基于K-ATP通道研究埃他卡林和美金刚对缺血性脑损伤的保护作用
|
摘要
脑卒中严重危害着人类健康。预计至2020年，脑卒中将成为致死的首要因素。在过去十年里，旨在寻找合适的神经保护药物来减轻脑卒中损伤的临床实验几乎都以失败告终。因此，寻找和发现新的脑卒中神经保护剂对于临床医生和科研工作者来说都是一项挑战。脑卒中神经损伤的机制包括：能量耗竭、胞内钙离子水平上升、兴奋性毒性、扩布性抑制、自由基产生、血脑屏障（Brain-blood-barrier, BBB）破坏、炎症和凋亡等。研究表明，脑神经功能的恢复不能仅限于单一神经元的保护，而应着眼于包括神经元、星形胶质细胞以及内皮细胞在内的整个神经血管单元。
ATP敏感性钾通道（ATP-sensitive potassium channel, K-ATP）是一种偶联细胞代谢和电活动的非电压依赖性的特殊钾离子通道。它由内向整流钾通道（inwardly rectifying potassium channel, Kir）和磺酰脲受体（sulphonylureareceptor，SUR）亚单位以4:4组合形成异构八聚体。平常该通道处于关闭状态，当细胞内ATP/ADP比例下调后，能被迅速激活。既往研究发现，当局部脑血流降低后，能量代谢发生障碍，细胞膜过度去极化，启动兴奋性毒性级联反应，形成钙超载，进而造成组织损伤。开放K-ATP通道后，细胞膜超级化，进而调节谷氨酸受体，尤其是谷氨酸受体最主要的亚型NMDA受体，抑制NMDA受体通道的打开，从而对抗脑缺血缺氧损伤。已有报道表明，Kir6.2敲除后，神经元在氧糖剥夺刺激下发生严重去极化，出现大量凋亡和坏死；而过表达Kir6.2将有助于减轻局部缺血性脑损伤。这些研究结果都证明K-ATP通道调节在拮抗局部缺血性脑损伤过程中至关重要。目前针对K-ATP通道开放产生脑保护效应的研究主要局限于单一神经元的保护。然而，神经血管单元各组分均在脑卒中病理生理机制中发挥重要作用。如胶质细胞能调节能量稳态；维持水和离子的平衡；释放神经营养因子和致炎因子，促进血管发生和神经再生。内皮细胞不仅参与维持血脑屏障的完整性，还释放神经营养因子，在病理情况下参与炎性反应。而K-ATP通道在脑内广泛分布，不仅在神经元上主要表达Kir6.2亚基构成的K-ATP通道,在星形胶质细胞、小胶质细胞和周细胞上也表达Kir6.1构成的K-ATP通道。脑内皮细胞表达何种亚型K-ATP通道，尚不明确。综上所述,我们推测：K-ATP通道开放产生的脑保护效应不仅依赖于神经元的保护。
因此，本文第一部分工作应用Kir6.2敲除鼠建立缺血缺氧（大脑中动脉闭塞Middlle Cerebral Artery Occlusion，MCAO）模型，并在此基础上应用新型K-ATP通道开放剂埃他卡林（Iptakalim，IPT）研究开放K-ATP通道在局部缺血性脑损伤中的保护作用，揭示K-ATP通道在缺血性脑损伤发生发展中的重要作用。第二部分探讨Kir6.2敲除对NMDA受体阻断剂美金刚治疗缺血性脑损伤作用的影响，阐明K-ATP通道是脑卒中神经保护药物的重要靶标。
第一部分Kir6.2敲除对埃他卡林缺血性脑损伤神经保护作用的影响
目的：研究、阐明Kir6.2敲除和K-ATP通道开放在小鼠缺血性脑损伤中的作用。
方法：1）整体研究：应用3月龄Kir6.2野生型（wildtype，Kir6.2+/+）及Kir6.2敲除（Kir6.2 knockout，Kir6.2-/-）雄性C57小鼠，线栓法建立大脑中动脉栓塞（middle cerebral artery occlusion，MCAO）模型，缺血前30分钟给予IPT10mg/kg腹腔注射统计死亡率并行神经功能缺陷评分；2，3，5-氯化三苯基四氮唑（2,3,5-triphenyltetrazoliumchloride，TTC）染色观察脑梗死体积变化；免疫荧光双标鉴定内皮细胞表达K-ATP通道亚型并观察内皮细胞及基底膜形态学改变；免疫组织化学法观察神经元、星形胶质、小胶质细胞数量及形态学改变；免疫荧光法检测胶质递质D-SerineD-Serine、MMP-9的含量；western blotting法测定星形胶质细胞缝隙连接connexin43、MMP-9、PSD-95、AQP4的表达；ELISA法测定TNF-α的水平。2）离体研究：分离、培养孕14/15天Kir6.2+/+、Kir6.2-/-胚胎小鼠皮层神经元，建立氧糖剥夺损伤（OGD）模型。模型前对神经元给予10μM IPT或10μM AMPK抑制剂compound C药物处理。通过Hoechst33342核染色以及MTT（3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide）比色法观察原代培养的皮层神经元在OGD模型下的损伤。应用western blotting方法检测氧糖剥夺刺激后各组神经元p-AMPK的表达变化。
结果：整体研究：1）KirKir6.2敲除和IPT预保护对小鼠脑缺血后生存率、神经功能缺陷及脑梗死体积的影响：Kir6.2-/-小鼠随缺血时间延长死亡例数逐渐增加,而Kir6.2+/+小鼠几乎未出现死亡。IPT预保护可以提高Kir6.2-/-小鼠生存率。两种基因型小鼠在脑缺血损伤后均出现神经运动功能障碍及脑组织梗死。Kir6.2-/-小鼠神经功能受损更为严重，脑梗死体积较Kir6.2+/+小鼠显著增加。IPT可显著改善Kir6.2+/+小鼠神经运动功能障碍，部分改善Kir6.2-/-小鼠神经运动功能障碍，并显著降低两种基因型小鼠脑梗死体积。2）Kir6.2敲除和IPT预保护对小鼠脑缺血后神经血管单元结构和功能的影响：在野生型小鼠，内皮细胞同时表达Kir6.1和Kir6.2构成的K-ATP通道。脑缺血后3小时，Kir6.2+/+小鼠内皮细胞MMP-9生成水平显著增加，随即降解其紧密连接蛋白occludin和基底膜蛋白collagenIV，突触后致密物PSD-95表达减弱。至脑缺血后24小时，小鼠海马CA1区神经元胞外间隙增大，星形胶质细胞和小胶质细胞活化增殖，胶质递质D-SerineD-Serine表达增强，星形胶质细胞水通道蛋白AQP4表达降低。Kir6.2-/-小鼠脑缺血后内皮细胞MMP-9表达水平及occludin和collagenIV的降解程度显著高于Kir6.2+/+小鼠；PSD-95表达水平显著降低；海马CA1区神经元损伤程度显著增加；星形胶质细胞胞体崩解突起断裂，不表达D-SerineD-Serine且connexin43表达下调，AQP4表达水平随缺血时间延长先升高再降低；小胶质细胞剧烈活化，程度较Kir6.2+/+小鼠显著，TNF-α生成水平显著增高。IPT能降低Kir6.2+/+小鼠脑缺血早期MMP-9的表达，进而抑制occludin和collagenIV的降解；逆转PSD-95表达水平的降低，显著保护其CA1区神经元；抑制星形胶质细胞和小胶质细胞活化，下调D-SerineD-Serine的表达并升高connexin43的表达。IPT也能降低Kir6.2-/-小鼠脑内MMP-9的生成并抑制occludin和collagenIV的降解；上调PSD-95的表达，促进小鼠神经元的存活；抑制星形胶质细胞胞体崩解和突起断裂，恢复D-SerineD-Serine的表达并上调connexin43的表达，且下调早期AQP4的表达水平；减轻小胶质细胞的活化并抑制其TNF-α的生成。
离体研究：1）基础状态下，Kir6.2+/+和Kir6.2-/-原代培养的皮层神经元凋亡率、细胞活力没有差异。氧糖剥夺引起Kir6.2+/+和Kir6.2-/-神经元损伤且Kir6.2-/-神经元损伤较Kir6.2+/+神经元显著增加。IPT能抑制两种基因型神经元的损伤。2）氧糖剥夺30分钟复氧2小时后，Kir6.2+/+和Kir6.2-/-神经元p-AMPK表达水平增高，Kir6.2-/-神经元p-AMPK增高水平更为显著。两种基因型神经元p-AMPK表达水平能被IPT显著抑制。3）APMK抑制剂compound C能减轻两种基因型神经元氧糖剥夺损伤。
结论：
1、Kir6.2敲除加重脑缺血后内皮细胞结构损伤及功能障碍；引起突触后致密蛋白表达水平显著降低；海马CA1区神经元大量缺失；其周围星形胶质细胞崩解；小胶质细胞剧烈活化，从而使Kir6.2-/-小鼠缺血性脑损伤更为严重。
2、K-ATP通道开放剂IPT显著减轻脑缺血对两种基因型小鼠神经血管单元的损伤；改善小鼠神经血管单元的功能，进而对两种基因型小鼠缺血性脑损伤均发挥神经保护作用。
3、不仅神经元和胶质细胞分别主要表达Kir6.1和Kir6.2构成的K-ATP通道，脑内皮细胞也同时表达Kir6.1和Kir6.2构成的K-ATP通道。开放K-ATP通道通过调节神经血管单元各组分功能在缺血性脑损伤中发挥神经保护作用。
第二部分Kir6.2敲除对美金刚缺血性脑损伤神经保护作用的影响
目的：探讨Kir6.2敲除对美金刚治疗缺血性脑损伤作用的影响及其机制，阐明K-ATP通道是脑卒中神经保护药物的重要靶标。
方法：在体应用3月龄Kir6.2+/+及Kir6.2-/-雄性C57小鼠，线栓法建立大脑中动脉栓塞（middle cerebral artery occlusion，MCAO）模型，缺血前30分钟给予美金刚20mg/kg，腹腔注射。统计死亡率并行神经功能缺陷评分；2，3，5-氯化三苯基四氮唑（2,3,5-triphenyltetrazoliumchloride，TTC）染色观察脑梗死体积变化；免疫组织化学法观察内皮细胞及基底膜形态学改变；神经元、星形胶质、小胶质细胞数量及形态学改变；缺血后第二天起经腹腔给予5-溴-2-脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine，BrdU，100mg/kg，每天一次，至第7天），最后一次给予BrdU 24小时后麻醉、灌注固定、取脑、冰冻切片，行BrdU免疫组化。缺血后14天行doublecortin (DCX)免疫组化。离体分离、培养孕14/15天野生型（Kir6.2+/+）、敲除型（Kir6.2-/-）胚胎小鼠皮层神经元，建立氧糖剥夺损伤（OGD）模型。模型前对神经元给予20μM美金刚药物处理。通过Hoechst33342核染色以及MTT（3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromid）比色法观察原代培养的皮层神经元在OGD模型下的损伤。
结果：1）Kir6.2敲除和美金刚预保护对小鼠脑缺血后神经运动功能障碍和脑梗死体积的影响：脑缺血后，两种小鼠都出现神经运动功能障碍和脑组织梗死。Kir6.2-/-小鼠神经运功能障碍及脑梗死体积较Kir6.2+/+小鼠显著增加。美金刚能改善Kir6.2+/+小鼠神经运动功能障碍并降低其脑梗死体积，但对Kir6.2-/-小鼠却无此保护作用。2）Kir6.2敲除和美金刚预保护对小鼠脑缺血后神经血管单元结构和功能的影响：脑缺血后3小时，Kir6.2+/+小鼠内皮细胞MMP-9表达水平显著增加，紧密连接蛋白occludin和基底膜蛋白collagenIV表达水平降低；PSD-95表达减弱。至脑缺血后24小时，其CA1区神经元排列稀疏，胞外间隙增大，星形胶质细胞和小胶质细胞活化增殖，AQP4表达降低。Kir6.2-/-小鼠MMP-9表达水平及occludin和collagenIV降解程度高于Kir6.2+/+小鼠；脑内PSD-95表达水平显著低于Kir6.2+/+小鼠；CA1区神经元损伤较Kir6.2+/+小鼠显著增加；星形胶质细胞胞体崩解突起断裂，AQP4表达水平随缺血时间延长先升高再降低；小胶质细胞剧烈活化。美金刚能降低Kir6.2+/+小鼠MMP-9的表达水平，抑制occludin和collagenIV的降解；逆转其PSD-95表达水平的降低；显著促进CA1区神经元的存活；减轻星形胶质细胞和小胶质细胞的活化。在Kir6.2-/-小鼠，美金刚虽然能下调其缺血性脑损伤早期脑内MMP-9的表达进而抑制occludin和collagenIV的降解，但不能上调缺血早期PSD-95的表达进而无法促进脑缺血24小时后神经元的存活。离体实验表明：美金刚不能减轻Kir6.2-/-神经元氧糖剥夺损伤。整体实验显示，Kir6.2-/-小鼠脑缺血24小时后星形胶质细胞的崩解和小胶质细胞的活化也无法被改善。3）KirKir6.2敲除和美金刚对小鼠缺血性脑损伤后神经再生的影响：脑缺血损伤后，美金刚能促进Kir6.2+/+和Kir6.2-/-小鼠SVZ区神经干细胞增殖。至脑缺血损伤后14天，Kir6.2-/-小鼠全部死亡，美金刚能促进存活的Kir6.2+/+小鼠SVZ区神经干细胞的迁移。
结论：Kir6.2敲除取消美金刚急性缺血性脑损伤的神经保护作用，提示美金刚对小鼠急性缺血性脑损伤的保护作用依赖于Kir6.2构成的K-ATP通道，其机制可能与美金刚无法抑制PSD-95在脑缺血早期的降解相关。美金刚可以促进脑缺血后的神经再生过程，有利于组织损伤及修复，该效应不依赖于Kir6.2构成的K-ATP通道。
综上所述，本文研究工作的主要创新之处在于：
1、K-ATP通道是缺血性脑损伤神经血管单元保护的重要靶点发现内皮细胞同时表达Kir6.1和Kir6.2构成的K-ATP通道，Kir6.2敲除通过加重脑缺血后内皮细胞功能障碍进而促进神经血管单元各组分的损伤，这些结果阐明K-ATP通道是神经血管单元保护的重要靶点，为研发理想的神经保护药物提供了新的靶标。
2、K-ATP通道通过调节神经血管单元功能在缺血性脑损伤中发挥神经保护作用发现开放K-ATP通道通过调节神经血管单元各组分功能在缺血性脑损伤中发挥神经保护作用。研究结果为进一步探讨K-ATP通道在脑卒中神经损伤中的作用机制积累了学术基础。
3、Kir6.2敲除取消美金刚的神经保护作用Kir6.2敲除取消美金刚急性缺血性脑损伤的神经保护作用，表明美金刚对急性缺血性脑损伤的保护作用依赖于Kir6.2构成的K-ATP通道。美金刚可以促进脑缺血后的神经再生过程，有利于组织损伤及修复，该效应不依赖于Kir6.2构成的K-ATP通道。
Ischemic stroke constitutes a significant human health hazard. It has beenpredicted that, by the year 2020, stroke would become one of the primary causes ofdeath. In the past ten years, clinical trials aiming to figure out suitableneuroprotectants against the debilitating effects of stroke have met with no success.Identifying novel neuroprotectants which can reverse the effects against stroke isbecoming a challenge to both clinicians and scientists. Leading pathogenicmechanisms of ischemic cascade include energy failure, elevation of intracellularCa2+, excitotoxicity, spreading depression, generation of free radicals, BBB disruption,inflammation, and apoptosis. In order to yield sufficient neuroprotection, an idealtherapeutic method for stroke would be the one which rescues multiple brain cells.Therefore, the new targets should be proved to protect the entire neurovascular unit.
ATP-sensitive potassium (K-ATP) channels provide a unique link betweencellular energetics and electrical excitability. They are heteromultimers composedof four inwardly rectifier potassium channel pore subunits (Kir6.x) and foursulfonylurea receptors (SURs). These channels are usually closed in normalconditions but are activated rapidly in response to the decreases in intracellularATP/ADP ratio under ischemic conditions. Thus, the opening of K-ATP channelscan get intrinsic protection by regulating membrane potential. Opening K-ATPchannels can hyperpolarize the cell membrane, limiting neuron excitability and Ca2+ influx and thus blocking the subsequent neurotoxic biochemical cascade throughinhibiting activation of N-methyl-D-aspartate receptor-gated ion channel activation.Recent reports have shown that Kir6.2-/- mice displayed enhanced neuronal damageafter ischemic insults and over-expression of Kir6.2 ameliorates the brain ischemicinjury. These findings indicated that K-ATP channels are directly involved inneuroprotection against acute hypoxic or ischemic insult. However, most ofmechanistic studies about the neuroprotection of K-ATP against cerebral ischemiahave been only focused on neurons protection. In fact, accumulating evidences nowshow that glial cells and endothelial cells play an active and important role in thepathophysiology of stroke. Brain energetics, water and ion homeostasis,inflammation, trophic factor production, vascular regulation, neurongenesis, andvasculogenesis are all under the control of glial cells. Endothelial cells form theBrain-blood brarrier (BBB) and participate in inflammatory and immune reactions byproducing solubale mediators. K-ATP channels are widely distributed in brain. Ithas been documented that neurons mainly express Kir6.2-containing K-ATP channelswhereas Kir6.1 is the principal pore-forming subunit of astrocytes and microglia.The recent studies have showed that pericytes also express Kir6.1-based channels.However, it is uncertain that what kind of subunit of K-ATP channel endothelial cellsexpress. Therefore, we proposed that the neuroprotection by K-ATP opening didnot only apply on neurons. However, the exactly mechanisms are still unknown.
Therefore, we established the ischemia model by middle cerebral arteryocclusion (MCAO) in both genotypes. In the first part of present study, we used anovel and unselective K-ATP channel opener—iptakalim (IPT) and Kir6.2 knockoutmice to investigated the neuroprotection by opening K-ATP channels. In the secondpart, the neuroprotective effects of NMDA receptor antagonist---memantine againstcerebral ischemia were studied in Kir6.2 knockout mice. Finally, it was illustratedthat K-ATP was an important target of neuroprotectants in stroke.
Part I Effects of Kir6.2 knockout on iptakalim-inducedneuroprotectioneuroprotection after cerebral ischemia
AIM: To investigate the effects of Kir6.2 knockout and the opening of K-ATPchannels in ischemia-induced cerebral injury in mice.
METHODS: 1）In vivo, focal ischemia was achieved in three-month-oldwildtype (Kir6.2+/+) and Kir6.2 knockout (Kir6.2-/-) male mice by occlusion of middlecerebral artery (MCAO) with a modified intralumenal filament technique as describedpreviously. IPT was injected intraperitoneally in a dose of 10mg/kg, 30 minutesbefore middle cerebral artery occlusion (MCAO). Mortality, neurological deficitsand infarct volume were measured in each group 24 hours after ischemia. Doubleimmunofluorescence was used for identifying the subtype of K-ATP channel inendothelial cells and to assess the contents of glia-transmitter D-SerineD-Serine andMMP-9. Immunostaining was taken for observation of endothelial cells, basalmembrane neurons, astrocytes and microglia. Fresh hippocampal homogenateswere used to determine the expressions of connexin43 and MMP-9. The TNF-αproduction was also analyzed by enzyme-linked immunosorbent assay (ELISA). 2)In vitro, primary cortical neuron cultures were prepared from the cortical tissues ofembryonic day 14/15 Kir6.2+/+ and Kir6.2-/- C57BL/6J mice. Cultures were usedafter 7 days in vitro. Oxygen and glucose (OGD) were deprived to induce theneuronal injury. Cortical neurons were incubated with 10μM IPT or 10μM AMPKinhibitor compound c for thirty minutes before OGD stimulation.3-(4,5-dimethylthiazol-2-yl)- -2,5-diphenyltetrazolium bromide (MTT) was used toevaluate the cell viability. Staining with Hoechst 33342 was used to determineneuronal apoptosis. Western blotting was taken for the analysis of p-AMPKexpression.
RESULTS: In vivo，1) Effects of Kir6.2 knockout and IPT pretreatmenton mortality, neurological deficits and infarct volume induced by MCAO Afterischemia, Kir6.2-/- mice exhibited higher mortality while nearly all of Kir6.2+/+ micesurvived. Pretreatment with IPT could increase the survival ratio of Kir6.2-/- mice.Server neurological deficits and larger infarct volume were observed in Kir6.2-/- mice.IPT could attenuate the neurological deficits and decrease the infarct volume in both genotypes. 2) EffectEffects of Kir6.2 knockout and IPT on the structure and functionof neurovasucular unit after ischemia. Endothelial cells expressed Kir6.1 andKir6.2- containing K-ATP channels. Higher production of MMP-9 by endothelialcells was observed in Kir6.2-/- mice three hours after ischemia. Occludin andcollagenIV were then dramatically disrupted in Kir6.2-/- mice. The level of PSD-95expression was significantly decreased. Till 24 hours after ischemia, there was aserver decrease in the number of CA1 pyramidal cells in the Kir6.2-/- group comparedwith that in the Kir6.2+/+ group. Astrocytes displayed reactive changes surroundingthe neurons in CA1 region of Kir6.2+/+ group. The expression of D-SerineD-Serinewas increased while the expression of AQP4 was decreased. However, astrocytesshowed damaged features in CA1 region in Kir6.2-/- mice. The expression ofconnexin43 and D-SerineD-Serine were significantly reduced. AQP4 expressionwas initially dramatically increased and then decreased. Severer microglial wereactivated and much more TNF-αproduction was induced by ischemia in Kir6.2-/-genotype. Pretreatment with IPT inhibited MMP-9 secretion and then prevented thedegradation of occludin and collagenIV and in both genotypes；markedly increasedthe expression of PSD-95 and promoted the neuronal survival in both genotypes;inhibited the activation of astrocytes with the decreased the expression ofD-SerineD-Serine, enhancement the expression of connexin43 and inhibition ofAQP4 expression in Kir6.2+/+ mice; protected astrocytes against ischemia withupregulation of connexin43, D-SerineD-Serine and AQP4 expression in Kir6.2-/- mice;inhibited microglia activation in both genotypes and decreased the production ofTNF-αin Kir6.2-/- genotye;
In vitro, Kir6.2 knockout does not altered the level of apoptosis, neuronalsurvival under normal conditions but increased the injury of OGD. IPT increasedthe neuronal survival of two genotypes induced by OGD stimulaiton. After OGDstimulation, the expression of p-AMPK was increased in both genotypes. Higherexpression level of p-AMPK was observed in Kir6.2-/- neurons. IPT inhibited thephosphorylation of AMPK induced by OGD stimulaiton. Inhibition of p-AMPKexpression by compound C could decrease the injury of both genotypes.
CONCLUSION:
1. Lack of Kir6.2- containing K-ATP channels aggravates endothelial cellsmalfunction, obviously induces neuron apoptosis and astrocyte damage, daramaticallyactivates microglia, which ultimately cause enhancement of cerebral ischemicdamage in Kir6.2-/- mice.
2. K-ATP channel opener IPT exerts evident neuroprotective effects in bothgenotype mice through improving the function of entire neurovascular unit.
3. All components of neurovascular unit express K-ATP channel. Endothelialcells express Kir6.1- and Kir6.2-containing K-ATP channels. Targeting K-ATPchannels provides a promising therapeutic strategy for protection of neurovascularunit in brain ischemic lesion.
Part II Effects of Kir6.2 knockout on memantine-inducedneuroprotection after ischemia
AIM: To investigate whether Kir6.2-containing K-ATP channel participate inmemantine-induced neuroprotection after ischemia and prove that K-ATP is animportant target of neuroprotectants aginast cerebral ischemia.
METHODS: In vivo, focal ischemia was achieved in three-month-oldwildtype (Kir6.2+/+) and Kir6.2 knockout (Kir6.2-/-) male mice by occlusion of middlecerebral artery (MCAO) with a modified intralumenal filament technique as describedpreviously. Memantine was injected intraperitoneally in a dose of 20mg/kg, 30minutes before MCAO. Since the second day after ischemia, BrdU (100mg, once aday, ip) was administrated to both genotypes followed by 7-day-memantine treatment.Neurological deficits and infarct volume were measured in each group 24 hours afterischemia. Immunostaining was taken for observation of the morphological changesof endothelial cells，basal membrane，neurons, astrocytes, microglia and countingBrdU-positive cells. Immunofluorescence was used to evaluating PSD-95 anddoublecortin (DCX) expression. Primary cortical neuron cultures were preparedfrom the cortical tissues of embryonic day 14/15 Kir6.2+/+ and Kir6.2-/- C57BL/6J mice. In vitro, oxygen and glucose were deprived to induce the neuronal injury.Cortical neurons were incubated with 10μM memantine before OGD stimulation.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used toevaluate the cell viability. Staining with Hoechst 33342 was used to determineneuronal apoptosis.
RESULTS: 1) Effects of Kir6.2 knockout and memantine pretreatment onneurological deficits and infarct volume induced by MCAO After ischemia,Kir6.2-/- mice exhibited server neurological deficits and larger cerebral infarct volume.Pretreatment with memantine could attenuate the neurological deficits and decreasethe infarct volume but have no impact on Kir6.2-/- mice. 2) Effects of Kir6.2knockout and memantine pretretment on the structure and function ofneurovasucular unit after stroke. Three hours after ischemia, higher productionof MMP-9 by endothelial cells was observed in Kir6.2-/- mice. Occludin andcollagenIV were dramatically disrupted in Kir6.2-/- mice. The expression of PSD-95was much lower in Kir6.2-/- mice. Twanty-four hours after ischemia, there was amuch more decrease in the number of CA1 pyramidal cells in the Kir6.2-/- group.Astrocytes displayed reactive changes in CA1 region of Kir6.2+/+ group. Theexpression of AQP4 was decreased. However, astrocytes showed damaged featuresin Kir6.2-/- mice. AQP4 expression was initially dramatically increased and thendecreased. Severer activation of microglial was observed in Kir6.2-/- genotype. InKir6.2+/+ mice, pretreatment with memantine inhibited MMP-9 secretion and thenprevented the degradation of occludin and collagenIV; prevented the decrease ofPSD-95 in CA1 regions and markedly increased the neuronal survival; inhibited theactivation of astrocytes and microglia; decreased AQP4 expression. In Kir6.2-/- mice,pretreatment with memantine decreased the initial secretion of MMP-9 anddegradation of occludin and collagenIV, but had no effect on the decrease of PSD-95.Till 24 hours after ischemia, the neuronal survival, astrocytes damage and microgliawere not improved. In vitro, the anti-apoptosis effect of memantine on neurons wasabolished by Kir6.2 knockout during OGD stimulation. 3) Effects of Kir6.2knockout and memantine on the neurogenesis after stroke. Daily injection of memantine promoted the proliferation of stem cells located in SVZ in both genotypesafter ischemia. Fourteen days after stroke, all of the Kir6.2-/- mice died.Memantine promoted the migration of stem cells in SVZ of Kir6.2+/+ mice.
CONCLUSION: Memantine have no neuroprotective effects on actue cerebralischemic injury of Kir6.2-/- mice. The neuroprotection of memantine in ischemicstroke is dependent on Kir6.2-containing K-ATP channels. The underlyingmechanism is attributed to that memantine can not inhibite the degradation of PSD-95induced by ischemia after Kir6.2 knockout. Memantine could promote neurogenesisafter stroke which will benefit the intrinsic revovery, which is independent onKir6.2-containing K-ATP channels.
In summary, our present study has the following new concerns:
1. K-ATP channels are promising therapeutic targets for the protection ofneurovascular unit in brain ischemic lesion. Endothelia cells express Kir6.1- andKir6.2- containing K-ATP channels. Kir6.2 knockout damaged the entireneurovascular unit during ischemia. The therapeutic strategy targeted to K-ATP mayoffer a new perspective for the development of new options in ischemic stroketreatment.
2. Opening K-ATP channels inhibit the ischemic brain damage throughneurovascular unit protection. Opening K-ATP channels modulate the integrityand function of the neurovascular to play significant neuroprotection in ischemicstroke. The results obtained in present study substantially improve ourunderstanding of K-ATP in stroke.
3. Kir6.2 knockout abolishes the neuroprotective effects of memantine.Memantine have no neuroprotective effects on acute cerebral ischemic injury ofKir6.2-/- mice. The underlying mechanism is attributed to that memantine can notinhibite the degradation of PSD-95 induced by ischemia after Kir6.2 knockout.Kir6.2-containing K-ATP channels are involved in the neuroprotection of memantinein cerebral ischemia. Memantine can promote neurogenesis after stroke which willbenefit the intrinsinc revovery. This effect is independent on Kir6.2-containing K-ATP channels.
ATP敏感性钾通道（ATP-sensitive potassium channel, K-ATP）是一种偶联细胞代谢和电活动的非电压依赖性的特殊钾离子通道。它由内向整流钾通道（inwardly rectifying potassium channel, Kir）和磺酰脲受体（sulphonylureareceptor，SUR）亚单位以4:4组合形成异构八聚体。平常该通道处于关闭状态，当细胞内ATP/ADP比例下调后，能被迅速激活。既往研究发现，当局部脑血流降低后，能量代谢发生障碍，细胞膜过度去极化，启动兴奋性毒性级联反应，形成钙超载，进而造成组织损伤。开放K-ATP通道后，细胞膜超级化，进而调节谷氨酸受体，尤其是谷氨酸受体最主要的亚型NMDA受体，抑制NMDA受体通道的打开，从而对抗脑缺血缺氧损伤。已有报道表明，Kir6.2敲除后，神经元在氧糖剥夺刺激下发生严重去极化，出现大量凋亡和坏死；而过表达Kir6.2将有助于减轻局部缺血性脑损伤。这些研究结果都证明K-ATP通道调节在拮抗局部缺血性脑损伤过程中至关重要。目前针对K-ATP通道开放产生脑保护效应的研究主要局限于单一神经元的保护。然而，神经血管单元各组分均在脑卒中病理生理机制中发挥重要作用。如胶质细胞能调节能量稳态；维持水和离子的平衡；释放神经营养因子和致炎因子，促进血管发生和神经再生。内皮细胞不仅参与维持血脑屏障的完整性，还释放神经营养因子，在病理情况下参与炎性反应。而K-ATP通道在脑内广泛分布，不仅在神经元上主要表达Kir6.2亚基构成的K-ATP通道,在星形胶质细胞、小胶质细胞和周细胞上也表达Kir6.1构成的K-ATP通道。脑内皮细胞表达何种亚型K-ATP通道，尚不明确。综上所述,我们推测：K-ATP通道开放产生的脑保护效应不仅依赖于神经元的保护。
因此，本文第一部分工作应用Kir6.2敲除鼠建立缺血缺氧（大脑中动脉闭塞Middlle Cerebral Artery Occlusion，MCAO）模型，并在此基础上应用新型K-ATP通道开放剂埃他卡林（Iptakalim，IPT）研究开放K-ATP通道在局部缺血性脑损伤中的保护作用，揭示K-ATP通道在缺血性脑损伤发生发展中的重要作用。第二部分探讨Kir6.2敲除对NMDA受体阻断剂美金刚治疗缺血性脑损伤作用的影响，阐明K-ATP通道是脑卒中神经保护药物的重要靶标。
第一部分Kir6.2敲除对埃他卡林缺血性脑损伤神经保护作用的影响
目的：研究、阐明Kir6.2敲除和K-ATP通道开放在小鼠缺血性脑损伤中的作用。
方法：1）整体研究：应用3月龄Kir6.2野生型（wildtype，Kir6.2+/+）及Kir6.2敲除（Kir6.2 knockout，Kir6.2-/-）雄性C57小鼠，线栓法建立大脑中动脉栓塞（middle cerebral artery occlusion，MCAO）模型，缺血前30分钟给予IPT10mg/kg腹腔注射统计死亡率并行神经功能缺陷评分；2，3，5-氯化三苯基四氮唑（2,3,5-triphenyltetrazoliumchloride，TTC）染色观察脑梗死体积变化；免疫荧光双标鉴定内皮细胞表达K-ATP通道亚型并观察内皮细胞及基底膜形态学改变；免疫组织化学法观察神经元、星形胶质、小胶质细胞数量及形态学改变；免疫荧光法检测胶质递质D-SerineD-Serine、MMP-9的含量；western blotting法测定星形胶质细胞缝隙连接connexin43、MMP-9、PSD-95、AQP4的表达；ELISA法测定TNF-α的水平。2）离体研究：分离、培养孕14/15天Kir6.2+/+、Kir6.2-/-胚胎小鼠皮层神经元，建立氧糖剥夺损伤（OGD）模型。模型前对神经元给予10μM IPT或10μM AMPK抑制剂compound C药物处理。通过Hoechst33342核染色以及MTT（3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide）比色法观察原代培养的皮层神经元在OGD模型下的损伤。应用western blotting方法检测氧糖剥夺刺激后各组神经元p-AMPK的表达变化。
结果：整体研究：1）KirKir6.2敲除和IPT预保护对小鼠脑缺血后生存率、神经功能缺陷及脑梗死体积的影响：Kir6.2-/-小鼠随缺血时间延长死亡例数逐渐增加,而Kir6.2+/+小鼠几乎未出现死亡。IPT预保护可以提高Kir6.2-/-小鼠生存率。两种基因型小鼠在脑缺血损伤后均出现神经运动功能障碍及脑组织梗死。Kir6.2-/-小鼠神经功能受损更为严重，脑梗死体积较Kir6.2+/+小鼠显著增加。IPT可显著改善Kir6.2+/+小鼠神经运动功能障碍，部分改善Kir6.2-/-小鼠神经运动功能障碍，并显著降低两种基因型小鼠脑梗死体积。2）Kir6.2敲除和IPT预保护对小鼠脑缺血后神经血管单元结构和功能的影响：在野生型小鼠，内皮细胞同时表达Kir6.1和Kir6.2构成的K-ATP通道。脑缺血后3小时，Kir6.2+/+小鼠内皮细胞MMP-9生成水平显著增加，随即降解其紧密连接蛋白occludin和基底膜蛋白collagenIV，突触后致密物PSD-95表达减弱。至脑缺血后24小时，小鼠海马CA1区神经元胞外间隙增大，星形胶质细胞和小胶质细胞活化增殖，胶质递质D-SerineD-Serine表达增强，星形胶质细胞水通道蛋白AQP4表达降低。Kir6.2-/-小鼠脑缺血后内皮细胞MMP-9表达水平及occludin和collagenIV的降解程度显著高于Kir6.2+/+小鼠；PSD-95表达水平显著降低；海马CA1区神经元损伤程度显著增加；星形胶质细胞胞体崩解突起断裂，不表达D-SerineD-Serine且connexin43表达下调，AQP4表达水平随缺血时间延长先升高再降低；小胶质细胞剧烈活化，程度较Kir6.2+/+小鼠显著，TNF-α生成水平显著增高。IPT能降低Kir6.2+/+小鼠脑缺血早期MMP-9的表达，进而抑制occludin和collagenIV的降解；逆转PSD-95表达水平的降低，显著保护其CA1区神经元；抑制星形胶质细胞和小胶质细胞活化，下调D-SerineD-Serine的表达并升高connexin43的表达。IPT也能降低Kir6.2-/-小鼠脑内MMP-9的生成并抑制occludin和collagenIV的降解；上调PSD-95的表达，促进小鼠神经元的存活；抑制星形胶质细胞胞体崩解和突起断裂，恢复D-SerineD-Serine的表达并上调connexin43的表达，且下调早期AQP4的表达水平；减轻小胶质细胞的活化并抑制其TNF-α的生成。
离体研究：1）基础状态下，Kir6.2+/+和Kir6.2-/-原代培养的皮层神经元凋亡率、细胞活力没有差异。氧糖剥夺引起Kir6.2+/+和Kir6.2-/-神经元损伤且Kir6.2-/-神经元损伤较Kir6.2+/+神经元显著增加。IPT能抑制两种基因型神经元的损伤。2）氧糖剥夺30分钟复氧2小时后，Kir6.2+/+和Kir6.2-/-神经元p-AMPK表达水平增高，Kir6.2-/-神经元p-AMPK增高水平更为显著。两种基因型神经元p-AMPK表达水平能被IPT显著抑制。3）APMK抑制剂compound C能减轻两种基因型神经元氧糖剥夺损伤。
结论：
1、Kir6.2敲除加重脑缺血后内皮细胞结构损伤及功能障碍；引起突触后致密蛋白表达水平显著降低；海马CA1区神经元大量缺失；其周围星形胶质细胞崩解；小胶质细胞剧烈活化，从而使Kir6.2-/-小鼠缺血性脑损伤更为严重。
2、K-ATP通道开放剂IPT显著减轻脑缺血对两种基因型小鼠神经血管单元的损伤；改善小鼠神经血管单元的功能，进而对两种基因型小鼠缺血性脑损伤均发挥神经保护作用。
3、不仅神经元和胶质细胞分别主要表达Kir6.1和Kir6.2构成的K-ATP通道，脑内皮细胞也同时表达Kir6.1和Kir6.2构成的K-ATP通道。开放K-ATP通道通过调节神经血管单元各组分功能在缺血性脑损伤中发挥神经保护作用。
第二部分Kir6.2敲除对美金刚缺血性脑损伤神经保护作用的影响
目的：探讨Kir6.2敲除对美金刚治疗缺血性脑损伤作用的影响及其机制，阐明K-ATP通道是脑卒中神经保护药物的重要靶标。
方法：在体应用3月龄Kir6.2+/+及Kir6.2-/-雄性C57小鼠，线栓法建立大脑中动脉栓塞（middle cerebral artery occlusion，MCAO）模型，缺血前30分钟给予美金刚20mg/kg，腹腔注射。统计死亡率并行神经功能缺陷评分；2，3，5-氯化三苯基四氮唑（2,3,5-triphenyltetrazoliumchloride，TTC）染色观察脑梗死体积变化；免疫组织化学法观察内皮细胞及基底膜形态学改变；神经元、星形胶质、小胶质细胞数量及形态学改变；缺血后第二天起经腹腔给予5-溴-2-脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine，BrdU，100mg/kg，每天一次，至第7天），最后一次给予BrdU 24小时后麻醉、灌注固定、取脑、冰冻切片，行BrdU免疫组化。缺血后14天行doublecortin (DCX)免疫组化。离体分离、培养孕14/15天野生型（Kir6.2+/+）、敲除型（Kir6.2-/-）胚胎小鼠皮层神经元，建立氧糖剥夺损伤（OGD）模型。模型前对神经元给予20μM美金刚药物处理。通过Hoechst33342核染色以及MTT（3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromid）比色法观察原代培养的皮层神经元在OGD模型下的损伤。
结果：1）Kir6.2敲除和美金刚预保护对小鼠脑缺血后神经运动功能障碍和脑梗死体积的影响：脑缺血后，两种小鼠都出现神经运动功能障碍和脑组织梗死。Kir6.2-/-小鼠神经运功能障碍及脑梗死体积较Kir6.2+/+小鼠显著增加。美金刚能改善Kir6.2+/+小鼠神经运动功能障碍并降低其脑梗死体积，但对Kir6.2-/-小鼠却无此保护作用。2）Kir6.2敲除和美金刚预保护对小鼠脑缺血后神经血管单元结构和功能的影响：脑缺血后3小时，Kir6.2+/+小鼠内皮细胞MMP-9表达水平显著增加，紧密连接蛋白occludin和基底膜蛋白collagenIV表达水平降低；PSD-95表达减弱。至脑缺血后24小时，其CA1区神经元排列稀疏，胞外间隙增大，星形胶质细胞和小胶质细胞活化增殖，AQP4表达降低。Kir6.2-/-小鼠MMP-9表达水平及occludin和collagenIV降解程度高于Kir6.2+/+小鼠；脑内PSD-95表达水平显著低于Kir6.2+/+小鼠；CA1区神经元损伤较Kir6.2+/+小鼠显著增加；星形胶质细胞胞体崩解突起断裂，AQP4表达水平随缺血时间延长先升高再降低；小胶质细胞剧烈活化。美金刚能降低Kir6.2+/+小鼠MMP-9的表达水平，抑制occludin和collagenIV的降解；逆转其PSD-95表达水平的降低；显著促进CA1区神经元的存活；减轻星形胶质细胞和小胶质细胞的活化。在Kir6.2-/-小鼠，美金刚虽然能下调其缺血性脑损伤早期脑内MMP-9的表达进而抑制occludin和collagenIV的降解，但不能上调缺血早期PSD-95的表达进而无法促进脑缺血24小时后神经元的存活。离体实验表明：美金刚不能减轻Kir6.2-/-神经元氧糖剥夺损伤。整体实验显示，Kir6.2-/-小鼠脑缺血24小时后星形胶质细胞的崩解和小胶质细胞的活化也无法被改善。3）KirKir6.2敲除和美金刚对小鼠缺血性脑损伤后神经再生的影响：脑缺血损伤后，美金刚能促进Kir6.2+/+和Kir6.2-/-小鼠SVZ区神经干细胞增殖。至脑缺血损伤后14天，Kir6.2-/-小鼠全部死亡，美金刚能促进存活的Kir6.2+/+小鼠SVZ区神经干细胞的迁移。
结论：Kir6.2敲除取消美金刚急性缺血性脑损伤的神经保护作用，提示美金刚对小鼠急性缺血性脑损伤的保护作用依赖于Kir6.2构成的K-ATP通道，其机制可能与美金刚无法抑制PSD-95在脑缺血早期的降解相关。美金刚可以促进脑缺血后的神经再生过程，有利于组织损伤及修复，该效应不依赖于Kir6.2构成的K-ATP通道。
综上所述，本文研究工作的主要创新之处在于：
1、K-ATP通道是缺血性脑损伤神经血管单元保护的重要靶点发现内皮细胞同时表达Kir6.1和Kir6.2构成的K-ATP通道，Kir6.2敲除通过加重脑缺血后内皮细胞功能障碍进而促进神经血管单元各组分的损伤，这些结果阐明K-ATP通道是神经血管单元保护的重要靶点，为研发理想的神经保护药物提供了新的靶标。
2、K-ATP通道通过调节神经血管单元功能在缺血性脑损伤中发挥神经保护作用发现开放K-ATP通道通过调节神经血管单元各组分功能在缺血性脑损伤中发挥神经保护作用。研究结果为进一步探讨K-ATP通道在脑卒中神经损伤中的作用机制积累了学术基础。
3、Kir6.2敲除取消美金刚的神经保护作用Kir6.2敲除取消美金刚急性缺血性脑损伤的神经保护作用，表明美金刚对急性缺血性脑损伤的保护作用依赖于Kir6.2构成的K-ATP通道。美金刚可以促进脑缺血后的神经再生过程，有利于组织损伤及修复，该效应不依赖于Kir6.2构成的K-ATP通道。
Ischemic stroke constitutes a significant human health hazard. It has beenpredicted that, by the year 2020, stroke would become one of the primary causes ofdeath. In the past ten years, clinical trials aiming to figure out suitableneuroprotectants against the debilitating effects of stroke have met with no success.Identifying novel neuroprotectants which can reverse the effects against stroke isbecoming a challenge to both clinicians and scientists. Leading pathogenicmechanisms of ischemic cascade include energy failure, elevation of intracellularCa2+, excitotoxicity, spreading depression, generation of free radicals, BBB disruption,inflammation, and apoptosis. In order to yield sufficient neuroprotection, an idealtherapeutic method for stroke would be the one which rescues multiple brain cells.Therefore, the new targets should be proved to protect the entire neurovascular unit.
ATP-sensitive potassium (K-ATP) channels provide a unique link betweencellular energetics and electrical excitability. They are heteromultimers composedof four inwardly rectifier potassium channel pore subunits (Kir6.x) and foursulfonylurea receptors (SURs). These channels are usually closed in normalconditions but are activated rapidly in response to the decreases in intracellularATP/ADP ratio under ischemic conditions. Thus, the opening of K-ATP channelscan get intrinsic protection by regulating membrane potential. Opening K-ATPchannels can hyperpolarize the cell membrane, limiting neuron excitability and Ca2+ influx and thus blocking the subsequent neurotoxic biochemical cascade throughinhibiting activation of N-methyl-D-aspartate receptor-gated ion channel activation.Recent reports have shown that Kir6.2-/- mice displayed enhanced neuronal damageafter ischemic insults and over-expression of Kir6.2 ameliorates the brain ischemicinjury. These findings indicated that K-ATP channels are directly involved inneuroprotection against acute hypoxic or ischemic insult. However, most ofmechanistic studies about the neuroprotection of K-ATP against cerebral ischemiahave been only focused on neurons protection. In fact, accumulating evidences nowshow that glial cells and endothelial cells play an active and important role in thepathophysiology of stroke. Brain energetics, water and ion homeostasis,inflammation, trophic factor production, vascular regulation, neurongenesis, andvasculogenesis are all under the control of glial cells. Endothelial cells form theBrain-blood brarrier (BBB) and participate in inflammatory and immune reactions byproducing solubale mediators. K-ATP channels are widely distributed in brain. Ithas been documented that neurons mainly express Kir6.2-containing K-ATP channelswhereas Kir6.1 is the principal pore-forming subunit of astrocytes and microglia.The recent studies have showed that pericytes also express Kir6.1-based channels.However, it is uncertain that what kind of subunit of K-ATP channel endothelial cellsexpress. Therefore, we proposed that the neuroprotection by K-ATP opening didnot only apply on neurons. However, the exactly mechanisms are still unknown.
Therefore, we established the ischemia model by middle cerebral arteryocclusion (MCAO) in both genotypes. In the first part of present study, we used anovel and unselective K-ATP channel opener—iptakalim (IPT) and Kir6.2 knockoutmice to investigated the neuroprotection by opening K-ATP channels. In the secondpart, the neuroprotective effects of NMDA receptor antagonist---memantine againstcerebral ischemia were studied in Kir6.2 knockout mice. Finally, it was illustratedthat K-ATP was an important target of neuroprotectants in stroke.
Part I Effects of Kir6.2 knockout on iptakalim-inducedneuroprotectioneuroprotection after cerebral ischemia
AIM: To investigate the effects of Kir6.2 knockout and the opening of K-ATPchannels in ischemia-induced cerebral injury in mice.
METHODS: 1）In vivo, focal ischemia was achieved in three-month-oldwildtype (Kir6.2+/+) and Kir6.2 knockout (Kir6.2-/-) male mice by occlusion of middlecerebral artery (MCAO) with a modified intralumenal filament technique as describedpreviously. IPT was injected intraperitoneally in a dose of 10mg/kg, 30 minutesbefore middle cerebral artery occlusion (MCAO). Mortality, neurological deficitsand infarct volume were measured in each group 24 hours after ischemia. Doubleimmunofluorescence was used for identifying the subtype of K-ATP channel inendothelial cells and to assess the contents of glia-transmitter D-SerineD-Serine andMMP-9. Immunostaining was taken for observation of endothelial cells, basalmembrane neurons, astrocytes and microglia. Fresh hippocampal homogenateswere used to determine the expressions of connexin43 and MMP-9. The TNF-αproduction was also analyzed by enzyme-linked immunosorbent assay (ELISA). 2)In vitro, primary cortical neuron cultures were prepared from the cortical tissues ofembryonic day 14/15 Kir6.2+/+ and Kir6.2-/- C57BL/6J mice. Cultures were usedafter 7 days in vitro. Oxygen and glucose (OGD) were deprived to induce theneuronal injury. Cortical neurons were incubated with 10μM IPT or 10μM AMPKinhibitor compound c for thirty minutes before OGD stimulation.3-(4,5-dimethylthiazol-2-yl)- -2,5-diphenyltetrazolium bromide (MTT) was used toevaluate the cell viability. Staining with Hoechst 33342 was used to determineneuronal apoptosis. Western blotting was taken for the analysis of p-AMPKexpression.
RESULTS: In vivo，1) Effects of Kir6.2 knockout and IPT pretreatmenton mortality, neurological deficits and infarct volume induced by MCAO Afterischemia, Kir6.2-/- mice exhibited higher mortality while nearly all of Kir6.2+/+ micesurvived. Pretreatment with IPT could increase the survival ratio of Kir6.2-/- mice.Server neurological deficits and larger infarct volume were observed in Kir6.2-/- mice.IPT could attenuate the neurological deficits and decrease the infarct volume in both genotypes. 2) EffectEffects of Kir6.2 knockout and IPT on the structure and functionof neurovasucular unit after ischemia. Endothelial cells expressed Kir6.1 andKir6.2- containing K-ATP channels. Higher production of MMP-9 by endothelialcells was observed in Kir6.2-/- mice three hours after ischemia. Occludin andcollagenIV were then dramatically disrupted in Kir6.2-/- mice. The level of PSD-95expression was significantly decreased. Till 24 hours after ischemia, there was aserver decrease in the number of CA1 pyramidal cells in the Kir6.2-/- group comparedwith that in the Kir6.2+/+ group. Astrocytes displayed reactive changes surroundingthe neurons in CA1 region of Kir6.2+/+ group. The expression of D-SerineD-Serinewas increased while the expression of AQP4 was decreased. However, astrocytesshowed damaged features in CA1 region in Kir6.2-/- mice. The expression ofconnexin43 and D-SerineD-Serine were significantly reduced. AQP4 expressionwas initially dramatically increased and then decreased. Severer microglial wereactivated and much more TNF-αproduction was induced by ischemia in Kir6.2-/-genotype. Pretreatment with IPT inhibited MMP-9 secretion and then prevented thedegradation of occludin and collagenIV and in both genotypes；markedly increasedthe expression of PSD-95 and promoted the neuronal survival in both genotypes;inhibited the activation of astrocytes with the decreased the expression ofD-SerineD-Serine, enhancement the expression of connexin43 and inhibition ofAQP4 expression in Kir6.2+/+ mice; protected astrocytes against ischemia withupregulation of connexin43, D-SerineD-Serine and AQP4 expression in Kir6.2-/- mice;inhibited microglia activation in both genotypes and decreased the production ofTNF-αin Kir6.2-/- genotye;
In vitro, Kir6.2 knockout does not altered the level of apoptosis, neuronalsurvival under normal conditions but increased the injury of OGD. IPT increasedthe neuronal survival of two genotypes induced by OGD stimulaiton. After OGDstimulation, the expression of p-AMPK was increased in both genotypes. Higherexpression level of p-AMPK was observed in Kir6.2-/- neurons. IPT inhibited thephosphorylation of AMPK induced by OGD stimulaiton. Inhibition of p-AMPKexpression by compound C could decrease the injury of both genotypes.
CONCLUSION:
1. Lack of Kir6.2- containing K-ATP channels aggravates endothelial cellsmalfunction, obviously induces neuron apoptosis and astrocyte damage, daramaticallyactivates microglia, which ultimately cause enhancement of cerebral ischemicdamage in Kir6.2-/- mice.
2. K-ATP channel opener IPT exerts evident neuroprotective effects in bothgenotype mice through improving the function of entire neurovascular unit.
3. All components of neurovascular unit express K-ATP channel. Endothelialcells express Kir6.1- and Kir6.2-containing K-ATP channels. Targeting K-ATPchannels provides a promising therapeutic strategy for protection of neurovascularunit in brain ischemic lesion.
Part II Effects of Kir6.2 knockout on memantine-inducedneuroprotection after ischemia
AIM: To investigate whether Kir6.2-containing K-ATP channel participate inmemantine-induced neuroprotection after ischemia and prove that K-ATP is animportant target of neuroprotectants aginast cerebral ischemia.
METHODS: In vivo, focal ischemia was achieved in three-month-oldwildtype (Kir6.2+/+) and Kir6.2 knockout (Kir6.2-/-) male mice by occlusion of middlecerebral artery (MCAO) with a modified intralumenal filament technique as describedpreviously. Memantine was injected intraperitoneally in a dose of 20mg/kg, 30minutes before MCAO. Since the second day after ischemia, BrdU (100mg, once aday, ip) was administrated to both genotypes followed by 7-day-memantine treatment.Neurological deficits and infarct volume were measured in each group 24 hours afterischemia. Immunostaining was taken for observation of the morphological changesof endothelial cells，basal membrane，neurons, astrocytes, microglia and countingBrdU-positive cells. Immunofluorescence was used to evaluating PSD-95 anddoublecortin (DCX) expression. Primary cortical neuron cultures were preparedfrom the cortical tissues of embryonic day 14/15 Kir6.2+/+ and Kir6.2-/- C57BL/6J mice. In vitro, oxygen and glucose were deprived to induce the neuronal injury.Cortical neurons were incubated with 10μM memantine before OGD stimulation.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used toevaluate the cell viability. Staining with Hoechst 33342 was used to determineneuronal apoptosis.
RESULTS: 1) Effects of Kir6.2 knockout and memantine pretreatment onneurological deficits and infarct volume induced by MCAO After ischemia,Kir6.2-/- mice exhibited server neurological deficits and larger cerebral infarct volume.Pretreatment with memantine could attenuate the neurological deficits and decreasethe infarct volume but have no impact on Kir6.2-/- mice. 2) Effects of Kir6.2knockout and memantine pretretment on the structure and function ofneurovasucular unit after stroke. Three hours after ischemia, higher productionof MMP-9 by endothelial cells was observed in Kir6.2-/- mice. Occludin andcollagenIV were dramatically disrupted in Kir6.2-/- mice. The expression of PSD-95was much lower in Kir6.2-/- mice. Twanty-four hours after ischemia, there was amuch more decrease in the number of CA1 pyramidal cells in the Kir6.2-/- group.Astrocytes displayed reactive changes in CA1 region of Kir6.2+/+ group. Theexpression of AQP4 was decreased. However, astrocytes showed damaged featuresin Kir6.2-/- mice. AQP4 expression was initially dramatically increased and thendecreased. Severer activation of microglial was observed in Kir6.2-/- genotype. InKir6.2+/+ mice, pretreatment with memantine inhibited MMP-9 secretion and thenprevented the degradation of occludin and collagenIV; prevented the decrease ofPSD-95 in CA1 regions and markedly increased the neuronal survival; inhibited theactivation of astrocytes and microglia; decreased AQP4 expression. In Kir6.2-/- mice,pretreatment with memantine decreased the initial secretion of MMP-9 anddegradation of occludin and collagenIV, but had no effect on the decrease of PSD-95.Till 24 hours after ischemia, the neuronal survival, astrocytes damage and microgliawere not improved. In vitro, the anti-apoptosis effect of memantine on neurons wasabolished by Kir6.2 knockout during OGD stimulation. 3) Effects of Kir6.2knockout and memantine on the neurogenesis after stroke. Daily injection of memantine promoted the proliferation of stem cells located in SVZ in both genotypesafter ischemia. Fourteen days after stroke, all of the Kir6.2-/- mice died.Memantine promoted the migration of stem cells in SVZ of Kir6.2+/+ mice.
CONCLUSION: Memantine have no neuroprotective effects on actue cerebralischemic injury of Kir6.2-/- mice. The neuroprotection of memantine in ischemicstroke is dependent on Kir6.2-containing K-ATP channels. The underlyingmechanism is attributed to that memantine can not inhibite the degradation of PSD-95induced by ischemia after Kir6.2 knockout. Memantine could promote neurogenesisafter stroke which will benefit the intrinsic revovery, which is independent onKir6.2-containing K-ATP channels.
In summary, our present study has the following new concerns:
1. K-ATP channels are promising therapeutic targets for the protection ofneurovascular unit in brain ischemic lesion. Endothelia cells express Kir6.1- andKir6.2- containing K-ATP channels. Kir6.2 knockout damaged the entireneurovascular unit during ischemia. The therapeutic strategy targeted to K-ATP mayoffer a new perspective for the development of new options in ischemic stroketreatment.
2. Opening K-ATP channels inhibit the ischemic brain damage throughneurovascular unit protection. Opening K-ATP channels modulate the integrityand function of the neurovascular to play significant neuroprotection in ischemicstroke. The results obtained in present study substantially improve ourunderstanding of K-ATP in stroke.
3. Kir6.2 knockout abolishes the neuroprotective effects of memantine.Memantine have no neuroprotective effects on acute cerebral ischemic injury ofKir6.2-/- mice. The underlying mechanism is attributed to that memantine can notinhibite the degradation of PSD-95 induced by ischemia after Kir6.2 knockout.Kir6.2-containing K-ATP channels are involved in the neuroprotection of memantinein cerebral ischemia. Memantine can promote neurogenesis after stroke which willbenefit the intrinsinc revovery. This effect is independent on Kir6.2-containing K-ATP channels.
引文
[1] Jeyaseelan K, Lim KY, Armugam A. Neuroprotectants in stroke therapy. Expert OpinPharmacother, 2008, 9(6):887-900.
[2] Lo EH. Experimental models, neurovascular mechanisms and translational issues instroke research. Br J Pharmacol, 2008, 153 Suppl 1:S396-405.
[3] Lo EH, Dalkara T, Moskowitz MA. Mechanisms, challenges and opportunities in stroke.Nat Rev Neurosci, 2003, 4(5):399-415.
[4] Nakase T, Fushiki S, Naus CC. Astrocytic gap junctions composed of connexin43 reduceapoptotic neuronal damage in cerebral ischemia. Stroke, 2003, 34(8):1987-1993.
[5] Dyker AG, Lees KR. The rationale for new therapies in acute ischaemic stroke. J ClinPharm Ther, 1996, 21(6):377-391.
[6] Rosell A, Ortega-Aznar A, Alvarez-Sabin J, Fernandez-Cadenas I, Ribo M, Molina CA,Lo EH, Montaner J. Increased brain expression of matrix metalloproteinase-9 afterischemic and hemorrhagic human stroke. Stroke, 2006, 37(6):1399-1406.
[7] Mayer TE, Hamann GF, Baranczyk J, Rosengarten B, Klotz E, Wiesmann M, Missler U,Schulte-Altedorneburg G, Brueckmann HJ. Dynamic CT perfusion imaging of acutestroke. AJNR Am J Neuroradiol, 2000, 21(8):1441-1449.
[8] Richard Green A, Odergren T, Ashwood T. Animal models of stroke: do they have valuefor discovering neuroprotective agents? Trends Pharmacol Sci, 2003, 24(8):402-408.
[9] Smith WS. Pathophysiology of focal cerebral ischemia: a therapeutic perspective. J VascInterv Radiol, 2004, 15(1 Pt 2):S3-12.
[10] MacManus JP, Buchan AM. Apoptosis after experimental stroke: fact or fashion? JNeurotrauma, 2000, 17(10):899-914.
[11] Unal-Cevik I, Kilinc M, Can A, Gursoy-Ozdemir Y, Dalkara T. Apoptotic and necroticdeath mechanisms are concomitantly activated in the same cell after cerebral ischemia.Stroke, 2004, 35(9):2189-2194.
[12] Durukan A, Tatlisumak T. Acute ischemic stroke: overview of major experimental rodentmodels, pathophysiology, and therapy of focal cerebral ischemia. Pharmacol BiochemBehav, 2007, 87(1):179-197.
[13] Siesjo BK, Katsura K, Kristian T. Acidosis-related damage. Adv Neurol, 1996,71:209-233; discussion 234-206.
[14] Huang Y, McNamara JO. Ischemic stroke: "acidotoxicity" is a perpetrator. Cell, 2004,118(6):665-666.
[15] Mergenthaler P, Dirnagl U, Meisel A. Pathophysiology of stroke: lessons from animalmodels. Metab Brain Dis, 2004, 19(3-4):151-167.
[16] Phan TG, Wright PM, Markus R, Howells DW, Davis SM, Donnan GA. Salvaging theischaemic penumbra: more than just reperfusion? Clin Exp Pharmacol Physiol, 2002,29(1-2):1-10.
[17] Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic stroke: an integratedview. Trends Neurosci, 1999, 22(9):391-397.
[18] Hartings JA, Williams AJ, Tortella FC. Occurrence of nonconvulsive seizures, periodicepileptiform discharges, and intermittent rhythmic delta activity in rat focal ischemia.Exp Neurol, 2003, 179(2):139-149.
[19] Dreier JP, Woitzik J, Fabricius M, Bhatia R, Major S, Drenckhahn C, Lehmann TN,Sarrafzadeh A, Willumsen L, Hartings JA, Sakowitz OW, Seemann JH, Thieme A,Lauritzen M, Strong AJ. Delayed ischaemic neurological deficits after subarachnoidhaemorrhage are associated with clusters of spreading depolarizations. Brain, 2006,129(Pt 12):3224-3237.
[20] Dijkhuizen RM, Beekwilder JP, van der Worp HB, Berkelbach van der Sprenkel JW,Tulleken KA, Nicolay K. Correlation between tissue depolarizations and damage in focalischemic rat brain. Brain Res, 1999, 840(1-2):194-205.
[21] Mackert BM. The discovery of slowness--recent progress in DC-MEG research. NeurolClin Neurophysiol, 2004, 2004:41.
[22] McIlvoy LH. The effect of hypothermia and hyperthermia on acute brain injury. AACNClin Issues, 2005, 16(4):488-500.
[23] Garcia JH, Liu KF, Ho KL. Neuronal necrosis after middle cerebral artery occlusion inWistar rats progresses at different time intervals in the caudoputamen and the cortex.Stroke, 1995, 26(4):636-642; discussion 643.
[24] Yang GY, Gong C, Qin Z, Liu XH, Lorris Betz A. Tumor necrosis factor alpha expressionproduces increased blood-brain barrier permeability following temporary focal cerebralischemia in mice. Brain Res Mol Brain Res, 1999, 69(1):135-143.
[25] Emerich DF, Dean RL, 3rd, Bartus RT. The role of leukocytes following cerebralischemia: pathogenic variable or bystander reaction to emerging infarct? Exp Neurol,2002, 173(1):168-181.
[26] Danton GH, Dietrich WD. Inflammatory mechanisms after ischemia and stroke. JNeuropathol Exp Neurol, 2003, 62(2):127-136.
[27] Schilling M, Besselmann M, Leonhard C, Mueller M, Ringelstein EB, Kiefer R.Microglial activation precedes and predominates over macrophage infiltration in transientfocal cerebral ischemia: a study in green fluorescent protein transgenic bone marrowchimeric mice. Exp Neurol, 2003, 183(1):25-33.
[28] Dienel GA, Hertz L. Astrocytic contributions to bioenergetics of cerebral ischemia. Glia,2005, 50(4):362-388.
[29] Trendelenburg G, Dirnagl U. Neuroprotective role of astrocytes in cerebral ischemia:focus on ischemic preconditioning. Glia, 2005, 50(4):307-320.
[30] Allen GV, Cheung RT, Cechetto DF. Neurochemical changes following occlusion of themiddle cerebral artery in rats. Neuroscience, 1995, 68(4):1037-1050.
[31] Matsumoto K, Graf R, Rosner G, Taguchi J, Heiss WD. Elevation of neuroactivesubstances in the cortex of cats during prolonged focal ischemia. J Cereb Blood FlowMetab, 1993, 13(4):586-594.
[32] Fisher M, Schaebitz W. An overview of acute stroke therapy: past, present, and future.Arch Intern Med, 2000, 160(21):3196-3206.
[33] Sugawara T, Fujimura M, Noshita N, Kim GW, Saito A, Hayashi T, Narasimhan P, MaierCM, Chan PH. Neuronal death/survival signaling pathways in cerebral ischemia.NeuroRx, 2004, 1(1):17-25.
[34] Schaller B, Graf R. Cerebral ischemia and reperfusion: the pathophysiologic concept as abasis for clinical therapy. J Cereb Blood Flow Metab, 2004, 24(4):351-371.
[35] Love S. Apoptosis and brain ischaemia. Prog Neuropsychopharmacol Biol Psychiatry,2003, 27(2):267-282.
[36] Guglielmo MA, Chan PT, Cortez S, Stopa EG, McMillan P, Johanson CE, Epstein M,Doberstein CE. The temporal profile and morphologic features of neuronal death inhuman stroke resemble those observed in experimental forebrain ischemia: the potentialrole of apoptosis. Neurol Res, 1998, 20(4):283-296.
[37] Love S, Barber R, Wilcock GK. Neuronal death in brain infarcts in man. NeuropatholAppl Neurobiol, 2000, 26(1):55-66.
[38] Sairanen T, Karjalainen-Lindsberg ML, Paetau A, Ijas P, Lindsberg PJ. Apoptosisdominant in the periinfarct area of human ischaemic stroke--a possible target ofantiapoptotic treatments. Brain, 2006, 129(Pt 1):189-199.
[39] Iadecola C. Neurovascular regulation in the normal brain and in Alzheimer's disease. NatRev Neurosci, 2004, 5(5):347-360.
[40] Zlokovic BV. Neurovascular mechanisms of Alzheimer's neurodegeneration. TrendsNeurosci, 2005, 28(4):202-208.
[41] Abbott NJ, Ronnback L, Hansson E. Astrocyte-endothelial interactions at the blood-brainbarrier. Nat Rev Neurosci, 2006, 7(1):41-53.
[42] Wang YF, Tsirka SE, Strickland S, Stieg PE, Soriano SG, Lipton SA. Tissue plasminogenactivator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type andtPA-deficient mice. Nat Med, 1998, 4(2):228-231.
[43] Nicole O, Docagne F, Ali C, Margaill I, Carmeliet P, MacKenzie ET, Vivien D, Buisson A.The proteolytic activity of tissue-plasminogen activator enhances NMDAreceptor-mediated signaling. Nat Med, 2001, 7(1):59-64.
[44] Chen ZL, Strickland S. Neuronal death in the hippocampus is promoted byplasmin-catalyzed degradation of laminin. Cell, 1997, 91(7):917-925.
[45] Melchor JP, Strickland S. Tissue plasminogen activator in central nervous systemphysiology and pathology. Thromb Haemost, 2005, 93(4):655-660.
[46] Wang X, Lee SR, Arai K, Lee SR, Tsuji K, Rebeck GW, Lo EH. Lipoproteinreceptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator.Nat Med, 2003, 9(10):1313-1317.
[47] Lo EH, Broderick JP, Moskowitz MA. tPA and proteolysis in the neurovascular unit.Stroke, 2004, 35(2):354-356.
[48] Aoki T, Sumii T, Mori T, Wang X, Lo EH. Blood-brain barrier disruption and matrixmetalloproteinase-9 expression during reperfusion injury: mechanical versus embolicfocal ischemia in spontaneously hypertensive rats. Stroke, 2002, 33(11):2711-2717.
[49] Sumii T, Lo EH. Involvement of matrix metalloproteinase in thrombolysis-associatedhemorrhagic transformation after embolic focal ischemia in rats. Stroke, 2002,33(3):831-836.
[50] Tsuji K, Aoki T, Tejima E, Arai K, Lee SR, Atochin DN, Huang PL, Wang X, Montaner J,Lo EH. Tissue plasminogen activator promotes matrix metalloproteinase-9 upregulationafter focal cerebral ischemia. Stroke, 2005, 36(9):1954-1959.
[51] Montaner J, Fernandez-Cadenas I, Molina CA, Monasterio J, Arenillas JF, Ribo M,Quintana M, Chacon P, Andreu AL, Alvarez-Sabin J. Safety profile of tissue plasminogenactivator treatment among stroke patients carrying a common polymorphism (C-1562T)in the promoter region of the matrix metalloproteinase-9 gene. Stroke, 2003,34(12):2851-2855.
[52] Ning M, Furie KL, Koroshetz WJ, Lee H, Barron M, Lederer M, Wang X, Zhu M,Sorensen AG, Lo EH, Kelly PJ. Association between tPA therapy and raised early matrixmetalloproteinase-9 in acute stroke. Neurology, 2006, 66(10):1550-1555.
[53] Seino S, Miki T. Physiological and pathophysiological roles of ATP-sensitive K+channels. Prog Biophys Mol Biol, 2003, 81(2):133-176.
[54] Nichols CG. KATP channels as molecular sensors of cellular metabolism. Nature, 2006,440(7083):470-476.
[55] Dunn-Meynell AA, Rawson NE, Levin BE. Distribution and phenotype of neuronscontaining the ATP-sensitive K+ channel in rat brain. Brain Res, 1998, 814(1-2):41-54.
[56] Thomzig A, Laube G, Pruss H, Veh RW. Pore-forming subunits of K-ATP channels,Kir6.1 and Kir6.2, display prominent differences in regional and cellular distribution inthe rat brain. J Comp Neurol, 2005, 484(3):313-330.
[57] Wang Y, Haider HK, Ahmad N, Ashraf M. Mechanisms by which K(ATP) channelopeners produce acute and delayed cardioprotection. Vascul Pharmacol, 2005,42(5-6):253-264.
[58] Rodrigo GC, Standen NB. ATP-sensitive potassium channels. Curr Pharm Des, 2005,11(15):1915-1940.
[59] Raval AP, Dave KR, DeFazio RA, Perez-Pinzon MA. epsilonPKC phosphorylates themitochondrial K(+) (ATP) channel during induction of ischemic preconditioning in therat hippocampus. Brain Res, 2007, 1184:345-353.
[60] Gaspar T, Snipes JA, Busija AR, Kis B, Domoki F, Bari F, Busija DW. ROS-independentpreconditioning in neurons via activation of mitoK(ATP) channels by BMS-191095. JCereb Blood Flow Metab, 2008, 28(6):1090-1103.
[61] Kis B, Rajapakse NC, Snipes JA, Nagy K, Horiguchi T, Busija DW. Diazoxide inducesdelayed pre-conditioning in cultured rat cortical neurons. J Neurochem, 2003,87(4):969-980.
[62] Yao Z, Gross GJ. A comparison of adenosine-induced cardioprotection and ischemicpreconditioning in dogs. Efficacy, time course, and role of KATP channels. Circulation,1994, 89(3):1229-1236.
[63] Sargent CA, Sleph PG, Dzwonczyk S, Normandin D, Antonaccio MJ, Grover GJ.Cardioprotective effects of the cyanoguanidine potassium channel opener P-1075. JCardiovasc Pharmacol, 1993, 22(4):564-570.
[64] Sun XL, Zeng XN, Zhou F, Dai CP, Ding JH, Hu G. KATP channel openers facilitateglutamate uptake by GluTs in rat primary cultured astrocytes. Neuropsychopharmacology,2008, 33(6):1336-1342.
[65] Zhou F, Wu JY, Sun XL, Yao HH, Ding JH, Hu G. Iptakalim alleviates rotenone-induceddegeneration of dopaminergic neurons through inhibiting microglia-mediatedneuroinflammation. Neuropsychopharmacology, 2007, 32(12):2570-2580.
[66] Ikonomidou C, Turski L. Why did NMDA receptor antagonists fail clinical trials forstroke and traumatic brain injury? Lancet Neurol, 2002, 1(6):383-386.
[67] Plosker GL, Lyseng-Williamson KA. Memantine: a pharmacoeconomic review of its usein moderate-to-severe Alzheimer's disease. Pharmacoeconomics, 2005, 23(2):193-206.
[68] Orgogozo JM, Rigaud AS, Stoffler A, Mobius HJ, Forette F. Efficacy and safety ofmemantine in patients with mild to moderate vascular dementia: a randomized,placebo-controlled trial (MMM 300). Stroke, 2002, 33(7):1834-1839.
[69] Babenko AP, Aguilar-Bryan L, Bryan J. A view of sur/KIR6.X, KATP channels. AnnuRev Physiol, 1998, 60:667-687.
[70] Wang H, Zhang YL, Tang XC, Feng HS, Hu G. Targeting ischemic stroke with a novelopener of ATP-sensitive potassium channels in the brain. Mol Pharmacol, 2004,66(5):1160-1168.
[71] Sun HS, Feng ZP, Miki T, Seino S, French RJ. Enhanced neuronal damage after ischemicinsults in mice lacking Kir6.2-containing ATP-sensitive K+ channels. J Neurophysiol,2006, 95(4):2590-2601.
[72] Heron-Milhavet L, Xue-Jun Y, Vannucci SJ, Wood TL, Willing LB, Stannard B,Hernandez-Sanchez C, Mobbs C, Virsolvy A, LeRoith D. Protection againsthypoxic-ischemic injury in transgenic mice overexpressing Kir6.2 channel pore inforebrain. Mol Cell Neurosci, 2004, 25(4):585-593.
[73] Brown RC, Morris AP, O'Neil RG. Tight junction protein expression and barrierproperties of immortalized mouse brain microvessel endothelial cells. Brain Res, 2007,1130(1):17-30.
[74] Hartmann C, Zozulya A, Wegener J, Galla HJ. The impact of glia-derived extracellularmatrices on the barrier function of cerebral endothelial cells: an in vitro study. Exp CellRes, 2007, 313(7):1318-1325.
[75] Lin JH, Lou N, Kang N, Takano T, Hu F, Han X, Xu Q, Lovatt D, Torres A, Willecke K,Yang J, Kang J, Nedergaard M. A central role of connexin43 in hypoxic preconditioning.J Neurosci, 2008, 28(3):681-695.
[76] Panickar KS, Norenberg MD. Astrocytes in cerebral ischemic injury: morphological andgeneral considerations. Glia, 2005, 50(4):287-298.
[77] Bondjers C, He L, Takemoto M, Norlin J, Asker N, Hellstrom M, Lindahl P, Betsholtz C.Microarray analysis of blood microvessels from PDGF-B and PDGF-Rbeta mutant miceidentifies novel markers for brain pericytes. Faseb J, 2006, 20(10):1703-1705.
[78] Cuzner ML, Opdenakker G. Plasminogen activators and matrix metalloproteases,mediators of extracellular proteolysis in inflammatory demyelination of the centralnervous system. J Neuroimmunol, 1999, 94(1-2):1-14.
[79] Yong VW, Krekoski CA, Forsyth PA, Bell R, Edwards DR. Matrix metalloproteinasesand diseases of the CNS. Trends Neurosci, 1998, 21(2):75-80.
[80] Zhao BQ, Wang S, Kim HY, Storrie H, Rosen BR, Mooney DJ, Wang X, Lo EH. Role ofmatrix metalloproteinases in delayed cortical responses after stroke. Nat Med, 2006,12(4):441-445.
[81] Yamashita T, Kamiya T, Deguchi K, Inaba T, Zhang H, Shang J, Miyazaki K, Ohtsuka A,Katayama Y, Abe K. Dissociation and protection of the neurovascular unit afterthrombolysis and reperfusion in ischemic rat brain. J Cereb Blood Flow Metab, 2009,29(4):715-725.
[82] Asahi M, Wang X, Mori T, Sumii T, Jung JC, Moskowitz MA, Fini ME, Lo EH. Effectsof matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrierand white matter components after cerebral ischemia. J Neurosci, 2001,21(19):7724-7732.
[83] Papadopoulos MC, Verkman AS. Aquaporin-4 and brain edema. Pediatr Nephrol, 2007,22(6):778-784.
[84] Gary DS, Mattson MP. Integrin signaling via the PI3-kinase-Akt pathway increasesneuronal resistance to glutamate-induced apoptosis. J Neurochem, 2001,76(5):1485-1496.
[85] Gu Z, Kaul M, Yan B, Kridel SJ, Cui J, Strongin A, Smith JW, Liddington RC, Lipton SA.S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death.Science, 2002, 297(5584):1186-1190.
[86] Ziff EB. Enlightening the postsynaptic density. Neuron, 1997, 19(6):1163-1174.
[87] Hu BR, Park M, Martone ME, Fischer WH, Ellisman MH, Zivin JA. Assembly ofproteins to postsynaptic densities after transient cerebral ischemia. J Neurosci, 1998,18(2):625-633.
[88] Ramamurthy S, Ronnett GV. Developing a head for energy sensing: AMP-activatedprotein kinase as a multifunctional metabolic sensor in the brain. J Physiol, 2006, 574(Pt1):85-93.
[89] Ronnett GV, Ramamurthy S, Kleman AM, Landree LE, Aja S. AMPK in the brain: itsroles in energy balance and neuroprotection. J Neurochem, 2009, 109 Suppl 1:17-23.
[90] Li J, McCullough LD. Effects of AMP-activated protein kinase in cerebral ischemia. JCereb Blood Flow Metab, 30(3):480-492.
[91] Culmsee C, Monnig J, Kemp BE, Mattson MP. AMP-activated protein kinase is highlyexpressed in neurons in the developing rat brain and promotes neuronal survivalfollowing glucose deprivation. J Mol Neurosci, 2001, 17(1):45-58.
[92] Garcia-Gil M, Pesi R, Perna S, Allegrini S, Giannecchini M, Camici M, Tozzi MG.5'-aminoimidazole-4-carboxamide riboside induces apoptosis in human neuroblastomacells. Neuroscience, 2003, 117(4):811-820.
[93] Du L, Hickey RW, Bayir H, Watkins SC, Tyurin VA, Guo F, Kochanek PM, Jenkins LW,Ren J, Gibson G, Chu CT, Kagan VE, Clark RS. Starving neurons show sex difference inautophagy. J Biol Chem, 2009, 284(4):2383-2396.
[94] Wang W, Guan KL. AMP-activated protein kinase and cancer. Acta Physiol (Oxf), 2009,196(1):55-63.
[95] Wolosker H. NMDA receptor regulation by D-SerineD-Serine: new findings andperspectives. Mol Neurobiol, 2007, 36(2):152-164.
[96] Katsuki H, Nonaka M, Shirakawa H, Kume T, Akaike A. Endogenous D-SerineD-Serineis involved in induction of neuronal death by N-methyl-D-aspartate and simulatedischemia in rat cerebrocortical slices. J Pharmacol Exp Ther, 2004, 311(2):836-844.
[97] Billard JM. D-SerineD-Serine signalling as a prominent determinant of neuronal-glialdialogue in the healthy and diseased brain. J Cell Mol Med, 2008, 12(5B):1872-1884.
[98] Fatemi SH, Folsom TD, Reutiman TJ, Pandian T, Braun NN, Haug K. Chronicpsychotropic drug treatment causes differential expression of connexin43 and GFAP infrontal cortex of rats. Schizophr Res, 2008, 104(1-3):127-134.
[99] Jain SK, Schuessler RB, Saffitz JE. Mechanisms of delayed electrical uncoupling inducedby ischemic preconditioning. Circ Res, 2003, 92(10):1138-1144.
[100] Liu T, Clark RK, McDonnell PC, Young PR, White RF, Barone FC, Feuerstein GZ.Tumor necrosis factor-alpha expression in ischemic neurons. Stroke, 1994,25(7):1481-1488.
[101] Takuma K, Baba A, Matsuda T. Astrocyte apoptosis: implications for neuroprotection.Prog Neurobiol, 2004, 72(2):111-127.
[102] Kaushal V, Schlichter LC. Mechanisms of microglia-mediated neurotoxicity in a newmodel of the stroke penumbra. J Neurosci, 2008, 28(9):2221-2230.
[103] Hazell AS. Excitotoxic mechanisms in stroke: an update of concepts and treatmentstrategies. Neurochem Int, 2007, 50(7-8):941-953.
[104] Waxman EA, Lynch DR. N-methyl-D-aspartate receptor subtypes: multiple roles inexcitotoxicity and neurological disease. Neuroscientist, 2005, 11(1):37-49.
[105] Muir KW. Glutamate-based therapeutic approaches: clinical trials with NMDAantagonists. Curr Opin Pharmacol, 2006, 6(1):53-60.
[106] Rabey JM, Nissipeanu P, Korczyn AD. Efficacy of memantine, an NMDA receptorantagonist, in the treatment of Parkinson's disease. J Neural Transm Park Dis DementSect, 1992, 4:277-282.
[107] Miguel-Hidalgo JJ, Alvarez XA, Cacabelos R, Quack G. Neuroprotection by memantineagainst neurodegeneration induced by beta-amyloid(1-40). Brain Res, 2002,958(1):210-221.
[108] Ditzler K. Efficacy and tolerability of memantine in patients with dementia syndrome. Adouble-blind, placebo controlled trial. Arzneimittelforschung, 1991, 41(8):773-780.
[109] Sonkusare SK, Kaul CL, Ramarao P. Dementia of Alzheimer's disease and otherneurodegenerative disorders--memantine, a new hope. Pharmacol Res, 2005, 51(1):1-17.
[110] Volbracht C, van Beek J, Zhu C, Blomgren K, Leist M. Neuroprotective properties ofmemantine in different in vitro and in vivo models of excitotoxicity. Eur J Neurosci, 2006,23(10):2611-2622.
[111] Gilling KE, Jatzke C, Hechenberger M, Parsons CG. Potency, voltage-dependency,agonist concentration-dependency, blocking kinetics and partial untrapping of theuncompetitive N-methyl-D-aspartate (NMDA) channel blocker memantine at humanNMDA (GluN1/GluN2A) receptors. Neuropharmacology, 2009, 56(5):866-875.
[112] Netzer R, Bigalke H. Memantine reduces repetitive action potential firing in spinal cordnerve cell cultures. Eur J Pharmacol, 1990, 186(2-3):149-155.
[113] Reiser G, Binmoller FJ, Koch R. Memantine (1-amino-3,5-dimethyladamantane) blocksthe serotonin-induced depolarization response in a neuronal cell line. Brain Res, 1988,443(1-2):338-344.
[114] Giustizieri M, Cucchiaroni ML, Guatteo E, Bernardi G, Mercuri NB, Berretta N.Memantine inhibits ATP-dependent K+ conductances in dopamine neurons of the ratsubstantia nigra pars compacta. J Pharmacol Exp Ther, 2007, 322(2):721-729.
[115] Liu C, Lin N, Wu B, Qiu Y. Neuroprotective effect of memantine combined withtopiramate in hypoxic-ischemic brain injury. Brain Res, 2009, 1282:173-182.
[116] Choi DW, Rothman SM. The role of glutamate neurotoxicity in hypoxic-ischemicneuronal death. Annu Rev Neurosci, 1990, 13:171-182.
[117] Yang XL. Characterization of receptors for glutamate and GABA in retinal neurons. ProgNeurobiol, 2004, 73(2):127-150.
[118] Juurlink BH, Sweeney MI. Mechanisms that result in damage during and followingcerebral ischemia. Neurosci Biobehav Rev, 1997, 21(2):121-128.
[119] Marvanova M, Lakso M, Pirhonen J, Nawa H, Wong G, Castren E. The neuroprotectiveagent memantine induces brain-derived neurotrophic factor and trkB receptor expressionin rat brain. Mol Cell Neurosci, 2001, 18(3):247-258.
[120] Rossi C, Angelucci A, Costantin L, Braschi C, Mazzantini M, Babbini F, Fabbri ME,Tessarollo L, Maffei L, Berardi N, Caleo M. Brain-derived neurotrophic factor (BDNF) isrequired for the enhancement of hippocampal neurogenesis following environmentalenrichment. Eur J Neurosci, 2006, 24(7):1850-1856.
[121] Witt A, Macdonald N, Kirkpatrick P. Memantine hydrochloride. Nat Rev Drug Discov,2004, 3(2):109-110.
[122] Johnson JW, Kotermanski SE. Mechanism of action of memantine. Curr Opin Pharmacol,2006, 6(1):61-67.
[123] Gascon S, Sobrado M, Roda JM, Rodriguez Pena A, Diaz-Guerra M. Excitotoxicity andfocal cerebral ischemia induce truncation of the nr2a and nr2b subunits of the nmdareceptor and cleavage of the scaffolding protein psd-95. Mol Psychiatry.2008,13(1):99-114
[1] Ludwin SK, Kosek JC, Eng LF. The topographical distribution of S-100 and GFAproteins in the adult rat brain: an immunohistochemical study using horseradishperoxidase-labelled antibodies. J Comp Neurol, 1976, 165(2):197-207.
[2] Mayer TE, Hamann GF, Baranczyk J, Rosengarten B, Klotz E, Wiesmann M, Missler U,Schulte-Altedorneburg G, Brueckmann HJ. Dynamic CT perfusion imaging of acutestroke. AJNR Am J Neuroradiol, 2000, 21(8):1441-1449.
[3] Walz W. Role of astrocytes in the clearance of excess extracellular potassium.Neurochem Int, 2000, 36(4-5):291-300.
[4] Brannen CL, Sugaya K. In vitro differentiation of multipotent human neural progenitorsin serum-free medium. Neuroreport, 2000, 11(5):1123-1128.
[5] Benarroch EE. Aquaporin-4, homeostasis, and neurologic disease. Neurology, 2007,69(24):2266-2268.
[6] Drukarch B, Schepens E, Stoof JC, Langeveld CH, Van Muiswinkel FL.Astrocyte-enhanced neuronal survival is mediated by scavenging of extracellular reactiveoxygen species. Free Radic Biol Med, 1998, 25(2):217-220.
[7] Maragakis NJ, Rothstein JD. Mechanisms of Disease: astrocytes in neurodegenerativedisease. Nat Clin Pract Neurol, 2006, 2(12):679-689.
[8] Ginsberg MD, Pulsinelli WA. The ischemic penumbra, injury thresholds, and thetherapeutic window for acute stroke. Ann Neurol, 1994, 36(4):553-554.
[9] Suh SW, Bergher JP, Anderson CM, Treadway JL, Fosgerau K, Swanson RA. Astrocyteglycogen sustains neuronal activity during hypoglycemia: studies with the glycogenphosphorylase inhibitor CP-316,819 ([R-R*,S*]-5-chloro-N-[2-hydroxy-3-(methoxy--methylamino)-3-oxo-1-(phenylmet hyl)propyl]-1H-indole-2-carboxamide). J PharmacolExp Ther, 2007, 321(1):45-50.
[10] Bak LK, Schousboe A, Waagepetersen HS. The glutamate/GABA-glutamine cycle:aspects of transport, neurotransmitter homeostasis and ammonia transfer. J Neurochem,2006, 98(3):641-653.
[11] Miller BR, Dorner JL, Shou M, Sari Y, Barton SJ, Sengelaub DR, Kennedy RT, RebecGV. Up-regulation of GLT1 expression increases glutamate uptake and attenuates theHuntington's disease phenotype in the R6/2 mouse. Neuroscience, 2008, 153(1):329-337.
[12] Haberg A, Qu H, Saether O, Unsgard G, Haraldseth O, Sonnewald U. Differences inneurotransmitter synthesis and intermediary metabolism between glutamatergic andGABAergic neurons during 4 hours of middle cerebral artery occlusion in the rat: the roleof astrocytes in neuronal survival. J Cereb Blood Flow Metab, 2001, 21(12):1451-1463.
[13] Kawahara K, Kosugi T, Tanaka M, Nakajima T, Yamada T. Reversed operation ofglutamate transporter GLT-1 is crucial to the development of preconditioning-inducedischemic tolerance of neurons in neuron/astrocyte co-cultures. Glia, 2005, 49(3):349-359.
[14] Shleper M, Kartvelishvily E, Wolosker H. D-SerineD-Serine is the dominant endogenouscoagonist for NMDA receptor neurotoxicity in organotypic hippocampal slices. JNeurosci, 2005, 25(41):9413-9417.
[15] Pow DV, Hendrickson AE. Distribution of the glycine transporter glyt-1 in mammalianand nonmammalian retinae. Vis Neurosci, 1999, 16(2):231-239.
[16] Borden LA. GABA transporter heterogeneity: pharmacology and cellular localization.Neurochem Int, 1996, 29(4):335-356.
[17] Contreras JE, Sanchez HA, Veliz LP, Bukauskas FF, Bennett MV, Saez JC. Role ofconnexin-based gap junction channels and hemichannels in ischemia-induced cell deathin nervous tissue. Brain Res Brain Res Rev, 2004, 47(1-3):290-303.
[18] Vyskocil F, Kritz N, Bures J. Potassium-selective microelectrodes used for measuring theextracellular brain potassium during spreading depression and anoxic depolarization inrats. Brain Res, 1972, 39(1):255-259.
[19] Nakase T, Sohl G, Theis M, Willecke K, Naus CC. Increased apoptosis and inflammationafter focal brain ischemia in mice lacking connexin43 in astrocytes. Am J Pathol, 2004,164(6):2067-2075.
[20] Lin JH, Lou N, Kang N, Takano T, Hu F, Han X, Xu Q, Lovatt D, Torres A, Willecke K,Yang J, Kang J, Nedergaard M. A central role of connexin43 in hypoxic preconditioning.J Neurosci, 2008, 28(3):681-695.
[21] Papadopoulos MC, Verkman AS. Aquaporin-4 and brain edema. Pediatr Nephrol, 2007,22(6):778-784.
[22] Mizui T, Kinouchi H, Chan PH. Depletion of brain glutathione by buthionine sulfoximineenhances cerebral ischemic injury in rats. Am J Physiol, 1992, 262(2 Pt 2):H313-317.
[23] Tsacopoulos M, Magistretti PJ. Metabolic coupling between glia and neurons. J Neurosci,1996, 16(3):877-885.
[24] Swanson RA, Ying W, Kauppinen TM. Astrocyte influences on ischemic neuronal death.Curr Mol Med, 2004, 4(2):193-205.
[25] Wilson JX. Antioxidant defense of the brain: a role for astrocytes. Can J PhysiolPharmacol, 1997, 75(10-11):1149-1163.
[26] Mitsui A, Hamuro J, Nakamura H, Kondo N, Hirabayashi Y, Ishizaki-Koizumi S,Hirakawa T, Inoue T, Yodoi J. Overexpression of human thioredoxin in transgenic micecontrols oxidative stress and life span. Antioxid Redox Signal, 2002, 4(4):693-696.
[27] Wang H, Zhang YL, Tang XC, Feng HS, Hu G. Targeting ischemic stroke with a novelopener of ATP-sensitive potassium channels in the brain. Mol Pharmacol, 2004,66(5):1160-1168.
[28] Du L, Hickey RW, Bayir H, Watkins SC, Tyurin VA, Guo F, Kochanek PM, Jenkins LW,Ren J, Gibson G, Chu CT, Kagan VE, Clark RS. Starving neurons show sex difference inautophagy. J Biol Chem, 2009, 284(4):2383-2396.
[29] Wang X, Lee SR, Arai K, Lee SR, Tsuji K, Rebeck GW, Lo EH. Lipoproteinreceptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator.Nat Med, 2003, 9(10):1313-1317.
[30] Gu Z, Kaul M, Yan B, Kridel SJ, Cui J, Strongin A, Smith JW, Liddington RC, Lipton SA.S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death.Science, 2002, 297(5584):1186-1190.
[30] Asahi M, Wang X, Mori T, Sumii T, Jung JC, Moskowitz MA, Fini ME, Lo EH. Effectsof matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrierand white matter components after cerebral ischemia. J Neurosci, 2001,21(19):7724-7732.
[31] Ren X, Wang Y, Jones WK. TNF-alpha is required for late ischemic preconditioning butnot for remote preconditioning of trauma. J Surg Res, 2004, 121(1):120-129.
[32] Pradillo JM, Hurtado O, Romera C, Cardenas A, Fernandez-Tome P, Alonso-Escolano D,Lorenzo P, Moro MA, Lizasoain I. TNFR1 mediates increased neuronal membraneEAAT3 expression after in vivo cerebral ischemic preconditioning. Neuroscience, 2006,138(4):1171-1178.
[33] Morganti-Kossmann MC, Kossmann T, Brandes ME, Mergenhagen SE, Wahl SM.Autocrine and paracrine regulation of astrocyte function by transforming growthfactor-beta. J Neuroimmunol, 1992, 39(1-2):163-173.
[34] Henrich-Noack P, Prehn JH, Krieglstein J. TGF-beta 1 protects hippocampal neuronsagainst degeneration caused by transient global ischemia. Dose-response relationship andpotential neuroprotective mechanisms. Stroke, 1996, 27(9):1609-1614; discussion 1615.
[35] Trendelenburg G, Dirnagl U. Neuroprotective role of astrocytes in cerebral ischemia:focus on ischemic preconditioning. Glia, 2005, 50(4):307-320.
[36] Bruccoleri A, Harry GJ. Chemical-induced hippocampal neurodegeneration andelevations in TNFalpha, TNFbeta, IL-1alpha, IP-10, and MCP-1 mRNA in osteopetrotic(op/op) mice. J Neurosci Res, 2000, 62(1):146-155.
[37] Hughes PM, Allegrini PR, Rudin M, Perry VH, Mir AK, Wiessner C. Monocytechemoattractant protein-1 deficiency is protective in a murine stroke model. J CerebBlood Flow Metab, 2002, 22(3):308-317.
[38] Iadecola C. Bright and dark sides of nitric oxide in ischemic brain injury. TrendsNeurosci, 1997, 20(3):132-139.
[39] Lo EH, Dalkara T, Moskowitz MA. Mechanisms, challenges and opportunities in stroke.Nat Rev Neurosci, 2003, 4(5):399-415.
[40] Kapinya KJ, Lowl D, Futterer C, Maurer M, Waschke KF, Isaev NK, Dirnagl U.Tolerance against ischemic neuronal injury can be induced by volatile anesthetics and isinducible NO synthase dependent. Stroke, 2002, 33(7):1889-1898.
[41] Muchowski PJ, Wacker JL. Modulation of neurodegeneration by molecular chaperones.Nat Rev Neurosci, 2005, 6(1):11-22.
[42] Mariucci G, Tantucci M, Giuditta A, Ambrosini MV. Permanent brain ischemia inducesmarked increments in hsp72 expression and local protein synthesis in synapses of theischemic hemisphere. Neurosci Lett, 2007, 415(1):77-80.
[43] Bechtold DA, Brown IR. Induction of Hsp27 and Hsp32 stress proteins and vimentin inglial cells of the rat hippocampus following hyperthermia. Neurochem Res, 2003,28(8):1163-1173.
[44] Blochl A, Thoenen H. Characterization of nerve growth factor (NGF) release fromhippocampal neurons: evidence for a constitutive and an unconventionalsodium-dependent regulated pathway. Eur J Neurosci, 1995, 7(6):1220-1228.
[45] Almeida RD, Manadas BJ, Melo CV, Gomes JR, Mendes CS, Graos MM, Carvalho RF,Carvalho AP, Duarte CB. Neuroprotection by BDNF against glutamate-induced apoptoticcell death is mediated by ERK and PI3-kinase pathways. Cell Death Differ, 2005,12(10):1329-1343.
[46] Kurozumi K, Nakamura K, Tamiya T, Kawano Y, Kobune M, Hirai S, Uchida H, SasakiK, Ito Y, Kato K, Honmou O, Houkin K, Date I, Hamada H. BDNF gene-modifiedmesenchymal stem cells promote functional recovery and reduce infarct size in the ratmiddle cerebral artery occlusion model. Mol Ther, 2004, 9(2):189-197.
[47] Geremia NM, Pettersson LM, Hasmatali JC, Hryciw T, Danielsen N, Schreyer DJ, VergeVM. Endogenous BDNF regulates induction of intrinsic neuronal growth programs ininjured sensory neurons. Exp Neurol, 2009.
[48] Chu LF, Wang WT, Ghanta VK, Lin CH, Chiang YY, Hsueh CM. Ischemic braincell-derived conditioned medium protects astrocytes against ischemia throughGDNF/ERK/NF-kB signaling pathway. Brain Res, 2008.
[49] Yamagata K, Tagami M, Ikeda K, Tsumagari S, Yamori Y, Nara Y. Differential regulationof glial cell line-derived neurotrophic factor (GDNF) mRNA expression during hypoxiaand reoxygenation in astrocytes isolated from stroke-prone spontaneously hypertensiverats. Glia, 2002, 37(1):1-7.
[50] Mattson MP. Glutamate and neurotrophic factors in neuronal plasticity and disease. AnnN Y Acad Sci, 2008, 1144:97-112.
[51] Vangeison G, Rempe DA. The Janus-Faced Effects of Hypoxia on Astrocyte Function.Neuroscientist, 2009.
[52] Blanco M, Lizasoain I, Sobrino T, Vivancos J, Castillo J. Ischemic preconditioning: anovel target for neuroprotective therapy. Cerebrovasc Dis, 2006, 21 Suppl 2:38-47.
[53] Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involvescross-talk between Jak2 and NF-kappaB signalling cascades. Nature, 2001,412(6847):641-647.
[54] Abdel-Mageed AB, Zhao F, Rider BJ, Agrawal KC. Erythropoietin-inducedmetallothionein gene expression: role in proliferation of K562 cells. Exp Biol Med(Maywood), 2003, 228(9):1033-1039.
[55] Matsui T, Mori T, Tateishi N, Kagamiishi Y, Satoh S, Katsube N, Morikawa E, MorimotoT, Ikuta F, Asano T. Astrocytic activation and delayed infarct expansion after permanentfocal ischemia in rats. Part I: enhanced astrocytic synthesis of s-100beta in the periinfarctarea precedes delayed infarct expansion. J Cereb Blood Flow Metab, 2002,22(6):711-722.
[56] Song H, Stevens CF, Gage FH. Astroglia induce neurogenesis from adult neural stemcells. Nature, 2002, 417(6884):39-44.
[57] Tekkok SB, Brown AM, Westenbroek R, Pellerin L, Ransom BR. Transfer ofglycogen-derived lactate from astrocytes to axons via specific monocarboxylatetransporters supports mouse optic nerve activity. J Neurosci Res, 2005, 81(5):644-652.
[58] Beck H, Semisch M, Culmsee C, Plesnila N, Hatzopoulos AK. Egr-1 regulates expressionof the glial scar component phosphacan in astrocytes after experimental stroke. Am JPathol, 2008, 173(1):77-92.
[2] Lo EH. Experimental models, neurovascular mechanisms and translational issues instroke research. Br J Pharmacol, 2008, 153 Suppl 1:S396-405.
[3] Lo EH, Dalkara T, Moskowitz MA. Mechanisms, challenges and opportunities in stroke.Nat Rev Neurosci, 2003, 4(5):399-415.
[4] Nakase T, Fushiki S, Naus CC. Astrocytic gap junctions composed of connexin43 reduceapoptotic neuronal damage in cerebral ischemia. Stroke, 2003, 34(8):1987-1993.
[5] Dyker AG, Lees KR. The rationale for new therapies in acute ischaemic stroke. J ClinPharm Ther, 1996, 21(6):377-391.
[6] Rosell A, Ortega-Aznar A, Alvarez-Sabin J, Fernandez-Cadenas I, Ribo M, Molina CA,Lo EH, Montaner J. Increased brain expression of matrix metalloproteinase-9 afterischemic and hemorrhagic human stroke. Stroke, 2006, 37(6):1399-1406.
[7] Mayer TE, Hamann GF, Baranczyk J, Rosengarten B, Klotz E, Wiesmann M, Missler U,Schulte-Altedorneburg G, Brueckmann HJ. Dynamic CT perfusion imaging of acutestroke. AJNR Am J Neuroradiol, 2000, 21(8):1441-1449.
[8] Richard Green A, Odergren T, Ashwood T. Animal models of stroke: do they have valuefor discovering neuroprotective agents? Trends Pharmacol Sci, 2003, 24(8):402-408.
[9] Smith WS. Pathophysiology of focal cerebral ischemia: a therapeutic perspective. J VascInterv Radiol, 2004, 15(1 Pt 2):S3-12.
[10] MacManus JP, Buchan AM. Apoptosis after experimental stroke: fact or fashion? JNeurotrauma, 2000, 17(10):899-914.
[11] Unal-Cevik I, Kilinc M, Can A, Gursoy-Ozdemir Y, Dalkara T. Apoptotic and necroticdeath mechanisms are concomitantly activated in the same cell after cerebral ischemia.Stroke, 2004, 35(9):2189-2194.
[12] Durukan A, Tatlisumak T. Acute ischemic stroke: overview of major experimental rodentmodels, pathophysiology, and therapy of focal cerebral ischemia. Pharmacol BiochemBehav, 2007, 87(1):179-197.
[13] Siesjo BK, Katsura K, Kristian T. Acidosis-related damage. Adv Neurol, 1996,71:209-233; discussion 234-206.
[14] Huang Y, McNamara JO. Ischemic stroke: "acidotoxicity" is a perpetrator. Cell, 2004,118(6):665-666.
[15] Mergenthaler P, Dirnagl U, Meisel A. Pathophysiology of stroke: lessons from animalmodels. Metab Brain Dis, 2004, 19(3-4):151-167.
[16] Phan TG, Wright PM, Markus R, Howells DW, Davis SM, Donnan GA. Salvaging theischaemic penumbra: more than just reperfusion? Clin Exp Pharmacol Physiol, 2002,29(1-2):1-10.
[17] Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic stroke: an integratedview. Trends Neurosci, 1999, 22(9):391-397.
[18] Hartings JA, Williams AJ, Tortella FC. Occurrence of nonconvulsive seizures, periodicepileptiform discharges, and intermittent rhythmic delta activity in rat focal ischemia.Exp Neurol, 2003, 179(2):139-149.
[19] Dreier JP, Woitzik J, Fabricius M, Bhatia R, Major S, Drenckhahn C, Lehmann TN,Sarrafzadeh A, Willumsen L, Hartings JA, Sakowitz OW, Seemann JH, Thieme A,Lauritzen M, Strong AJ. Delayed ischaemic neurological deficits after subarachnoidhaemorrhage are associated with clusters of spreading depolarizations. Brain, 2006,129(Pt 12):3224-3237.
[20] Dijkhuizen RM, Beekwilder JP, van der Worp HB, Berkelbach van der Sprenkel JW,Tulleken KA, Nicolay K. Correlation between tissue depolarizations and damage in focalischemic rat brain. Brain Res, 1999, 840(1-2):194-205.
[21] Mackert BM. The discovery of slowness--recent progress in DC-MEG research. NeurolClin Neurophysiol, 2004, 2004:41.
[22] McIlvoy LH. The effect of hypothermia and hyperthermia on acute brain injury. AACNClin Issues, 2005, 16(4):488-500.
[23] Garcia JH, Liu KF, Ho KL. Neuronal necrosis after middle cerebral artery occlusion inWistar rats progresses at different time intervals in the caudoputamen and the cortex.Stroke, 1995, 26(4):636-642; discussion 643.
[24] Yang GY, Gong C, Qin Z, Liu XH, Lorris Betz A. Tumor necrosis factor alpha expressionproduces increased blood-brain barrier permeability following temporary focal cerebralischemia in mice. Brain Res Mol Brain Res, 1999, 69(1):135-143.
[25] Emerich DF, Dean RL, 3rd, Bartus RT. The role of leukocytes following cerebralischemia: pathogenic variable or bystander reaction to emerging infarct? Exp Neurol,2002, 173(1):168-181.
[26] Danton GH, Dietrich WD. Inflammatory mechanisms after ischemia and stroke. JNeuropathol Exp Neurol, 2003, 62(2):127-136.
[27] Schilling M, Besselmann M, Leonhard C, Mueller M, Ringelstein EB, Kiefer R.Microglial activation precedes and predominates over macrophage infiltration in transientfocal cerebral ischemia: a study in green fluorescent protein transgenic bone marrowchimeric mice. Exp Neurol, 2003, 183(1):25-33.
[28] Dienel GA, Hertz L. Astrocytic contributions to bioenergetics of cerebral ischemia. Glia,2005, 50(4):362-388.
[29] Trendelenburg G, Dirnagl U. Neuroprotective role of astrocytes in cerebral ischemia:focus on ischemic preconditioning. Glia, 2005, 50(4):307-320.
[30] Allen GV, Cheung RT, Cechetto DF. Neurochemical changes following occlusion of themiddle cerebral artery in rats. Neuroscience, 1995, 68(4):1037-1050.
[31] Matsumoto K, Graf R, Rosner G, Taguchi J, Heiss WD. Elevation of neuroactivesubstances in the cortex of cats during prolonged focal ischemia. J Cereb Blood FlowMetab, 1993, 13(4):586-594.
[32] Fisher M, Schaebitz W. An overview of acute stroke therapy: past, present, and future.Arch Intern Med, 2000, 160(21):3196-3206.
[33] Sugawara T, Fujimura M, Noshita N, Kim GW, Saito A, Hayashi T, Narasimhan P, MaierCM, Chan PH. Neuronal death/survival signaling pathways in cerebral ischemia.NeuroRx, 2004, 1(1):17-25.
[34] Schaller B, Graf R. Cerebral ischemia and reperfusion: the pathophysiologic concept as abasis for clinical therapy. J Cereb Blood Flow Metab, 2004, 24(4):351-371.
[35] Love S. Apoptosis and brain ischaemia. Prog Neuropsychopharmacol Biol Psychiatry,2003, 27(2):267-282.
[36] Guglielmo MA, Chan PT, Cortez S, Stopa EG, McMillan P, Johanson CE, Epstein M,Doberstein CE. The temporal profile and morphologic features of neuronal death inhuman stroke resemble those observed in experimental forebrain ischemia: the potentialrole of apoptosis. Neurol Res, 1998, 20(4):283-296.
[37] Love S, Barber R, Wilcock GK. Neuronal death in brain infarcts in man. NeuropatholAppl Neurobiol, 2000, 26(1):55-66.
[38] Sairanen T, Karjalainen-Lindsberg ML, Paetau A, Ijas P, Lindsberg PJ. Apoptosisdominant in the periinfarct area of human ischaemic stroke--a possible target ofantiapoptotic treatments. Brain, 2006, 129(Pt 1):189-199.
[39] Iadecola C. Neurovascular regulation in the normal brain and in Alzheimer's disease. NatRev Neurosci, 2004, 5(5):347-360.
[40] Zlokovic BV. Neurovascular mechanisms of Alzheimer's neurodegeneration. TrendsNeurosci, 2005, 28(4):202-208.
[41] Abbott NJ, Ronnback L, Hansson E. Astrocyte-endothelial interactions at the blood-brainbarrier. Nat Rev Neurosci, 2006, 7(1):41-53.
[42] Wang YF, Tsirka SE, Strickland S, Stieg PE, Soriano SG, Lipton SA. Tissue plasminogenactivator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type andtPA-deficient mice. Nat Med, 1998, 4(2):228-231.
[43] Nicole O, Docagne F, Ali C, Margaill I, Carmeliet P, MacKenzie ET, Vivien D, Buisson A.The proteolytic activity of tissue-plasminogen activator enhances NMDAreceptor-mediated signaling. Nat Med, 2001, 7(1):59-64.
[44] Chen ZL, Strickland S. Neuronal death in the hippocampus is promoted byplasmin-catalyzed degradation of laminin. Cell, 1997, 91(7):917-925.
[45] Melchor JP, Strickland S. Tissue plasminogen activator in central nervous systemphysiology and pathology. Thromb Haemost, 2005, 93(4):655-660.
[46] Wang X, Lee SR, Arai K, Lee SR, Tsuji K, Rebeck GW, Lo EH. Lipoproteinreceptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator.Nat Med, 2003, 9(10):1313-1317.
[47] Lo EH, Broderick JP, Moskowitz MA. tPA and proteolysis in the neurovascular unit.Stroke, 2004, 35(2):354-356.
[48] Aoki T, Sumii T, Mori T, Wang X, Lo EH. Blood-brain barrier disruption and matrixmetalloproteinase-9 expression during reperfusion injury: mechanical versus embolicfocal ischemia in spontaneously hypertensive rats. Stroke, 2002, 33(11):2711-2717.
[49] Sumii T, Lo EH. Involvement of matrix metalloproteinase in thrombolysis-associatedhemorrhagic transformation after embolic focal ischemia in rats. Stroke, 2002,33(3):831-836.
[50] Tsuji K, Aoki T, Tejima E, Arai K, Lee SR, Atochin DN, Huang PL, Wang X, Montaner J,Lo EH. Tissue plasminogen activator promotes matrix metalloproteinase-9 upregulationafter focal cerebral ischemia. Stroke, 2005, 36(9):1954-1959.
[51] Montaner J, Fernandez-Cadenas I, Molina CA, Monasterio J, Arenillas JF, Ribo M,Quintana M, Chacon P, Andreu AL, Alvarez-Sabin J. Safety profile of tissue plasminogenactivator treatment among stroke patients carrying a common polymorphism (C-1562T)in the promoter region of the matrix metalloproteinase-9 gene. Stroke, 2003,34(12):2851-2855.
[52] Ning M, Furie KL, Koroshetz WJ, Lee H, Barron M, Lederer M, Wang X, Zhu M,Sorensen AG, Lo EH, Kelly PJ. Association between tPA therapy and raised early matrixmetalloproteinase-9 in acute stroke. Neurology, 2006, 66(10):1550-1555.
[53] Seino S, Miki T. Physiological and pathophysiological roles of ATP-sensitive K+channels. Prog Biophys Mol Biol, 2003, 81(2):133-176.
[54] Nichols CG. KATP channels as molecular sensors of cellular metabolism. Nature, 2006,440(7083):470-476.
[55] Dunn-Meynell AA, Rawson NE, Levin BE. Distribution and phenotype of neuronscontaining the ATP-sensitive K+ channel in rat brain. Brain Res, 1998, 814(1-2):41-54.
[56] Thomzig A, Laube G, Pruss H, Veh RW. Pore-forming subunits of K-ATP channels,Kir6.1 and Kir6.2, display prominent differences in regional and cellular distribution inthe rat brain. J Comp Neurol, 2005, 484(3):313-330.
[57] Wang Y, Haider HK, Ahmad N, Ashraf M. Mechanisms by which K(ATP) channelopeners produce acute and delayed cardioprotection. Vascul Pharmacol, 2005,42(5-6):253-264.
[58] Rodrigo GC, Standen NB. ATP-sensitive potassium channels. Curr Pharm Des, 2005,11(15):1915-1940.
[59] Raval AP, Dave KR, DeFazio RA, Perez-Pinzon MA. epsilonPKC phosphorylates themitochondrial K(+) (ATP) channel during induction of ischemic preconditioning in therat hippocampus. Brain Res, 2007, 1184:345-353.
[60] Gaspar T, Snipes JA, Busija AR, Kis B, Domoki F, Bari F, Busija DW. ROS-independentpreconditioning in neurons via activation of mitoK(ATP) channels by BMS-191095. JCereb Blood Flow Metab, 2008, 28(6):1090-1103.
[61] Kis B, Rajapakse NC, Snipes JA, Nagy K, Horiguchi T, Busija DW. Diazoxide inducesdelayed pre-conditioning in cultured rat cortical neurons. J Neurochem, 2003,87(4):969-980.
[62] Yao Z, Gross GJ. A comparison of adenosine-induced cardioprotection and ischemicpreconditioning in dogs. Efficacy, time course, and role of KATP channels. Circulation,1994, 89(3):1229-1236.
[63] Sargent CA, Sleph PG, Dzwonczyk S, Normandin D, Antonaccio MJ, Grover GJ.Cardioprotective effects of the cyanoguanidine potassium channel opener P-1075. JCardiovasc Pharmacol, 1993, 22(4):564-570.
[64] Sun XL, Zeng XN, Zhou F, Dai CP, Ding JH, Hu G. KATP channel openers facilitateglutamate uptake by GluTs in rat primary cultured astrocytes. Neuropsychopharmacology,2008, 33(6):1336-1342.
[65] Zhou F, Wu JY, Sun XL, Yao HH, Ding JH, Hu G. Iptakalim alleviates rotenone-induceddegeneration of dopaminergic neurons through inhibiting microglia-mediatedneuroinflammation. Neuropsychopharmacology, 2007, 32(12):2570-2580.
[66] Ikonomidou C, Turski L. Why did NMDA receptor antagonists fail clinical trials forstroke and traumatic brain injury? Lancet Neurol, 2002, 1(6):383-386.
[67] Plosker GL, Lyseng-Williamson KA. Memantine: a pharmacoeconomic review of its usein moderate-to-severe Alzheimer's disease. Pharmacoeconomics, 2005, 23(2):193-206.
[68] Orgogozo JM, Rigaud AS, Stoffler A, Mobius HJ, Forette F. Efficacy and safety ofmemantine in patients with mild to moderate vascular dementia: a randomized,placebo-controlled trial (MMM 300). Stroke, 2002, 33(7):1834-1839.
[69] Babenko AP, Aguilar-Bryan L, Bryan J. A view of sur/KIR6.X, KATP channels. AnnuRev Physiol, 1998, 60:667-687.
[70] Wang H, Zhang YL, Tang XC, Feng HS, Hu G. Targeting ischemic stroke with a novelopener of ATP-sensitive potassium channels in the brain. Mol Pharmacol, 2004,66(5):1160-1168.
[71] Sun HS, Feng ZP, Miki T, Seino S, French RJ. Enhanced neuronal damage after ischemicinsults in mice lacking Kir6.2-containing ATP-sensitive K+ channels. J Neurophysiol,2006, 95(4):2590-2601.
[72] Heron-Milhavet L, Xue-Jun Y, Vannucci SJ, Wood TL, Willing LB, Stannard B,Hernandez-Sanchez C, Mobbs C, Virsolvy A, LeRoith D. Protection againsthypoxic-ischemic injury in transgenic mice overexpressing Kir6.2 channel pore inforebrain. Mol Cell Neurosci, 2004, 25(4):585-593.
[73] Brown RC, Morris AP, O'Neil RG. Tight junction protein expression and barrierproperties of immortalized mouse brain microvessel endothelial cells. Brain Res, 2007,1130(1):17-30.
[74] Hartmann C, Zozulya A, Wegener J, Galla HJ. The impact of glia-derived extracellularmatrices on the barrier function of cerebral endothelial cells: an in vitro study. Exp CellRes, 2007, 313(7):1318-1325.
[75] Lin JH, Lou N, Kang N, Takano T, Hu F, Han X, Xu Q, Lovatt D, Torres A, Willecke K,Yang J, Kang J, Nedergaard M. A central role of connexin43 in hypoxic preconditioning.J Neurosci, 2008, 28(3):681-695.
[76] Panickar KS, Norenberg MD. Astrocytes in cerebral ischemic injury: morphological andgeneral considerations. Glia, 2005, 50(4):287-298.
[77] Bondjers C, He L, Takemoto M, Norlin J, Asker N, Hellstrom M, Lindahl P, Betsholtz C.Microarray analysis of blood microvessels from PDGF-B and PDGF-Rbeta mutant miceidentifies novel markers for brain pericytes. Faseb J, 2006, 20(10):1703-1705.
[78] Cuzner ML, Opdenakker G. Plasminogen activators and matrix metalloproteases,mediators of extracellular proteolysis in inflammatory demyelination of the centralnervous system. J Neuroimmunol, 1999, 94(1-2):1-14.
[79] Yong VW, Krekoski CA, Forsyth PA, Bell R, Edwards DR. Matrix metalloproteinasesand diseases of the CNS. Trends Neurosci, 1998, 21(2):75-80.
[80] Zhao BQ, Wang S, Kim HY, Storrie H, Rosen BR, Mooney DJ, Wang X, Lo EH. Role ofmatrix metalloproteinases in delayed cortical responses after stroke. Nat Med, 2006,12(4):441-445.
[81] Yamashita T, Kamiya T, Deguchi K, Inaba T, Zhang H, Shang J, Miyazaki K, Ohtsuka A,Katayama Y, Abe K. Dissociation and protection of the neurovascular unit afterthrombolysis and reperfusion in ischemic rat brain. J Cereb Blood Flow Metab, 2009,29(4):715-725.
[82] Asahi M, Wang X, Mori T, Sumii T, Jung JC, Moskowitz MA, Fini ME, Lo EH. Effectsof matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrierand white matter components after cerebral ischemia. J Neurosci, 2001,21(19):7724-7732.
[83] Papadopoulos MC, Verkman AS. Aquaporin-4 and brain edema. Pediatr Nephrol, 2007,22(6):778-784.
[84] Gary DS, Mattson MP. Integrin signaling via the PI3-kinase-Akt pathway increasesneuronal resistance to glutamate-induced apoptosis. J Neurochem, 2001,76(5):1485-1496.
[85] Gu Z, Kaul M, Yan B, Kridel SJ, Cui J, Strongin A, Smith JW, Liddington RC, Lipton SA.S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death.Science, 2002, 297(5584):1186-1190.
[86] Ziff EB. Enlightening the postsynaptic density. Neuron, 1997, 19(6):1163-1174.
[87] Hu BR, Park M, Martone ME, Fischer WH, Ellisman MH, Zivin JA. Assembly ofproteins to postsynaptic densities after transient cerebral ischemia. J Neurosci, 1998,18(2):625-633.
[88] Ramamurthy S, Ronnett GV. Developing a head for energy sensing: AMP-activatedprotein kinase as a multifunctional metabolic sensor in the brain. J Physiol, 2006, 574(Pt1):85-93.
[89] Ronnett GV, Ramamurthy S, Kleman AM, Landree LE, Aja S. AMPK in the brain: itsroles in energy balance and neuroprotection. J Neurochem, 2009, 109 Suppl 1:17-23.
[90] Li J, McCullough LD. Effects of AMP-activated protein kinase in cerebral ischemia. JCereb Blood Flow Metab, 30(3):480-492.
[91] Culmsee C, Monnig J, Kemp BE, Mattson MP. AMP-activated protein kinase is highlyexpressed in neurons in the developing rat brain and promotes neuronal survivalfollowing glucose deprivation. J Mol Neurosci, 2001, 17(1):45-58.
[92] Garcia-Gil M, Pesi R, Perna S, Allegrini S, Giannecchini M, Camici M, Tozzi MG.5'-aminoimidazole-4-carboxamide riboside induces apoptosis in human neuroblastomacells. Neuroscience, 2003, 117(4):811-820.
[93] Du L, Hickey RW, Bayir H, Watkins SC, Tyurin VA, Guo F, Kochanek PM, Jenkins LW,Ren J, Gibson G, Chu CT, Kagan VE, Clark RS. Starving neurons show sex difference inautophagy. J Biol Chem, 2009, 284(4):2383-2396.
[94] Wang W, Guan KL. AMP-activated protein kinase and cancer. Acta Physiol (Oxf), 2009,196(1):55-63.
[95] Wolosker H. NMDA receptor regulation by D-SerineD-Serine: new findings andperspectives. Mol Neurobiol, 2007, 36(2):152-164.
[96] Katsuki H, Nonaka M, Shirakawa H, Kume T, Akaike A. Endogenous D-SerineD-Serineis involved in induction of neuronal death by N-methyl-D-aspartate and simulatedischemia in rat cerebrocortical slices. J Pharmacol Exp Ther, 2004, 311(2):836-844.
[97] Billard JM. D-SerineD-Serine signalling as a prominent determinant of neuronal-glialdialogue in the healthy and diseased brain. J Cell Mol Med, 2008, 12(5B):1872-1884.
[98] Fatemi SH, Folsom TD, Reutiman TJ, Pandian T, Braun NN, Haug K. Chronicpsychotropic drug treatment causes differential expression of connexin43 and GFAP infrontal cortex of rats. Schizophr Res, 2008, 104(1-3):127-134.
[99] Jain SK, Schuessler RB, Saffitz JE. Mechanisms of delayed electrical uncoupling inducedby ischemic preconditioning. Circ Res, 2003, 92(10):1138-1144.
[100] Liu T, Clark RK, McDonnell PC, Young PR, White RF, Barone FC, Feuerstein GZ.Tumor necrosis factor-alpha expression in ischemic neurons. Stroke, 1994,25(7):1481-1488.
[101] Takuma K, Baba A, Matsuda T. Astrocyte apoptosis: implications for neuroprotection.Prog Neurobiol, 2004, 72(2):111-127.
[102] Kaushal V, Schlichter LC. Mechanisms of microglia-mediated neurotoxicity in a newmodel of the stroke penumbra. J Neurosci, 2008, 28(9):2221-2230.
[103] Hazell AS. Excitotoxic mechanisms in stroke: an update of concepts and treatmentstrategies. Neurochem Int, 2007, 50(7-8):941-953.
[104] Waxman EA, Lynch DR. N-methyl-D-aspartate receptor subtypes: multiple roles inexcitotoxicity and neurological disease. Neuroscientist, 2005, 11(1):37-49.
[105] Muir KW. Glutamate-based therapeutic approaches: clinical trials with NMDAantagonists. Curr Opin Pharmacol, 2006, 6(1):53-60.
[106] Rabey JM, Nissipeanu P, Korczyn AD. Efficacy of memantine, an NMDA receptorantagonist, in the treatment of Parkinson's disease. J Neural Transm Park Dis DementSect, 1992, 4:277-282.
[107] Miguel-Hidalgo JJ, Alvarez XA, Cacabelos R, Quack G. Neuroprotection by memantineagainst neurodegeneration induced by beta-amyloid(1-40). Brain Res, 2002,958(1):210-221.
[108] Ditzler K. Efficacy and tolerability of memantine in patients with dementia syndrome. Adouble-blind, placebo controlled trial. Arzneimittelforschung, 1991, 41(8):773-780.
[109] Sonkusare SK, Kaul CL, Ramarao P. Dementia of Alzheimer's disease and otherneurodegenerative disorders--memantine, a new hope. Pharmacol Res, 2005, 51(1):1-17.
[110] Volbracht C, van Beek J, Zhu C, Blomgren K, Leist M. Neuroprotective properties ofmemantine in different in vitro and in vivo models of excitotoxicity. Eur J Neurosci, 2006,23(10):2611-2622.
[111] Gilling KE, Jatzke C, Hechenberger M, Parsons CG. Potency, voltage-dependency,agonist concentration-dependency, blocking kinetics and partial untrapping of theuncompetitive N-methyl-D-aspartate (NMDA) channel blocker memantine at humanNMDA (GluN1/GluN2A) receptors. Neuropharmacology, 2009, 56(5):866-875.
[112] Netzer R, Bigalke H. Memantine reduces repetitive action potential firing in spinal cordnerve cell cultures. Eur J Pharmacol, 1990, 186(2-3):149-155.
[113] Reiser G, Binmoller FJ, Koch R. Memantine (1-amino-3,5-dimethyladamantane) blocksthe serotonin-induced depolarization response in a neuronal cell line. Brain Res, 1988,443(1-2):338-344.
[114] Giustizieri M, Cucchiaroni ML, Guatteo E, Bernardi G, Mercuri NB, Berretta N.Memantine inhibits ATP-dependent K+ conductances in dopamine neurons of the ratsubstantia nigra pars compacta. J Pharmacol Exp Ther, 2007, 322(2):721-729.
[115] Liu C, Lin N, Wu B, Qiu Y. Neuroprotective effect of memantine combined withtopiramate in hypoxic-ischemic brain injury. Brain Res, 2009, 1282:173-182.
[116] Choi DW, Rothman SM. The role of glutamate neurotoxicity in hypoxic-ischemicneuronal death. Annu Rev Neurosci, 1990, 13:171-182.
[117] Yang XL. Characterization of receptors for glutamate and GABA in retinal neurons. ProgNeurobiol, 2004, 73(2):127-150.
[118] Juurlink BH, Sweeney MI. Mechanisms that result in damage during and followingcerebral ischemia. Neurosci Biobehav Rev, 1997, 21(2):121-128.
[119] Marvanova M, Lakso M, Pirhonen J, Nawa H, Wong G, Castren E. The neuroprotectiveagent memantine induces brain-derived neurotrophic factor and trkB receptor expressionin rat brain. Mol Cell Neurosci, 2001, 18(3):247-258.
[120] Rossi C, Angelucci A, Costantin L, Braschi C, Mazzantini M, Babbini F, Fabbri ME,Tessarollo L, Maffei L, Berardi N, Caleo M. Brain-derived neurotrophic factor (BDNF) isrequired for the enhancement of hippocampal neurogenesis following environmentalenrichment. Eur J Neurosci, 2006, 24(7):1850-1856.
[121] Witt A, Macdonald N, Kirkpatrick P. Memantine hydrochloride. Nat Rev Drug Discov,2004, 3(2):109-110.
[122] Johnson JW, Kotermanski SE. Mechanism of action of memantine. Curr Opin Pharmacol,2006, 6(1):61-67.
[123] Gascon S, Sobrado M, Roda JM, Rodriguez Pena A, Diaz-Guerra M. Excitotoxicity andfocal cerebral ischemia induce truncation of the nr2a and nr2b subunits of the nmdareceptor and cleavage of the scaffolding protein psd-95. Mol Psychiatry.2008,13(1):99-114
[1] Ludwin SK, Kosek JC, Eng LF. The topographical distribution of S-100 and GFAproteins in the adult rat brain: an immunohistochemical study using horseradishperoxidase-labelled antibodies. J Comp Neurol, 1976, 165(2):197-207.
[2] Mayer TE, Hamann GF, Baranczyk J, Rosengarten B, Klotz E, Wiesmann M, Missler U,Schulte-Altedorneburg G, Brueckmann HJ. Dynamic CT perfusion imaging of acutestroke. AJNR Am J Neuroradiol, 2000, 21(8):1441-1449.
[3] Walz W. Role of astrocytes in the clearance of excess extracellular potassium.Neurochem Int, 2000, 36(4-5):291-300.
[4] Brannen CL, Sugaya K. In vitro differentiation of multipotent human neural progenitorsin serum-free medium. Neuroreport, 2000, 11(5):1123-1128.
[5] Benarroch EE. Aquaporin-4, homeostasis, and neurologic disease. Neurology, 2007,69(24):2266-2268.
[6] Drukarch B, Schepens E, Stoof JC, Langeveld CH, Van Muiswinkel FL.Astrocyte-enhanced neuronal survival is mediated by scavenging of extracellular reactiveoxygen species. Free Radic Biol Med, 1998, 25(2):217-220.
[7] Maragakis NJ, Rothstein JD. Mechanisms of Disease: astrocytes in neurodegenerativedisease. Nat Clin Pract Neurol, 2006, 2(12):679-689.
[8] Ginsberg MD, Pulsinelli WA. The ischemic penumbra, injury thresholds, and thetherapeutic window for acute stroke. Ann Neurol, 1994, 36(4):553-554.
[9] Suh SW, Bergher JP, Anderson CM, Treadway JL, Fosgerau K, Swanson RA. Astrocyteglycogen sustains neuronal activity during hypoglycemia: studies with the glycogenphosphorylase inhibitor CP-316,819 ([R-R*,S*]-5-chloro-N-[2-hydroxy-3-(methoxy--methylamino)-3-oxo-1-(phenylmet hyl)propyl]-1H-indole-2-carboxamide). J PharmacolExp Ther, 2007, 321(1):45-50.
[10] Bak LK, Schousboe A, Waagepetersen HS. The glutamate/GABA-glutamine cycle:aspects of transport, neurotransmitter homeostasis and ammonia transfer. J Neurochem,2006, 98(3):641-653.
[11] Miller BR, Dorner JL, Shou M, Sari Y, Barton SJ, Sengelaub DR, Kennedy RT, RebecGV. Up-regulation of GLT1 expression increases glutamate uptake and attenuates theHuntington's disease phenotype in the R6/2 mouse. Neuroscience, 2008, 153(1):329-337.
[12] Haberg A, Qu H, Saether O, Unsgard G, Haraldseth O, Sonnewald U. Differences inneurotransmitter synthesis and intermediary metabolism between glutamatergic andGABAergic neurons during 4 hours of middle cerebral artery occlusion in the rat: the roleof astrocytes in neuronal survival. J Cereb Blood Flow Metab, 2001, 21(12):1451-1463.
[13] Kawahara K, Kosugi T, Tanaka M, Nakajima T, Yamada T. Reversed operation ofglutamate transporter GLT-1 is crucial to the development of preconditioning-inducedischemic tolerance of neurons in neuron/astrocyte co-cultures. Glia, 2005, 49(3):349-359.
[14] Shleper M, Kartvelishvily E, Wolosker H. D-SerineD-Serine is the dominant endogenouscoagonist for NMDA receptor neurotoxicity in organotypic hippocampal slices. JNeurosci, 2005, 25(41):9413-9417.
[15] Pow DV, Hendrickson AE. Distribution of the glycine transporter glyt-1 in mammalianand nonmammalian retinae. Vis Neurosci, 1999, 16(2):231-239.
[16] Borden LA. GABA transporter heterogeneity: pharmacology and cellular localization.Neurochem Int, 1996, 29(4):335-356.
[17] Contreras JE, Sanchez HA, Veliz LP, Bukauskas FF, Bennett MV, Saez JC. Role ofconnexin-based gap junction channels and hemichannels in ischemia-induced cell deathin nervous tissue. Brain Res Brain Res Rev, 2004, 47(1-3):290-303.
[18] Vyskocil F, Kritz N, Bures J. Potassium-selective microelectrodes used for measuring theextracellular brain potassium during spreading depression and anoxic depolarization inrats. Brain Res, 1972, 39(1):255-259.
[19] Nakase T, Sohl G, Theis M, Willecke K, Naus CC. Increased apoptosis and inflammationafter focal brain ischemia in mice lacking connexin43 in astrocytes. Am J Pathol, 2004,164(6):2067-2075.
[20] Lin JH, Lou N, Kang N, Takano T, Hu F, Han X, Xu Q, Lovatt D, Torres A, Willecke K,Yang J, Kang J, Nedergaard M. A central role of connexin43 in hypoxic preconditioning.J Neurosci, 2008, 28(3):681-695.
[21] Papadopoulos MC, Verkman AS. Aquaporin-4 and brain edema. Pediatr Nephrol, 2007,22(6):778-784.
[22] Mizui T, Kinouchi H, Chan PH. Depletion of brain glutathione by buthionine sulfoximineenhances cerebral ischemic injury in rats. Am J Physiol, 1992, 262(2 Pt 2):H313-317.
[23] Tsacopoulos M, Magistretti PJ. Metabolic coupling between glia and neurons. J Neurosci,1996, 16(3):877-885.
[24] Swanson RA, Ying W, Kauppinen TM. Astrocyte influences on ischemic neuronal death.Curr Mol Med, 2004, 4(2):193-205.
[25] Wilson JX. Antioxidant defense of the brain: a role for astrocytes. Can J PhysiolPharmacol, 1997, 75(10-11):1149-1163.
[26] Mitsui A, Hamuro J, Nakamura H, Kondo N, Hirabayashi Y, Ishizaki-Koizumi S,Hirakawa T, Inoue T, Yodoi J. Overexpression of human thioredoxin in transgenic micecontrols oxidative stress and life span. Antioxid Redox Signal, 2002, 4(4):693-696.
[27] Wang H, Zhang YL, Tang XC, Feng HS, Hu G. Targeting ischemic stroke with a novelopener of ATP-sensitive potassium channels in the brain. Mol Pharmacol, 2004,66(5):1160-1168.
[28] Du L, Hickey RW, Bayir H, Watkins SC, Tyurin VA, Guo F, Kochanek PM, Jenkins LW,Ren J, Gibson G, Chu CT, Kagan VE, Clark RS. Starving neurons show sex difference inautophagy. J Biol Chem, 2009, 284(4):2383-2396.
[29] Wang X, Lee SR, Arai K, Lee SR, Tsuji K, Rebeck GW, Lo EH. Lipoproteinreceptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator.Nat Med, 2003, 9(10):1313-1317.
[30] Gu Z, Kaul M, Yan B, Kridel SJ, Cui J, Strongin A, Smith JW, Liddington RC, Lipton SA.S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death.Science, 2002, 297(5584):1186-1190.
[30] Asahi M, Wang X, Mori T, Sumii T, Jung JC, Moskowitz MA, Fini ME, Lo EH. Effectsof matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrierand white matter components after cerebral ischemia. J Neurosci, 2001,21(19):7724-7732.
[31] Ren X, Wang Y, Jones WK. TNF-alpha is required for late ischemic preconditioning butnot for remote preconditioning of trauma. J Surg Res, 2004, 121(1):120-129.
[32] Pradillo JM, Hurtado O, Romera C, Cardenas A, Fernandez-Tome P, Alonso-Escolano D,Lorenzo P, Moro MA, Lizasoain I. TNFR1 mediates increased neuronal membraneEAAT3 expression after in vivo cerebral ischemic preconditioning. Neuroscience, 2006,138(4):1171-1178.
[33] Morganti-Kossmann MC, Kossmann T, Brandes ME, Mergenhagen SE, Wahl SM.Autocrine and paracrine regulation of astrocyte function by transforming growthfactor-beta. J Neuroimmunol, 1992, 39(1-2):163-173.
[34] Henrich-Noack P, Prehn JH, Krieglstein J. TGF-beta 1 protects hippocampal neuronsagainst degeneration caused by transient global ischemia. Dose-response relationship andpotential neuroprotective mechanisms. Stroke, 1996, 27(9):1609-1614; discussion 1615.
[35] Trendelenburg G, Dirnagl U. Neuroprotective role of astrocytes in cerebral ischemia:focus on ischemic preconditioning. Glia, 2005, 50(4):307-320.
[36] Bruccoleri A, Harry GJ. Chemical-induced hippocampal neurodegeneration andelevations in TNFalpha, TNFbeta, IL-1alpha, IP-10, and MCP-1 mRNA in osteopetrotic(op/op) mice. J Neurosci Res, 2000, 62(1):146-155.
[37] Hughes PM, Allegrini PR, Rudin M, Perry VH, Mir AK, Wiessner C. Monocytechemoattractant protein-1 deficiency is protective in a murine stroke model. J CerebBlood Flow Metab, 2002, 22(3):308-317.
[38] Iadecola C. Bright and dark sides of nitric oxide in ischemic brain injury. TrendsNeurosci, 1997, 20(3):132-139.
[39] Lo EH, Dalkara T, Moskowitz MA. Mechanisms, challenges and opportunities in stroke.Nat Rev Neurosci, 2003, 4(5):399-415.
[40] Kapinya KJ, Lowl D, Futterer C, Maurer M, Waschke KF, Isaev NK, Dirnagl U.Tolerance against ischemic neuronal injury can be induced by volatile anesthetics and isinducible NO synthase dependent. Stroke, 2002, 33(7):1889-1898.
[41] Muchowski PJ, Wacker JL. Modulation of neurodegeneration by molecular chaperones.Nat Rev Neurosci, 2005, 6(1):11-22.
[42] Mariucci G, Tantucci M, Giuditta A, Ambrosini MV. Permanent brain ischemia inducesmarked increments in hsp72 expression and local protein synthesis in synapses of theischemic hemisphere. Neurosci Lett, 2007, 415(1):77-80.
[43] Bechtold DA, Brown IR. Induction of Hsp27 and Hsp32 stress proteins and vimentin inglial cells of the rat hippocampus following hyperthermia. Neurochem Res, 2003,28(8):1163-1173.
[44] Blochl A, Thoenen H. Characterization of nerve growth factor (NGF) release fromhippocampal neurons: evidence for a constitutive and an unconventionalsodium-dependent regulated pathway. Eur J Neurosci, 1995, 7(6):1220-1228.
[45] Almeida RD, Manadas BJ, Melo CV, Gomes JR, Mendes CS, Graos MM, Carvalho RF,Carvalho AP, Duarte CB. Neuroprotection by BDNF against glutamate-induced apoptoticcell death is mediated by ERK and PI3-kinase pathways. Cell Death Differ, 2005,12(10):1329-1343.
[46] Kurozumi K, Nakamura K, Tamiya T, Kawano Y, Kobune M, Hirai S, Uchida H, SasakiK, Ito Y, Kato K, Honmou O, Houkin K, Date I, Hamada H. BDNF gene-modifiedmesenchymal stem cells promote functional recovery and reduce infarct size in the ratmiddle cerebral artery occlusion model. Mol Ther, 2004, 9(2):189-197.
[47] Geremia NM, Pettersson LM, Hasmatali JC, Hryciw T, Danielsen N, Schreyer DJ, VergeVM. Endogenous BDNF regulates induction of intrinsic neuronal growth programs ininjured sensory neurons. Exp Neurol, 2009.
[48] Chu LF, Wang WT, Ghanta VK, Lin CH, Chiang YY, Hsueh CM. Ischemic braincell-derived conditioned medium protects astrocytes against ischemia throughGDNF/ERK/NF-kB signaling pathway. Brain Res, 2008.
[49] Yamagata K, Tagami M, Ikeda K, Tsumagari S, Yamori Y, Nara Y. Differential regulationof glial cell line-derived neurotrophic factor (GDNF) mRNA expression during hypoxiaand reoxygenation in astrocytes isolated from stroke-prone spontaneously hypertensiverats. Glia, 2002, 37(1):1-7.
[50] Mattson MP. Glutamate and neurotrophic factors in neuronal plasticity and disease. AnnN Y Acad Sci, 2008, 1144:97-112.
[51] Vangeison G, Rempe DA. The Janus-Faced Effects of Hypoxia on Astrocyte Function.Neuroscientist, 2009.
[52] Blanco M, Lizasoain I, Sobrino T, Vivancos J, Castillo J. Ischemic preconditioning: anovel target for neuroprotective therapy. Cerebrovasc Dis, 2006, 21 Suppl 2:38-47.
[53] Digicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotection involvescross-talk between Jak2 and NF-kappaB signalling cascades. Nature, 2001,412(6847):641-647.
[54] Abdel-Mageed AB, Zhao F, Rider BJ, Agrawal KC. Erythropoietin-inducedmetallothionein gene expression: role in proliferation of K562 cells. Exp Biol Med(Maywood), 2003, 228(9):1033-1039.
[55] Matsui T, Mori T, Tateishi N, Kagamiishi Y, Satoh S, Katsube N, Morikawa E, MorimotoT, Ikuta F, Asano T. Astrocytic activation and delayed infarct expansion after permanentfocal ischemia in rats. Part I: enhanced astrocytic synthesis of s-100beta in the periinfarctarea precedes delayed infarct expansion. J Cereb Blood Flow Metab, 2002,22(6):711-722.
[56] Song H, Stevens CF, Gage FH. Astroglia induce neurogenesis from adult neural stemcells. Nature, 2002, 417(6884):39-44.
[57] Tekkok SB, Brown AM, Westenbroek R, Pellerin L, Ransom BR. Transfer ofglycogen-derived lactate from astrocytes to axons via specific monocarboxylatetransporters supports mouse optic nerve activity. J Neurosci Res, 2005, 81(5):644-652.
[58] Beck H, Semisch M, Culmsee C, Plesnila N, Hatzopoulos AK. Egr-1 regulates expressionof the glial scar component phosphacan in astrocytes after experimental stroke. Am JPathol, 2008, 173(1):77-92.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |