【作者】 张敏；

【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】 实验发现,给动物突然造成较严重的脑缺血,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,可保护神经元,使其能够耐受在该轻微脑缺血后给予的通常会引起神经元严重损伤的较严重脑缺血。预先给予的轻微、短时脑缺血称为脑缺血预处理(cerebral ischemic preconditioning, CIP),所产生的这种保护作用称为脑缺血耐受(brain ischemic tolerance, BIT)。自1990年Kitagawa首先发现这一现象以来,大量研究证实了它的存在。阐明CIP脑保护作用的机制,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。大量研究表明,严重脑缺血缺氧可导致脑内谷氨酸等兴奋性氨基酸(excitatory amino acids, EAAs)的浓度异常升高,从而产生兴奋性神经毒作用。细胞膜上的高亲和性兴奋性氨基酸转运体(excitatory amino acid transporters, EAATs)可将兴奋性氨基酸从突触间隙转运至细胞内,在及时终止兴奋性突触传递以及维持细胞外液兴奋性氨基酸的正常水平中发挥重要作用。目前已发现五种高亲和性EAATs,包括EAAT1 (又称为glutamate/aspartate transporter, GLAST)、EAAT2 (又称为glail glutamate transporter-1, GLT-1)、EAAT3 (又称为excitatory amino acid carrier 1, EAAC1)、EAAT4和EAAT5。一般认为,GLAST和GLT-1为胶质细胞转运体,主要分布在星型胶质细胞;EAAC1、EAAT4和EAAT5为神经元转运体,其中EAAC1主要分布在海马神经元,EAAT4主要分布在小脑神经元,EAAT5主要分布在视网膜。EAATs主要由钠、钾离子浓度梯度驱动转运。当离子梯度降低或膜电位降低时,如缺血、癫痫发作时,EAATs摄取EAAs的功能减弱,甚至可以将细胞内的谷氨酸反向转运至细胞外,导致细胞外谷氨酸浓度异常升高,而产生神经毒作用。尽管神经元和胶质细胞都表达EAATs,但是普遍认为胶质细胞的EAATs对谷氨酸的转运能力比神经元要强大的多,尤其是星形胶质细胞谷氨酸转运体亚型GLT-1在调节细胞外液谷氨酸浓度方面发挥主要作用。据此,我们推测,GLT-1可能在脑缺血耐受诱导过程中发挥作用。然而,到目前为止,仅有的几篇关于GLT-1是否在脑缺血耐受诱导过程中发挥作用的研究均为离体实验,且所得结果互相矛盾。为了探讨GLT-1是否在脑缺血耐受诱导过程中发挥保护作用,本实验应用脑组织病理学、免疫组织化学、western blot分析、脑内微透析以及高效液相色谱等方法,研究了在体(in vivo)脑缺血耐受诱导过程中大鼠海马GLT-1和胶质纤维酸性蛋白(Glial fibrillary acidic protein, GFAP)的表达以及谷氨酸、门冬氨酸、甘氨酸以和γ-氨基丁酸(γ-Aminobutyric acid, GABA)浓度的变化。1 CIP诱导大鼠海马CA1区GLT-1和GFAP蛋白表达上调采用大鼠四血管闭塞(4 vessel occlusion, 4VO)全脑缺血模型,应用脑组织病理学评价、免疫组织化学以及western blot分析等方法,观察脑缺血耐受诱导过程中大鼠海马CA1区GLT-1和GFAP蛋白表达的变化,探讨GLT-1在脑缺血耐受诱导过程中的作用。1.1脑组织病理学评价145只大鼠随机分为以下5组。①正常对照组(n=5);②凝闭椎动脉组(n=35):凝闭双侧椎动脉;③CIP组(n=35):全脑缺血3 min;④损伤性脑缺血组(n=35):全脑缺血8 min;⑤CIP+损伤性脑缺血组(n=35):CIP后2天全脑缺血8 min。除正常对照组之外,其余各组根据动物末次手术后取材时间,分为0 h(即刻)、3 h、1 d、2 d、3 d、5 d、7 d共7个亚组(每个时间点n=5)。各组动物均于预定时间点取脑,连续切片,硫堇染色下观察海马CA1区迟发性神经元死亡(delayed neuronal death, DND)情况。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。计数海马CA1区每1 mm区段内细胞膜完整、胞核饱满、核仁清晰的锥体神经元的数目,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)。硫堇染色显示,正常对照组大鼠海马CA1区锥体神经元排列整齐致密,可见2～3层,细胞形态完整、边界清晰、尼氏小体丰富,胞核大而圆、核仁清晰, HG为0级, ND为210±5.7。凝闭椎动脉组大鼠各时间点海马CA1区均未见明显损伤,与control组相比,HG、ND均无明显变化。CIP组大鼠在各时间点海马CA1区均未见明显损伤,与凝闭椎动脉组大鼠相应时间点相比,HG、ND均无明显变化。损伤性脑缺血组大鼠在2天内未见明显的锥体神经元损伤;3天时可见部分神经元死亡,细胞形态发生明显改变,可见胞体缩小,形态不规则,呈多角型或梭型,胞膜皱缩,胞核固缩、浓染,核仁模糊不清或消失,突起明显深染变长;至损伤性脑缺血后5 d和7 d时,神经元几乎全部死亡,与凝闭椎动脉组大鼠相比,HG明显升高,ND明显减少。CIP+损伤性脑缺血组大鼠在各时间点海马CA1区锥体细胞排列整齐致密,胞核饱满,核仁较清晰,仅个别锥体细胞胞核固缩,无明显细胞缺失,与损伤性脑缺血后7 d组相比,HG明显降低,ND明显升高(p<0.01)。这些结果表明,CIP对2天后发生的严重脑缺血再灌注损伤有明显的对抗作用,表明脑缺血耐受诱导成功。1.2免疫组织化学检测动物分组及实验程序与上述脑组织病理学评价相同。GLT-1免疫组织化学染色显示,对照组可见在海马CA1区有一定量GLT-1阳性标记物。凝闭椎动脉组几乎各时间点均可以见到GLT-1明显上调,以3h点表达最高,并且隐约可以见到胶质细胞样轮廓。CIP组与凝闭椎动脉组各相应时间点比较,早期明显上调,以1d时较为明显;另外可见在环绕锥体细胞的周围区域,GLT-1表达有所上调,形成一定的“网格”状。与凝闭椎动脉组相比,损伤性脑缺血组各相应时间点GLT-1的表达均下调,尤其以死亡的锥体细胞周围下调的最为明显,甚至表现为GLT-1大片缺失。CIP+损伤性脑缺血组大鼠与损伤性缺血组相比,各相应时间点GLT-1均明显上调,GLT-1阳性标记物紧紧包绕在锥体细胞周围,并形成明显的“网格”状。GFAP免疫组化染色显示形态完整的星形胶质细胞呈星形或蜘蛛状,有明显的突起。control组海马CA1区可见星形胶质细胞散在均匀分布。凝闭椎动脉组与control组相比,所有时间点GFAP阳性标记物均明显减少。CIP与凝闭椎动脉组相比,CA1区星形胶质细胞的突起有所延长,在环绕锥体细胞的周围区域可见一定程度的GFAP免疫颗粒分布;GFAP阳性细胞数、总面积、平均光密度均明显上调(P<0.01),以1~3天较为明显。损伤性脑缺血组海马CA1区GFAP阳性细胞数目明显增多,胞体肥大,突起变长、增粗,但并不包绕锥体神经元胞体;GFAP阳性细胞数、总面积、平均光密度均显著升高(P<0.01);上述变化在损伤性脑缺血后3 d和5 d最为明显。CIP+损伤性脑缺血组,海马CA1区GFAP阳性细胞胞体并不明显增大,但突起明显延长并环绕锥体神经元胞体,形成非常明显的网格状;上述变化在损伤性脑缺血后2 d达高峰,一直持续至7 d。1.3 western blot分析205只大鼠随机分为以下6组:①正常对照组(n=5);②sham组(n=20):根据sham手术后取材时间,分为0 h(即刻)、3 h、12h、2d共4个时间点(每个时间点n=5);③椎动脉凝闭组(n=45);④CIP组(n=45);⑤损伤性脑缺血组(n=45);⑥CIP+损伤性脑缺血组(n=45)。除正常对照组及sham组之外,其余各组根据动物末次手术后的取材时间,分为0 h(即刻)、3 h、6h、12h、1 d、2 d、3 d、5 d、7 d共9个亚组(每个时间点n=5)。Sham组动物暴露椎动脉和颈总动脉,但不阻断其血流。其余各组动物的处理与1.1相同。GLT-1的western blot分析显示,与对照组相比,凝闭椎动脉组GLT-1的表达在多个时间点明显上调,第一次高峰在3 h和6 h,第二次高峰在5 d,以3 h最高。与sham组相比,凝闭椎动脉组GLT-1的表达在即刻、3 h、2 d均显著升高,表明凝闭椎动脉后GLT-1的表达上调不是麻醉和手术导致的,而是凝闭椎动脉所引起的。CIP组中,与CIP后即刻或凝闭椎动脉组相比,除CIP后3 h、3 d GLT-1的表达明显下调外,其余各时间点GLT-1的表达均显著上调(P<0.05)。损伤性脑缺血组中,与该组的即刻时间点、或凝闭椎动脉组相比,除损伤性缺血后12 h外,其余所有时间点GLT-1的表达均下调。CIP+损伤性脑缺血组中,与该组的即刻取材时间点相比,GLT-1的表达在3 h、12 h明显上调;与损伤性脑缺血组相比,除1 d之外,其余各时间点GLT-1的表达均显著上调。GFAP的western blot分析显示,与对照组相比,凝闭椎动脉组GFAP的表达于各时间点均明显下调。与对照相比,sham组所有时间点GFAP的表达均未见明显的变化。凝闭椎动脉组与sham相比,GFAP的表达在即刻、12 h、2 d均显著减少,表明凝闭椎动脉后GFAP的表达下调不是麻醉和手术导致的,而是凝闭椎动脉所引起的。CIP组中,与该组的即刻时间点相比,CIP后GFAP的表达在12 h、7 d明显上调;与凝闭椎动脉组相比,CIP后GFAP的表达在所有时间点均明显升高。损伤性脑缺血组中,与该组的即刻时间点相比,损伤性脑缺血后GFAP蛋白的表达在3 h、1 d、2 d、3 d、5 d、7 d均明显上调;与凝闭椎动脉组相比,损伤性脑缺血后GFAP的表达除即刻时间点之外,其余所有时间点均明显升高。CIP+损伤性脑缺血组中,与该组的即刻时间点相比,GFAP蛋白的表达在3 h、2 d、5 d、7 d明显上调;与损伤性脑缺血组相比,CIP+损伤性缺血后GFAP的表达在即刻时间点明显升高,3 h、1 d、7 d明显降低,其于各时间点无明显差异。以上结果表明:3 min CIP在对抗8 min缺血打击引起的DND的同时,可引起海马CA1区星形胶质细胞的突起延长、包绕锥体神经元,并且表达大量的GLT-1,提示CIP引起的GLT-1表达上调参与CIP的脑保护作用。2大鼠脑缺血耐受诱导过程中海马CA3区及齿状回GLT-1和GFAP蛋白表达的变化模型制备、动物分组、实验程序及实验方法与第一部分相同。采用神经元死亡率(死亡神经元数目与该区域神经元总数目之比)表示CA3区及齿状回的DND。2.1神经病理学评价与海马CA1区不同,全脑缺血打击后,仅个别动物海马CA3区和齿状回出现了少量锥体细胞或颗粒细胞的DND,细胞缺失率不超过20%。预先给予CIP,同样可防止上述DND的发生。其余各组的变化与CA1相同。由此可见,海马CA3区和齿状回的神经元对缺血的耐受能力较强,CIP对这些神经元同样具有保护作用。2.2 GLT-1和GFAP蛋白的表达海马CA3区及齿状回GLT-1、GFAP蛋白的表达与海马CA1区有所不同。在正常对照组海马CA3区和齿状回,特别是CA3区即可见较多的星型胶质细胞的突起伸入锥体神经元层,并且围绕在锥体神经元周围,同时在锥体神经元之间的区域可以见到一定量的GLT-1免疫阳性颗粒;单纯凝闭椎动脉,即可使这些免疫阳性颗粒明显增多,形成了较为明显的“网格”现象;8 min全脑缺血打击后,大部分区域,特别是锥体神经元之间的部位的GLT-1和GFAP的表达进一步增强,使其所形成的锥体细胞层内的“网格”现象更为明显。但CA3及齿状回的个别神经元缺失区的GLT-1和GFAP的表达下降;单纯CIP以及CIP+脑缺血打击后,海马CA3区和齿状回GLT-1和GFAP的表达与CA1区相类似,但GLT-1和GFAP表达的上调及其所形成的“网格”现象更为明显。这些现象提示,海马CA3区和齿状回GLT-1的基础表达较高,并且受到缺血刺激时,GLT-1的反应性表达上调更显著。这些特点可能是海马CA3区和齿状回对缺血耐受性较强的原因之一。3大鼠脑缺血耐受诱导过程中海马CA1区氨基酸浓度的变化应用清醒动物脑内微透析和高效液相色谱技术,分析大鼠脑缺血耐受诱导过程中海马CA1区细胞外液中谷氨酸、门冬氨酸、甘氨酸和γ-氨基丁酸(GABA)浓度的变化,探讨氨基酸平衡的变化在脑缺血耐受诱导中的作用。24只大鼠随机分为4组(n=6):①凝闭椎动脉组;②CIP组;③损伤性脑缺血组;④CIP+损伤性脑缺血组。各组模型的制备与1.1相同。各组动物均在清醒状态下行背侧海马CA1区微透析。按预定程序收集透析液,高效液相色谱法测定透析液中上述氨基酸的浓度。各组动物于微透析结束后,常规饲养7天,断头取材行脑组织病理学评价,确定微透析探头的位置和海马CA1区组织病理学改变。脑组织病理学观察显示,微透析探头被准确地植入了每例大鼠的背侧海马CA1区。除8 min脑缺血打击引起了海马CA1区几乎全部锥体神经元死亡之外,其余各组均未见到明显的神经元损伤。上述结果与我们以前的研究结果相一致。在VAO组的所有透析标本之间,未观察到谷氨酸、门冬氨酸、甘氨酸及GABA浓度的明显变化。3分钟的全脑缺血引起谷氨酸、门冬氨酸、甘氨酸及GABA浓度迅速升高,分别达到其正常对照水平的1.5、2、1.9和2.3倍,随着血液再灌注,其浓度迅速降至正常水平。8分钟的全脑缺血引起谷氨酸、门冬氨酸、甘氨酸和GABA浓度迅速升高。甘氨酸和GABA的升高呈单峰、出现于脑缺血打击末,峰值为其正常对照水平的3~4倍,再灌注后很快恢复至对照水平。而谷氨酸和门冬氨酸的升高呈双峰状,其第一个高峰出现的时相及幅度与甘氨酸和GABA相似;第二个峰分别出现于再灌注后7分钟和19分钟,其峰值较第一个峰更高,分别为其正常对照水平的5~7倍。CIP+损伤性脑缺血组中,谷氨酸、门冬氨酸、甘氨酸及GABA的浓度呈单峰状升高,于损伤性脑缺血末达到峰顶,分别为其自身对照水平的1.7、2.5、7和4倍,再灌注后迅速降低至正常水平,并持续到实验结束。上述结果表明,缺血打击引起谷氨酸、门冬氨酸与GABA之间失衡;CIP可防止缺血打击所引起的谷氨酸、门冬氨酸与GABA之间的失衡,提示CIP所诱导的脑缺血耐受可能与其防止脑缺血打击引起的氨基酸失平衡有关。此外,我们还对四血管闭塞法大鼠全脑缺血模型的制备进行了进一步探讨,发现凝闭双侧椎动脉本身也具有脑缺血预处理样作用,能够在一定程度上减轻其后48 h内较严重的全脑缺血所造成的损伤;椎动脉经寰椎的横突孔进入寰椎,行经寰椎内的翼状管,经翼状孔内口进入椎管。4结论(1) CIP引起大鼠海马CA1区星形胶质细胞的突起延长,伸入到锥体神经元之间并包绕锥体神经元,这些延长的突起上表达大量的GLT-1,此变化可以保护锥体神经元,使其能够耐受较严重的、通常会导致锥体神经元迟发性死亡的缺血打击。(2)大鼠海马CA3区和齿状回的神经元对缺血的耐受能力较强,可能与该区GLT-1和GFAP的基础表达较高,以及受到缺血刺激时反应性表达上调更明显有关。这些发现进一步说明了GLT-1在BIT诱导中的作用。(3)脑缺血打击引起大鼠海马CA1区谷氨酸、门冬氨酸与GABA之间失衡;CIP可通过防止缺血打击引起谷氨酸、门冬氨酸与GABA之间的失衡,从而诱导脑缺血耐受。更多还原

【Abstract】 Transient sublethal cerebral ischemia could protect hippocampal neurons against delayed neuronal death (DND) induced normally by lethal ischemic insult. The transient cerebral ischemia is usually referred to as cerebral ischemic preconditioning (CIP), and the protective role is named as brain ischemic tolerance (BIT). The phenomenon was first found by Kitagawa in 1990 and proved by many other studies. It is very important to clearify the mechanisms of BIT induced by CIP for developing new therapeutic methods to enhance the tolerance of neurons to ischemia and hypoxia.Many studies have proved that excitotoxicity of glutamate is an important mechanism for DND induced by transient global ischemic insult. Glutamate uptake is transiently reduced and the extracellular glutamate is increased after hypoxia-ischemia insults in the brain. Excitatory amino acid transporters (EAATs) are essential for maintaining normal extracellular level of glutamate. Five distinct high-affinity, sodium-dependent EAATs are identified in the rat brain, which include EAAT1 (glutamate/aspartate transporter, GLAST), EAAT2 (glial glutamate transpor-1, GLT-1), EAAT3 (excitatory amino acid carrier 1, EAAC1), EAAT4 and EAAT5. GLAST and GLT-1 are localized primarily in astrocytes. EAAC1 is widely distributed in hippocampal neurons. EAAT4 is localized mainly in cerebellar Purkinje cells. EAAT5 is mainly localized in retina. EAATs normally remove glutamate into cells and are driven by sodium, potassium and possibly by hydroxide ion gradients. Conversely, When the ion gradient or membrane potential drops, for instance during ischemia or epileptic activity, EAATs may decrease the uptake of glutamate, or even reverse and release glutamate into the extracellular space in a calcium-independent manner. Therefore, extracellular glutamate becomes abnormally high and leads to excitotoxicity.Although both neurons and glia contain EAATs, it is generally accepted that the uptake capacity of astrocytes is much higher than that of neurons. Many studies have shown that GLT-1 plays a principal role in removing the released glutamate from the extracellular space and maintaining the extracellular glutamate below neurotoxic level in the brain. Considering the importance of GLT-1 in removing glutamate, it is reasonable to hypothesize that GLT-1 maybe play an important role in the acquisition of the brain ischemic tolerance induced by CIP. Unfortinately, there were limited reports until now concerning the role of GLT-1 in the induction of brain ischemic tolerance. Although they obtained a similar conclusion that GLT-1 participated in the induction of brain ischemic tolerance, the mechanisms underlying were just reversal. Additionally, they just performed the experiments in vitro. Little is known whether GLT-1 plays a role during the induction of brain ischemic tolerance in vivo. Therefore, the present study was undertaken to study whether GLT-1 participates in the induction of brain ischemic tolerance in vivo by observeing the expression of GLT-1 and glial fibrillary acidic protein (GFAP), a specific protein expressed by astrocytes, using immunohisto- chemistry and western blot analysis, and changes in concentrations of extracellular glutamate, aspartate, glycine andγ-aminobutyric acid (GABA) using brain microdialysis and high performance liquid chromatography (HPLC) during the induction of brain ischemic tolerance in rats.1 The up-regulation of GLT-1 and GFAP in the rat hippocampal CA1 subfield induced by CIPThe rat global cerebral ischemic model was established by four-vessel occlusion. To clarify the role of GLT-1 during the induction of BIT in vivo, the expression of GLT-1 and GFAP in the CA1 hippocampus during the induction of brain ischemic tolerance in rats was observed by immunohistochemistry and western blotting.1.1 Neuropathological evaluationOne hundred and forty five adult male Wistar rats were divided into 5 groups randomly:①control group (n=5);②vertebral artery occluding group (n=35): the bilateral vertebral arteries were electrocauterized permanently;③CIP group (n=35): a global brain ischemia for 3 min was given;④brain ischemic insult group (n=35): a global brain ischemic insult for 8 min was given;⑤CIP+ischemic insult group (n=35): a CIP was performed first and then a lethal global ischemic insult for 8 min was given 2 days after the CIP. The observations were performed at time 0 (immediate), 3 h, 1 d, 2 d, 3 d, 5 d and 7 d, after the last operation or treatment (n = 5 in each time point), except for the control group. At the determined endpoint of the experiment, the animals were sacrificed and the brain was removed for the neuropathological evaluation. The brain tissues were sectioned, and the delayed neuronal death (DND) was observed under staining with thionin. The histological changes of the hippocampal CA1 subfield were divided into 4 histological grade (HG) under the light microscope according to the following standard: grade 0, no neuron death; gradeⅠ, scattered single neuron death; gradeⅡ, mass neuron death; gradeⅢ, almost complete neuron death. The neuronal density (ND) of the hippocampal CA1 subfield 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. The average of number of pyramidal neurons in 3 areas of the hippocampal CA1 subfield was calculated as value of ND.Neuropathological evaluation showed that in the control rats, pyramidal neurons in the CA1 hippocampus were arranged in order with 2 to 3 cell layers, the outline of the neurons was intact, nucleus was full and nucleolus was clear. The HG was 0 and ND was 210±5.7 mm-1. No significant neuronal damage was observed in the CA1 subfield at all time points observed after VAO. Neither the HG nor the ND was different from that of the control group. No change in ND or HG was found at all time points after CIP compared with that of the control or VAO group. During the first two days after the lethal ischemic insult for 8 min, no significant pyramidal neuronal damage was observed in the hippocampal CA1 subfield. However, obvious DND was observed from the third day after the ischemic insult, such as decrease in ND and increase in HG. The damage deteriorated with time manifested as pyknosis of cell bodies, karyopyknosis of the nucleus, disappearance of the nucleolus. Almost complete neurons died on the fifth and seventh day after the lethal ischemic insult, represented by more significant decrease in ND and increase in HG compared with that of the third day after the lethal ischemic insult. When the animals were pretreated with the CIP 2 days before the lethal ischemic insult, the above injured changes were prevented clearly, which indicated that the CIP protected the pyramidal neurons in the CA1 hippocampus against the DND induced normally by the lethal ischemic insult.1.2 Immunohistochemistry assayAnimals, grouping and protocols of the experiment were the same as those in neuropathological evaluation.There were very weak, but diffuse immunoparticles distributed in the peri-pyramidal neuronal structure of the hippocampal CA1 subfield in the control group. The staining pattern is consistent with other reports. Compared with the control group, the intensity of GLT-1 immunoreactivity was markedly increased at almost all time points and showed morphological characteristics of astrocytes in the VAO group. The intensity of GLT-1 immunoreactivity was further increased after the CIP compared with that of the VAO group. Very interestingly, some GLT-1 immunoreactive particles were observed in the area between the pyramidal neurons, which tightly surrounded pyramidal neurons and made the pyramidal layer looked like“shaped grade”. Compared with the VAO group, the GLT-1 expression was markedly decreased at all time points after the lethal ischemic insult for 8 min, especially in the area where almost complete pyramidal neurons died, and the neighboring area of the pyramidal layer, even appeared as a sheet absence of GLT-1 immunoreactivity. But when the animals were pretreated with the CIP 2 days before the lethal ischemic insult, the decrease of the GLT-1 immunoreactivity induced by the lethal ischemic insult was prevented thoroughly. Moreover, there were more GLT-1 immunoreactive particles tightly surrounded the pyramidal neurons, which made the“shaped grid”observed in the CIP group to be more clear.In the control group, GFAP immunostaining showed that astrocytes took on star or spider-like shape with prominent processes. Very few GFAP immunoreactive particles were observed in the area between the pyramidal neurons. Compared with the control group, significant down-regulation of GFAP immunoreactivity was observed at all time points in the VAO group, and few GFAP immunoreactive particles were observed in the area tightly surrounded the pyramidal neurons. After a CIP for 3 min, The GFAP expression was significantly up-regulated at almost all time points compared with that of the VAO group. Some GFAP immunoreactive particles were observed, like GLT-1, in the area between the pyramidal neurons, which tightly surrounded the pyramidal neurons and made the pyramidal layer look like“shaped grade”. The characteristics of immunostaining were similar with those of GLT-1 immunostaining mentioned above. Moreover, both the total area and average optical density of GFAP immunostaining after the CIP were significantly up-regulated compared with those of the VAO group. After the lethal brain ischemic insult for 8 min, astrocytes hypertrophied in soma with thickened processes and more intensive staining, which reached peak on 3 d after the lethal ischemia. However, no GFAP immunoreactive particles were observed in the area between the pyramidal neurons or the neighboring area of the pyramidal layer at all. From the fifth day after the lethal ischemic insult, the body of the astrocytes became more hypertrophic, whereas the processes of the hypertrophic astrocytes became collapsing and fragmenting. Although the number, total area and average optical density of immunoreactive cells were increased significantly after the lethal ischemic insult compared with those of the VAO group. When the animals were pretreated with a CIP 2 days before the lethal ischemic insult, there were many GFAP immunoreactive particles tightly surrounded the pyramidal neurons thoroughly, which made the“shaped grid”observed in the CIP group to be more clear. The phenomenon reached peak on 2 d and constantly lasted to 7 d (the end of the observed period in the experiment). However, both the number and average optical density of the GFAP immunoreactive cells were significantly decreased compared with those of the ischemic insult group.1.3 Western blotting analysisTwo hundred and five adult male Wistar rats were divided into 6 groups randomly:①control group (n=5);②sham group (n=20);③VAO group (n=45);④CIP group (n=45);⑤brain ischemic insult group (n=45);⑥CIP+brain ischemic insult group (n=45). The western blotting analysis in the sham group was performed at time 0 (immediate), 3 h, 12h and 2 d, while in the other groups was performed at time 0 (immediate), 3 h, 6h, 12h, 1 d, 2 d, 3 d, 5 d and 7 d, after the last operation or treatment (n = 5 in each time point). In our preliminary experiment, some changes in expression of GLT-1 and GFAP were observed after VAO. To clarify whether the changes were induced by surgical procedures or by VAO, the sham group was designed in western blotting analysis. Rats in the sham group were subjected to a sham operation consisting of exposing of bilateral vertebral artery and bilateral common carotid arteries, but neither vertebral arteries nor common carotid arteries were occluded. The protocols of the rats in the other groups were the same as that in part 1.1.Compared with the control group, no difference of GLT-1 levels was found at each time point observed in the sham group. However, the levels of GLT-1 expression were significantly up-regulated in CA1 subfield at almost all time points in the VAO group compared with that of the control group. The above results indicated that the GLT-1 up-regulation in the VAO group was induced by the occluding of vertebral arteries itself other than the anesthesia or surgical procedures. In the CIP group, the GLT-1 level at immediate time point was much higher than that of the control group. On the base of the up-regulated level of GLT-1 at the immediate time point, the expression of GLT-1 was further up-regulated after CIP for 3 min. In the brain ischemic insult group, the GLT-1 expression was significantly down-regulated at almost all time points compared with that of the VAO group. When the animals were pretreated with a CIP for 3 min 2 days before the lethal ischemic insult, the down-regulation of GLT-1 induced by lethal brain ischemic insult was prevented thoroughly by the CIP.No difference of GFAP levels was found at every time points in the sham group compared with that of the control group. However, compared with the control group, the GFAP levels were significantly down-regulated at all time points after the occluding of the vertebral arteries. The above indicated that the GFAP down-regulation in the VAO group was induced by the occluding of vertebral arteries itself other than anesthesia or surgical operation. Compared with the VAO group, the GFAP levels were significantly up-regulated at almost all time points except the immediate time point after CIP. Compared with the VAO group, the GFAP levels were significantly up-regulated at almost all time points except the immediate time point after lethal brain ischemic insult for 8 min. Compared with the ischemic insult group, the GFAP levels were significantly down-regulated on 5 d and 7 d, whereas up-regulated at time 0 and 6 h in CIP+brain ischemic insult group.Summary These results indicated that the surrounding of pyramidal neurons by astrocytes and up-regulation of GLT-1 induced by CIP played an important role in the acquisition of the brain ischemic tolerance induced by CIP.2 Changes in the expression of GLT-1 and GFAP in the hippocampal CA3 subfield and dentate gyrus during the induction of brain ischemic tolerance in ratsThe preparation of the model, grouping, protocols and methods were the same as those in part 1, except for that the neuropathological evaluation was performed by calculating percentages of injured neurons in the CA3 and DG.2.1 Neuropathological evaluationLethal ischemic insult for 8 min induced mild DND in the CA3 subfield and dentate gyrus (DG) in some rats. The rate of neuronal death was approximately 5% in the CA3 subfield and 19% in DG. The CIP 2 days before the lethal brain ischemic insult could also protect the neurons in the CA3 subfield and DG against the DND induced normally by the lethal ischemic insult. All the above indicated that the neurons in the CA3 subfield and DG were relatively tolerated to ischemia, and CIP could also provide protection to the neurons in the CA3 subfield and DG.2.2 The expression of GLT-1 and GFAPSome differences in the expression of GLT-1 and GFAP were found in the CA3 subfield and DG compared with the CA1 subfield in the rat hippocampus. Processes of astrocytes in the CA3 subfield and DG, especially in the CA3 subfield, extended into the area between the neurons even in the control rats, which tightly surrounded neurons and made the neuronal layer looked like“shaped grade”. At the same time, many GLT-1 immunoreactive particles were observed in the same area. In the VAO group, both GFAP and GLT-1 immunoreactive particles in the area between the neurons in CA3 subfield and DG were significantly up-regulated by the VAO, which tightly surrounded neurons and made the“shaped grade”observed in the control group to be clearer. In the brain ischemic insult group, the GLT-1 expression was significantly increased and the“shaped grade”became more clearly in large area of the CA3 subfield and DG after the ischemic insult for 8 min, except for the significant decrease in the area where neurons died, and the neighboring area of the dead neurons. Compared with the CA1 subfield, the up-regulation of the expression of GLT-1 and GFAP to CIP for 3 min was also observed in CA3 subfield and DG, whereas the level of the up-regulation in CA3 subfield and DG was much higher than that in the CA1 subfield. In the CIP+ischemic insult group, similar response was observed among different subfields in the hippocampus, and the“shaped grade”existed in the CA3 subfield was very clear.Summary The tolerance of the neurons in the CA3 subfield and DG to ischemia insult maybe related to the relatively higher basal expression and stronger responsive upregulation of GLT-1 and GFAP to ischemic stimulation in the area between neurons in the both subfields. These findings further illustrated the involvement of GLT-1 in the induction of BIT. 3. Changes in concentrations of amino acids in the extracellular fluid in the rat CA1 hippocampus during the induction of BITTo investigate the role of the balance between excitatory amino acids (EAAs) and inhibitory amino acid in the induction of BIT, the concentration of glutamate, aspartate, glycine and GABA was analyzed during the induction of BIT by microdialysis combined with HPLC. Twenty four rats were divided into four groups randomly: vertebral artery occluding (VAO) group (n = 6), CIP group (n = 6), brain ischemic insult (II) group (n = 6), and CIP + brain ischemic insult group (n = 6). The preparations of the model were the same as those in part 1. The extracellular fluid in the rat CA1 region of the dorsal hippocampus was collected by intracerebral microdialysis in conscious rats. The concentration of amino acids in the dialysate was detected by HPLC. All rats were sacrificed on 7th day after the microdialysis for checking the position of the microdialysis probe and neuropathological evaluation.In all cases the microdialysis probe was correctly positioned in the CA1 subfield of the rat dorsal hippocampus. Obvious DND was observed after the brain ischemic insult for 8 min, while no obvious neuronal damage was observed in other groups. The results were consistent with our previous study.No changes in the concentration of each amino acid analyzed were observed among all samples from the VAO rats. In the CIP group, CIP for 3 min caused a significant acute increase coincident in both concentrations and time course of glutamate, aspartate, glycine and GABA, in which the peak values of the concentration were about 1.5, 2, 1.9, and 2.3 fold of their own average control level, respectively. In the brain ischemic insult group, lethal ischemic insult for 8 min evoked a significant increase in glutamate, aspartate, glycine and GABA. The acute increase of glycine and GABA appeared mono-peak at the end of the lethal ischemic insult. The peak value was about 3~4 folds of the average control level of its own. The increase of glutamate and aspartate showed a double-peak pattern. The first peaks were coincident in time course and magnitude with those in glycine and GABA. While the second peaks, which were about 5~7 fold of the control level, were higher in magnitude and appeared respectively at 7 min and 19 min after the reperfusion. In the CIP+ brain ischemic insult group, when the animals were pretreated with the CIP 2 days before the lethal ischemic insult, a significant acute increase of glutamate, aspartate, glycine and GABA coincident in both concentrations and time course was observed, in which the peak values were about 1.7, 2.5, 7, and 4 fold of their own average control level, respectively. The second higher peaks in glutamate and aspartate normally induced by brain ischemic insult were completely inhibited by the CIP. The results have shown that the lethal global brain ischemic insult for 8 min caused the imbalance between the EAAs such as glutamate as well as aspartate and GABA, the inhibitory amino acid; Whereas, when the animals were pretreated with the CIP for 3 min 2 days before the lethal ischemic insult, the increase among glutamate, aspartate, and GABA was coincident and kept balance.Summary The lethal brain ischemic insult for 8 min caused imbalance between glutamate as well as aspartate and GABA in the CA1 hippocampus, which especially manifested as a delayed but more obvious increase in the concentration of glutamate and aspartate. The imbalance could be prevented by a preceded CIP, which could protect the pyramidal neurons of the CA1 hippocampus from DND normally induced by lethal brain ischemia. These findings indicated that preventing of the imbalance between the glutamate as well as aspartate and GABA maybe one of mechanisms involved in the neuroopretection of CIP.In addition, we found that the prior occlusion of the bilateral vertebral arteries during producing 4VO global cerebral ischemic model might play a protective effect like cerebral ischemic preconditioning that can protect to some extent pyramidal neurons of the hippocampus against severe ischemic insult induced by occlusion of bilateral common carotid arteries within 48 h. The vertebral artery enters into the atlas via the transverse foramina, travels through the external aperture of the alar foramina, and then passes through the internal aperture of the alar foramina before entering into the vertebral canal. 4 Conclusions(1) The surrounding of pyramidal neurons by astrocytes and up- regulation of GLT-1 induced by CIP played an important role in the acquisition of the brain ischemic tolerance induced by CIP(2) The tolerance of the neurons in the CA3 subfield and DG to ischemia insult maybe related to the relatively higher basal expression and stronger responsive upregulation of GLT-1 and GFAP to ischemic stimulation in the area between neurons in the areas. These findings further illustrated the involvement of GLT-1 in the induction of BIT.(3) The lethal brain ischemic insult for 8 min caused imbalance between glutamate as well as aspartate and GABA in the CA1 hippocampus, which especially manifested as a delayed but more obvious increase in the concentration of glutamate and aspartate. The imbalance could be prevented by a preceded CIP, which could protect the pyramidal neurons of the CA1 hippocampus from DND normally induced by lethal brain ischemia. These findings indicated that preventing of the imbalance between the glutamate as well as aspartate and GABA maybe one of mechanisms involved in the neuroopretection of CIP.更多还原