【作者】 耿进霞；

【导师】 李文斌；

【作者基本信息】 河北医科大学 ， 生理学， 2007， 博士

【摘要】 脑缺血疾病是严重危害人类健康的疾病之一。神经细胞对缺血性危害极为敏感,如果缺血时间较长,神经细胞会大量死亡,即使恢复血液供应,神经细胞也难以再生,甚至会留下严重的后遗症。随着对脑缺血疾病研究的深入,脑缺血耐受(brain ischemic tolerance, BIT)现象越来越普遍受到关注。实验发现,给动物突然造成较严重的脑缺血后,海马CA1区的神经元会大量死亡。若在此之前,预先给动物造成轻微、短时、不至于引起神经元死亡的脑缺血,间隔一定时间后,再给动物造成较严重的、通常会引起大量神经元死亡的脑缺血,此时海马CA1区的神经元则基本不死亡,即神经元对缺血性损害产生了抵抗力,这一现象被称为脑缺血耐受。预先给予的轻微、短时脑缺血称为脑缺血预处理(cerebral ischemic preconditioning,CIP)。阐明脑缺血预处理脑保护作用的发生机制,对临床上研究、开发提高神经元对缺血缺氧耐受性的治疗方法具有重要意义。各种疾病引起的脑组织缺血、缺氧均可引起神经元能量代谢障碍,抑制Na+/K+依赖式ATP酶的活动,使细胞外K+浓度明显增高,Na+浓度相应降低,导致神经元去极化,引起兴奋性神经末梢释放谷氨酸;此外,细胞外高K+能够逆转高亲合力谷氨酸转运体的活动,把谷氨酸从细胞内转运至细胞外。这些原因引起细胞外谷氨酸等兴奋性氨基酸增多。这些增多的谷氨酸与神经细胞膜相应的受体结合,引起Na+、Ca2+内流以及细胞内Ca2+释放,导致Na+、Ca2+超载,进而使神经细胞死亡。因此,将这些氨基酸称为兴奋性神经毒素。降低脑缺血时细胞外液中谷氨酸的浓度,减少其与突触后膜特异性受体的结合,是防治其兴奋性毒性作用、减轻缺血时神经元损伤的重要手段。兴奋性氨基酸转运体(excitatory amino acid transports, EAATs)是调控脑内细胞外液谷氨酸浓度的重要机制。其中星形胶质细胞谷氨酸转运体亚型GLT-1在终止谷氨酸能神经传递,维持细胞外液谷氨酸浓度处于正常水平方面发挥重要作用。一些研究对星形胶质细胞GLT-1在缺血缺氧耐受中的作用给予了关注。例如,Douen等研究表明,皮层扩散性抑制预处理可以下调星形胶质细胞谷氨酸转运体(EAAT1和EAAT2)的表达,防止缺血引起的谷氨酸逆转运;Romera等应用离体(in vitro)模型发现,缺血或缺氧预处理可以使谷氨酸转运体表达增加。这些结果提示GLT-1可能与脑缺血耐受有关。但星形胶质细胞GLT-1在整体情况下(in vivo)是否参与CIP的脑保护作用?调制GLT-1是否可增强脑细胞对缺血性损害的耐受性?等尚未见报道。因此,本实验应用大鼠全脑缺血模型、GLT-1抑制剂Dihydrokainate (DHK)、GLT-1反义及正义寡核苷酸,在整体水平探讨调制GLT-1对脑缺血耐受诱导的影响,为阐明GLT-1在脑缺血耐受形成中的作用提供实验依据。1 DHK抑制GLT-1的功能阻断CIP诱导的脑缺血耐受应用大鼠四血管闭塞(4-vessel occlusion,4VO)全脑缺血模型,观察GLT-1特异性抑制剂DHK对CIP脑保护作用的影响,探讨GLT-1在脑缺血耐受中的作用。将96只凝闭双侧椎动脉2d后的Wistar大鼠随机分为8组:①sham组(n=6):只暴露双侧颈总动脉,不阻断血流;②CIP组(n=6):夹闭双侧颈总动脉3 min;③脑缺血打击组(n=6):夹闭双侧颈总动脉8 min;④CIP+脑缺血打击组(n=6):夹闭双侧颈总动脉3 min作为CIP,再灌注2 d后再夹闭双侧颈总动脉8 min;⑤双蒸水组(n=6):右侧脑室注射双蒸水20μl;⑥DHK组(n=30):右侧脑室注射DHK溶液20μl,根据DHK的剂量进一步分为10、100、200、500和1000 nmol 5个亚组,每组6只动物;⑦DHK+CIP组(n=18):右侧脑室注射DHK溶液20μl,20 min后夹闭双侧颈总动脉3 min,根据DHK的剂量进一步分为10、100和200 nmol 3个亚组,每组6只动物;⑧DHK+CIP+脑缺血打击组(n=18):右侧脑室注射DHK溶液20μl ,20 min后夹闭双侧颈总动脉3 min,2 d后夹闭双侧颈总动脉8 min。根据DHK剂量不同分为10、100、200 nmol 3个亚组,每组6只动物。以上各组动物于sham手术、侧脑室注射或末次脑缺血后7 d断头取脑,常规脑组织切片(5μm厚),硫堇染色,光学显微镜下观察海马CA1区组织学形态,确定锥体神经元迟发性死亡(delayed neuronal death, DND)情况。计数海马CA1区每1 mm区段内细胞膜完整、胞核饱满、核仁清晰的锥体细胞数目,每张切片双侧海马各计数3个区段取平均数为神经元密度(Neuronal density, ND)。根据以下标准确定组织学分级(Histological grade, HG):0级,无神经元死亡;Ⅰ级,散在神经元死亡;Ⅱ级,成片神经元死亡;Ⅲ级,几乎全部的神经元死亡。结果发现,4VO过程中,大鼠翻正反射消失,瞳孔散大,脑电波频率变慢,波幅逐渐变小甚至成等电位线,说明产生了全脑缺血。硫堇结果显示,Sham组和CIP组大鼠海马CA1区无明显的DND,HG为0～Ⅰ级,ND值分别为208.25±5.97和202.93±4.32。脑缺血打击组大鼠海马CA1区出现明显的DND,与sham组和CIP组相比,HG(Ⅱ～Ⅲ级)明显升高(P<0.01),ND值(45.86±21.93)显著降低(P<0.01)。CIP+脑缺血打击组海马CA1区DND不明显,与脑缺血打击组相比,HG(0～Ⅰ级)明显降低(P<0.01),ND值(208.07±5.87)显著升高(P<0.01),表明CIP可以诱导海马CA1区神经元产生缺血性耐受,对抗缺血打击引起的DND。右侧脑室注射双蒸水后海马CA1区组织形态与Sham组和CIP组基本一致。单纯侧脑室给予DHK组中,10、100、200 nmol剂量组海马CA1区神经元少量死亡,三组的HG均为Ⅰ级,ND值分别为172.76±17.31、162.64±6.12和155.43±9.82;而500、1000 nmol剂量组的HG分别为为Ⅱ级和Ⅲ级,ND值分别为105.35±3.84和6±1.39;与10、100、200 nmol组相比,HG和ND的变化具有显著性(P<0.05),表明大剂量的DHK可引起显著的海马CA1区锥体神经元死亡。因此,以下各组中,仅观察DHK 10、100和200 nmol的影响。DHK+CIP组中海马CA1区组织形态与Sham组和CIP组基本一致(10、100和200 nmol组HG均为为0～Ⅰ级,ND值分别为192.15±6.25、188.65±3.90和191.35±3.88)。DHK+CIP+脑缺血打击组中,大鼠海马CA1区出现了显著的DND,随着DHK剂量的增加,其HG级逐渐升高(10 nmol组为Ⅰ级,100 nmol组为Ⅰ～Ⅱ级,200 nmol组为Ⅲ级),ND逐渐下降(10 nmol组为160.87±13.55,100 nmol组为117.07±10.11,200 nmol组为4.87±2.02);与CIP+脑缺血打击组相比,上述HG和ND的变化具有显著性(P<0.01);此外,与单独DHK组相比,DHK+CIP+脑缺血打击组的DHK的量效曲线明显左移,并且斜率变陡。这些结果表明,DHK+CIP+脑缺血打击组中,除去DHK本身引起的少量神经元死亡以外,DHK还阻断CIP抗脑缺血打击的作用,从而引起更多的锥体细胞死亡。以上结果表明, GLT-1特异性抑制剂DHK可剂量依赖性地阻断CIP抗脑缺血打击的作用,提示GLT-1参与CIP诱导的脑缺血耐受。2 GLT-1反义寡核苷酸抑制GLT-1表达减弱CIP抗脑缺血打击的作用应用GLT-1反义寡核苷酸(antisense oligodeoxynucleotides,AS-ODNs)抑制GLT-1蛋白的表达,观察其对CIP抗脑缺血打击作用的影响,进一步探讨GLT-1在脑缺血耐受中的作用。2.1 GLT-1 AS-ODNs对GLT-1蛋白表达的影响将42只凝闭双侧椎动脉36 h的Wistar大鼠随机分为3组:①对照组(n=6):右侧脑室注射双蒸水5μl,注射后12 h取材;②AS-ODNs 9 nmol组(n=18):右侧脑室注射AS-ODNs溶液5μl(9 nmol),根据AS-ODNs注射后取材的时间又分为三个亚组:12 h、24 h、36 h组;③AS-ODNs 18 nmol组(n=18):右侧脑室注射AS-ODNs溶液5μl(18 nmol),根据AS-ODNs注射后取材的时间又分为三个亚组:12 h、24 h、36 h组。所有大鼠在预定时间断头取材,低温条件下分离海马CA1区,采用Western blotting方法测定GLT-1的蛋白表达,应用凝胶图像分析系统对Western免疫反应阳性条带进行积分光密度(integrated optical density,IOD)测定,以GLT-1的IOD值与β-actin的IOD值的比值代表GLT-1表达的相对水平。结果显示,双蒸水对照组的IOD的比值为0.65±0.22。注射AS-ODNs 9 nmol组中,12 h组的IOD比值为0.37±0.07,24 h组为0.20±0.05, 36 h组为0.25±0.07。注射AS-ODNs 18 nmol组中,12 h组的IOD比值为0.11±0.05,24 h组为0.05±0.02,36 h组为0.17±0.16。与双蒸水对照组相比,注射AS-ODNs后IOD的比值显著降低(P<0.05),且呈现一定的剂量依赖性。以上结果表明,侧脑室注射AS-ODNs溶液可以剂量依赖性地抑制GLT-1蛋白表达,这种抑制作用在注射AS-ODNs后24 h最为显著。2.2 GLT-1 AS-ODNs抑制CIP对海马CA1区锥体神经元的保护作用将54只凝闭双侧椎动脉的Wistar大鼠随机分为7组:①sham组(n=6):只暴露双侧颈总动脉,不阻断血流;②CIP组(n=6):夹闭双侧颈总动脉3 min;③脑缺血打击组(n=6):夹闭双侧颈总动脉8 min;④CIP+脑缺血打击组(n=6):夹闭双侧颈总动脉3 min作为CIP,再灌注2 d后再夹闭双侧颈总动脉8 min。⑤双蒸水组(n=6):于分离暴露双侧颈总动脉(但不夹闭)前12 h、后12 h及后36 h右侧脑室注射双蒸水,每次5μl,其他同sham组;⑥AS-ODNs组(n=12):于分离暴露双侧颈总动脉(但不夹闭)前12 h、后12 h及后36 h右侧脑室注射AS-ODNs溶液,每次5μl,其他同sham组。根据AS-ODNs的剂量进一步分为9和18 nmol 2个亚组,每组6只动物;⑦AS-ODNs+CIP+脑缺血打击组(n=12):于CIP前12 h、后12 h及后36 h右侧脑室注射AS-ODNs溶液,每次5μl,其它同CIP+脑缺血打击组。根据AS-ODNs的剂量进一步分为9 nmol和18 nmol 2个亚组,每组6只动物。以上各组动物均在sham手术、侧脑室注射或末次脑缺血后7d断头取材,常规脑组织切片(5μm厚),硫堇染色下观察海马CA1区DND的发生情况(方法同前)。结果显示,Sham组和CIP组大鼠海马CA1区无明显的DND,HG为0～Ⅰ级,ND值分别为208.25±5.97和202.86±4.28。脑缺血打击组大鼠海马CA1区出现明显的DND,HG(Ⅱ～Ⅲ级)明显升高(P<0.01),ND值(45.86±21.93)显著降低(P<0.01)。CIP+脑缺血打击组海马CA1区DND不明显,与脑缺血组相比,HG(0～Ⅰ级)明显降低(P<0.01),ND值(208.98±5.9)显著升高(P<0.01),表明CIP可以诱导海马CA1区神经元产生缺血性耐受,对抗缺血打击引起的DND。侧脑室注射双蒸水和AS-ODNs (9 nmol and 18 nmol)后海马CA1区组织形态与Sham组和CIP组基本一致,HG为0～Ⅰ级,ND值略有减少(分别为176.22±6.29、176.89±2.88和175.32±2.77(P<0.01)),表明侧脑室注射以及单纯AS-ODNs不会对海马CA1区神经元产生明显损伤。AS-ODNs+CIP+脑缺血打击组中,与CIP+脑缺血打击组相比,其HG显著升高(Ⅱ～Ⅲ级)(P<0.01),ND值显著下降(分别为133.56±3.42、70.94±7.38)(P<0.01),表明AS-ODNs抑制了GLT-1表达从而减弱了CIP对海马CA1区锥体神经元的保护作用。以上结果显示,单纯侧脑室注射AS-ODNs可引起海马CA1区GLT-1表达下调,但不引起明显的DND;而预先侧脑室注射AS-ODNs可减弱CIP对海马CA1区锥体神经元的保护作用。这些结果表明AS-ODNs通过抑制GLT-1表达从而减弱了CIP对海马CA1区锥体神经元的保护作用,进一步证实了GLT-1在CIP对脑缺血耐受诱导中的作用。3 GLT-1正义寡核苷酸上调GLT-1的表达增强脑对缺血打击的耐受应用GLT-1正义寡核苷酸(sense oligodeoxynucleotides,S-ODNs)上调GLT-1的表达后,观察海马CA1区锥体神经元对脑缺血打击的耐受情况,进一步确定GLT-1在脑缺血耐受中的作用。3.1 GLT-1 S-ODNs上调GLT-1的表达将42只凝闭双侧椎动脉36 h的Wistar大鼠随机分为3组:①对照组(n=6):右侧脑室注射双蒸水5μl,注射后12 h取材;②S-ODNs 9 nmol组(n=18):右侧脑室注射S-ODNs溶液5μl(9 nmol),根据S-ODNs注射后取材的时间又分为三个亚组:12 h、24 h、36 h组;③S-ODNs 18 nmol组(n=18):右侧脑室注射S-ODNs溶液5μl(18 nmol),根据S-ODNs注射后取材的时间又分为三个亚组:12 h、24 h、36 h组。所有大鼠在预定时间断头取材,低温条件下分离海马CA1区,采用Western blotting方法测定GLT-1的蛋白表达(方法同前)。结果显示,双蒸水对照组的IOD的比值为0.64±0.19。注射S-ODNs 9 nmol组中,12 h组的IOD比值为0.78±0.02,24 h组为1.01±0.04, 36 h组为0.68±0.06。注射S-ODNs 18 nmol组中,12 h组的IOD比值为0.84±0.02,24 h组为1.23±0.03,36 h组为0.62±0.02。与双蒸水对照组相比,注射S-ODNs后IOD的比值显著增加(P<0.01),且呈现剂量依赖性。以上结果表明,侧脑室注射S-ODNs溶液可以剂量依赖性地上调GLT-1蛋白表达,这种上调作用在注射S-ODNs后24 h最为显著。3.2 GLT-1 S-ODNs增强海马CA1区锥体神经元对缺血打击的耐受性将凝闭双侧椎动脉的Wistar大鼠54只随机分为7组:①sham组(n=6):只暴露双侧颈总动脉,不阻断血流;②CIP组(n=6):夹闭双侧颈总动脉3 min;③脑缺血打击组(n=6):夹闭双侧颈总动脉8 min;④CIP+脑缺血打击组(n=6):夹闭双侧颈总动脉3 min作为CIP,再灌注2 d后再夹闭双侧颈总动脉8 min;⑤双蒸水组(n=6):于分离暴露双侧颈总动脉(但不夹闭)前12 h、后12 h及后36 h右侧脑室注射双蒸水,每次5μl,其他同sham组;⑥S-ODNs组(n=12):于分离暴露双侧颈总动脉(但不夹闭)前12 h、后12 h及后36 h右侧脑室注射S-ODNs溶液,每次5μl,其他同sham组。根据S-ODNs的剂量进一步分为9和18 nmol 2个亚组,每组6只动物;⑦S-ODNs+脑缺血打击组(n=12):于分离暴露双侧颈总动脉(但不夹闭)前12 h、后12 h及后36 h右侧脑室注射S-ODNs溶液,每次5μl,并于暴露双侧颈总动脉2 d后夹闭双侧颈总动脉8 min。根据S-ODNs的剂量进一步分为9和18 nmol 2个亚组,每组6只动物。以上各组动物均在sham手术或末次手术后7d断头取材,常规脑组织切片(5μm厚),硫堇染色下观察海马CA1区DND的发生情况(方法同前)。结果显示,Sham组和CIP组大鼠海马CA1区无明显的DND,HG为0～Ⅰ级,ND值分别为208.25±5.97和202.86±4.28。脑缺血打击组大鼠海马CA1区出现明显的DND,HG(Ⅱ～Ⅲ级)明显升高(P<0.01),ND值(45.86±21.93)显著降低(P<0.01)。CIP+脑缺血打击组海马CA1区DND不明显,与脑缺血组相比,HG(0～Ⅰ级)明显降低(P<0.01),ND值(208.98±5.9)显著升高(P<0.01),表明CIP可以诱导海马CA1区神经元产生缺血性耐受,对抗缺血打击引起的DND。侧脑室注射双蒸水和S-ODNs后海马CA1区组织形态与Sham组和CIP组基本一致,HG为0～Ⅰ级,ND值为176.22±6.29(双蒸水)、186.53±5.64(9 nmol S-ODNs)和204.4±12.99(18 nmol S-ODNs),与sham组或CIP组相比无显著性差异(P>0.05)。S-ODNs +脑缺血打击组的HG为Ⅰ级,与脑缺血打击组相比显著降低(P<0.01);ND值分别为148.53±9.1(9 nmol S-ODNs组)和166.67±8.31(18 nmol S-ODNs组),与脑缺血打击组相比显著增加(P<0.01);表明S-ODNs通过上调GLT-1蛋白表达从而减弱了脑缺血对海马CA1区锥体神经元的损伤。以上结果显示,侧脑室注射S-ODNs引起海马CA1区GLT-1表达明显上调,预先注射S-ODNs减弱了脑缺血打击对海马CA1区锥体神经元的损伤。这些结果证实S-ODNs通过增加GLT-1的蛋白表达从而减弱了脑缺血对海马CA1区锥体神经元的损伤,产生了与CIP相同的效果。4结语(1)谷氨酸转运体GLT-1抑制剂DHK通过抑制GLT-1的功能而减弱了CIP对海马CA1区锥体神经元的保护作用;(2)侧脑室注射谷氨酸转运体GLT-1 AS-ODNs可抑制GLT-1的表达,同时减弱了CIP对海马CA1区锥体神经元的保护作用;(3)侧脑室注射GLT-1 S-ODNs上调GLT-1表达的同时,增强了海马CA1区锥体神经元对脑缺血打击的耐受性,产生了与CIP相同的效果。(4)以上结果表明GLT-1参与了CIP诱导的BIT。更多还原

【Abstract】 Ischemic cerebrovascular disease is one kind of diseases which severely impair human health. Since neuronal cells are very sensitive to ischemia, long time brain ischemic insult results in neuronal death, which usually can not revive and frequently causes severe sequelae even if blood supply recovers. With the progress of the studies on the ischemic cerebrovascular disease, more and more attention has been paid to the phenomenon of brain ischemic tolerance (BIT). It is found in animal experiment that suddenly severe cerebral ischemic insult causes severe delayed neuronal death (DND) in the CA1 hippocampus. But when the ischemic insult was preceded by a transient sublethal cerebral ischemic preconditioning, the DND normally induced by the ischemic insult could be significantly prevented. This phenomenon was called brain ischemic tolerance (BIT), and the sublethal cerebral ischemia given in advance was called cerebral ischemic preconditioning (CIP). It is important to clarify the mechanism of the neuro-protection induced by CIP for developing new therapeutic methods to enhance the tolerance of neurons to ischemia and hypoxia.Ischemia and hypoxia in brain tissue induced by various kinds of diseases can result in dysmetabolism in energy and inhibit the action of Na+-K+-ATPase, which would lead to an obvious increase of extracellular K+ concentration and intracellular Na+ concentration. The changes facilitate depolarization of neurons and release of glutamate in the nerve terminal. In addition, the high concentration of extracellular K+ and intracellular Na+ can reverse the activity of high-affinity glutamate transporter, which would lead to reversal transporting of glutamate from intracellular to extracellular space. These reasons result in an increase in extracellular glutamate concentration. The increased extracellular glutamate binds with and activates NMDA or non-NMDA receptors, and then induces influx of Na+ and Ca2+ and release of intracellular Ca2+ to cytoplasm. The neurons die of the overload of Na+ and Ca2+. Thus, the glutamate has been referred to as excitotoxin. Therefore, it is an impotant strategy to decrease the concentration of extracellular glutamate or to reduce the combination of glutamate with its specific receptor for preventing the excitotoxicity of the increased glutamate in brain ischemia. Excitatory amino acid transports (EAATs) are important mechanism for regulating the extracellular concentration of glutamate. It has been well-known that the glial glutamate transporter subtype GLT-1 plays a dominant role in terminating glutamate neurotransmission, and maintaining the extracellular glutamate below neurotoxic levels. Some studies paid attention to roles of GLT-1 in induction of tolerance of brain to ischemic or hypoxic insults. For example, Douen et al reported that preconditioning with cortical spreading depression could down-regulate the expression of glutamate transporters EAAT1 and EAAT2 of astrocytes to prevent the reversal transporting of glutamate during cerebral ischemia. Romera et al found that preconditioning with ischemia or hypoxia in vitro could increase the expression of glutamate transports. These results suggested that GLT-1 might associate with the induction of BIT. But there is no report yet about whether GLT-1 of astrocytes participates in the neuro-protection of CIP in vivo and whether the modulation of GLT-1 may enhance the tolerance of neuronal cells to ischemic insult. Therefore, the present study was undertaken to explore in vivo the effect of modulation of GLT-1 by Dihydrokainate (DHK), a selective inhibitor of GLT-1, GLT-1 antisense oligodeoxynucleotides (AS-ODNs) or sense oligodeoxynucleotides (S-ODNs) on the induction of BIT using rat global cerebral ischemic model. The results to be obtained would provide experimental evidence for clarifying the role of GLT-1 in the induction of BIT.1 Inhibiting function of GLT-1 with DHK blocked BIT induced by CIPThe role of GLT-1 in BIT was investigated in rat 4-vessel occlusion (4VO) global brain ischemia model by observing the effect of Dihydrokainate (DHK), a selective inhibitor of GLT-1, on the neuro-protection of CIP against delayed pyramidal neuronal death in the CA1 hippocampus normally induced by severe brain ischemic insult.Ninety-six Wistar rats with permanently occluded bilateral vertebral arteries for 2d were randomly assigned to eight groups:①Sham group (n=6): the bilateral common carotid arteries (BCCA) were separated, but without occluding the blood flow;②CIP group (n=6): the BCCA were clamped for 3 min;③Brain ischemic insult group (n=6): the BCCA were clamped for 8 min;④CIP+Brain ischemic insult group (n=6): a CIP for 3 min was preformed first, and then a brain ischemic insult for 8 min was given 2d after the CIP;⑤Distilled water group (n=6): 20μl distilled water was injected into the right lateral ventricle;⑥DHK group(n=30): 20μl DHK solution was injected into the right lateral ventricle. This group was further divided into 10 nmol, 100 nmol, 200 nmol, 500 nmol and 1000 nmol subgroups according to the doses of DHK;⑦DHK+CIP group(n=18): 20μl DHK solution was injected into the right lateral ventricle 20 min before CIP. This group was further divided into 10 nmol, 100 nmol and 200 nmol subgroups according to the doses of DHK;⑧DHK+CIP+brain ischemic insult group (n=18): 20μl DHK solution was injected into the right lateral ventricle 20 min before CIP. Other procedures were the same as those in CIP+brain ischemic insult group. According to the doses of DHK used, this group was also further divided into 10 nmol, 100 nmol and 200 nmol subgroups. The animals were sacrificed by decapitation at 7d after the sham operations or the last operations. The brain was sectioned in a thickness of 5μm and stained with thionin. Histological changes of the CA1 hippocampus were examined under light microscope to determine delayed neuronal death (DND) by neuronal density (ND) and histological grade (HG) of the CA1 hippocampus. The ND was determined by counting the number of surviving pyramidal neurons with intact cell membrane, full nucleus and clear nucleolus within 1 mm linear length of the CA1 hippocampus. The average of the number in 3 areas of the CA1 hippocampus was calculated as value of ND. HG was divided into the following 4 grades: grade 0, no neuron death; grade Ⅰ, scattered single neuron death; gradeⅡ, death of many neurons; gradeⅢ, death of almost complete neurons. The average HG of the bilateral hippocampus was counted as statistical data.The results showed that during the 4VO, the pupils of rats enlarged. EEG showed decreases in frequency and amplitude, even approached isoelectric level, and the righting reflex disappeared. These manifestations indicated that the rats were really subjected to global cerebral ischemia.It was found by thionine staining that there was no significant neuronal damage in the CA1 subfield of the hippocampus in the sham and CIP groups. HG was 0～Ⅰ, and values of ND were 208.25±5.97 and 202.93±4.32 in the two groups, respectively. Obvious destruction of the CA1 subfield was found in brain ischemic insult for 8 min group. The value of ND was 45.86±21.93, which was much low compared with that in the sham and CIP groups (P<0.01). HG was gradeⅡ～Ⅲ, which was much high compared with that in the sham and CIP. In CIP+ischemic insult group, no obvious neuronal damage in the hippocampal CA1 region was found. HG (grade 0～Ⅰ) was significantly lower than that in brain ischemic insult group (P<0.01), and the value of ND (208.07±5.87) was much higher than that in brain ischemic insult group (P<0.01). The results indicated that CIP protected pyramidal neurons of the CA1 hippocampus against DND induced by brain ischemic insult. There was no apparent DND in distilled water group, in which histological characteristics such as HG and ND were similar with those in the sham group. DHK in doses of 10, 100 and 200 nmol just caused slight DND of pyramidal neurons in the CA1 hippocampus. HG was gradeⅠin each dose, and the values of ND were 172.76±17.31, 162.64±6.12 and 155.43±9.82, respectively. While obvious DND was found in 500 and 1000 nmol groups; the value of ND was significantly decreased (105.35±3.84 and 6±1.39, respectively); HG was significantly increased (ⅡandⅢ, respectively) compared with 10, 100 and 200 nmol DHK groups (P<0.05). These results indicated that large dose DHK could lead to significant pyramidal neuron damage in the hippocampal CA1 region. Thus, we only observed effects of 10, 100 and 200 nmol DHK in the subsequent experiment. There was no apparent DND was found in DHK+CIP group in each dose of 10, 100 and 200 nmol DHK, in which histological characteristics such as HG and ND were similar with those in the sham group. Obvious DND was found in DHK+CIP+brain ischemic insult group. HG wereⅠ,Ⅰ～ⅡandⅢ, and the values of ND were 160.87±13.55, 117.07±10.11 and 4.87±2.02, respectively, in the doses of 10 nmol, 100 nmol and 200 nmol. The changes above in DHK+CIP+brain ischemic insult group were significant compared with CIP+brain ischemic insult group (P<0.01). Additionally, the correlative curve between the doses and effectiveness in DHK+CIP+brain ischemic insult groups shifted to left siginifantly, and the slope rate became sharper than that in DHK group. These results indicated that administration of DHK blocked in a dose dependent manner the neuroprotection of CIP against DND normally induced by severe brain ischemic insult in the CA1 hippoacmpus.The above results suggested the involvement of GLT-1 in the neuroprotection of CIP against DND normally induced by severe brain ischemic insult in the CA1 hippoacmpus.2 Inhibition of the expression of GLT-1 with GLT-1 AS-ODNs attenuated the neuro-protection of CIP against brain ischemic insultThe role of GLT-1 in BIT was investigated by observing the effect of antisense oligodeoxynucleotides (AS-ODNs) of GLT-1, which can inhibit the protein expression of GLT-1, on the neuro-protection of CIP against brain ischemic insult.2.1 The effect of AS-ODNs on the protein expression of GLT-1Forty-two Wistar rats with permanently occluded bilateral vertebral arteries for 36 h were randomly assigned to three groups.①control group (n=6): 5μl distilled water was injected into the right lateral ventricle and the animals were sacrificed by decapitation 12 h later.②AS-ODNs 9 nmol group(n=18):5μl AS-ODNs (9 nmol) solution was injected into the right lateral ventricle. This group was further divided into 12h, 24h and 36h subgroups according to the time of reperfusion after injection.③AS-ODNs 18 nmol group(n=18):5μl AS-ODNs (18 nmol) solution was injected into the right lateral ventricle. This group was also further divided into 12h, 24h and 36h subgroups according to the time of reperfusion after injection. The animals were sacrificed by decapitation at the determined endpoint of the experiment. The hippocampal CA1 was dissected out quickly on ice and determine the expression of GLT-1 using Western blotting. Integrated optical density (IOD) of each band was measured using image analysis system. The changes of GLT-1 expression were represented with the ratio of IOD of the aim protein band toβ-actin.The results of Western blotting showed that IOD in the control group was 0.65±0.22. In 9 nmol AS-ODNs groups, IOD were 0.37±0.07, 0.20±0.05 and 0.25±0.07, respectively. In 18 nmol AS-ODNs groups, IOD were 0.11±0.05, 0.05±0.02 and 0.17±0.16, respectively. IOD in AS-ODNs groups were decreased significantly compared with control group (P<0.05), and these changes were in a dose dependent manner.The results indicatedthatAS-ODNs injected into the lateral ventricle inhibited expression of GLT-1 in a dose dependent manner, and the inhibiting effect was much significant at 24h.2.2 AS-ODNs of GLT-1 inhibit the neuro-protection on pyramidal neurons of the CA1 hippocampus induced by CIPFifty-four Wistar rats with permanently occluded bilateral vertebral arteries were randomly assigned to seven groups.①Sham group (n=6): the bilateral common carotid arteries (BCCA) were separated, but without occluding the blood flow.②CIP group (n=6): the BCCA were clamped for 3 min.③Brain ischemic insult group (n=6): the BCCA were clamped for 8 min.④CIP+Brain ischemic insult group (n=6): 3 min CIP was preformed 2d prior to 8 min ischemic insult.⑤D istilled water group (n=6): 5μl distilled water was injected into the right lateral ventricle 12 h before, 12 h and 36 h after the BCCA were separated (but without occluding the blood flow), respectively.⑥AS-ODNs group(n=12): 5μl AS-ODNs solution was injected into the right lateral ventricle 12 h before, 12 h and 36 h after the BCCA were separated (but without occluding the blood flow), respectively. This group was further divided into 9 nmol and 18 nmol subgroups according to the doses of AS-ODNs (n=6 in each group).⑦A S-ODNs+CIP+Ischemic insult group (n=12): 5μl AS-ODNs solution was injected into the right lateral ventricle 12h before, 12h and 36h after CIP, respectively. This group was also further divided into 9 nmol and 18 nmol subgroups according to the doses of AS-ODNs. The other treatment was the same as CIP+Brain ischemic insult group, with six rats in each group. The animals were sacrificed by decapitation 7 d after sham operations or the last operations. Histological changes of the CA1 region of the hippocampus were examined using thionin staining (the methods were the same as that in part 1).Thionin staining showed that there was no significant neuronal damage in the CA1 subfield of the hippocampus in the sham and CIP groups. HG was 0～Ⅰ, and values of ND were 208.25±5.97 and 202.86±4.28 in the two groups, respectively. Obvious DND in the hippocampal CA1 region was found in ischemic insult for 8 min group. The value of ND was 45.86±21.93, which was much low compared with that in the sham and CIP groups (P<0.01). HG was gradeⅡ～Ⅲ, which was much high compared with that in the sham and CIP groups. In CIP+ischemic insult group, no obvious neuronal damage in the hippocampal CA1 region was found. HG (grade 0～Ⅰ) was significantly lower than that in 8 min brain ischemia group (P<0.01), and the value of ND was 208.98±5.9, which was much higher than that in 8 min brain ischemic insult group (P<0.01). The results indicated that CIP induced BIT of pyramidal neurons of the CA1 hippocampus against DND caused by brain ischemic insult. There was no apparent DND was found when distilled water and AS-ODNs (9 nmol and 18 nmol) were administrated into right lateral cerebral ventricle, and the histological changes were the same as those in sham and CIP groups. HG was grade 0～Ⅰand the values of ND were 176.22±6.29, 176.89±2.88 and 175.32±2.77, respectively. These results indicated that the injection of distilled water and AS-ODNs into the right lateral cerebral ventricle could not lead to apparent pyramidal neuron damage in the hippocampal CA1 region. In AS-ODNs +CIP + brain ischemic insult groups, DND were significantly obvious compared with that in CIP+ ischemic insult group. HG were gradeⅡ～Ⅲ, and ND (9 nmol and 18 nmol groups) were 133.56±3.42 and 70.94±7.38, respectively (P<0.01), Which indicated that AS-ODNs attenuated the neuro-protection on pyramidal neurons of the CA1 hippocampus induced by CIP via inhibiting the expression of GLT-1.The results indicated that the injection of AS-ODNs into the lateral cerebral ventricle induced the down-regulation of GLT-1 but no obvious DND in hippocampal CA1 region. However, the protective effect of CIP on pyramidal neurons of the CA1 hippocampus was attenuated by the injection of AS-ODNs. These results further suggested the association of GLT-1 with the induction of BIT by CIP in rats.3 Up-regulating the expression of GLT-1 with GLT-1 S-ODNs enhanced the tolerance to brain ischemic insult in the hippocampal CA1 regionThe role of GLT-1 in the brain ischemic tolerance was investigated by observing the effect of sense oligodeoxynucleotides (S-ODNs) of GLT-1 on tolerance to brain ischemic insult in the hippocampal CA1 region via up-regulating the protein expression of GLT-1.3.1 The effect of S-ODNs on the protein expression of GLT-1Forty-two Wistar rats with permanently occluded bilateral vertebral arteries for 36 h were randomly assigned to three groups.①control group (n=6): 5μl distilled water was injected into the right lateral cerebral ventricle and the animals were sacrificed by decapitation 12 h later.②S-ODNs 9 nmol group(n=18):5μl S-ODNs (9 nmol) solution was injected into the right lateral cerebral ventricle. This group was further divided into 12 h, 24 h and 36 h subgroups according to the time of reperfusion after injection.③S-ODNs 18 nmol group (n=18):5μl S-ODNs (18 nmol) solution was injected into the right lateral cerebral ventricle. This group was further divided into 12 h, 24 h and 36 h subgroups according to the time of reperfusion after injection. The animals were sacrificed by decapitation at the determined endpoint of the experiment. The hippocampal CA1 was dissected out quickly on ice and detected the expression of GLT-1 using Western blotting. IOD of each band was measured using image analysis system. The changes of GLT-1 expression were represented with the ratio of IOD of the aim protein toβ-actin.The results of Western blotting showed that IOD in the control group was 0.64±0.19. In 9 nmol S-ODNs groups, IOD were 0.78±0.02, 1.01±0.04 and 0.68±0.06, respectively. In 18 nmol S-ODNs groups, IOD were 0.84±0.02, 1.23±0.03 and 0.62±0.02, respectively. IOD in S-ODNs groups were up-regulated significantly compared with control group (P<0.05), and these changes were in a dose dependent manner.The results indicated that S-ODNs injected into the lateral ventricle up-regulated the protein expression of glutamate transporter subtype GLT-1 in a dose dependent manner, and this effect was much significant at 24 h. 3.2 S-ODNs of GLT-1 attenuated the injury of pyramidal neurons in the CA1 hippocampus induced by brain cerebral ischemiaFifty-four Wistar rats with permanently occluded bilateral vertebral arteries were randomly assigned to seven groups.①Sham group (n=6): the bilateral common carotid arteries (BCCA) were separated, but without occluding the blood flow.②CIP group (n=6): the BCCA were clamped for 3 min.③Brain ischemic insult group (n=6): the BCCA were clamped for 8 min.④CIP+Brain ischemic insult group (n=6): 3 min CIP was preformed 2d prior to 8 min ischemic insult.⑤D istilled water group (n=6): 5μl distilled water was injected into the right lateral cerebral ventricle 12h before, 12h and 36h after the BCCA were separated (but without occluding the blood flow) , respectively.⑥S-ODNs group(n=12): 5μl S-ODNs solution was injected into the right lateral cerebral ventricle 12h before, 12h and 36h after the BCCA were separated (but without occluding the blood flow), respectively. This group was further divided into 9 nmol and 18 nmol subgroups according to the doses of S-ODNs (n=6 in each group).⑦S -ODNs + Ischemic insult group (n=12): 5μl S-ODNs solution was injected into the right lateral cerebral ventricle 12 h before, 12 h and 36 h after the BCCA were separated (but without occluding the blood flow), respectively. The BCCA were clamped for 8 min after the BCCA were separated for 2 d. This group was also further divided into 9 nmol and 18 nmol subgroups according to the doses of S-ODNs. The animals were sacrificed by decapitation 7 d after sham operations or the last operations. Histological changes of the CA1 hippocampus were examined using thionin staining (the method was the same as that in part 1).Thionin staining showed that there was no significant neuronal damage in the CA1 subfield of the hippocampus in the sham and CIP groups. HG was 0～Ⅰ, and values of ND were 208.25±5.97 and 202.86±4.28 in the two groups, respectively. Obvious DND in the hippocampal CA1 region was found in ischemic insult for 8 min group and the value of ND was 45.86±21.93, which was much low compared with that in the sham and CIP groups (P<0.01). HG was gradeⅡ～Ⅲ, which was much high compared with that in the sham and CIP groups. In CIP+ischemic insult group, no obvious neuronal damage in the hippocampal CA1 region was found. HG (grade 0～Ⅰ) was significantly lower than that in 8 min brain ischemia group (P<0.01) and the value of ND was 208.98±5.9, which was much higher than that in 8 min brain ischemic insult group (P<0.01). The results indicated that CIP induced BIT of pyramidal neurons of the CA1 hippocampus against DND caused by brain ischemic insult. There was no apparent DND when distilled water and S-ODNs (9 nmol and 18 nmol) were administrated into right lateral cerebral ventricle, and the histological changes were the same as those in sham and CIP groups. HG was grade 0～Ⅰand the values of ND were 176.22±6.29, 186.53±5.64 and 204.4±12.99, respectively (P>0.05). In S-ODNs + Ischemic insult group, ND (they were 148.53±9.1 in 9 nmol group and 166.67±8.31 in 18 nmol group) was significantly increased compared with that in brain ischemic insult group (P<0.01). HG was gradeⅠwhich was significantly decreased compared with that in brain ischemic insult group. These results indicated that S-ODNs attenuate the damage of pyramidal neurons in the CA1 hippocampus induced by brain ischemic insult through up-regulating the expression of GLT-1.The results indicated that the injection of S-ODNs into the lateral cerebral ventricle led to the up-regulation of GLT-1 in hippocampal CA1 region. The damage induced by brain ischemic insult on pyramidal neurons of the CA1 hippocampus was attenuated by the injection of S-ODNs. These results further suggested the role of GLT-1 in the induction of BIT by CIP in rats.4 Conclusions(1) DHK, an inhibitor selective for glial glutamate transporter GLT-1, inhibited the protective effect induced by CIP on pyramidal neurons in the CA1 subfield of the hippocampus via inhibiting the function of GLT-1 in rats.(2) The injection of AS-ODNs of GLT-1 into the lateral cerebral ventricle inhibited the expression of glutamate transporter subtype GLT-1. At the same time, AS-ODNs of GLT-1 attenuated the neuro-protection on pyramidal neurons of the CA1 hippocampus induced by CIP.(3) The injection of S-ODNs of GLT-1 into the lateral cerebral ventricle enhanced the tolerance to brain ischemic insult in the hippocampal CA1 region as wll as up-regulating the protein expression of GLT-1.(4) These results indicated that GLT-1 participates in BIT induced by CIP.更多还原