细胞红蛋白对新生大鼠缺氧缺血性脑损伤的神经保护作用

目的 探讨细胞红蛋白(cytoglobin,CYGB)在缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)中的神经保护作用. 方法 健康7日龄新生Sprague-Dawlay大鼠随机分为假手术组、HIBD组、HIBD+氯化高铁血红素(Hemin)组和HIBD+锌原卟啉(zinc protoporphyrin,ZnPP)组.HIBD组、HIBD+ Hemin组及HIBD+ ZnPP组大鼠分别腹腔注射0.5 ml生理盐水、Hemin(50 mg/kg)及ZnPP(50 mg/kg),12h后结扎并剪断左侧颈总动脉,缺氧2h,制成HIBD动物模型.建立模型后0、24、48 h免疫组织化学法检测各组大鼠海马及皮质中CYGB的表达情况,每组10只.HE染色观察建立模型后48 h的大鼠脑组织病理变化,每组8只.称重法检测建立模型后72h新生大鼠脑含水量,每组8只.建立模型后28 d采用Morris水迷宫实验检测海马学习记忆功能变化,每组10只.组间差异比较采用方差分析,组间两两比较使用Bonferroni法. 结果 (1)大鼠脑组织CYGB的表达水平:用平均灰度值(与蛋白表达水平成反比)表示.在0h,HIBD+ Hemin组和HIBD组皮质CYGB的平均灰度值分别为166.7±5.1和207.1±5.1,低于假手术组(232.3±3.4),而HIBD+ ZnPP组(234.9±4.5)高于假手术组(P＜0.05).随时间的增加,各HIBD组CYGB的平均灰度值递减,至48 h时,CYGB的平均灰度值HIBD+Hemin组最低(126.0±2.6),其次是HIBD组(150.9±4.5)和HIBD+ ZnPP组(163.7=6.3),最高是假手术组(232.1±5.8),差异均有统计学意义(P均＜0.01).CYGB在海马组织的表达变化与皮质一致.(2)各组脑组织病理变化:建立模型后48 h,HIBD组、HIBD+ Hemin组和HIBD+ ZnPP组新生大鼠脑组织可见典型的脑梗死和出血,HIBD+Hemin组梗死灶明显较HIBD+ZnPP组小,出血减轻.(3)脑组织含水量变化:建立模型后72 h,HIBD组和HIBD+ZnPP组左侧脑组织含水量分别为(86.5±0.4)％和(87.3±0.3)％,明显高于右脑[(85.6±0.2)％和(85.9±0.2)％],差异有统计学意义(t分别为12.57和11.32,P均＜0.01).(4)海马学习记忆功能变化:水迷宫实验中,5d总平均逃逸潜伏期HIBD+ ZnPP组最长[(76.7±29.8)s],其次是HIBD组[(71.0±30.5)s]和HIBD+ Hemin组[(46.7±34.0)s],最短为假手术组[(38.3±30.3)s],差异均有统计学意义(P均＜0.05).(5)远期脑组织病理变化:建立模型后34 d,脑组织萎缩率HIBD+ ZnPP组最高[(34.07±6.75)％],其次是HIBD组[(29.73±6.53)％],再次是HIBD+Hemin组[(18.33±4.52)％],均高于假手术组[(1.55±1.32)％],差异均有统计学意义(P均＜0.01).HIBD组及HIBD+ZnPP组大鼠脑组织可见锥体细胞层变薄,大片神经元缺失,HIBD+Hemin组仅见少量神经元缺失. 结论 CYGB的高表达可减轻HIBD脑组织近期及远期的病理损伤,保护远期的海马学习记忆功能.     

胰岛素样生长因子1对新生大鼠缺氧缺血性脑损伤保护机制的研究

目的　建立单侧缺氧缺血性脑损伤 (HIBD)动物模型 ,研究胰岛素样生长因子 1(IGF 1)对HIBD的影响和可能机制。　方法　选择健康 7日龄Wistar大鼠 12 0只 ,建立HIBD模型 ,随机分成假手术组、HIBD组、HIBD后 0 .2mg/kg人基因重组IGF 1干预组 (RH IGF 1组 )、0 .0 6 6mg/kg人基因重组IGF 1干预组 (SRH IGF 1组 )及盐水对照组 (对照组 )。各组按观察时段进一步分为 2 4、4 8、72h组 ,每组 8只。各组于规定时刻观测脑形态学改变、谷氨酸 (Glu)含量、凋亡细胞计数、Bcl 2蛋白表达。　结果　 (1)HIBD 4 8h组Glu(116 2 .2± 10 8.1)mg/kg ,较假手术组(75 0 .9± 5 3.4 )mg/kg明显升高 (P <0 .0 5 ) ;HIBD组凋亡细胞计数 [2 4h :(7.6± 1.9) % ,4 8h(12 .6±1.2 ) % ,72h :(13.8± 0 .9) % ],较假手术组 [2 4h(2 .0± 0 .2 ) % ,4 8h(2 .0± 0 .3) % ,72h(2 .0±0 .2 ) % ]明显增加 (P均 <0 .0 5 )。 (2 )与对照组相比 ,RH IGF 1组脑组织病变减轻 ;干预 4 8h组Glu[SRH IGF 1组 (781.4± 5 4 .2 )mg/kg ,RH IGF 1组 (74 0 .5± 4 6 .6 )mg/kg],较对照组 (112 6 .6± 4 8.0 )mg/kg明显降低 (P均 <0 .0 5 ) ;RH IGF 1组凋亡细胞计数 [2 4h :(3.6± 0 .9) % ,4 8h(8.2± 2 .2 ) % ,72h(9.4± 1.4 ) % ],较对     

血小板生长因子对缺氧缺血性脑损伤新生鼠脑细胞凋亡率和血清神经元特异性烯醇化酶的影响

目的 研究血小板源性生长因子(platelet derived growth factor,PDGF)对缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)的新生鼠神经细胞凋亡率及血清神经元特异性烯醇化酶(neuron-specific enolase,NSE)浓度的影响,进而探讨其对HIBD的神经保护作用. 方法 7日龄新生Wistar大鼠48只制备HIBD模型,并分为PDGF治疗组和生理盐水对照组,每组各24只.另取24只为假手术组.治疗组在缺氧缺血后即刻给PDGF-BB 50 ng/kg腹腔注射.对照组和假手术组腹腔注射等体积的生理盐水.每组于处置后12、24和72 h随机取8只处死,留血清标本,酶联免疫吸附法检测大鼠血清标本NSE浓度;取右侧大脑组织制备脑细胞悬液,双染法流式细胞仪检测脑细胞凋亡率.采用单因素方差分析及q检验进行统计学分析. 结果 (1)脑细胞凋亡率:治疗组[(6.09±0.70)％、(9.67±1.52)％和(14.15±1.52)％]和对照组[(8.00±1.10)％、(11.45±2.42)％和(22.90±2.03)％]3个时点的脑细胞凋亡率均较假手术组(2.11±0.54)％、(2.34±0.46)％和(2.21±0.49)％]显著增加(P均＜0.01或＜0.05),治疗组较对照组各时点脑细胞凋亡率均明显降低(P均＜0.01或＜0.05),3组大鼠在12、24、72 h时的组间比较差异均有统计学意义(F=39.01、66.60、194.20,P均＜0.01).(2)血清NSE浓度:各时点对照组[(10.04±0.19) μg/L、(9.33±0.15)μg/L和(8.36±0.16)μg/L]和治疗组[(8.43±0.17)μg/L、(6.73±0.16) μg/L和(6.12±0.13)μg/L]较假手术组[(4.22±0.53)μg/L、(3.96±0.60) μg/L和(3.59±0.55) μg/L]NSE浓度增加(P均＜0.01),治疗组较对照组各时点NSE浓度降低(P均＜0.01),3组大鼠在12、24、72h组间比较差异均有统计学意义(F=371.25、245.61、236.22,P均＜0.01). 结论 PDGF能抑制新生大鼠HIBD后神经细胞凋亡及降低血清NSE浓度,对HIBD新生大鼠有神经保护作用.     

新生大鼠缺氧缺血性脑损伤后caspase-3蛋白表达与神经元变性的关系

目的探讨新生大鼠缺氧缺血性脑损伤（hypoxic ischemic brain damage，HIBD）后半胱氨酸天冬氨酸特异性蛋白酶-3（cysteinyl asparate-specific proteinase-3，caspase-3）蛋白表达及其与神经元变性的关系。方法将35只7日龄Wistar大鼠随机分为正常对照组、假手术组、HIBD后6、12、24、72和120h组，每组5只。HIBD组按Rice法制成模型。采用免疫组化法及Fluoro-Jade B（FJB）荧光染色分别观察caspase-3蛋白表达及神经元变性情况。结果缺氧缺血（hypoxic ischemia，HI）后12h皮质caspase-3表达（0．405±0．021）高于正常对照组（0．367±0．023）（P〈0．05），HI后24h皮质、纹状体及海马caspase-3表达达高峰（P〈0．05）；至HI后120h皮质、纹状体和海马caspase-3表达均与对照组差异无统计学意义（P〉0．05）。正常对照组大鼠各脑区内FJB阳性细胞罕见，HI后24h各部位脑组织FJB阳性细胞明显增多（P〈0．05），72h进一步增多（P〈0．05），120h时FJB阳性细胞数达高峰（P〈0．05）。结论新生大鼠HIBD后脑caspase-3蛋白表达与变性神经元在空间与时间分布上存在一致性，但变性神经元分布得更广泛、更持久。提示caspase-3蛋白可能在缺氧缺血所致神经元变性中起到一定作用。     

三日龄大鼠缺氧缺血性脑损伤对大脑MRI影像和学习记忆能力的远期影响

目的 观察缺氧缺血(HI)对3日龄大鼠脑细胞凋亡及成年后头部MRI影像和学习记忆能力的影响.方法 应用凋亡基因芯片研究3日龄大鼠缺氧缺血性脑损伤后12 h与损伤7 d凋亡基因表达的差异.42日龄时进行大脑MRI检查,并在44日龄时采用水迷宫测试其学习和记忆能力.组间比较采用t检验和秩和检验.结果 与HI脑损伤后12 h相比,损伤后7 d表达上调的基因包括TNF及其受体家族中的Tnfsf10(TRAIL)、Tnfsf13(CD30)、Tnfrsf21、Tnfrsf11b;Caspase家族中的Caspase1、2、3和6;Bcl2家族中的促凋亡基因Bak1、Becn1、Bcl10和Bid3;死亡域TRADD和Myd88.而Caspase 8和抗凋亡基因Mapk8ip表达下调.42日龄时头部MRI检查显示HI组右侧大脑皮质面积较左侧和假手术组显著减小[侧脑室层(23.5±3.6)mm2、(33.0±4.3)mm2和(34.5±3.9)mm2(F=17.09,P<0.01);海马层(18.9±4.4)mm2、(29.1±5.0)mm2和(30.8±4.5)mm2(F=14.44,P<0.01)].HI组水迷宫试验上台时间在第4天为(52.7±35.9)s,明显长于假手术组的(17.8±8.9)s(P<0.01).记忆测试中,HI组大鼠经过站台的次数显著少于假手术组(T=292.5,P<0.05).结论 3日龄SD大鼠HI脑损伤后,神经细胞凋亡基因激活持续到损伤后7 d,涉及凋亡的外源性和内源性通路.神经细胞的凋亡会导致大脑皮层萎缩,可能影响动物成年后的空间学习和记忆能力.     

缺氧对大鼠肺泡上皮细胞凋亡及缺氧诱导因子-1α表达的影响

目的 探讨氯化钴(cobalt chloride,CoCl2)化学缺氧对大鼠肺泡上皮细胞(alveolar epithelial cell,AEC)凋亡的影响及相关凋亡蛋白缺氧诱导因子-1α(hypoxia inducible factor-talpha,HIF-1α)的表达变化及意义.方法 将AEC进行体外培养,分4组,每组6个样本.缺氧4 h组:选用化学缺氧物质CoCl2 500/μmol/L为作用浓度,诱导AEC缺氧,共培养4 h;缺氧24 h组:与CoCl2共培养24 h;缺氧48 h组:与CoCl2共培养48 h;常氧对照组:不做任何特殊处理.利用荧光显微镜及流式细胞仪检测细胞凋亡率,透射电子显微镜进行细胞凋亡的形态学观察,Western印记法检测HIF-1α蛋白的表达情况.结果 (1)荧光显微镜的结果 显示缺氧4、24、48 h组细胞凋亡率分别为(5.83±0.76)%、(15.00±3.28)%和(51.50±3.00)%,均高于常氧对照组[(1.50±0.50)%](P＜0.05);(2)流式细胞仪的结果 与荧光显微镜的结果一致;(3)透射电子显微镜可以观察到AEC凋亡的形态学改变,缺氧24 h显著;(4)缺氧4、24、48 h组HIF-1α蛋白的表达分别为0.69±0.035、1.02±0.044和0.71±0.046,均高于常氧对照组(0.21±0.026)(P＜0.01);(5)各组HIF-1α蛋白变化与荧光显微镜及流式细胞仪检测的细胞凋亡率变化均呈正相关(r=0.484,P=0.016;r=0.713,P=0.009).结论 缺氧对大鼠AEC的生存有抑制作用,这种抑制作用与缺氧引起的细胞凋亡有关;HIF-1α可能参与了缺氧诱导AEC凋亡的发生,而AEC凋亡可能又参与了缺氧肺损伤的发病.     

早期高压氧对缺血缺氧性脑损伤新生大鼠脑神经保护的效果探讨

目的探讨早期高压氧对缺血缺氧性脑损伤(HIBD)新生大鼠脑神经保护的效果。方法将7日龄新生Wistar大鼠120只随机分为假手术组、HIBD组、高压氧组,每组各40只。将高压氧组再随机分为早期高压氧亚组和晚期高压氧亚组,每亚组各20只。对所有大鼠建立HIBD模型后(假手术组采用假手术处理),早期高压氧亚组在建模后1h内,晚期高压氧亚组在建模后48h实行高压氧治疗(2ATA,每次90min,24h1次,连续治疗7d)。各亚组治疗结束后处死大鼠,假手术组与HIBD组处死等量大鼠。利用免疫组织化学法对大鼠海马中神经巢蛋白(Nestin)的含量进行检测,利用Morris水迷宫实验对大鼠学习记忆功能进行检测。结果假手术组Nestin阳性细胞检出数量为(10.26±1.37)个/HP,早期高压氧亚组Nestin阳性细胞检出数量为(15.82±2.19)个/HP,晚期高压氧亚组Nestin阳性细胞检出数量为(32.64±4.83)个/HP,HIBD组Nestin阳性细胞检出数量为(36.18±4.02)个/HP。高压氧组和HIBD组Nestin阳性细胞检出数量显著高于假手术组,差异有统计学意义(P0.05),晚期高压氧亚组显著高于早期高压氧亚组,差异有统计学意义(P0.05),晚期高压氧亚组与HIBD组比较,差异无统计学意义(P0.05);4组平均逃逸潜伏期差异有统计学意义(F=24.18,P0.05),假手术组早期高压氧组晚期高压氧组HIBD组;假手术组、早期高压氧组和晚期高压氧组3组大鼠在搜索耗时和搜索路程上组间比较,差异有统计学意义(F=21.05,P0.05),假手术组早期高压氧组晚期高压氧组,而晚期高压氧组和HIBD组组间比较差异无统计学意义(P0.05),各组搜索速度组间比较,差异无统计学意义(P0.05)。结论早期高压氧对HIBD脑损伤具有保护功能,Nestin蛋白可能参与了脑神经的修复过程,早期高压氧还对HIBD引起的远期学习记忆功能障碍有改善作用。     

促红细胞生成素对新生鼠缺氧缺血性脑损伤海马CA1区Caspase-3激活的影响

目的　探讨重组人促红细胞生成素 (rhEPO )对新生鼠缺氧缺血性脑损伤 (HIBD)的保护作用及对海马CA1区Caspase 3激活的影响。　方法　将 7日龄SD大鼠分为缺氧缺血模型组 (n= 11)、rhEPO治疗组 (n =11)和假手术对照组 (n =10 ) ,模型组和治疗组建立新生鼠缺氧缺血性脑损伤模型。观察模型组和治疗组缺氧后 0、6、12、2 4h不同时间点自发左旋、夹尾左旋、夹尾尖叫现象。用免疫组织化学方法检测活化的Caspase 3的表达 ,观察Caspase 3阳性细胞在脑组织内分布情况 ,计算各组海马CA1区单位面积内阳性细胞数 (个 / 1mm)。　结果　模型组 2只、治疗组 1只在缺氧过程中因持续抽搐死亡。缺氧后 0h两组动物均出现自发左旋 (治疗组 10 / 10 ,模型组 9/ 9) ,2 4h治疗组自发左旋发生率明显减少 (治疗组 1/ 10 ,模型组 6/ 9,P =0 .0 198)。免疫组化显示Caspase 3阳性细胞在对照组脑组织内散在分布 ,模型组和治疗组在海马、大脑皮层较为密集。模型组CA1区阳性细胞数显著高于对照组 (41.3 8± 2 .0 9vs 10 .52± 2 .70 ,P <0 .0 1)。治疗组阳性细胞数显著低于模型组(41.3 8± 2 .0 9vs 2 2 .63± 3 .17,P <0 .0 1)。　结论　Caspase 3在新生鼠HIBD中活性显著增高 ,应用rhEPO能有效抑制Caspase 3激活 ,提示rhEPO通过抑制Casp     

Nogo-A mRNA在缺氧缺血性脑损伤新生大鼠脑组织中的表达

目的研究神经生长抑制因子Nogo-AmRNA在缺氧缺血性脑损伤(hypoxic-ischemicbraindamage,HIBD)新生大鼠脑组织的动态变化,探讨Nogo-A基因对HIBD新生大鼠中枢神经再生的影响。方法将180只SD新生大鼠随机分为对照组(n=90)和实验组(n=90),实验组制成HIBD模型,根据处死动物的时间不同随机分为HIBD后3、7、14、21和28d共5个亚组,每组18只;对照组也在相应时间点取材;采用原位杂交法检测新生大鼠脑组织Nogo-AmRNA在不同时间的表达量。结果原位杂交法显示Nogo-AmRNA在实验组新生大鼠的皮层呈强阳性表达,阳性细胞分布密集,对照组阳性细胞分布较实验组稀疏;实验组新生大鼠脑组织Nogo-AmRNA阳性细胞数量在缺血缺氧后7d(34.73±3.34)比14d(36.67±5.33)和21d(42.33±2·21)均较对照组7d(28·67±2·91)、14d(32·17±5·06)和21d(36·83±4·90)明显增高(P<0·05或0·01)。结论脑组织Nogo-AmRNA表达在脑损伤急性期(3d)变化不明显,而在1周后较为明显,提示缺氧缺血性脑损伤后期Nogo-AmRNA表达明显升高,它可能是造成新生大鼠脑损伤后神经再生障碍的重要原因之一。     

缺氧、缺血对新生鼠大脑半胱氨酸蛋白酶活性的影响

目的探讨缺氧、缺血对新生鼠大脑组织天冬氨酸特异酶切的半胱氨酸蛋白酶-3（简称caspase-3）活性的影响及意义。方法选择7d龄Sprague-Dawley大鼠33只，结扎左侧颈总动脉后，吸入8%氧气2h，制成缺氧、缺血性脑损伤（HIBD）动物模型，HIBD后0.5、12、24、48h取两侧大脑组织制成组织匀浆，用显色底物天冬氨酸-谷氨酸-缬氨酸-天冬氨酸-对硝基苯胺复合物测定caspase-3活性。结果缺氧、缺血侧大脑组织caspase-3活性逐渐增高，24h后吸光度值达1.0047±0.4390，48h后降至0.4569±0.2285，各时点间差异有极显著性（P<0.001）。对侧大脑组织各时间点caspase-3活性未见明显变化（P>0.05）。结论新生鼠大脑缺氧、缺血后caspase-3明显激活，支持细胞凋亡参与HIBD，应用caspases抑制剂或其他抗凋亡药物，有可能为HIBD的治疗开辟新的途径。     

地塞米松干预对缺氧缺血性脑损伤脑组织髓鞘碱性蛋白的影响

目的 探讨地塞米松(dexamethasone,Dex)在缺氧缺血性脑损伤(hypoxic ischemicbrain damage,HIBD)中的作用及可能机制.方法 通过建立大鼠HIBD模型,分为Dex大剂量预处理组、Dex小剂量预处理组、Dex大剂量治疗组、Dex小剂量治疗组、生理盐水组和正常对照组,检测血清及脑组织髓鞘碱性蛋白(myelin basic protein,MBP)、白介素-1β(interleukin-1β IL-1β)、凋亡细胞计数和脑组织病理改变.结果 (1)与生理盐水组比较,不同剂量Dex干预组和治疗组脑组织病变均减轻,表现神经元树突复旧,尼氏体增多;(2)HIBD后3 d生理盐水组血清、脑组织MBP、IL-1β的含量(5.88±0.46,34.25±4.65;127.97±16.60,1060.33±42.22)及脑组织凋亡细胞计数(13.27±0.90,11.05±1.23)较正常对照组(2.01±0.12,10.24±1.75;41.21±4.02,221.10±30.57及0.75±0.17)显著增加,P<0.05;HIBD后7 d生理盐水组上述指标有所降低,但较正常对照组仍显著增高,P<0.05;(3)Dex干预后血清及脑组织MBP、IL-1β的含量:预处理组(4.30±0.73,24.10±3.36和59.89±6.16,393.68±31.46)、治疗组(4.48±0.49,16.98±1.32及50.81±8.32,405.15 ±29.02)较生理盐水组(5.88±0.46,34.25±4.65和127.97±16.60,1060.33±42.22)显著下降,P<0.05;预处理组与治疗组无显著性差异,P>0.05;大剂量组较小剂量组作用更明显;(4)Dex预处理组、治疗组的脑组织凋亡细胞计数(7.92±1.64和8.97±0.81)显著低于生理盐水组(13.27±0.90),P<0.05;预处理组较治疗组下降更为明显,P<0.05;大剂量组较小剂量组作用更明显.结论 血清MBP含量的变化可反映脑白质损伤的程度;Dex可能是通过抑制IL-1β的过度表达,减轻炎症级联反应从而起到脑损伤的保护作用.     

Heat shock protein 72 expression and microtubule-associated protein 2 disappearance after hypoxia-ischemia in the developing rat brain.

OBJECTIVE: This study was intended to investigate the temporal changes in heat shock protein 72 expression and microtubule-associated protein 2 disappearance in rat brain at 2 different ages after hypoxic-ischemic insult. STUDY DESIGN: Both 5-day-old and 14-day-old Wistar rats were subjected to unilateral common carotid artery ligation and hypoxia in 8% oxygen for 2 hours at 33 degrees C. Brain sections were examined sequentially for heat shock protein 72 expression at 0.5, 3, 6, 12, 24, 48, and 72 hours of recovery after hypoxia-ischemia and for microtubule-associated protein 2 disappearance at 0, 24, 48, and 72 hours of recovery and at 7 days of recovery after hypoxia-ischemia. Results of immunohistochemical staining for heat shock protein 72 and microtubule-associated protein 2 were used as markers for detection of early hypoxic-ischemic brain damage. Permanent neuronal damage was assessed with hematoxylin and eosin staining at 7 days after hypoxia. RESULTS: In 5-day-old rats microtubule-associated protein 2 expression was lost as early as 0 hours after hypoxia-ischemia in the cerebral cortex and hippocampus, with a peak at 48 hours after which expression recovered. Expression of heat shock protein 72 was detected in the ligated hemisphere at 0.5 hours after hypoxia-ischemia and peaked at 6 to 24 hours of recovery. In 14-day-old rats microtubule-associated protein 2 was stained in the cortex at 0 hours after hypoxia-ischemia but gradually disappeared in the cerebral cortex and hippocampus after 24 hours of recovery. The expression of heat shock protein 72 was not detected by 6 hours of recovery in the cerebral cortex and by 3 to 12 hours of recovery in the hippocampus, but heat shock protein 72 was persistently expressed in the cortex and hippocampus after 48 hours of recovery. Neuronal damage was significantly less in 5-day-old rats than in 14-day-old rats. CONCLUSION: In 5-day-old rats hypoxia-ischemia causes earlier changes in heat shock protein 72 and microtubule-associated protein 2 immunostaining results and causes less severe brain damage than in 14-day-old rats.     

细胞外信号调节激酶信号转导通路活化在新生大鼠缺氧缺血性脑损伤中的作用

目的 观察细胞外信号调节激酶(extracellular-signal regulated kinase,ERK)在新生大鼠缺氧缺血性脑损伤中的作用.方法 Wistar大鼠随机分为假手术组、缺氧缺血组和PD98059(ERK抑制剂)组,通过结扎右侧颈总动脉,恢复2 h后,吸入含8%氧气的氧氮混合气体2 h的方法 建立新生大鼠缺氧缺血性脑损伤模型,PD98059组结扎右侧颈总动脉前10 min股静脉注射PD980592 mg/kg.各组新生大鼠于模型制作后6 h剥离结扎侧海马及皮层脑组织,分别测定脑组织丙二醛(malondialdehyde,MDA)含量、超氧化物歧化酶(superoxide dismutase,SOD)活性和细胞凋亡水平.Western印迹法检测ERK1和ERK2,Bcl-2和Bax蛋白的表达.组间差异比较采用方差分析及q检验.结果 缺氧缺血组细胞凋亡率明显高于假手术组[(18.80±1.37)%和(3.53±0.34)%](q=6.06,P＜0.01),PD98059组细胞凋亡率[(15.53±0.64)%]低于缺氧缺血组(q=3.87,P＜0.01).缺氧缺血组MDA含量为(342.9±10.8)μmol/L,明显高于假手术组[(181.5±17.0)μmol/L](q=6.35,P＜0.01)和PD98059组[(252.0±17.1)μmol/L](q=5.28,P＜0.01),而SOD活性[(34.8±4.3)U/ml]明显低于假手术组[(63.4±4.3)U/ml](q=4.99,P＜0.01)和PD98059组[(51.5±3.8)U/ml](q=4.17,P＜0.01).3组总ERK1和ERK2蛋白表达差异无统计学意义.缺氧缺血组磷酸化ERK1和磷酸化ERK2蛋白表达明显高于假手术组(q分别=3.82和4.08,P均＜0.01)和PD98059组(q分别=4.79和5.12,P均＜0.01).缺氧缺血后Bcl-2和Bax蛋白表达明显高于假手术组(q分别=3.55和3.42,P均＜0.01);PD98059组较缺氧缺血组抗凋亡蛋白Bcl-2表达增加,促凋亡蛋白Bax表达降低(q分别=3.71和5.86,P均＜0.01).结论 ERK激活通过调节凋亡蛋白表达参与了新生大鼠缺氧缺血性脑损伤的发生.

Abstract：

Objective To investigate the effects of extracellular-signal regulated kinase (ERK) on hypoxic-ischemic brain damage of neonatal rats.Methods The hypoxic-ischemic brain damage model of neonatal Wistar rats was established as following:first the right common carotid artery of the rats was ligated;2 h after operation,the rats began to inhale 8%-oxygen oxygen-nitrogen gas mixture lasting for 2 h.Rats were randomly divided into three groups:sham-group,hypoxic-ischemic group and ERK inhibitor PD98059 group (the rats were injected PD98059 2 mg/kg 10 min before the ligation).Six hours after the models were done,hippocampi and cortex of the ligation side of rats in the three groups were collected,and the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured.Apoptosis of neuron was assessed by TUNEL staining.The expression of ERK1,ERK 2,Bcl-2 and Bax were examined by Western blot.The differences among the groups were analyzed with ANOVA and q test.Results Compared with the sham-group,the MDA level [(342.9± 10.8) μmol/L vs (181.5± 17.0) μmol/L,q= 6.35,P＜0.01) and the apoptosis rate of neuron [(18.80±1.37)% vs (3.53±0.34)%,q=6.06,P＜0.01) of hypoxic-ischemic group was higher,and SOD level was lower [(34.8±4.3) U/ml vs (63.4±4.3) U/ml,q=4.99,P＜0.01].While the apoptosis rate of neuron [(15.53±0.64) %] and MDA level [(252.0± 17.1) μmol/L] of PD98059 group were lower than those of hypoxic-ischemic group(q=3.87 and 5.28,P＜0.01respectively),the SOD level [(51.5 ± 3.8) U/ml] was higher than that of hypoxic-ischemic group (q=4.17,P＜0.01).There were no differences of ERK1 and ERK2 expressions among the three groups.The phosphorylated ERK1 and ERK2 levels of hypoxic-ischemic group were higher than those of sham-group (q=3.82 and 4.08,P＜0.01) and PD98059 group (q=4.79 and 5.12,P＜0.01).The expression of Bcl-2 and Bax of hypoxic-ischemic group were higher than those of sham-group (q=3.55 and 3.42,P＜0.01).Compared with hypoxic-ischemic group,Bcl-2 expression (q=3.71,P＜0.01) of PD98059 group was higher,and Bax expression (q=5.86,P ＜ 0.01) was lower.Conclusions ERK is involved in hypoxic-ischemic brain damage of neonatal rats through regulating the expression of apoptosis protein.     

Hypoxic-ischemic tolerance induced by hyperthermic pretreatment in newborn rats

OBJECTIVE: We investigated the effect of hyperthermic pretreatment 24 hours before hypoxic-ischemic exposure on neuronal cell damage in 7-day-old rats. METHODS: Newborn rats were separated on postnatal day 7 into two groups: 1) preheated (those exposed to 2 hours of hyperthermic pretreatment at 42-43C) (n = 29), and 2) nonheated (n = 20). At 24 hours after the hyperthermic stress, rats from both groups were subjected to left carotid artery ligation followed by 2 hours of hypoxia (8% oxygen/92% nitrogen) at 33C. All rats were killed 1 week after hypoxia-ischemia, and brains were extracted for histologic study. A different group of 7-day-old rats (n = 8) was placed in the same hyperthermic environment as mentioned above for 2 hours, and 24 hours after heat exposure brains were extracted for immunohistochemistry of heat-shock protein 70. RESULTS: The total incidence of hypoxic-ischemic brain damage significantly decreased in the preheated group (12 of 25 [48%]) compared with the nonheated group (17 of 20 [85%]; P < .03). The induction of immunoreactive heat-shock protein 70 was observed mainly in glial and vascular endothelial cells and, in a lesser amount, in neuronal cells of the cerebral cortex and hippocampus. CONCLUSION: Incidence of hypoxic-ischemic brain damage is consistently reduced by 2 hours of hyperthermic pretreatment in 7-day-old rats.     

Important role of 72-kd heat shock protein expression in the endothelial cell in acquisition of hypoxic-ischemic tolerance in the immature rat

OBJECTIVES: Hypoxic-ischemic tolerance can be induced in neonatal rats through hyperthermic preconditioning. The purposes of this study were to determine the interval between hyperthermic preconditioning and a subsequent hypoxic-ischemic insult that would provide optimal neuroprotection against the insult and to examine the relationship between tolerance induction and heat shock protein expression. STUDY DESIGN: On postnatal day 7 Wistar rat pups were separated into the following 2 groups: a heated group (those exposed to 15 minutes of hyperthermic pretreatment at a brain temperature of 41.5 degrees C-42.0 degrees C) and an unheated control group. At 6, 12, 24, 48, and 72 hours after the hyperthermic stress, rats from both groups were exposed to left carotid artery ligation followed by 2 hours of hypoxia (8% oxygen and 92% nitrogen) at 33 degrees C. Twenty animals from each group were used at each time point. All rats were killed at 1 week after hypoxia-ischemia, at which time the brains were processed and neuronal damage in the cortex and hippocampus was assessed histologically. Another set of 7-day-old rats (n = 30) was studied immunohistochemically at 6, 12, 24, 48, and 72 hours after the same hyperthermic treatment. Expression of 72-kd heat shock protein was measured in neuronal, glial, and vascular endothelial cells. RESULTS: Hyperthermia-induced hypoxic-ischemic tolerance was observed at 6, 12, and 24 hours but not at 48 and 72 hours after hyperthermic preconditioning. Heat shock protein 72 expression in the vascular endothelial cells, rather than in the glial or neuronal cells, was most strongly associated with hypoxic-ischemic tolerance. CONCLUSION: These findings suggest that heat shock protein 72 in endothelial cells plays an important role in the acquisition of hypoxic-ischemic tolerance at postnatal day 7, a time when maximal angiogenesis occurs and the blood-brain barrier is still immature.     

碱性成纤维细胞生长因子对新生大鼠脑缺血后神经新生的影响

目的 研究碱性成纤维细胞生长因子(bFGF)对新生大鼠脑缺血后神经新生的影响.方法 通过结扎3日龄新生SD大鼠双侧颈总动脉制备脑缺血模型,随机分为治疗组(36只):给予bFGF 10 ng/g侧脑室注射;对照组(36只)给予相同体积的生理盐水.另取36只新生大鼠,仅分离双侧颈总动脉,不结扎,不给予药物,作为假手术组.三组大鼠分别于术后第4、7、14天处死,获取脑组织标本,采用免疫荧光染色、Western印记和实时PCR方法,观察三组大鼠不同时点脑室下区巢蛋白(nestin)、Ⅲ型β-微管蛋白(Tuj1)、胶质纤维酸性蛋白(GFAP)和少突胶质细胞NG2蛋白及其mRNA的表达变化.结果 (1)免疫荧光染色:治疗组BrdU+/nestin+细胞数目术后7 d达高峰[(48.7±5.9)个/视野],高于同时点对照组[(32.2±3.1)个/视野]和假手术组[(17.3±1.6)个/视野],差异有统计学意义(P<0.01);治疗组BrdU+/Tuj1+细胞、BrdU+/GFAP+细胞、BrdU+/NG2+细胞数目术后14 d[分别为(92.6±9.7)、(58.2±6.1)、(57.3±5.4)个/视野]达高峰,高于同时点对照组[分别为(65.8±7.1)、(42.1±4.4)、(37.8±3.2)个/视野]和假手术组[分别为(35.3±3.1)、(33.6±3.4)、(22.4±2.1)个/视野],差异有统计学意义(P<0.01).(2)Western印迹和实时PCR:与免疫荧光染色结果一致,即治疗组nestin蛋白和mRNA表达术后7 d达高峰,高于同时点对照组和假手术组,差异有统计学意义(P<0.01);治疗组Tuj1、GFAP和NG2蛋白和mRNA术后14 d达高峰,高于同时点对照组和假手术组,差异有统计学意义(P<0.01).结论 bFGF可促进新生大鼠脑缺血后脑室下区神经干细胞的增殖以及向功能神经细胞(神经元、星形胶质细胞、少突胶质细胞)的分化,可能对新生大鼠脑缺血后神经细胞的再生具有促进作用.     

电刺激小脑顶核对缺氧缺血新生大鼠学习记忆能力的影响

目的探讨电刺激小脑顶核对缺氧缺血新生大鼠学习记忆功能的影响。方法 7日龄Wist-ar大鼠（36只）随机分为缺氧缺血性脑损伤组和假手术组,缺氧缺血性脑损伤组又分为模型组和电刺激组,采用结扎左侧颈总动脉并吸入氮氧混合气体2h制作新生大鼠缺氧缺血性脑损伤动物模型,分别于手术第2天相同时间点给予无电刺激装置（假手术组）,连接装置但不接通（模型组）,连接装置并且接通（电刺激组）。各组12只动物,随机分为7d干预组（6只）和14d干预组（6只）,两组分别于7、14d干预后,（1）用Y-型迷宫检测脑损伤鼠的学习记忆功能;（2）苏木精-伊红染色法光镜下观察脑神经细胞和神经纤维的病理变化;（3）免疫组织化学检测脑皮质和海马区STAT3蛋白水平的表达。结果 （1）Y-型迷宫测试结果模型组总时间较电刺激组增多,模型组主动回避反应率和正确反应率低于电刺激组（P〈0.05）;模型组总时间与假手术组相比,显著增加;模型组主动回避反应率和正确反应率也明显低于假手术组,差异均有统计学意义（P〈0.05）。（2）假手术组脑组织各部位的结构及细胞层次清楚、形态正常,未见明显损伤性改变。模型组脑组织可见明显的毛细血管出血,细胞核固缩、核碎裂明显,模型建立成功。电刺激组神经细胞变性坏死较少,多数细胞形态相对正常。（3）电刺激组子鼠脑皮质及海马周围信号转导和转录激活蛋白3（STAT3）蛋白较模型组增多（P〈0.05）。结论电刺激小脑顶核可促进脑损伤鼠学习记忆能力的恢复。     

脑缺血对新生大鼠脑室下区神经新生的影响

目的 观察3日龄新生大鼠缺血性脑损伤后脑室下区(subventricular zone,SVZ)新生细胞的变化,探讨未成熟脑缺血性损伤后的内源性修复机制. 方法 3日龄新生SD大鼠32只随机分为实验组和对照组,每组16只.实验组结扎双侧颈总动脉,对照组不结扎.两组大鼠均于术后5～7 d给予5-溴脱氧尿嘧啶(5-bromodeoxyuridine,BrdU),每次50 mg/kg腹腔注射,每12 h 1次×6次.两组大鼠分别于术后14 d、28 d处死取脑,采用免疫荧光染色方法检测BrdU、Ⅲ型β-微管蛋白(class Ⅲ β-tubulin,TuJ1)、少突胶质细胞O抗原-4(oligodendroeyte O antigen-4,O4)以及胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达变化,观察SVZ各类新生细胞的变化. 结果 实验组术后14 d和28 d新生神经元(BrdU+/TuJ1+双标阳性细胞)分别为(7800±800)个/视野和(10 700±1400)个/视野、新生少突胶质细胞(BrdU+/O4+双标阳性细胞)分别为(6100±1000)个/视野和(7300±1400)个/视野,新生星形胶质细胞(Brdu+/GFAP+双标阳性细胞)分别为(4500±700)个/视野和(6700±1100)个/视野,均随着时点的延长而增加(P<0.01);且各新生神经元数目较同时点对照组明显增多,差异有统计学意义(P均<0.01). 结论 新生大鼠缺血性脑损伤后,SVZ新生神经元、新生少突胶质细胞、新生星形胶质细胞增多,提示未成熟脑SVZ在缺血性损伤后有修复作用.