrin 10 μmol/L for 24 h in group D + H + T.After 24 h of culture,the neuronal viability,apoptosis rate,and expression of HO-1 mRNA and protein,and CDK5,ATM and P53 protein were detected.ResultsCompared with group C,the expression of HO-1 mRNA,and HO-1,CDK5,ATM and P53 protein was up-regulated,the neuronal viability was significantly decreased,and the apoptosis rate was significantly increased in group D (P ＜ 0.01 ).Compared with group D,the expression of HO-1 mRNA and protein was up-regulated,the expression of CDK5,ATM and P53 protein was down-regulated,the neuronal viability was significantly increased,and the apoptosis rate was significanlly decreased in group D + H ( P ＜ 0.01 ).Compared with group D + H,the expression of HO-1 mRNA and protein was down-regulated,the expression of CDK5,ATM and P53 protein was up-regulated,the neuronal viability was significantly decreased,and the apoptosis rate was significantly increased in group D + H + T ( P ＜ 0.01 ).ConclusionHO-1 can inhibit neuronal apoptosis through blocking CDK5-ATM-P53 signal transduction pathway in rat hippocampal neurons subjected to OGD injury.
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Objective To investigate the role of HO-1 in inhibition of oxygen-glucose deprivation (OGD)-induced apoptosis in rat hippocampal neurons by sevoflurane preconditioning.Methods Hippoeanlpal neurons of newborn Wistar rats (<48 h) were cultured in vitro.Tne neurons were randomly divided into 6 groups with 108 wells in each group:control group(group C),2% sevoflurane preconditioning group (group S1),OGD group,S1 +OGD group,4% sevoflurane preconditioning+OGD group (group S2+OGD),and 4% sevoflurane preconditioning+ZnPPⅨ+OGD group(group Z).Group C received no treatment.The neurons were cultured for 24 h after 2% sevoflurane preconditioning in group S1.For OGD experiments,the neurons were placed in deoxygenated glucose-free medium and sealed under 95% N2-5% CO2 in an anaerobic chamber equilibrated to 37℃ and 100%humidity for 45 min.then OGD was terminated by replacement of the stored medium and returning the cultures to a standard incubator maintained at 37℃ in 5% C02 and the neurons were cultured for 24 h as described by Ray et al. The OGD model was established after 2% and 4% sevoflurane preconditioning in group S1 + OGD and S2 + OGD respectively. In group Z, when the neurons were preconditioned with 4% sevoflurane, ZnPPⅨ was added to the culture medium at the same time, and the other procedures were the same as those in group S2 + OGD. The neuron viability, apoptesis rate, and expression of HO-I protein and mRNA were detected at 24 h of culture. Results Compared with group C, neuron viability was significantly decreased,apoptosis rate was significantly increased, and expression of HO-1 protein and mRNA was up-regulated in group OGD, S1 + OGD, S2 + OGD and Z, expression of HO-1 protein and mRNA was up-regulated in group S1 ( P < 0.01 ), but no significant change was found in neuron viability and apoptosis rate in group S1 ( P > 0.05). Compared with group OGD, neuron viability was significantly increased, apoptosis rate was significantly decreased, and expression of HO-1 protein and mRNA was up-regulated in group S1 + OGD and S2 + OGD ( P < 0.01), but no significant change was found in the indexes mentioned above in group Z ( P > 0.05 ). Neuron viability was significantly higher, apoptosis rate lower and expression of HO-1 protein and mRNA higher in group S2 + OGD than in group S1 + OGD ( P < 0.01). Neuron viability was significantly lower, apoptosis rate higher and expression of HO-1 protein and mRNA lower in group Z than in group S2+OGD(P<0.01).Conclusion HO-1 is involved in the inhibition of OGD-indueed apoptosis in rat hippocampal neurons by sevoflurane preconditioning.
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Aim To investigate effects of Sevoflurane on PI_3-K/Akt/P~(70S6K) cell-survival signal transduction pathways after neuron ischemia-reperfusion,and explore neuroprotection mechanisms of sevoflurane.Methods Newborn(24～48 h)Wister rats were decapitated and hippocampus tissue was dissected and cut into 1 mm?1 mm?1 mm pieces.Then digestion with 0.125% trypsin,centrifuged at 800 r?min~(-1) for 5 min at 4℃,and suspended in a medium containing DMEM supplemented to 25 mmol?L~(-1) glucose,10% fetal bovine serum,10% horse serum,and 2 mmol?L~(-1) glutamine.Cells were plated at 1.0?10~5?ml~(-1) on poly-Dlysine-treated 96-well(100 ?l/well)plates as well 6-well(2 ml/well) plates.Cultures were treated with 10 ?mol?L~(-1) cytosine arabinoside on day 4 in culture to minimize glial growth.One-half of the medium was replaced twice a week with medium containing DMEM(4.5 g?L~(-1) glucose)/F12(1 ∶1),5% fetal bovine serum and 5% horse serum.Cells were used after 7 days. For ischemia-reperfusion(oxygen glucose deprivation,OGD)experiments,cultures were washed three times in a glucose-free balanced salt solution(BSS)and placed in deoxygenated glucose-free medium and sealed under 95% N_2-5% CO_2 in an anaerobic chamber equilibrated to 37℃ and 100% humidity for 45 min.OGD was terminated by replacement of stored medium and by returning the cultures to a standard incubator maintained at 37℃ in 95% O_2-5% CO_2.Experimental group cells were respectively carried out OGD,OGD+2% Sevoflurane,OGD+2% Sevoflurane +10 ?mol?L~(-1) LY294002,OGD+2% Sevoflurane +10 ?mol?L~(-1) Triciribin,and OGD+2% Sevoflurane +10 nmol?L~(-1) Rapamycin.Control cells were cultured normally.Group Sevo was carried out OGD meanwhile anesthesized with 2% sevoflurane.Group LY,Tri and Rap cells was carried out OGD meanwhile culture medium was added 10 ?mol?L~(-1) LY294002,10 ?mol?L~(-1) Triciribin or 10 nmol?L~(-1) Rapamycin,and anesthesized with 2% sevoflurane.Compound remained present throughout the duration of the experiment until analysis 24 h later.Neuron viability and apoptosis were measured.The protein expression of PI_3-K,Akt and P~(70S6K) were detected.Results Sevoflurane enhanced expression of PI_3-K,Akt and P~(70S6K),meanwhile increased neuron viability and decreased neuron apoptosis(vs group I/R,P
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Objective To investigate the role of endogenous cystathionine beta synthase (CBS)/hydrogen sulfide (H2S) and heme oxygenase-1 (HO-1)/carbon monoxide (CO) during global cerebral ischemia-reperfusion (I/R).Methods Thirty male Wistar rats weighing 180-220g were anesthetized with intraperitoneal (IP) 3% pentobarbiral 40 mg?kg-1. Global cerebral I/R was produced by 4-vessel occlusion. Bilateral vertebral arteries were cauterized and bilateral common carotid arteries were occluded with atraumatic clamps for 20 min. The clamps were then released to allow reperfusion. The animals were randomly divided into 5 groups (n=6 each) : group Ⅰ sham operation(C); groupⅡ I/R; in group Ⅲ (Z+I/R),Ⅳ(H+I/R) andⅤ(Z + H + I/R) zinc protoporphyrin (HO-1 inhibitor) 45 ?mol?kg-1 or/and 1 ml of 5 mmol?L-1 hydroxylamine (CBS inhibitor) were given IP 30 min before I/R. The animals were killed at 6h of reperfusion. Brains were removed immediately for determination of H2S, CO, GSH, MDA level and SOD activity and expression of CBS mRNA and HO-1 mRNA in hippocampus and histologic examination with electron microscope. Results H2S, CO, MDA content and CBS mRNA and HO-1 mRNA expression were significantly higher while GSH content and SOD activity were significantly lower in I/R group than in control group (sham operation) . CO content and HO-1 mRNA expression were significantly lower while H2S, GSH content and CBS mRNA expression were significantly higher in Z + I/R group than in I/R group. H2S, GSH content and CBS mRNA expression were significantly lower and CO content and HO-1 mRNA expression were significantly higher in H + I/R group than in I/R group. In Z + H + I/R group H2S, CO, GSH content, SOD activity and expression of HO-1 mRNA and CBS mRNA were significantly decreased whereas MDA content was significantly increased as compared with I/R group. Mitochondria in hippocampal neurons were severely damaged in group Ⅱ-Ⅴ and the damage was worst in group Z + H + I/R. Conclusion CBS/H2S and HO-1/CO systems can protect brain against I/R injury.
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Objective To investigate the effect of acute hyperventilation on the cerebral function of dogs with acute intracranial hypertension.Methods A total of 16 dogs with acute epidural hematoma were randomly divided into four groups.Group A,the group control,were ventilated and PETCO2 was maintained at 35-45mmHg.Groups B,C and D were hyperventilated and PETCO2 was maintained at 28-35,20-27 and below 20mmHg,respectively,for 1h.Jugular vein blood was taken to determine S100B and CRP content at preoperation(T1),2h(T2) post-ventilation,6h(T3),12h(T4),24h(T5) and 48h(T6).Results S100B and CRP contents in experimental group dogs increased,with a significant difference from that before operation(P