大鼠窒息性心跳停止复苏后脑缺血损伤模型的建立

目的建立稳定的窒息性心跳停止复苏后脑缺血损伤大鼠模型。方法 72只健康雄性SD大鼠随机均分为六组:假手术组(S组)和窒息组(C5、C6、C7、C8和C9组)。C5、C6、C7、C8、C9组分别在窒息5、6、7、8、9min后进行心肺复苏。记录各窒息组窒息到停循环时间、循环停止时间、自主循环恢复时间、复苏成功率、复苏后生命体征变化、神经功能缺损评分。复苏后第9天,观察海马CA1区神经元存活情况。结果窒息组均发生了循环停止,随着窒息时间延长,循环停止时间与自主循环恢复时间逐渐延长;C5组大鼠全部存活,C6、C7、C8和C9组大鼠复苏成功率分别为91.7%、83.3%、75.0%和33.3%,C9组复苏成功率低于其他各组(P<0.05);C5、C6、C7组复苏后各时点神经功能缺损评分高于C8和C9组(P<0.05)。复苏后第9天,与S组比较,C5、C6组海马CA1区神经元损伤不明显,C7组有少量神经元坏死,C8组和C9组大量神经元坏死(P<0.01)。结论窒息8min后进行心肺复苏能够建立稳定的脑损伤模型。     

七氟醚后处理对大鼠窒息性心脏骤停心肺复苏后全脑损伤的保护作用

目的评价不同浓度七氟醚后处理(SPC)对大鼠窒息性心脏骤停(CA)复苏后脑缺血损伤的保护作用。方法 80只健康雄性SD大鼠随机均分为五组:假手术组(S组)、模型组(C组)、0.5、1.0和1.5倍最低肺泡有效浓度(MAC)SPC组(P1、P2、P3组)。C、P1、P2和P3组建立8min窒息性CA模型,后给予心肺复苏抢救,并在复苏即刻给予间断吸入七氟醚2次,每次5min,间隔10min,确保浓度分别为1.25%、2.5%和3.75%。记录各组大鼠复苏后72h血糖值、血清神经元特异性烯醇化酶(NSE)含量、海马CA1区神经元形态学变化及海马区P53蛋白的表达,复苏后24、72h和7d的神经功能缺损评分(NDS评分),及复苏后7d至11d的大鼠空间学习记忆能力。结果P2、P3组复苏后血糖值、NSE值明显低于C组(P0.05);S、P2、P3组海马CA1区存活神经元数量明显多于,p53蛋白表达明显少于C组(P0.05);神经功能和空间学习记忆能力明显高于C组(P0.05)。结论 1.0和1.5MAC浓度的SPC可以显著改善窒息性CA所致的大鼠全脑损伤。     

δ阿片受体在窒息性心跳骤停-复苏大鼠脑损伤中的作用

目的 评价δ阿片受体在窒息性心跳骤停-复苏大鼠脑损伤中的作用.方法 清洁级成年雄性SD大鼠96只,体重300 ～ 350 g,采用随机数字表法,将大鼠随机分为4组(n=24):假手术组(S组)、模型组(M组)、δ阿片受体激动剂BW373U86组(B组)和δ阿片受体拮抗剂Naltrindole组(N组).采用夹闭气管导管法诱导窒息性心跳骤停,窒息8 min后行纯氧机械通气,同时人工胸外心脏按压,并快速静脉注射肾上腺素0.02 mg/kg和5％碳酸氢钠1 mg/kg,出现自主呼吸、MAP＞50 mm Hg,持续10 min为循环恢复(ROSC).于ROSC即刻B组和N组分别经股静脉注射1 mg/kg BW373U86和Naltrindole;S组和M组以等容量生理盐水替代.于ROSC 3、24、72 h时行神经系统评分(NDS).评分后处死取脑组织,采用RT-PCR法检测海马脑源性神经营养因子(BDNF) mRNA和酪氨酸激酶受体B(TrkB) mRNA表达水平.于ROSC 72 h时采用HE染色观察海马CA1区神经元组织学分级、计数存活神经元数量.结果 与S组比较,M组、B组和N组BDNF mRNA、TrkB mRNA表达上调,神经元组织学分级升高,NDS降低,存活神经元减少(P＜0.05);与M组比较,B组BDNF mRNA、TrkB mRNA表达上调,N组BDNFmRNA、TrkB mRNA表达下调,B组和N组神经元组织学分级降低,NDS升高,存活神经元增多(P＜ 0.05).与B组相比,N组NDS降低,存活神经元减少,BDNF mRNA、TrkB mRNA表达下调,神经元组织学分级升高(P＜0.05).结论 激活δ阿片受体可减轻窒息性心跳骤停-复苏大鼠脑损伤,其作用机制可能与δ阿片受体激活后促进BDNF及其受体TrkB表达上调有关.     

异丙酚对大鼠窒息性心脏停搏复苏后海马c-JUN氨基末端激酶活化的影响

目的 探讨异丙酚对大鼠窒息性心脏停搏复苏后海马c-JUN氨基末端激酶(JNK)活化的影响.方法 雄性SD大鼠40只,6月龄,体重350 ～ 380 g,采用随机数字表法,将其分为4组(n=10),假手术组(S组)仅动静脉置管和气管插管而不制备窒息性心脏停搏;窒息性心脏停搏复苏组(CA-CPR组)采用窒息法建立CA-CPR模型;异丙酚组(P组)于窒息前30 min静脉注射异丙酚20mg/kg,继之以40 mg·kg-1 ·h-1的速率静脉输注至复苏开始;生理盐水组(NS组)以等容量生理盐水替代异丙酚,余处理同P组.成功复苏后12 h时,处死大鼠,取脑组织,测定湿/干重(W/D)比;采用免疫组化法和Western blot法测定海马磷酸化JNK(p-JNK)的表达水平,并观察海马病理学结果.结果 与S组比较,CA-CPR组、P组和NS组脑W/D比升高,海马p-JNK表达水平上调(P＜0.05或0.01);与CA-CPR组比较,P组脑W/D比降低,海马p-JNK表达水平下调(P＜0.05或0.01),NS组上述指标差异无统计学意义(P＞0.05).P组海马病理学损伤较CA-CPR组减轻.结论 异丙酚可抑制大鼠窒息性心脏停搏复苏后脑组织JNK的活化,从而减轻脑损伤.     

丙泊酚对新生大鼠海马神经元形态结构及生长相关蛋白表达的影响

目的观察丙泊酚麻醉对新生大鼠海马神经元形态结构及生长相关蛋白表达的影响。方法雄性SD大鼠175只,日龄7d,体重8~15g,随机分为对照组(A组)、丙泊酚25mg/kg组(B组)、丙泊酚50mg/kg组(C组)、丙泊酚100mg/kg组(D组)和丙泊酚200mg/kg组(E组),每组35只。A组不注射任何药物,B、C、D和E组分别腹腔注射丙泊酚25、50、100和200mg/kg。每组取5只大鼠,于苏醒后即刻抽取动脉血样进行血气分析。各组其余大鼠又随机分成等份的两个亚组:A1和A2组、B1和B2组、C1和C2组、D1和D2组、E1和E2组。A1、B1、C1、D1和E1组大鼠于苏醒后2h用透射电镜观察海马神经元的形态学变化,采用半定量逆转录-聚合酶链反应(RT-PCR)和免疫印迹法(Western blot)检测海马脑源性神经营养因子(BDNF)、Bcl-2mRNA和蛋白的表达;A2、B2、C2、D2和E2组大鼠苏醒后放回笼中继续饲养至9w时,再行上述相同实验。结果五组大鼠动脉血pH值、PaO2、PaCO2、碳酸氢根离子浓度(HCO-3)、剩余碱(BE)、SaO2差异无统计学意义。A1、A2、B1和B2组大鼠海马神经元基本正常,C1、C2、D1和D2组大鼠海马神经元细胞核肿胀、染色质减少,E1和E2组大鼠海马神经元核碎裂、染色质边集甚至出现凋亡小体。与A1组比较,B1、C1、D1和E1组大鼠海马组织BDNF mRNA、Bcl-2mRNA、BDNF蛋白和Bcl-2蛋白表达均下调(P0.05);与A2组比较,B2、C2、D2和E2组大鼠海马组织BDNF mRNA、Bcl-2mRNA、BDNF蛋白和Bcl-2蛋白表达也均下调(P0.05)。结论丙泊酚麻醉破坏海马神经元的形态结构,其机制可能与其抑制海马BDNF、Bcl-2的表达有关。     

血红素加氧酶-1对心跳骤停复苏大鼠神经功能的影响

目的 探讨血红素加氧酶-1(HO-1)对心跳骤停复苏大鼠神经功能的影响.方法 雄性SD大鼠65只,随机分为四组:心跳骤停复苏组(Ⅰ组,20只)、氯化高铁血红素组(Ⅱ组,20只)、锡原卟啉组(Ⅲ组,20只)和假手术组(Ⅳ组,5只).Ⅰ组在心跳骤停后进行标准的心肺复苏直至恢复自主循环( ROSC);Ⅱ和Ⅲ组分别在心跳骤停前12h或1h经腹腔注射15 mg/kg的氯化高铁血红素或30 μmol/kg锡原卟啉Ⅸ,并进行心肺复苏;Ⅳ组不进行窒息心跳骤停.记录心跳骤停复苏各组的复苏参数,于ROSC后4d对各组进行神经功能缺陷评分(NDS),随后断头取脑,计数海马CA1区存活神经元数、TUNEL阳性细胞数及caspase 3阳性细胞数.结果 心跳骤停复苏各组的复苏参数差异无统计学意义.与Ⅳ组比较,Ⅰ、Ⅱ和Ⅲ组NDS评分明显升高(P＜0.05);与Ⅰ组比较,Ⅱ组NDS评分显著降低(P＜0.05).海马CA1区染色显示:与Ⅳ组比较,Ⅰ、Ⅱ和Ⅲ组存活神经元数显著减少,TUNEL阳性细胞数和caspase 3阳性细胞数明显增加(P＜0.05);与Ⅰ组比较,Ⅱ组TUNEL阳性细胞数和caspase 3阳性细胞数减少(P＜0.05).结论 诱导HO-1的表达可改善心跳骤停复苏后大鼠的神经功能,其机制可能与改善海马CA1区神经元的存活,抑制神经元凋亡有关.     

N受体α4β2亚型在异氟醚抑制大鼠海马突触长时程增强中的作用

目的 评价海马神经元N受体α4β2亚型在异氟醚抑制大鼠海马突触长时程增强(LTP)中的作用.方法 健康成年雄性SD大鼠,取海马组织,制备海马脑片.取70张脑片,随机分为10组(n=7):各组用正常人工脑脊液(aCSF)灌流海马脑片,记录稳定正常的细胞外群峰电位(PS)30 min,LTP组继续给予正常的aCSF灌流,其余各组分别用含异氟醚0.125 mmol/L(I1组)、0.25 mmol/L(I2组)、0.5 mmol/L(I3组)、地棘蛙素0.1 mmol/L(E1组)、1.0 μmol/L(E2组)、地棘蛙素0.1 μmol/L+异氟醚0.25 mmol/L(E1+I2组)、地棘蛙素1.0 μmol/L+异氟醚0.25 mmol/L(E2+I2组)、双氢β-刺酮碱(DHβE)0.1μmol/L(D组)、DHβE0.1μmol/L+异氟醚0.125 mmol/L(D+I1组)的aCSF灌流.采用细胞外微电极记录技术,记录海马脑片CA1区细胞外PS 30 min后,施以高频强直刺激(HFS)15 min,诱发LTP,记录各组HFS结束后5、10、15、20、25、30、40、50、60 min时的PS幅值.结果 与LTP组比较,I1.2.3组、D组、D+I1组、E1+I2组HFS后PS幅值降低,E1.2组HFS后PS幅值升高(P＜0.05),E2+I2组HFS后PS幅值差异无统计学意义(P＞0.05).与I1组比较,D+I1组HFS后PS幅值降低(P＜0.05).与I2组比较,E1+I2组、E2+I2组HFS后PS幅值升高(P＜0.01).结论 异氟醚通过拮抗海马神经元N受体α4β2亚型从而抑制了突触LTP的形成.     

Ro20-1724对多次氯胺酮麻醉幼年大鼠成年后学习记忆功能的影响

目的探讨磷酸二酯酶-4抑制剂Ro20-1724对多次氯胺酮麻醉幼年大鼠成年后学习记忆功能的影响及可能机制。方法筛选合格的21日龄的SD大鼠60只,雌雄不拘。随机分为五组(n=12):空白对照组(A组)、生理盐水组(予以等容量生理盐水,B组)、氯胺酮组(腹腔注射70mg/kg氯胺酮,C组)、氯胺酮+Ro20-1724组(腹腔注射70mg/kg氯胺酮,30min后给予0.5mg/kg R020-1724,D组)、氯胺酮+Ro20-1724溶媒组(腹腔注射70 mg/kg氯胺酮,30 min后给予等量R020-1724的溶媒无水乙醇,E组),每天1次,连续7d。出生8周后利用Morris水迷宫进行定位航行试验和空间搜索试验,获取多次氯胺酮麻醉的幼年大鼠成年后对水迷宫的学习和记忆获取能力以及平台空间位置的记忆保持能力。同时用蛋白质印迹法检测成年后大鼠海马磷酸化环腺苷酸应答元件结合蛋白(Phosphorylated cAMP response element binding protein,p-CREB)的表达以及电子显微镜观察成年后大鼠海马神经元超微结构。结果 C组和E组逃避潜伏期明显长于A组和B组(P0.05),穿越平台次数明显少于A组和B组(P0.05),海马p-CREB表达明显少于A组和B组(P0.05);C组和E组逃避潜伏期明显长于D组(P0.05),穿越平台次数明显少于D组(P0.05),海马p-CREB表达明显少于D组(P0.05);而A组与B组、C组与E组差异无统计学意义。电子显微镜显示C组和E组海马神经元肿胀较明显和普遍,有核膜不完整,核糖体密度低,粗面内质网有脱颗粒现象。D组海马神经元稍肿胀,线粒体密度稍低,核膜基本完整。结论磷酸二酯酶-4抑制剂Ro20-1724(0.5mg/kg)可减轻多次氯胺酮(70mg/kg)麻醉幼年大鼠引起的成年后学习记忆的受损,其机制可能与磷酸二酯酶-4抑制剂Ro20-1724(0.5mg/kg)增强了海马中cAMP/CREB信号传导以及减轻了海马神经元的损伤有关。     

肝素对窒息性心跳骤停-复苏致大鼠心肌损伤的影响

目的 探讨窒息性心跳骤停大鼠心肺复苏时应用肝素抗凝对心肌损伤的影响.方法 健康雌性SD大鼠30只,12～16周龄,体重450～500 g,随机分为3组(n=10):假手术组(S组)、常规心肺复苏组(AD组)和常规心肺复苏+肝素抗凝组(AD+Hep组).制备窒息性心跳骤停模型,窒息8min后行心肺复苏(胸外心脏按压200次/min与药物复苏同时进行,复苏时间2min),AD+Hep组常规药物复苏同时静脉注射肝素0.5 mg/kg(0.1 ml/kg),AD组静脉注射与肝素等容量的生理盐水,S组只行动静脉置管和气管插管.自主循环恢复(ROSC)后2 h,取股动脉血2ml后处死大鼠,取心肌组织,测定血清心肌肌钙蛋白Ⅰ(cTnI)浓度、心肌丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性,电镜下观察心肌超微结构.记录从窒息到心跳骤停时间、心肺复苏开始到ROSC时间.结果 与S组比较,AD组和AD+Hep组心肌MDA含量升高,SOD活性降低,血清cTnI浓度升高(P<0.05或0.01),心肌病理损伤明显.与AD组比较,AD+Hep组心肺复苏开始到ROSC时间缩短,心肌MDA含量降低,SOD活性升高,血清cTnI浓度降低(P<0.05或0.01),心肌病理损伤减轻.结论 窒息性心跳骤停大鼠心肺复苏时应用肝素抗凝可减轻心跳骤停.复苏致心肌损伤,其机制可能与抑制脂质过氧化反应有关.     

SIRT1在内毒素性脑损伤小鼠线粒体功能障碍中的作用

目的评价海马沉默信息因子1(SIRT1)在内毒素性脑损伤小鼠线粒体功能障碍中的作用。方法清洁级雄性C57BL/6小鼠80只, 6～8周龄, 采用随机数字表法分为4组(n=20):对照组(C组)、内毒素性脑损伤组(LPS组)、内毒素性脑损伤+SIRT1抑制剂组(LPS+E组)和内毒素性脑损伤+SIRT1激动剂(LPS+S组)。静脉注射LPS 10 mg/kg制备小鼠内毒素性脑损伤模型。于注射LPS前72 h时, LPS+E组腹腔注射SIRT1抑制剂EX527 10 mg/kg, 其余3组腹腔注射等容量DMSO;于注射LPS前30 min时LPS+S组腹腔注射SIRT1激动剂SRT1720 100 mg/kg, 其余3组腹腔注射等容量DMSO。注射LPS 24 h时行新物体识别实验, 安乐处死小鼠, 取海马组织行尼氏染色, 光镜下观察病理学结果, 并计数海马CA1区正常神经元;采用分光光度法测定海马ATP含量和线粒体呼吸链复合物Ⅰ、Ⅱ、Ⅲ、Ⅳ的活性, Jc-1染色法检测线粒体膜电位(MMP), 透射电镜观察神经元线粒体超微结构。结果与C组比较, LPS组、LPS+S组和LPS+E组新物体识...     

Improved Resuscitation after Cardiac Arrest in Rats Expressing the Baculovirus Caspase Inhibitor Protein p35 in Central Neurons

Background: Global cerebral ischemia is associated with delayed neuronal death. Given the role of caspases in apoptosis, caspase inhibitors may provide neuronal protection after cardiac arrest. To this end, the authors generated a transgenic rat line expressing baculovirus p35, a broad-spectrum caspase inhibitor, in central neurons. Its effects were evaluated on neuronal cell death and outcome after global cerebral ischemia.

Methods: Global cerebral ischemia was induced by cardiocirculatory arrest. After 6 min, animals were resuscitated by controlled ventilation, extrathoracic cardiac massage, epinephrine, and electrical countershocks. Neuronal death was assessed after 7 days by histologic evaluation of the hippocampal cornu ammonis 1 sector. Postischemic outcome was assessed by determination of overall survival and according to neurologic deficit scores 24 h, 3 days, and 7 days after resuscitation.

Results: The rate of 7-day survival after cardiac arrest for the transgenic rats (85%) was significantly higher than that for the nontransgenic controls (52%;P < 0.05). However, no differences were observed either in the number of terminal deoxynucleotidyltransferase-mediated d-uracil triphosphate-biotin nick end-labeling-positive cells or viable neurons in the cornu ammonis 1 sector or in the neurologic deficit score when comparing surviving transgenic and nontransgenic rats. These findings suggest that neuronal apoptosis after cardiac arrest is not primarily initiated by activation of caspases.     

Isoflurane Prevents Delayed Cell Death in an Organotypic Slice Culture Model of Cerebral Ischemia

Background: General anesthetics reduce neuronal death caused by focal cerebral ischemia in rodents and by in vitro ischemia in cultured neurons and brain slices. However, in intact animals, the protective effect may enhance neuronal survival for only several days after an ischemic injury, possibly because anesthetics prevent acute but not delayed cell death. To further understand the mechanisms and limitations of volatile anesthetic neuroprotection, the authors developed a rat hippocampal slice culture model of cerebral ischemia that permits assessment of death and survival of neurons for at least 2 weeks after simulated ischemia.

Methods: Survival of CA1, CA3, and dentate gyrus neurons in cultured hippocampal slices (organotypic slice culture) was examined 2-14 days after 45 min of combined oxygen-glucose deprivation at 37[degrees]C (OGD). Delayed cell death was serially measured in each slice by quantifying the binding of propidium iodide to DNA with fluorescence microscopy.

Results: Neuronal death was greatest in the CA1 region, with maximal death occurring 3-5 days after OGD. In CA1, cell death was 80 +/- 18% (mean +/- SD) 3 days after OGD and was 80-100% after 1 week. Death of 70 +/- 16% of CA3 neurons and 48 +/- 28% of dentate gyrus neurons occurred by the third day after OGD. Both isoflurane (1%) and the N-methyl-d-aspartate antagonist MK-801 (10 [mu]m) reduced cell death to levels similar to controls (no OGD) for 14 days after the injury. Isoflurane also reduced cell death in CA1 and CA3 caused by application of 100 but not 500 [mu]m glutamate. Cellular viability (calcein fluorescence) and morphology were preserved in isoflurane-protected neurons.     

Improved resuscitation after cardiac arrest in rats expressing the baculovirus caspase inhibitor protein p35 in central neurons

BACKGROUND: Global cerebral ischemia is associated with delayed neuronal death. Given the role of caspases in apoptosis, caspase inhibitors may provide neuronal protection after cardiac arrest. To this end, the authors generated a transgenic rat line expressing baculovirus p35, a broad-spectrum caspase inhibitor, in central neurons. Its effects were evaluated on neuronal cell death and outcome after global cerebral ischemia. METHODS: Global cerebral ischemia was induced by cardiocirculatory arrest. After 6 min, animals were resuscitated by controlled ventilation, extrathoracic cardiac massage, epinephrine, and electrical countershocks. Neuronal death was assessed after 7 days by histologic evaluation of the hippocampal cornu ammonis 1 sector. Postischemic outcome was assessed by determination of overall survival and according to neurologic deficit scores 24 h, 3 days, and 7 days after resuscitation. RESULTS: The rate of 7-day survival after cardiac arrest for the transgenic rats (85%) was significantly higher than that for the nontransgenic controls (52%; P < 0.05). However, no differences were observed either in the number of terminal deoxynucleotidyltransferase-mediated d-uracil triphosphate-biotin nick end-labeling-positive cells or viable neurons in the cornu ammonis 1 sector or in the neurologic deficit score when comparing surviving transgenic and nontransgenic rats. These findings suggest that neuronal apoptosis after cardiac arrest is not primarily initiated by activation of caspases. CONCLUSION: Expression of baculovirus p35 can improve survival after cardiac arrest in rats, but the mode and site of action remain to be elucidated.     

gamma-Aminobutyric acid-A receptors contribute to isoflurane neuroprotection in organotypic hippocampal cultures

The mechanisms by which anesthetics such as isoflurane reduce cell death in rodent models of cerebral ischemia remain incompletely defined. Reduction in glutamate excitotoxicity explains some but not all of isoflurane's neuroprotection. Because isoflurane potentiates gamma-aminobutyric acid (GABA) receptor-mediated ion fluxes and GABA(A) receptor agonists have neuroprotective effects, we hypothesized that GABA(A) receptors contribute to isoflurane neuroprotection. As a model of cerebral ischemia and recovery, we used rat hippocampal slice cultures. Survival of CA1, CA3, and dentate neurons was examined 2 and 3 days after 1-h combined oxygen-glucose deprivation (OGD) at 37 degrees C. To define the role of GABA(A) receptors in mediating protection, the effect of 1% isoflurane on cell survival was examined in the presence of the GABA(A) antagonist bicuculline during OGD. Cell death was measured with propidium iodide fluorescence. Isoflurane and the selective GABA(A) agonist muscimol (25 micro M) reduced cell death after OGD to values similar to slices not exposed to OGD, with the exception that muscimol did not reduce cell death in CA3 neurons 2 days after OGD. The GABA(A) antagonist bicuculline reduced the neuroprotective effects of isoflurane on hippocampal neurons 2 and 3 days after OGD. We conclude that GABA(A) receptors contribute to neuroprotection against OGD produced by isoflurane in the hippocampal slice model. Based on this and other studies, it is likely that neuroprotection produced by isoflurane is multifactorial and includes actions at both GABA(A) and glutamate receptors and possibly other mechanisms. IMPLICATIONS: Isoflurane is neuroprotective in rodent brain ischemia models, but the mechanisms for this effect remain incompletely defined. In organotypic cultures of rat hippocampus, we show that protection of CA1, CA3, and dentate neurons by 1% isoflurane from death caused by oxygen and glucose deprivation involves GABA(A) receptors.     

Contribution of Ih to neuronal damage in the hippocampus after traumatic brain injury in rats

Traumatic brain injury (TBI) causes selective neuronal damage in the hippocampus; however, the underlying mechanisms are still unclear. Post-traumatic alterations of ion channel activity, which actively regulate neuronal excitability and thus impact on excitotoxicity, may be involved in TBI-induced neuronal injury. Here we report that hyperpolarization-activated cation current (I(h)) contributes to the distinct vulnerability of hippocampal neurons in TBI. In a rat model of controlled cortical injury, moderate TBI produced neuronal death of both hippocampal CA3 neurons and mossy cells in the hilus, but not CA1 pyramidal cells. Treatment with lamotrigine, which enhances dendritic I(h), ameliorated TBI-induced neuronal damage to CA3 neurons and mossy cells. In contrast, intraventricular administration of I(h) channel blocker caused cell death in the CA1 region after TBI. Whole-cell recordings revealed that, differently from CA3 neurons, CA1 pyramidal cells expressed larger I(h) and exhibited a post-traumatic increase of I(h) amplitude. Moreover, blocking I(h) led to an increase of neuronal excitability, with greater effects seen in post-traumatic CA1 pyramidal cells than in CA3 neurons. In addition, the I(h) in mossy cells was dramatically inhibited early after TBI. Our findings indicate that differential changes of I(h) in hippocampal neurons may be one of the mechanisms of selective cell death, and that an enhancement of functional I(h) may protect hippocampal neurons against TBI.     

茶氨酸在大鼠脑缺血/再灌注过程中对大鼠海马c-Jun氨基末端激酶和P38信号通路的作用

目的 探讨茶氨酸在大鼠脑缺血/再灌注(ischemia/reperfusion,I/R)过程中对大鼠海马c-Jun氨基末端激酶(c-Jun N-terminal kinases,JNK)和P38信号通路所发挥的作用.方法 雄性SD大鼠108只,体重290~310 g,采用随机数字表法分成假手术组(SH组)、I/R组、茶氨酸组(TH组),每组36只.每组根据再灌注时间分为2、6、12、24、48、72 h 6个亚组,每亚组6只.采用4-VO法建立SD大鼠全脑缺血模型,在预定时间点行灌注、固定、取脑、石蜡包埋切片,免疫组化检测磷酸化JNK(phosphorylate JNK,p-JNK)和磷酸化P38(phosphorylate P38,p-P38)的表达变化,光镜下计数CA1区存活细胞,TUNEL法检测CA1区凋亡细胞.结果 与SH组比较,I/R组海马CA1区各时点p-JNK和p-P38表达明显增加(P<0.01),于再灌注2 h时即明显升高[灰度值分别为(163.5±3.8)和(163.0±1.9)],24 h到高峰[灰度值分别为(132.3±4.9)和(141.0±5.7)].TH组p-JNK和p-P38的表达则无明显增高,各时点与I/R组比较差异均有统计学意义(P<0.05).海马CA1区神经元存活数目TH组明显高于I/R组(P<0.01),凋亡细胞数显著低于I/R组(P<0.01).结论 在大鼠全脑I/R损伤过程中,茶氨酸通过抑制JNK和P38信号通路的激活可对脑I/R损伤导致的细胞损伤起到保护作用,对临床脑缺血的治疗有一定的指导意义.     

Effects of global ischemia duration on neuronal, astroglial, oligodendroglial, and microglial reactions in the vulnerable hippocampal CA1 subregion in rats

The hippocampal CA1 neurons are selectively vulnerable to global ischemia, and neuronal death occurs in a delayed manner. The threshold of global ischemia duration that induces neuronal death has been studied, but the relationship between ischemia duration and glial death in the hippocampal CA1 area has not been fully studied. We examined neuronal/glial viability and morphological changes in the CA1 subregion after different durations of global ischemia. Global ischemia was induced in Sprague-Dawley rats by 10, 5, and 3 min of bilateral common carotid artery occlusion and hypotension. At 1-56 days after ischemia, the morphological reactions of neurons, astrocytes, oligodendrocytes, and microglia were immunohistochemically evaluated. Most of the hippocampal CA1 pyramidal neurons underwent delayed death at 3 days after 10/5 min of ischemia, but not after 3 min of ischemia. The number of astrocytes gradually declined after 10/5 min of ischemia, and viable astrocytes showed characteristic staged morphological reactions. Oligodendrocytes also showed morphological changes in their processes after 10/5 min of ischemia. Microglia transformed into a reactive form at 5 days only after 10/5 min of ischemia. These data suggest that some morphological changes in glial cells were not dependent on neuronal cell death, but their own reactions to the different severity of ischemia.     

Effects of dantrolene on extracellular glutamate concentration and neuronal death in the rat hippocampal CA1 region subjected to transient ischemia

BACKGROUND: Excessive extracellular glutamate produced by cerebral ischemia has been proposed to initiate the cascade toward neuronal cell death. Changes in extracellular glutamate concentration are closely linked to changes in intracellular calcium ion concentration. Dantrolene inhibits calcium release from intracellular calcium stores. In this study, the authors investigated the effects of dantrolene on extracellular glutamate accumulation and neuronal degeneration in a rat model of transient global forebrain ischemia. METHODS: Male Wistar rats weighing 230-290 g were anesthetized with halothane in nitrous oxide-oxygen and were subjected to 10 min of transient forebrain ischemia using a four-vessel occlusion technique. Fifteen minutes before ischemic injury, dantrolene sodium (5 mm), dimethyl sulfoxide as a vehicle for dantrolene, or artificial cerebrospinal fluid as a control was intracerebroventricularly administered (n = 8 in each group). In the hippocampal CA1 subfield, the extracellular glutamate concentration in vivo was measured during the periischemic period with a microdialysis biosensor, and the number of intact neurons was evaluated on day 7 after reperfusion. RESULTS: Both dantrolene and dimethyl sulfoxide significantly reduced the ischemia-induced increase in glutamate concentration to a similar extent, i.e., by 53 and 51%, respectively, compared with artificial cerebrospinal fluid (P < 0.01). The number of intact hippocampal CA1 neurons (mean +/- SD; cells/mm) in dantrolene-treated rats (78 +/- 21) was significantly higher than that in artificial cerebrospinal fluid- (35 +/- 14; P < 0.001) and dimethyl sulfoxide-treated (56 +/- 11; P < 0.05) animals. Dimethyl sulfoxide also significantly increased the number of preserved neurons in comparison with artificial cerebrospinal fluid (P < 0.05). CONCLUSIONS: Intracerebroventricular dantrolene prevents delayed neuronal loss in the rat hippocampal CA1 region subjected to transient ischemia; however, this neuroprotection cannot be accounted for only by the reduced concentrations of extracellular glutamate during ischemia.     

Isoflurane prevents delayed cell death in an organotypic slice culture model of cerebral ischemia.

BACKGROUND: General anesthetics reduce neuronal death caused by focal cerebral ischemia in rodents and by in vitro ischemia in cultured neurons and brain slices. However, in intact animals, the protective effect may enhance neuronal survival for only several days after an ischemic injury, possibly because anesthetics prevent acute but not delayed cell death. To further understand the mechanisms and limitations of volatile anesthetic neuroprotection, the authors developed a rat hippocampal slice culture model of cerebral ischemia that permits assessment of death and survival of neurons for at least 2 weeks after simulated ischemia. METHODS: Survival of CA1, CA3, and dentate gyrus neurons in cultured hippocampal slices (organotypic slice culture) was examined 2-14 days after 45 min of combined oxygen-glucose deprivation at 37 degrees C (OGD). Delayed cell death was serially measured in each slice by quantifying the binding of propidium iodide to DNA with fluorescence microscopy. RESULTS: Neuronal death was greatest in the CA1 region, with maximal death occurring 3-5 days after OGD. In CA1, cell death was 80 +/- 18% (mean +/- SD) 3 days after OGD and was 80-100% after 1 week. Death of 70 +/- 16% of CA3 neurons and 48 +/- 28% of dentate gyrus neurons occurred by the third day after OGD. Both isoflurane (1%) and the N-methyl-D-aspartate antagonist MK-801 (10 microm) reduced cell death to levels similar to controls (no OGD) for 14 days after the injury. Isoflurane also reduced cell death in CA1 and CA3 caused by application of 100 but not 500 microm glutamate. Cellular viability (calcein fluorescence) and morphology were preserved in isoflurane-protected neurons. CONCLUSIONS: In an in vitro model of simulated ischemia, 1% isoflurane is of similar potency to 10 microm MK-801 in preventing delayed cell death. Modulation of glutamate excitotoxicity may contribute to the protective mechanism.     

Study on cognition disorder and morphologic change of neurons in hippocampus area following traumatic brain injury in rats

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