水通道蛋白4在局部脑缺血再灌注损伤中的作用
|
摘要
脑卒中(stroke)作为当今世界人口的第二大致死性疾病,因其致残、复发率高,严重危害着人类健康。其中,急性缺血性卒中(acuteischemic stroke,AIS)约占脑卒中人群的80%,是由脑局部血供中断引起的神经功能损伤。大量研究发现,脑血管闭塞可致病变部位缺血缺氧,引起一系列复杂的瀑布式级联反应。尽管目前临床治疗措施很多,但多数疗效并不理想。究其原因,主要由于治疗靶点过于单一、无法切断AIS诱导的信号级联网络所致,因此无法避免病程的进一步发生、发展。
以往观点认为,治疗AIS除及时恢复血供外,靶向神经元本身进行保护、阻断神经元死亡过程中的一个或几个环节,便可避免神经元继发性损伤的发生。多年来,人们一直在努力寻求能够保护缺血脑组织免受缺血损伤的药物。针对神经元保护,人们开发了种类繁多的神经保护剂,进行了数以百计的动物及临床试验,包括谷氨酸受体(glutamate receptors,GluRs)拮抗剂、自由基清除剂、抗炎性药物、离子通道调节剂、Caspases抑制剂以及增强内源性神经保护机制的多种神经生长因子或营养因子等。然而,上述药物应用于卒中患者后却无一奏效。近年来随着认识的不断深入,愈来愈多的研究发现,任何致神经元损伤的因素均可能同时波及到脑内其他细胞,尤其是胶质细胞以及血管内皮细胞,其病理改变可进一步加速神经元走向死亡。脑内胶质细胞与神经元之间形成复杂的信号交谈网络,在诸多病理生理过程中发挥特殊作用。因此作为整体功能单位,脑的神经功能恢复不能仅限于单一神经元保护,而应着眼于包括胶质细胞在内的整个“神经血管单元(neurovascular unit)”功能的维护。
神经血管单元中,星形胶质细胞由于与神经元联系紧密,可将血管性信号传递至脑内,因此被认为在脑卒中的病理生理过程中发挥了决定性作用。星形胶质细胞通过释放胶质递质(gliotransmitters),参与脑内多种信息的加工处理,不但对神经元突触信号的传递产生影响,还可通过释放血管活性分子实现血管紧张度的神经调节功能。因此,靶向于神经血管单元中的胶质细胞、尤其是星形胶质细胞功能的调控,已成为脑卒中等多种神经系统疾病治疗的重要策略。
水通道蛋白4(aquaporin-4,AQP4)作为脑内含量最丰富、转运效率最高的水通道蛋白(aquaporins,AQPs)亚型,主要定位于神经血管单元中的星形胶质细胞以及室管膜上皮细胞,尤其富集于星形胶质细胞朝向血管面及软脑膜面的胞膜区。此分布特点提示,AQP4在脑内内环境稳态及星形胶质细胞功能维持中发挥重要作用,可能是胶质细胞参与整个脑功能调节的重要结构基础。大量研究结果显示,AQP4基因缺失可致星形胶质细胞多种重要生理功能异常,继而影响多种病理生理过程。研究发现,AQP4蛋白下调或缺失可导致细胞发育、增殖、迁移、修复等多项功能发生障碍,空间钾缓冲能力下降,细胞骨架蛋白结构异常。此外,AQP4还参与了小胶质细胞活化、血-脑屏障(blood-brain barrier,BBB)完整性调控等过程。已有资料表明,AQP4与脑水肿、脑损伤以及脑肿瘤的发生、发展密切相关。有研究认为,脑缺血过程中AQP4的作用仅限于介导脑内跨膜水转运过程,调节脑组织水肿程度。然而AQP4是否还通过影响神经血管单元功能、尤其是星形胶质细胞功能,参与脑卒中病程的发生、发展,目前尚未见报道。
本文工作应用本实验室自行制备的AQP4基因敲除鼠,首先发现AQP4参与局部脑缺血再灌注损伤,从整体、细胞及分子水平研究并阐明AQP4在脑卒中发生、发展中的重要地位,揭示AQP4可通过调制星形胶质细胞功能,尤其是谷氨酸转运体(glutamate transporters,GluTs)功能,参与脑卒中病理生理改变的作用机制。研究结果不仅为AQP4神经生物学的研究开拓新领域,深化对AQP4生理及病理功能的认识,而且揭示AQP4是星形胶质细胞功能调节的重要靶点,为脑卒中临床治疗学的突破和研发理想的神经保护剂提供有益靶标。
第一部分AQP4基因缺失对tMcAO模型小鼠局部脑缺血再灌注损伤的影响
目的:研究并阐明AQP4在局部脑缺血再灌注损伤中的作用,探讨氟西汀(fluoxetine,Flu)脑缺血保护与AQP4的相关性。
方法:应用3月龄成年野生型(wildtype,AQP4~(+/+))及AQP4基因缺失(AQP4 knockout,AQP4~(-/-))型雄性CD1小鼠,线栓法建立短暂性大脑中动脉栓塞(transient middle cerebral artery occlusion,tMCAO)模型,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神经元、星形胶质、小胶质细胞数量及形态学改变;干湿比重法检测脑含水量;Western blotting及免疫组织化学法测定AQP4表达。
结果:AQP4基因缺失后,1)tMCAO模型小鼠死亡率增高,神经功能缺陷加重,脑梗死体积增大;缺血再灌注(ischemiareperfusion,IR)损伤程度与缺血时间长短的相关效应消失;2)IR所致的海马CA1区神经元丢失严重,星形及小胶质细胞增殖活化障碍;3)tMCAO术后24 h AQP4蛋白表达降低,72 h表达增高;4)AQP4基因缺失后小鼠基础及病理条件下脑含水量均显著增高。腹腔注射Flu 40 mg/kg,5)显著降低AQP4~(+/+)型小鼠术后24 h死亡率、改善小鼠神经行为学障碍并减小小鼠脑梗死体积,对AQP4~(-/-)型小鼠无上述保护作用;6)显著提高AQP4~(+/+)型小鼠海马CA1区神经元存活率,缓解缺血核心区星形胶质细胞增殖(不影响半暗带星形胶质细胞增殖),抑制小胶质细胞活化,对AQP4~(-/-)型小鼠无显著影响;7)显著上调术后24 h AQP4表达,下调术后72 h AQP4表达;8)显著降低AQP4~(+/+)型小鼠术后72 h脑含水量,对AQP4~(-/-)型小鼠脑含水量无显著影响。
结论:AQP4基因缺失导致CD1小鼠局部脑缺血再灌注损伤加重,Flu神经保护作用与调节AQP4表达相关。
第二部分AQP4基因缺失对谷氨酸所致兴奋性神经毒性的影响及其机制研究
一、AQP4基因缺失对神经元-星形胶质细胞共培养体系中谷氨酸兴奋性神经毒性的影响
目的:研究并阐明AQP4在谷氨酸(glutamate,Glu)引起的兴奋性神经毒性中的作用,探讨其与星形胶质细胞功能调控的相关性。
方法:取孕13～15d及生后3d内AQP4~(+/+)及AQP4~(-/-)型CD1小鼠,分别培养原代神经元及星形胶质细胞,倒扣爬片法建立神经元-星形胶质细胞共培养体系,Glu 100μM制备兴奋性神经毒性模型,免疫细胞化学法观察细胞形态改变;LDH释放量测定评价细胞损伤情况;MTT比色法检测细胞活力。
结果:1)AQP4~(+/+)、AQP4~(-/-)型原代星形胶质细胞在形态学及细胞生长特性方面未见明显差异;2)100μM Glu可显著引起两种基因型神经元-星形胶质共培养体系中细胞总LDH释放增加,AQP4~(-/-)型释放量显著高于AQP4~(+/+)型;3)AQP4基因缺失后Glu所致星形胶质细胞LDH释放减少、存活率增加,提示共培养体系中增高的LDH主要由受损神经元释放。
结论:星形胶质细胞AQP4基因缺失导致神经元-星形胶质细胞共培养体系Glu诱导的兴奋性神经毒性损伤加重、星形胶质细胞自身对Glu毒性的敏感度降低,表明AQP4参与Glu兴奋性神经毒性损伤过程,揭示星形胶质细胞功能改变可能是加剧共培养体系中神经元损伤的关键因素。
二、AQP4基因缺失加重谷氨酸所致兴奋性神经毒性损伤机制的研究
目的:研究并阐明AQP4基因缺失导致Glu兴奋性神经毒性损伤加重的具体机制,探讨其与星形胶质细胞GluTs功能调控的相关性。
方法:取生后3d内AQP4~(+/+)及AQP4~(-/-)型CD1乳鼠,原代培养星形胶质细胞,RT-PCR及Western blotting测定细胞GluTs mRNA及蛋白表达;[~3H]-D,L-Glu同位素标记法检测细胞Glu摄取功能。
结果:AQP4基因缺失后,1)星形胶质细胞谷氨酸转运体1(glutamate transporter 1,GLT-1)mRNA及蛋白水平下调,谷氨酸/天冬氨酸转运体(glutamate/aspartate transporter,GLAST)水平无显著改变;2)星形胶质细胞摄取[~3H]-D,L-Glu能力降低。
结论:AQP4基因缺失下调星形胶质细胞GLT-1表达,降低细胞Glu摄取能力,表明AQP4参与星形胶质细胞功能调控,其缺失导致Glu兴奋性神经毒性损伤加重的机制可能与GluTs功能降低有关。
综上所述,本研究工作的主要创新之处在于:
1.应用AQP4基因缺失小鼠,从整体、细胞和分子水平系统研究并阐明AQP4与脑卒中的发生、发展相关,明确其机制在于对星形胶质细胞功能的调节。研究结果丰富并拓展了AQP4在脑卒中神经损伤中的作用机制,为疾病的临床治疗提供了新的思路。
2.揭示Flu的脑缺血保护作用与调节AQP4表达相关,为将AQP4调节剂发展成为脑卒中治疗的新型药物积累了必要的学术与实验基础。
3.首次发现AQP4参与调控Glu兴奋性神经毒性过程,阐明其机制在于调节星形胶质细胞GluTs表达及Glu摄取功能,揭示AQP4是星形胶质细胞功能调节的重要靶点,为研发理想的神经保护药物——胶质细胞功能调节剂提供了新的靶标,也为靶向于星形胶质细胞功能调控药物应用于脑卒中等神经系统疾病的临床治疗提供了理论依据。
Stroke,a disorder encompassing all cerebrovascular accidents,is a public health problem of immense proportions across the globe.As the second leading cause of death behind ischemic heart disease,it leads to serious long-term disability in adults.About 80%of strokes are caused by focal cerebral artery occlusion(acute ischemic stroke,AIS)and brain injury is thought to result from a cascade of events from energy depletion to cell death.Many efforts are directed in the management of AIS but few have proved effective.Numerous pharmaceutical agents targeting one or more cell death pathways showed no improvement in patients' prognosis for not "stopping the stroke clock".These factors highlight the urgent need for new therapeutic options for AIS treatment.
Over the past years,the knowledge of cerebral hypersensitivity to ischemia is the main reason for nihilism surrounding the development of remedies for AIS.Substantial clinical trials of thrombolytic and neuroprotecants have been conducted over the last two decades. Neurologists have searched for a long time to find a potential agent among N-methyl-D-aspartate(NMDA)antagonists,radical scavengers, antiinflammatory agents,calcium antagonists,sodium or potassium channel blockers,caspases inhibitors,and neurotrophic factors,but with no success so far.Initial attempts at translating the positive effects of these agents from animal models to patients have either failed.Current experimental support reveals that glia or endotheliocytes were suffered as well in AIS,suggesting the impairment of neurovascular unit may probably be the key point.As an integral unit serving multiple and diverse roles,rescue of neurovascular unit integrity has been considered as a promising strategy for AIS therapeutics.
It is believed that astrocytes are critical determinants in neurovascular unit since besides coupling to neuron,they link vascular signal to brain.These glial cells participate in actual information processing and neuronal control of vascular tone by releasing gliotransmitters or vasoactive molecules.They are critically affected in, and contribute to,the anoxic/ischemic process and changes in neurone-astrocyte-endothelial cell signalling pathway may contribute mostly to ischemia reperfusion(IR)injury to central nervous system (CNS)elements.Owing to the pivotal role of astrocytes in neurovascular unit,agents targeting astrocytes regulation open new perspectives for AIS management.
In the CNS,aquaporin-4(AQP4)is the predominant isoform which is localized on ependymal cells lining the ventricles and astrocytes membrane.Its expression in neurovascular unit is restricted to the perivascular glial processes,suggesting AQP4 involvement in the maintenance of cerebral homeostasis and may be the key point in astrocytes modulation to control diverse physiological processes. Accumulating data have shown that AQP4 depletion or down-regulation caused astrocytes dysfunction such as reduced membrane water permeability,impaired cell growth or migration,attenuated potassium buffering,and altered cytoskeleton rearrangement.It also serves as a component in microglia activation or blood-brain barrier(BBB)integrity with experimental support.By far,AQP4 has gained sufficient attraction in numerous cerebral disorders including edema,traumatic injury,tumor,infection,and epilepsy,whereas little is known about its contribution to AIS.Due to most studies are still dwelling on AQP4 as a water transporter,the illumination of its significance in neurovascular unit may offer prospective clinical therapeutic benefits.
In the present study,we investigate the role of AQP4 in the proceeding of IR in vivo and in vitro by using AQP4 gene knockout mice. The results revealed here will improve our understanding on the neurobiology of AQP4 and provide a new target for developing therapeutic options for AIS management.
PartⅠEffects of AQP4 deficiency on the brain injury induced by IR
AIM:To investigate the role of AQP4 in IR and the impact of AQP4 knockout on the protective actions of fluoxetine(Flu).
METHODS:Focal IR was achieved in three-month-old wildtype (AQP4~(+/+))and AQP4 knockout(AQP4~(-/-))male mice by transient occlusion of middle cerebral artery(tMCAO)with a modified intralumenal filament technique as described previously.Mortality, neurological deficits and infarct volume were measured in each group. Immunostaining was taken for NeuN,GFAP and MAC1 expression. Brain water content and AQP4 level were also analyzed.
RESULTS:Compared with wildtype mice,1)AQP4~(-/-)genotype exhibited larger increases in mortality,neurological deficits and infarct volume;The dependence on the duration of ischemia was cancelled by AQP4 deficiency;2)Severer loss of CA1 neurons in hippocampus and inhibited astroglial proliferation or microglial activation induced by IR were observed in AQP4~(-/-)genotype;3)Pool of AQP4 was down-regulated at 24 h and up-regulated at 72 h after tMCAO,exhibiting a time-course change;4)Brain water content was higher in ipsilateral hemisphere in AQP4~(-/-)mice.After administration of Flu(40 mg/kg,i.p.),5)Mortality, neurological deficits and infarct volume of AQP4~(+/+)genotype were decreased;6)The loss of CA1 neurons and astroglial proliferation or microglial activation in the core were alleviated;7)Up- and down-regulation of AQP4(at 24 or 72 h,respectively)were detected;8) Brain water content was decreased strikingly at 72 h in AQP4~(+/+)mice. All these effects of Flu were abolished in AQP4~(-/-)genotype.
CONCLUSION:AQP4 deficiency aggravates IR-induced injury, and the neuroprotection of Flu might be due to the regulation of AQP4 expression.
PartⅡEffects of AQP4 deficiency on glutamate-induced excitotoxicity
1.Effects of AQP4 deficiency on glutamate-induced excitotoxicity in astrocyte-neuron co-cultures
AIM:To investigate the effects of AQP4 knockout on glutamate (Glu)induced excitotoxicity.
METHODS:Primary astrocyte and neuron cultures were prepared from wildtype and homozygous mutant mice.The co-culture system was achieved by inverting the coverslips bearing astrocytes and all cells were exposed to Glu(100μM).Immunocytochemistry for GFAP was taken for cell morphology observation;LDH release in culture medium was measured using an LDH diagnostic kit;MTT analysis was used to assess the Glu-induced cytotoxicity.
RESULTS:1)There were no differences in cellular morphology and cell growth characteristics between AQP4~(+/+)and AQP4~(-/-)astrocytes;2) Sustained exposure to Glu for 24 h caused damage to co-cultures of two genotypes and even worse lesion occurred in AQP4~(-/-)system;3)Less excitotoxicity was detected in primary cultured AQP4~(-/-)astrocytes, indicating severer loss of neurons in AQP4~(-/-)co-culture.
CONCLUSION:Deficiency of AQP4 aggravates Glu-induced excitotoxicity in astrocyte-neuron co-cultures while decreases astrocytic susceptivity to Glu,suggesting that AQP4 is involved in excitotoxic damage to neurons and the functional impairment of astrocytes may be the key point.
2.Mechanisms involved in the increased excitotoxicity by AQP4 deficiency
AIM:To investigate the mechanisms of aggravated excitotoxicity by AQP4 knockout and to elucidate the regulative action of AQP4 on the astrocytic functions.
METHODS:Primary astrocyte cultures were prepared from wildtype and homozygous mutant mice.RT-PCR and Western blotting were taken for glutamate transporters(GluTs)expression; [~3H]-D,L-glutamate uptake analysis was performed for the assessment of GluTs function.
RESULTS:1)Lack of AQP4 down-regulated astrocytic expression of glutamate transporter 1(GLT-1)but not of glutamate / aspartate transporter(GLAST);2)A lower uptake capability in AQP4~(-/-)astrocytes was detected.
CONCLUSION:Deficiency of AQP4 down-regulates GLT-1 expression and Glu uptake in astrocytes,indicating AQP4 is actually involved in the modulation of astrocytes function and the mechanisms underlying the increased excitotoxicity may be the inhibition of Glu uptake.
In summary,the present work provides direct evidences for the first time that AQP4 plays an important role in the process of IR and the underlying mechanisms predominantly lie in the modulation of GluTs function.The results obtained here substantially improve our understanding of AQP4-glutamanergic biology and intensively suggest:1) AQP4 is tightly involved in the pathology of stroke and plays an important role in the regulation of astroglial function;2)The therapeutic strategy targeted to astrocytic modulation with AQP4 may offer a new perspective for the development of new options for AIS treatment.
以往观点认为,治疗AIS除及时恢复血供外,靶向神经元本身进行保护、阻断神经元死亡过程中的一个或几个环节,便可避免神经元继发性损伤的发生。多年来,人们一直在努力寻求能够保护缺血脑组织免受缺血损伤的药物。针对神经元保护,人们开发了种类繁多的神经保护剂,进行了数以百计的动物及临床试验,包括谷氨酸受体(glutamate receptors,GluRs)拮抗剂、自由基清除剂、抗炎性药物、离子通道调节剂、Caspases抑制剂以及增强内源性神经保护机制的多种神经生长因子或营养因子等。然而,上述药物应用于卒中患者后却无一奏效。近年来随着认识的不断深入,愈来愈多的研究发现,任何致神经元损伤的因素均可能同时波及到脑内其他细胞,尤其是胶质细胞以及血管内皮细胞,其病理改变可进一步加速神经元走向死亡。脑内胶质细胞与神经元之间形成复杂的信号交谈网络,在诸多病理生理过程中发挥特殊作用。因此作为整体功能单位,脑的神经功能恢复不能仅限于单一神经元保护,而应着眼于包括胶质细胞在内的整个“神经血管单元(neurovascular unit)”功能的维护。
神经血管单元中,星形胶质细胞由于与神经元联系紧密,可将血管性信号传递至脑内,因此被认为在脑卒中的病理生理过程中发挥了决定性作用。星形胶质细胞通过释放胶质递质(gliotransmitters),参与脑内多种信息的加工处理,不但对神经元突触信号的传递产生影响,还可通过释放血管活性分子实现血管紧张度的神经调节功能。因此,靶向于神经血管单元中的胶质细胞、尤其是星形胶质细胞功能的调控,已成为脑卒中等多种神经系统疾病治疗的重要策略。
水通道蛋白4(aquaporin-4,AQP4)作为脑内含量最丰富、转运效率最高的水通道蛋白(aquaporins,AQPs)亚型,主要定位于神经血管单元中的星形胶质细胞以及室管膜上皮细胞,尤其富集于星形胶质细胞朝向血管面及软脑膜面的胞膜区。此分布特点提示,AQP4在脑内内环境稳态及星形胶质细胞功能维持中发挥重要作用,可能是胶质细胞参与整个脑功能调节的重要结构基础。大量研究结果显示,AQP4基因缺失可致星形胶质细胞多种重要生理功能异常,继而影响多种病理生理过程。研究发现,AQP4蛋白下调或缺失可导致细胞发育、增殖、迁移、修复等多项功能发生障碍,空间钾缓冲能力下降,细胞骨架蛋白结构异常。此外,AQP4还参与了小胶质细胞活化、血-脑屏障(blood-brain barrier,BBB)完整性调控等过程。已有资料表明,AQP4与脑水肿、脑损伤以及脑肿瘤的发生、发展密切相关。有研究认为,脑缺血过程中AQP4的作用仅限于介导脑内跨膜水转运过程,调节脑组织水肿程度。然而AQP4是否还通过影响神经血管单元功能、尤其是星形胶质细胞功能,参与脑卒中病程的发生、发展,目前尚未见报道。
本文工作应用本实验室自行制备的AQP4基因敲除鼠,首先发现AQP4参与局部脑缺血再灌注损伤,从整体、细胞及分子水平研究并阐明AQP4在脑卒中发生、发展中的重要地位,揭示AQP4可通过调制星形胶质细胞功能,尤其是谷氨酸转运体(glutamate transporters,GluTs)功能,参与脑卒中病理生理改变的作用机制。研究结果不仅为AQP4神经生物学的研究开拓新领域,深化对AQP4生理及病理功能的认识,而且揭示AQP4是星形胶质细胞功能调节的重要靶点,为脑卒中临床治疗学的突破和研发理想的神经保护剂提供有益靶标。
第一部分AQP4基因缺失对tMcAO模型小鼠局部脑缺血再灌注损伤的影响
目的:研究并阐明AQP4在局部脑缺血再灌注损伤中的作用,探讨氟西汀(fluoxetine,Flu)脑缺血保护与AQP4的相关性。
方法:应用3月龄成年野生型(wildtype,AQP4~(+/+))及AQP4基因缺失(AQP4 knockout,AQP4~(-/-))型雄性CD1小鼠,线栓法建立短暂性大脑中动脉栓塞(transient middle cerebral artery occlusion,tMCAO)模型,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神经元、星形胶质、小胶质细胞数量及形态学改变;干湿比重法检测脑含水量;Western blotting及免疫组织化学法测定AQP4表达。
结果:AQP4基因缺失后,1)tMCAO模型小鼠死亡率增高,神经功能缺陷加重,脑梗死体积增大;缺血再灌注(ischemiareperfusion,IR)损伤程度与缺血时间长短的相关效应消失;2)IR所致的海马CA1区神经元丢失严重,星形及小胶质细胞增殖活化障碍;3)tMCAO术后24 h AQP4蛋白表达降低,72 h表达增高;4)AQP4基因缺失后小鼠基础及病理条件下脑含水量均显著增高。腹腔注射Flu 40 mg/kg,5)显著降低AQP4~(+/+)型小鼠术后24 h死亡率、改善小鼠神经行为学障碍并减小小鼠脑梗死体积,对AQP4~(-/-)型小鼠无上述保护作用;6)显著提高AQP4~(+/+)型小鼠海马CA1区神经元存活率,缓解缺血核心区星形胶质细胞增殖(不影响半暗带星形胶质细胞增殖),抑制小胶质细胞活化,对AQP4~(-/-)型小鼠无显著影响;7)显著上调术后24 h AQP4表达,下调术后72 h AQP4表达;8)显著降低AQP4~(+/+)型小鼠术后72 h脑含水量,对AQP4~(-/-)型小鼠脑含水量无显著影响。
结论:AQP4基因缺失导致CD1小鼠局部脑缺血再灌注损伤加重,Flu神经保护作用与调节AQP4表达相关。
第二部分AQP4基因缺失对谷氨酸所致兴奋性神经毒性的影响及其机制研究
一、AQP4基因缺失对神经元-星形胶质细胞共培养体系中谷氨酸兴奋性神经毒性的影响
目的:研究并阐明AQP4在谷氨酸(glutamate,Glu)引起的兴奋性神经毒性中的作用,探讨其与星形胶质细胞功能调控的相关性。
方法:取孕13～15d及生后3d内AQP4~(+/+)及AQP4~(-/-)型CD1小鼠,分别培养原代神经元及星形胶质细胞,倒扣爬片法建立神经元-星形胶质细胞共培养体系,Glu 100μM制备兴奋性神经毒性模型,免疫细胞化学法观察细胞形态改变;LDH释放量测定评价细胞损伤情况;MTT比色法检测细胞活力。
结果:1)AQP4~(+/+)、AQP4~(-/-)型原代星形胶质细胞在形态学及细胞生长特性方面未见明显差异;2)100μM Glu可显著引起两种基因型神经元-星形胶质共培养体系中细胞总LDH释放增加,AQP4~(-/-)型释放量显著高于AQP4~(+/+)型;3)AQP4基因缺失后Glu所致星形胶质细胞LDH释放减少、存活率增加,提示共培养体系中增高的LDH主要由受损神经元释放。
结论:星形胶质细胞AQP4基因缺失导致神经元-星形胶质细胞共培养体系Glu诱导的兴奋性神经毒性损伤加重、星形胶质细胞自身对Glu毒性的敏感度降低,表明AQP4参与Glu兴奋性神经毒性损伤过程,揭示星形胶质细胞功能改变可能是加剧共培养体系中神经元损伤的关键因素。
二、AQP4基因缺失加重谷氨酸所致兴奋性神经毒性损伤机制的研究
目的:研究并阐明AQP4基因缺失导致Glu兴奋性神经毒性损伤加重的具体机制,探讨其与星形胶质细胞GluTs功能调控的相关性。
方法:取生后3d内AQP4~(+/+)及AQP4~(-/-)型CD1乳鼠,原代培养星形胶质细胞,RT-PCR及Western blotting测定细胞GluTs mRNA及蛋白表达;[~3H]-D,L-Glu同位素标记法检测细胞Glu摄取功能。
结果:AQP4基因缺失后,1)星形胶质细胞谷氨酸转运体1(glutamate transporter 1,GLT-1)mRNA及蛋白水平下调,谷氨酸/天冬氨酸转运体(glutamate/aspartate transporter,GLAST)水平无显著改变;2)星形胶质细胞摄取[~3H]-D,L-Glu能力降低。
结论:AQP4基因缺失下调星形胶质细胞GLT-1表达,降低细胞Glu摄取能力,表明AQP4参与星形胶质细胞功能调控,其缺失导致Glu兴奋性神经毒性损伤加重的机制可能与GluTs功能降低有关。
综上所述,本研究工作的主要创新之处在于:
1.应用AQP4基因缺失小鼠,从整体、细胞和分子水平系统研究并阐明AQP4与脑卒中的发生、发展相关,明确其机制在于对星形胶质细胞功能的调节。研究结果丰富并拓展了AQP4在脑卒中神经损伤中的作用机制,为疾病的临床治疗提供了新的思路。
2.揭示Flu的脑缺血保护作用与调节AQP4表达相关,为将AQP4调节剂发展成为脑卒中治疗的新型药物积累了必要的学术与实验基础。
3.首次发现AQP4参与调控Glu兴奋性神经毒性过程,阐明其机制在于调节星形胶质细胞GluTs表达及Glu摄取功能,揭示AQP4是星形胶质细胞功能调节的重要靶点,为研发理想的神经保护药物——胶质细胞功能调节剂提供了新的靶标,也为靶向于星形胶质细胞功能调控药物应用于脑卒中等神经系统疾病的临床治疗提供了理论依据。
Stroke,a disorder encompassing all cerebrovascular accidents,is a public health problem of immense proportions across the globe.As the second leading cause of death behind ischemic heart disease,it leads to serious long-term disability in adults.About 80%of strokes are caused by focal cerebral artery occlusion(acute ischemic stroke,AIS)and brain injury is thought to result from a cascade of events from energy depletion to cell death.Many efforts are directed in the management of AIS but few have proved effective.Numerous pharmaceutical agents targeting one or more cell death pathways showed no improvement in patients' prognosis for not "stopping the stroke clock".These factors highlight the urgent need for new therapeutic options for AIS treatment.
Over the past years,the knowledge of cerebral hypersensitivity to ischemia is the main reason for nihilism surrounding the development of remedies for AIS.Substantial clinical trials of thrombolytic and neuroprotecants have been conducted over the last two decades. Neurologists have searched for a long time to find a potential agent among N-methyl-D-aspartate(NMDA)antagonists,radical scavengers, antiinflammatory agents,calcium antagonists,sodium or potassium channel blockers,caspases inhibitors,and neurotrophic factors,but with no success so far.Initial attempts at translating the positive effects of these agents from animal models to patients have either failed.Current experimental support reveals that glia or endotheliocytes were suffered as well in AIS,suggesting the impairment of neurovascular unit may probably be the key point.As an integral unit serving multiple and diverse roles,rescue of neurovascular unit integrity has been considered as a promising strategy for AIS therapeutics.
It is believed that astrocytes are critical determinants in neurovascular unit since besides coupling to neuron,they link vascular signal to brain.These glial cells participate in actual information processing and neuronal control of vascular tone by releasing gliotransmitters or vasoactive molecules.They are critically affected in, and contribute to,the anoxic/ischemic process and changes in neurone-astrocyte-endothelial cell signalling pathway may contribute mostly to ischemia reperfusion(IR)injury to central nervous system (CNS)elements.Owing to the pivotal role of astrocytes in neurovascular unit,agents targeting astrocytes regulation open new perspectives for AIS management.
In the CNS,aquaporin-4(AQP4)is the predominant isoform which is localized on ependymal cells lining the ventricles and astrocytes membrane.Its expression in neurovascular unit is restricted to the perivascular glial processes,suggesting AQP4 involvement in the maintenance of cerebral homeostasis and may be the key point in astrocytes modulation to control diverse physiological processes. Accumulating data have shown that AQP4 depletion or down-regulation caused astrocytes dysfunction such as reduced membrane water permeability,impaired cell growth or migration,attenuated potassium buffering,and altered cytoskeleton rearrangement.It also serves as a component in microglia activation or blood-brain barrier(BBB)integrity with experimental support.By far,AQP4 has gained sufficient attraction in numerous cerebral disorders including edema,traumatic injury,tumor,infection,and epilepsy,whereas little is known about its contribution to AIS.Due to most studies are still dwelling on AQP4 as a water transporter,the illumination of its significance in neurovascular unit may offer prospective clinical therapeutic benefits.
In the present study,we investigate the role of AQP4 in the proceeding of IR in vivo and in vitro by using AQP4 gene knockout mice. The results revealed here will improve our understanding on the neurobiology of AQP4 and provide a new target for developing therapeutic options for AIS management.
PartⅠEffects of AQP4 deficiency on the brain injury induced by IR
AIM:To investigate the role of AQP4 in IR and the impact of AQP4 knockout on the protective actions of fluoxetine(Flu).
METHODS:Focal IR was achieved in three-month-old wildtype (AQP4~(+/+))and AQP4 knockout(AQP4~(-/-))male mice by transient occlusion of middle cerebral artery(tMCAO)with a modified intralumenal filament technique as described previously.Mortality, neurological deficits and infarct volume were measured in each group. Immunostaining was taken for NeuN,GFAP and MAC1 expression. Brain water content and AQP4 level were also analyzed.
RESULTS:Compared with wildtype mice,1)AQP4~(-/-)genotype exhibited larger increases in mortality,neurological deficits and infarct volume;The dependence on the duration of ischemia was cancelled by AQP4 deficiency;2)Severer loss of CA1 neurons in hippocampus and inhibited astroglial proliferation or microglial activation induced by IR were observed in AQP4~(-/-)genotype;3)Pool of AQP4 was down-regulated at 24 h and up-regulated at 72 h after tMCAO,exhibiting a time-course change;4)Brain water content was higher in ipsilateral hemisphere in AQP4~(-/-)mice.After administration of Flu(40 mg/kg,i.p.),5)Mortality, neurological deficits and infarct volume of AQP4~(+/+)genotype were decreased;6)The loss of CA1 neurons and astroglial proliferation or microglial activation in the core were alleviated;7)Up- and down-regulation of AQP4(at 24 or 72 h,respectively)were detected;8) Brain water content was decreased strikingly at 72 h in AQP4~(+/+)mice. All these effects of Flu were abolished in AQP4~(-/-)genotype.
CONCLUSION:AQP4 deficiency aggravates IR-induced injury, and the neuroprotection of Flu might be due to the regulation of AQP4 expression.
PartⅡEffects of AQP4 deficiency on glutamate-induced excitotoxicity
1.Effects of AQP4 deficiency on glutamate-induced excitotoxicity in astrocyte-neuron co-cultures
AIM:To investigate the effects of AQP4 knockout on glutamate (Glu)induced excitotoxicity.
METHODS:Primary astrocyte and neuron cultures were prepared from wildtype and homozygous mutant mice.The co-culture system was achieved by inverting the coverslips bearing astrocytes and all cells were exposed to Glu(100μM).Immunocytochemistry for GFAP was taken for cell morphology observation;LDH release in culture medium was measured using an LDH diagnostic kit;MTT analysis was used to assess the Glu-induced cytotoxicity.
RESULTS:1)There were no differences in cellular morphology and cell growth characteristics between AQP4~(+/+)and AQP4~(-/-)astrocytes;2) Sustained exposure to Glu for 24 h caused damage to co-cultures of two genotypes and even worse lesion occurred in AQP4~(-/-)system;3)Less excitotoxicity was detected in primary cultured AQP4~(-/-)astrocytes, indicating severer loss of neurons in AQP4~(-/-)co-culture.
CONCLUSION:Deficiency of AQP4 aggravates Glu-induced excitotoxicity in astrocyte-neuron co-cultures while decreases astrocytic susceptivity to Glu,suggesting that AQP4 is involved in excitotoxic damage to neurons and the functional impairment of astrocytes may be the key point.
2.Mechanisms involved in the increased excitotoxicity by AQP4 deficiency
AIM:To investigate the mechanisms of aggravated excitotoxicity by AQP4 knockout and to elucidate the regulative action of AQP4 on the astrocytic functions.
METHODS:Primary astrocyte cultures were prepared from wildtype and homozygous mutant mice.RT-PCR and Western blotting were taken for glutamate transporters(GluTs)expression; [~3H]-D,L-glutamate uptake analysis was performed for the assessment of GluTs function.
RESULTS:1)Lack of AQP4 down-regulated astrocytic expression of glutamate transporter 1(GLT-1)but not of glutamate / aspartate transporter(GLAST);2)A lower uptake capability in AQP4~(-/-)astrocytes was detected.
CONCLUSION:Deficiency of AQP4 down-regulates GLT-1 expression and Glu uptake in astrocytes,indicating AQP4 is actually involved in the modulation of astrocytes function and the mechanisms underlying the increased excitotoxicity may be the inhibition of Glu uptake.
In summary,the present work provides direct evidences for the first time that AQP4 plays an important role in the process of IR and the underlying mechanisms predominantly lie in the modulation of GluTs function.The results obtained here substantially improve our understanding of AQP4-glutamanergic biology and intensively suggest:1) AQP4 is tightly involved in the pathology of stroke and plays an important role in the regulation of astroglial function;2)The therapeutic strategy targeted to astrocytic modulation with AQP4 may offer a new perspective for the development of new options for AIS treatment.
引文
1. Bonita R, Mendis S, Truelsen T, Bogousslavsky J, Toole J, Yatsu F. The global stroke initiative. Lancet Neurol. 2004; 3(7): 391-393.
2. Huang J, Upadhyay UM, Tamargo RJ. Inflammation in stroke and focal cerebral ischemia. Surg Neurol. 2006; 66(3): 232-245.
3. Markiewicz I, Lukomska B. The role of astrocytes in the physiology and pathology of the central nervous system. Acta Neurobiol Exp (Wars). 2006; 66(4): 343-358.
4. Araque A, Parpura V, Sanzgiri RP, Haydon PG. Glutamate-dependent astrocyte modulation of synaptic transmission between cultured hippocampal neurons. Eur J Neurosci. 1998; 10(6): 2129-2142.
5. Koizumi S, Fujishita K, Tsuda M, Shigemoto-Mogami Y, Inoue K. Dynamic inhibition of excitatory synaptic transmission by astrocyte-derived ATP in hippocampal cultures. Proc Natl Acad Sci USA. 2003; 100(19): 11023-11028.
6. Zhang J, Hoffert C, Vu HK, Groblewski T, Ahmad S, O'Donnell D. Induction of CB2 receptor expression in the rat spinal cord of neuropathic but not inflammatory chronic pain models. Eur J Neurosci. 2003; 17(12): 2750-2754.
7. Zonta M, Angulo MC, Gobbo S, Rosengarten B, Hossmann KA, Pozzan T, et al. Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation. Nat Neurosci. 2003; 6(1): 43-50.
8. Mulligan SJ, MacVicar BA. Calcium transients in astrocyte endfeet cause cerebrovascular constrictions. Nature. 2004; 431(7005): 195-199.
9. Takano T, Tian GF, Peng W, Lou N, Libionka W, Han X, et al. Astrocyte-mediated control of cerebral blood flow. Nat Neurosci. 2006; 9(2): 260-267.
10. Ferrari D, Villalba M, Chiozzi P, Falzoni S, Ricciardi-Castagnoli P, Di Virgilio F. Mouse microglial cells express a plasma membrane pore gated by extracellular ATP. J Immunol. 1996; 156(4): 1531-1539.
11. Norenberg W, Cordes A, Blohbaum G, Frohlich R, Illes P. Coexistence of purino- and pyrimidinoceptors on activated rat microglial cells. Br J Pharmacol. 1997; 121(6): 1087-1098.
12. Honda S, Sasaki Y, Ohsawa K, Imai Y, Nakamura Y, Inoue K, et al. Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors. J Neurosci. 2001; 21(6): 1975-1982.
13. Shigemoto-Mogami Y, Koizumi S, Tsuda M, Ohsawa K, Kohsaka S, Inoue K. Mechanisms underlying extracellular ATP-evoked interleukin-6 release in mouse microglial cell line, MG-5. J Neurochem. 2001; 78(6): 1339-1349.
14. Suzuki T, Hide I, Ido K, Kohsaka S, Inoue K, Nakata Y. Production and release of neuroprotective tumor necrosis factor by P2X7 receptor-activated microglia. J Neurosci. 2004; 24(1): 1-7.
15. Verderio C, Matteoli M. ATP mediates calcium signaling between astrocytes and microglial cells: modulation by IFN-gamma. J Immunol. 2001; 166(10): 6383-6391.
16. Hagg T. Molecular regulation of adult CNS neurogenesis: an integrated view. Trends Neurosci. 2005; 28(11): 589-595.
17. Laywell ED, Steindler DA, Silver DJ. Astrocytic stem cells in the adult brain. Neurosurg Clin N Am. 2007; 18(1): 21-30.
18. Chen LW, Yung KL, Chan YS. Reactive astrocytes as potential manipulation targets in novel cell replacement therapy of Parkinson's disease. Curr Drug Targets. 2005; 6(7): 821-833.
19. Alvarez-Buylla A, Lim DA. For the long run: maintaining germinal niches in the adult brain. Neuron. 2004; 41(5): 683-686.
20. Matute C, Alberdi E, Ibarretxe G, Sanchez-Gomez MV. Excitotoxicity in glial cells. Eur J Pharmacol. 2002; 447(2-3): 239-246.
21. Anderson CM, Swanson RA. Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia. 2000; 32(1): 1-14.
22. King LS, Kozono D, Agre P. From structure to disease: the evolving tale of aquaporin biology. Nat Rev Mol Cell Bio. 2004; 5(9): 687-698.
23. Yang B, Verkman AS. Water and glycerol permeabilities of aquaporins 1-5 and MIP determined quantitatively by expression of epitope-tagged constructs in Xenopus oocytes. J Biol Chem. 1997; 272(26): 16140-16146.
24. Hasegawa H, Ma T, Skach W, Matthay MA, Verkman AS. Molecular cloning of a mercurial-insensitive water channel expressed in selected water-transporting tissues. J Biol Chem. 1994; 269(8): 5497-5500.
25. Jung JS, Bhat RV, Preston GM, Guggino WB, Baraban JM, Agre P. Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance. Proc Natl Acad Sci USA. 1994; 91(26): 13052-13056.
26. Zeidel ML, Ambudkar SV, Smith BL, Agre P. Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein. Biochemistry. 1992; 31(33): 7436-7440.
27. Frigeri A, Gropper MA, Umenishi F, Kawashima M, Brown D, Verkman AS. Localization of MIWC and GLIP water channel homologs in neuromuscular, epithelial and glandular tissues. J Cell Sci. 1995; 108 (9): 2993-3002.
28. Amiry-Moghaddam M, Ottersen OP. The molecular basis of water transport in the brain. Nat Rev Neurosci. 2003; 4(12): 991-1001.
29. Amiry-Moghaddam M, Frydenlund DS, Ottersen OP. Anchoring of aquaporin-4 in brain: molecular mechanisms and implications for the physiology and pathophysiology of water transport. Neuroscience. 2004; 129(4): 999-1010.
30. Nicchia GP, Nico B, Camassa LM, Mola MG, Loh N, Dermietzel R, et al The role of aquaporin-4 in the blood-brain barrier development and integrity: studies in animal and cell culture models. Neuroscience. 2004; 129(4): 935-945.
31. Manley GT, Binder DK, Papadopoulos MC, Verkman AS. New insights into water transport and edema in the central nervous system from phenotype analysis of aquaporin-4 null mice. Neuroscience. 2004; 129(4): 983-991.
32. Papadopoulos MC, Krishna S, Verkman AS. Aquaporin water channels and brain edema. Mt Sinai J Med. 2002; 69(4): 242-248.
33. Verkman AS, Binder DK, Bloch O, Auguste K, Papadopoulos MC. Three distinct roles of aquaporin-4 in brain function revealed by knockout mice. Biochim Biophys Acta. 2006; 1758(8): 1085-1093.
34. Papadopoulos MC, Manley GT, Krishna S, Verkman AS. Aquaporin-4 facilitates reabsorption of excess fluid in vasogenic brain edema. FASEB J. 2004; 18(11): 1291-1293.
35. Misu T, Fujihara K, Nakamura M, Murakami K, Endo M, Konno H, et al. Loss of aquaporin-4 in active perivascular lesions in neuromyelitis optica: a case report. Tohoku J Exp Med. 2006; 209(3): 269-275.
36. Roemer SF, Parisi JE, Lennon VA, Benarroch EE, Lassmann H, Bruck W, et al. Pattern-specific loss of aquaporin-4 immunoreactivity distinguishes neuromyelitis optica from multiple sclerosis. Brain. 2007; 130(5): 1194-1205.
37. Matsuoka T, Matsushita T, Kawano Y, Osoegawa M, Ochi H, Ishizu T, et al. Heterogeneity of aquaporin-4 autoimmunity and spinal cord lesions in multiple sclerosis in Japanese. Brain. 2007; 130(5): 1206-1212.
38. Lennon VA, Kryzer TJ, Pittock SJ, Verkman AS, Hinson SR. IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med. 2005; 202(4): 473-477.
39. Salaria S, Chana G, Caldara F, Feltrin E, Altieri M, Faggioni F, et al. Microarray analysis of cultured human brain aggregates following cortisol exposure: implications for cellular functions relevant to mood disorders. Neurobiol Dis. 2006; 23(3): 630-636.
40. Li Z, Gao L, Liu Q, Cao C, Sun XL, Ding JH, et al. Aquaporin-4 knockout regulated cocaine-induced behavior and neurochemical changes in mice. Neurosci Lett. 2006; 403(3): 294-298.
41. La Porta CA, Gena P, Gritti A, Fascio U, Svelto M, Calamita G. Adult murine CNS stem cells express aquaporin channels. Biol Cell. 2006; 98(2): 89-94.
42. Cavazzin C, Ferrari D, Facchetti F, Russignan A, Vescovi AL, La Porta CA, et al. Unique expression and localization of aquaporin-4 and aquaporin-9 in murine and human neural stem cells and in their glial progeny. Glia. 2006; 53(2): 167-181.
43. Schwartz PH, Nethercott H, Kirov II, Ziaeian B, Young MJ, Klassen H. Expression of neurodevelopmental markers by cultured porcine neural precursor cells. Stem Cells. 2005; 23(9): 1286-1294.
44. Nicchia GP, Frigeri A, Liuzzi GM, Svelto M. Inhibition of aquaporin-4 expression in astrocytes by RNAi determines alteration in cell morphology, growth, and water transport and induces changes in ischemia-related genes. FASEB J. 2003; 17(11): 1508-1510.
45. Wen H, Nagelhus EA, Amiry-Moghaddam M, Agre P, Ottersen OP, Nielsen S. Ontogeny of water transport in rat brain: postnatal expression of the aquaporin-4 water channel. Eur J Neurosci. 1999; 11(3): 935-945.
46. Nagelhus EA, Mathiisen TM, Ottersen OP. Aquaporin-4 in the central nervous system: cellular and subcellular distribution and coexpression with KIR4.1. Neuroscience. 2004; 129(4): 905-913.
47. Verkman AS. Novel roles of aquaporins revealed by phenotype analysis of knockout mice. Rev Physiol Biochem Pharmacol. 2005; 155: 31-55.
48. Padmawar P, Yao X, Bloch O, Manley GT, Verkman AS. K~+ waves in brain cortex visualized using a long-wavelength K~+-sensing fluorescent indicator. Nat Methods. 2005; 2(11): 825-827.
49. Binder DK, Yao X, Zador Z, Sick TJ, Verkman AS, Manley GT. Increased seizure duration and slowed potassium kinetics in mice lacking aquaporin-4 water channels. Glia. 2006; 53(6): 631-636.
50. Saadoun S, Papadopoulos MC, Watanabe H, Yan D, Manley GT, Verkman AS. Involvement of aquaporin-4 in astroglial cell migration and glial scar formation. J CellSci. 2005; 118(Pt 24): 5691-5698.
51. Auguste KI, Jin S, Uchida K, Yan D, Manley GT, Papadopoulos MC, et al. Greatly impaired migration of implanted aquaporin-4-deficient astroglial cells in mouse brain toward a site of injury. FASEB J. 2007; 21(1): 108-116.
52. Nicchia GP, Srinivas M, Li W, Brosnan CF, Frigeri A, Spray DC. New possible roles for aquaporin-4 in astrocytes: cell cytoskeleton and functional relationship with connexin43.FASEB J. 2005; 19(12): 1674-1676.
53. Fan Y, Zhang J, Sun XL, Gao L, Zeng XN, Ding JH, et al. Sex- and region-specific alterations of basal amino acid and monoamine metabolism in the brain of aquaporin-4 knockout mice. J Neurosci Res. 2005; 82(4): 458-464.
54. Tomas-Camardiel M, Venero JL, de Pablos RM, Rite I, Machado A, Cano J. In vivo expression of aquaporin-4 by reactive microglia. J Neurochem. 2004; 91(4): 891-899.
55. Tomas-Camardiel M, Venero JL, Herrera AJ, De Pablos RM, Pintor-Toro JA, Machado A, et al. Blood-brain barrier disruption highly induces aquaporin-4 mRNA and protein in perivascular and parenchymal astrocytes: protective effect by estradiol treatment in ovariectomized animals. J Neurosci Res. 2005; 80(2): 235-246.
56. Zhou J, Kong H, Hua X, Xiao M, Ding J, Hu G. Altered blood-brain barrier integrity in adult aquaporin-4 knockout mice. Neuroreport. 2008; 19(1): 1-5.
57. Caplan LR. Stroke treatment: promising but still struggling. JAMA. 1998; 279(16): 1304-1306.
58. Fisher M, Bogousslavsky J. Further evolution toward effective therapy for acute ischemic stroke. JAMA. 1998; 279(16): 1298-1303.
59. Heiss WD, Thiel A, Grond M, Graf R. Which targets are relevant for therapy of acute ischemic stroke? Stroke. 1999; 30(7): 1486-1489.
60. Muir KW, Grosset DG. Neuroprotection for acute stroke: making clinical trials work. Stroke. 1999; 30(1): 180-182.
61. Hickenbottom SL, Barsan WG. Acute ischemic stroke therapy. Neurol Clin. 2000; 18(2): 379-397.
62. Manley GT, Fujimura M, Ma T, Noshita N, Filiz F, Bollen AW, et al. Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke. Nat Med. 2000; 6(2): 159-163.
63. Woitzik J, Schneider UC, Thome C, Schroeck H, Schilling L. Comparison of different intravascular thread occlusion models for experimental stroke in rats. J Neurosci Methods. 2006; 151(2): 224-231.
64. Ma T, Yang B, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS. Generation and phenotype of a transgenic knockout mouse lacking the mercurial-insensitive water channel aquaporin-4. J Clin Invest. 1997; 100(5): 957-962.
65. Koizumi J, Yoshida Y, Nakazawa T, Ooneda G. Experimental studies of ischemic brain edema: A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. Jpn Stroke J. 1986; 8: 1-8.
66. Ngai AC, Nguyen TS, Meno JR, Britz GW. Postischemic augmentation of conducted dilation in cerebral arterioles. Stroke. 2007; 38(1): 124-130.
67. Ribeiro Mde C, Hirt L, Bogousslavsky J, Regli L, Badaut J. Time course of aquaporin expression after transient focal cerebral ischemia in mice. J Neurosci Res. 2006; 83(7): 1231-1240.
68. Frydenlund DS, Bhardwaj A, Otsuka T, Mylonakou MN, Yasumura T, Davidson KG, et al. Temporary loss of perivascular aquaporin-4 in neocortex after transient middle cerebral artery occlusion in mice. Proc Natl Acad Sci USA. 2006; 103(36): 13532-13536.
69. Kleffner I, Bungeroth M, Schiffbauer H, Schabitz WR, Ringelstein EB, Kuhlenbaumer G. The role of aquaporin-4 polymorphisms in the development of brain edema after middle cerebral artery occlusion. Stroke. 2008; 39(4): 1333-1335.
70. Panickar KS, Norenberg MD. Astrocytes in cerebral ischemic injury: morphological and general considerations. Glia. 2005; 50(4): 287-298.
71. Lai AY, Todd KG. Microglia in cerebral ischemia: molecular actions and interactions. Can J Physiol Pharmacol. 2006; 84(1): 49-59.
72. Denes A, Vidyasagar R, Feng J, Narvainen J, McColl BW, Kauppinen RA, et al. Proliferating resident microglia after focal cerebral ischaemia in mice. J Cereb Blood Flow Metab. 2007; 27(12): 1941-1953.
73. Tanaka R, Komine-Kobayashi M, Mochizuki H, Yamada M, Furuya T, Migita M, et al. Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia. Neuroscience. 2003; 117(3): 531-539.
74. Schousboe A. Transport and metabolism of glutamate and GABA in neurons are glial cells. Int Rev Neurobiol. 1981; 22: 1-45.
75. Tanaka K, Watase K, Manabe T, Yamada K, Watanabe M, Takahashi K, et al. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science. 1997; 276(5319): 1699-1702.
76. Schousboe A, Divac I. Difference in glutamate uptake in astrocytes cultured from different brain regions. Brain Res. 1979; 177(2): 407-409.
77. Rosenberg PA, Amin S, Leitner M. Glutamate uptake disguises neurotoxic potency of glutamate agonists in cerebral cortex in dissociated cell culture. J Neurosci. 1992; 12(1): 56-61.
78. Frandsen A, Schousboe A. Development of excitatory amino acid induced cytotoxicity in cultured neurons. Int J Dev Neurosci. 1990; 8(2): 209-216.
79. Griffin S, Clark JB, Canevari L. Astrocyte-neurone communication following oxygen-glucose deprivation. J Neurochem. 2005; 95(4): 1015-1022.
80. Danbolt NC. Glutamate uptake. Prog Neurobiol. 2001; 65(1): 1-105.
81. Fiacco TA, McCarthy KD. Astrocyte calcium elevations: properties, propagation, and effects on brain signaling. Glia. 2006; 54(7): 676-690.
82. Araque A. Astrocyte-neuron signaling in the brain-implications for disease. Curr Opin Investig Drugs. 2006; 7(7): 619-624.
83. Benarroch EE. Neuron-astrocyte interactions: partnership for normal function and disease in the central nervous system. Mayo Clin Proc. 2005; 80(10): 1326-1338.
84. Bender AS, Schousboe A, Reichelt W, Norenberg MD. Ionic mechanisms in glutamate-induced astrocyte swelling: role of K~+ influx. J Neurosci Res. 1998; 52(3): 307-321.
85. Sass JB, Ang LC, Juurlink BH. Aluminum pretreatment impairs the ability of astrocytes to protect neurons from glutamate mediated toxicity. Brain Res. 1993; 621(2): 207-214.
86. Ozog MA, Siushansian R, Naus CC. Blocked gap junctional coupling increases glutamate-induced neurotoxicity in neuron-astrocyte co-cultures. J Neuropathol ExpNeurol. 2002; 61(2): 132-141.
87. Kosugi T, Kawahara K. Reversed actrocytic GLT-1 during ischemia is crucial to excitotoxic death of neurons, but contributes to the survival of astrocytes themselves. Neurochem Res. 2006; 31(7): 933-943.
88. Du Y, Chen CP, Tseng CY, Eisenberg Y, Firestein BL. Astroglia-mediated effects of uric acid to protect spinal cord neurons from glutamate toxicity. Glia. 2007; 55(5): 463-472.
89. Haugeto O, Ullensvang K, Levy LM, Chaudhry FA, Honore T, Nielsen M, et al. Brain glutamate transporter proteins form homomultimers. J Biol Chem. 1996; 271(44): 27715-27722.
90. Marcaggi P, Attwell D. Role of glial amino acid transporters in synaptic transmission and brain energetics. Glia. 2004; 47(3): 217-225.
91. Torres GE, Gainetdinov RR, Caron MG. Plasma membrane monoamine transporters: structure, regulation and function. Nat Rev Neurosci. 2003; 4(1): 13-25.
92. Munch C, Ebstein M, Seefried U, Zhu B, Stamm S, Landwehrmeyer GB, et al. Alternative splicing of the 5'-sequences of the mouse EAAT2 glutamate transporter and expression in a transgenic model for amyotrophic lateral sclerosis. J Neurochem. 2002; 82(3): 594-603.
93. Maragakis NJ, Dykes-Hoberg M, Rothstein JD. Altered expression of the glutamate transporter EAAT2b in neurological disease. Ann Neurol. 2004; 55(4): 469-477.
94. Ganel R, Ho T, Maragakis NJ, Jackson M, Steiner JP, Rothstein JD. Selective up-regulation of the glial Na~+-dependent glutamate transporter GLT1 by a neuroimmunophilin ligand results in neuroprotection. Neurobiol Dis. 2006; 21(3): 556-567.
95. Hoogland G, van Oort RJ, Proper EA, Jansen GH, van Rijen PC, van Veelen CW, et al. Alternative splicing of glutamate transporter EAAT2 RNA in neocortex and hippocampus of temporal lobe epilepsy patients. Epilepsy Res. 2004; 59(2-3): 75-82.
96. Matute C, Melone M, Vallejo-Illarramendi A, Conti F. Increased expression of the astrocytic glutamate transporter GLT-1 in the prefrontal cortex of schizophrenics. Glia. 2005; 49(3): 451-455.
97. Smith RE, Haroutunian V, Davis KL, Meador-Woodruff JH. Expression of excitatory amino acid transporter transcripts in the thalamus of subjects with schizophrenia. Am J Psychiatry. 2001; 158(9): 1393-1399.
98. Rothstein JD, Patel S, Regan MR, Haenggeli C, Huang YH, Bergles DE, et al. Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature. 2005; 433: 73-77.
99. Hurtado O, Pradillo JM, Fernandez-Lopez D, Morales JR, Sobrino T, Castillo J, et al. Delayed post-ischemic administration of CDP-choline increases EAAT2 association to lipid rafts and affords neuroprotection in experimental stroke. Neurobiol Dis. 2008; 29(1): 123-131.
100.Diamond JS, Jahr CE. Transporters buffer synaptically released glutamate on a submillisecond time scale. J Neurosci. 1997; 17(12): 4672-4687.
101.Takayasu Y, lino M, Shimamoto K, Tanaka K, Ozawa S. Glial glutamate transporters maintain one-to-one relationship at the climbing fiber-Purkinje cell synapse by preventing glutamate spillover. JNeurosci. 2006; 26(24): 6563-6572.
102.O'Shea RD. Roles and regulation of glutamate transporters in the central nervous system. Clin Exp Pharmacol Physiol. 2002; 29(11): 1018-1023.
103.Kanai Y, Hediger MA. The glutamate/neutral amino acid transporter family SLC1: molecular, physiological and pharmacological aspects. Pflugers Arch. 2004; 447(5): 469-479.
104.Rae C, Lawrance ML, Dias LS, Provis T, Bubb WA, Balcar VJ. Strategies for studies of neurotoxic mechanisms involving deficient transport of L-glutamate: antisense knockout in rat brain in vivo and changes in the neurotransmitter metabolism following inhibition of glutamate transport in guinea pig brain slices. Brain Res Bull. 2000; 53(4): 373-381.
105.Had-Aissouni L, Re DB, Nieoullon A, Kerkerian-Le Goff L. Importance of astrocytic Inactivation of synaptically released glutamate for cell survival in the central nervous system—are astrocytes vulnerable to low intracellular glutamate concentrations? J Physiol Paris. 2002; 96(3-4): 317-322.
106.Re DB, Boucraut J, Samuel D, Birman S, Kerkerian-Le Goff L, et al. Glutamate transport alteration triggers differentiation-state selective oxidative death of cultured astrocytes: a mechanism different from excitotoxicity depending on intracellular GSH contents. J Neurochem. 2003; 85(5): 1159-1170.
107.Aoyama K, Suh SW, Hamby AM, Liu J, Chan WY, Chen Y, et al. Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse. Nat Neurosci. 2006; 9(1): 119-126.
108.O'Shea RD, Lau CL, Farso MC, Diwakarla S, Zagami CJ, Svendsen BB, et al Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1. Neurochem Int. 2006; 48(6-7): 604-610.
109.Zhou J, Sutherland ML. Glutamate transporter cluster formation in astrocytic processes regulates glutamate uptake activity. J Neurosci 2004; 24(28): 6301-6306.
1. Fisher M, Brott TG. Emerging therapies for acute ischemic stroke: new therapies on trial. Stroke. 2003; 34(2): 359-361.
2. Kaur J, Zhao Z, Klein GM, Lo EH, Buchan AM. The neurotoxicity of tissue plasminogen activator? J Cereb Blood Flow Metab. 2004; 24(9): 945-963.
3. Gropen TI, Gagliano PJ, Blake CA, et al. Quality improvement in acute stroke: the New York State Stroke Center Designation Project. Neurology. 2006; 67(1): 88-93.
4. Nicole O, Docagne F, Ali C, Margaill I, Carmeliet P, MacKenzie ET, et al. The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling. Nat Med. 2001; 7(1): 59-64.
5. Liot G, Roussel BD, Lebeurrier N, Benchenane K, Lopez-Atalaya JP, Vivien D, et al. Tissue-type plasminogen activator rescues neurones from serum deprivation-induced apoptosis through a mechanism independent of its proteolytic activity. J Neurochem. 2006; 98(5): 1458-1464.
6. Zhuo M, Holtzman DM, Li Y, Osaka H, DeMaro J, Jacquin M, et al. Role of tissue plasminogen activator receptor LRP in hippocampal long-term potentiation. J Neurosci. 2000; 20(2): 542-549.
7. Siao CJ, Tsirka SE. Tissue plasminogen activator mediates microglial activation via its finger domain through annexin II. J Neurosci. 2002; 22(9): 3352-3358.
8. Benchenane K, Lopez-Atalaya JP, Fernandez-Monreal M, Touzani O, Vivien D. Equivocal roles of tissue-type plasminogen activator in stroke-induced injury. Trends Neurosci. 2004; 27(3): 155-160.
9. Lebeurrier N, Vivien D, Ali C. The complexity of tissue-type plasminogen activator: can serine protease inhibitors help in stroke management? Expert Opin Ther Targets. 2004; 8(4): 309-320.
10. Samson AL, Medcalf RL. Tissue-type plasminogen activator: a multifaceted modulator of neurotransmission and synaptic plasticity. Neuron. 2006; 50(5): 673-678.
11. Benchenane K, Berezowski V, Ali C, Fernandez-Monreal M, Lopez-Atalaya JP, Brillault J, et al. Tissue-type plasminogen activator crosses the intact blood-brain barrier by low-density lipoprotein receptor-related protein-mediated transcytosis. Circulation. 2005; 111(17): 2241-2249.
12. Fernandez-Monreal M, Lopez-Atalaya JP, Benchenane K, Cacquevel M, Dulin F, Le Caer JP, et al. Arginine 260 of the amino-terminal domain of NR1 subunit is critical for tissue-type plasminogen activator-mediated enhancement of N-methyl-D-aspartate receptor signaling. J Biol Chem. 2004; 279(49): 50850-50856.
13. Fernandez-Monreal M, Lopez-Atalaya JP, Benchenane K, Leveille F, Cacquevel M, Plawinski L, et al. Is tissue-type plasminogen activator a neuromodulator? Mol Cell Neurosci. 2004; 25(4): 594-601.
14. Benchenane K, Castel H, Boulouard M, Bluthe R, Fernandez-Monreal M, Roussel BD, et al. Anti-NR1 Nterminal-domain vaccination unmasks the crucial action of tPA on NMDA-receptor-mediated toxicity and spatial memory. J Cell Sci. 2007; 120(Pt 4): 578-585.
15. Liberatore GT, Samson A, Bladin C, Schleuning WD, Medcalf RL. Vampire bat salivary plasminogen activator (desmoteplase): a unique fibrinolytic enzyme that does not promote neurodegeneration. Stroke. 2003; 34(2): 537-543.
16. Lopez-Atalaya JP, Roussel BD, Ali C, Maubert E, Petersen KU, Berezowski V, et al. Recombinant Desmodus rotundus salivary plasminogen activator crosses the blood-brain barrier through a low-density lipoprotein receptor-related protein-dependent mechanism without exerting neurotoxic effects. Stroke. 2007; 38(2): 1036-1043.
17. Wahlgren NG, Ahmed N. Neuroprotection in cerebral ischaemia: facts and fancies-the need for new approaches. Cerebrovasc Dis. 2004; 17 Suppl 1: 153-166.
18. Gill R, Foster AC, Woodruff GN. MK-801 is neuroprotective in gerbils when administered during the post-ischaemic period. Neuroscience. 1988; 25(3): 847-855.
19. Rother J. Neuroprotection Does Not Work! Stroke. 2008; 39(2): 523-524.
20. Grotta J. Neuroprotection is unlikely to be effective in humans using current trial design. Stroke. 2002; 33(1): 306-307.
21. Faden AI. Neuroprotection and traumatic brain injury: theoretical option or realistic proposition. Curr OpinNeurol. 2002; 15(6): 707-712.
22. Faden AI, Stoica B. Neuroprotection: challenges and opportunities. Arch Neurol. 2007; 64(6): 794-800.
23. Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke. 1999; 30(12): 2752-2758.
24. Fisher M, Hanley DF, Howard G, Jauch EC, Warach S; STAIR Group. Recommendations from the STAIR V meeting on acute stroke trials, technology and outcomes. Stroke. 2007; 38(2): 245-248.
25. Shuaib A, Hussain MS. The past and future of neuroprotection in cerebral ischaemic stroke. Eur Neurol. 2008; 59(1-2): 4-14.
26. Edaravone Acute Infarction Study Group. Effect of a novel free radical scavenger, edaravone (MCI-186), on acute brain infarction. Randomized, placebo-controlled, double-blind study at multicenters. Cerebrovas Dis. 2003; 15(3): 222-229.
27. Noble M. Ethics in the trenches: a multifaceted analysis of the stem cell debate. Stem Cell Rev. 2005; 1(4): 345-376.
28. Tahashi K, Tanabe K, Ohruki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5): 861-872.
29. Meissner A, Wernig M, Jaenisch R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol. 2007; 25(10): 1177-1181.
30. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007; 318(5858): 1917-1920.
31. Kalra L, Ratan RR. Advances in Stroke Regenerative Medicine 2007. Stroke. 2008; 39(2): 273-275.
32. Ohab JJ, Carmichael ST. Post-stroke neurogenesis: Emerging priniciples of migration and localization of immature neurons. Neuroscientist. 2007. Epub ahead of print.
33. Ratan RR, Siddiq A, Smirnova N, Karpisheva K, Haskew-Layton R, McConoughey S, et al. Harnessing hypoxic adaptation to prevent, treat and repair stroke. J Mol Med. 2007; 85(12): 1331-1338.
34. Kleim JA, Chan S, Pringle E, Schallert K, Procaccio V, Jimenez R, et al. BDNF val66met polymorphism is associated with modified experience-dependent plasticity in human motor cortex. Nat Neurosci. 2006; 9(6): 735-737.
35. Young AR, Ali C, Duretete A, Vivien D. Neuroprotection and stroke: time for a compromise. J Neurochem. 2007; 103(4): 1302-1309.
2. Huang J, Upadhyay UM, Tamargo RJ. Inflammation in stroke and focal cerebral ischemia. Surg Neurol. 2006; 66(3): 232-245.
3. Markiewicz I, Lukomska B. The role of astrocytes in the physiology and pathology of the central nervous system. Acta Neurobiol Exp (Wars). 2006; 66(4): 343-358.
4. Araque A, Parpura V, Sanzgiri RP, Haydon PG. Glutamate-dependent astrocyte modulation of synaptic transmission between cultured hippocampal neurons. Eur J Neurosci. 1998; 10(6): 2129-2142.
5. Koizumi S, Fujishita K, Tsuda M, Shigemoto-Mogami Y, Inoue K. Dynamic inhibition of excitatory synaptic transmission by astrocyte-derived ATP in hippocampal cultures. Proc Natl Acad Sci USA. 2003; 100(19): 11023-11028.
6. Zhang J, Hoffert C, Vu HK, Groblewski T, Ahmad S, O'Donnell D. Induction of CB2 receptor expression in the rat spinal cord of neuropathic but not inflammatory chronic pain models. Eur J Neurosci. 2003; 17(12): 2750-2754.
7. Zonta M, Angulo MC, Gobbo S, Rosengarten B, Hossmann KA, Pozzan T, et al. Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation. Nat Neurosci. 2003; 6(1): 43-50.
8. Mulligan SJ, MacVicar BA. Calcium transients in astrocyte endfeet cause cerebrovascular constrictions. Nature. 2004; 431(7005): 195-199.
9. Takano T, Tian GF, Peng W, Lou N, Libionka W, Han X, et al. Astrocyte-mediated control of cerebral blood flow. Nat Neurosci. 2006; 9(2): 260-267.
10. Ferrari D, Villalba M, Chiozzi P, Falzoni S, Ricciardi-Castagnoli P, Di Virgilio F. Mouse microglial cells express a plasma membrane pore gated by extracellular ATP. J Immunol. 1996; 156(4): 1531-1539.
11. Norenberg W, Cordes A, Blohbaum G, Frohlich R, Illes P. Coexistence of purino- and pyrimidinoceptors on activated rat microglial cells. Br J Pharmacol. 1997; 121(6): 1087-1098.
12. Honda S, Sasaki Y, Ohsawa K, Imai Y, Nakamura Y, Inoue K, et al. Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors. J Neurosci. 2001; 21(6): 1975-1982.
13. Shigemoto-Mogami Y, Koizumi S, Tsuda M, Ohsawa K, Kohsaka S, Inoue K. Mechanisms underlying extracellular ATP-evoked interleukin-6 release in mouse microglial cell line, MG-5. J Neurochem. 2001; 78(6): 1339-1349.
14. Suzuki T, Hide I, Ido K, Kohsaka S, Inoue K, Nakata Y. Production and release of neuroprotective tumor necrosis factor by P2X7 receptor-activated microglia. J Neurosci. 2004; 24(1): 1-7.
15. Verderio C, Matteoli M. ATP mediates calcium signaling between astrocytes and microglial cells: modulation by IFN-gamma. J Immunol. 2001; 166(10): 6383-6391.
16. Hagg T. Molecular regulation of adult CNS neurogenesis: an integrated view. Trends Neurosci. 2005; 28(11): 589-595.
17. Laywell ED, Steindler DA, Silver DJ. Astrocytic stem cells in the adult brain. Neurosurg Clin N Am. 2007; 18(1): 21-30.
18. Chen LW, Yung KL, Chan YS. Reactive astrocytes as potential manipulation targets in novel cell replacement therapy of Parkinson's disease. Curr Drug Targets. 2005; 6(7): 821-833.
19. Alvarez-Buylla A, Lim DA. For the long run: maintaining germinal niches in the adult brain. Neuron. 2004; 41(5): 683-686.
20. Matute C, Alberdi E, Ibarretxe G, Sanchez-Gomez MV. Excitotoxicity in glial cells. Eur J Pharmacol. 2002; 447(2-3): 239-246.
21. Anderson CM, Swanson RA. Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia. 2000; 32(1): 1-14.
22. King LS, Kozono D, Agre P. From structure to disease: the evolving tale of aquaporin biology. Nat Rev Mol Cell Bio. 2004; 5(9): 687-698.
23. Yang B, Verkman AS. Water and glycerol permeabilities of aquaporins 1-5 and MIP determined quantitatively by expression of epitope-tagged constructs in Xenopus oocytes. J Biol Chem. 1997; 272(26): 16140-16146.
24. Hasegawa H, Ma T, Skach W, Matthay MA, Verkman AS. Molecular cloning of a mercurial-insensitive water channel expressed in selected water-transporting tissues. J Biol Chem. 1994; 269(8): 5497-5500.
25. Jung JS, Bhat RV, Preston GM, Guggino WB, Baraban JM, Agre P. Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance. Proc Natl Acad Sci USA. 1994; 91(26): 13052-13056.
26. Zeidel ML, Ambudkar SV, Smith BL, Agre P. Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein. Biochemistry. 1992; 31(33): 7436-7440.
27. Frigeri A, Gropper MA, Umenishi F, Kawashima M, Brown D, Verkman AS. Localization of MIWC and GLIP water channel homologs in neuromuscular, epithelial and glandular tissues. J Cell Sci. 1995; 108 (9): 2993-3002.
28. Amiry-Moghaddam M, Ottersen OP. The molecular basis of water transport in the brain. Nat Rev Neurosci. 2003; 4(12): 991-1001.
29. Amiry-Moghaddam M, Frydenlund DS, Ottersen OP. Anchoring of aquaporin-4 in brain: molecular mechanisms and implications for the physiology and pathophysiology of water transport. Neuroscience. 2004; 129(4): 999-1010.
30. Nicchia GP, Nico B, Camassa LM, Mola MG, Loh N, Dermietzel R, et al The role of aquaporin-4 in the blood-brain barrier development and integrity: studies in animal and cell culture models. Neuroscience. 2004; 129(4): 935-945.
31. Manley GT, Binder DK, Papadopoulos MC, Verkman AS. New insights into water transport and edema in the central nervous system from phenotype analysis of aquaporin-4 null mice. Neuroscience. 2004; 129(4): 983-991.
32. Papadopoulos MC, Krishna S, Verkman AS. Aquaporin water channels and brain edema. Mt Sinai J Med. 2002; 69(4): 242-248.
33. Verkman AS, Binder DK, Bloch O, Auguste K, Papadopoulos MC. Three distinct roles of aquaporin-4 in brain function revealed by knockout mice. Biochim Biophys Acta. 2006; 1758(8): 1085-1093.
34. Papadopoulos MC, Manley GT, Krishna S, Verkman AS. Aquaporin-4 facilitates reabsorption of excess fluid in vasogenic brain edema. FASEB J. 2004; 18(11): 1291-1293.
35. Misu T, Fujihara K, Nakamura M, Murakami K, Endo M, Konno H, et al. Loss of aquaporin-4 in active perivascular lesions in neuromyelitis optica: a case report. Tohoku J Exp Med. 2006; 209(3): 269-275.
36. Roemer SF, Parisi JE, Lennon VA, Benarroch EE, Lassmann H, Bruck W, et al. Pattern-specific loss of aquaporin-4 immunoreactivity distinguishes neuromyelitis optica from multiple sclerosis. Brain. 2007; 130(5): 1194-1205.
37. Matsuoka T, Matsushita T, Kawano Y, Osoegawa M, Ochi H, Ishizu T, et al. Heterogeneity of aquaporin-4 autoimmunity and spinal cord lesions in multiple sclerosis in Japanese. Brain. 2007; 130(5): 1206-1212.
38. Lennon VA, Kryzer TJ, Pittock SJ, Verkman AS, Hinson SR. IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J Exp Med. 2005; 202(4): 473-477.
39. Salaria S, Chana G, Caldara F, Feltrin E, Altieri M, Faggioni F, et al. Microarray analysis of cultured human brain aggregates following cortisol exposure: implications for cellular functions relevant to mood disorders. Neurobiol Dis. 2006; 23(3): 630-636.
40. Li Z, Gao L, Liu Q, Cao C, Sun XL, Ding JH, et al. Aquaporin-4 knockout regulated cocaine-induced behavior and neurochemical changes in mice. Neurosci Lett. 2006; 403(3): 294-298.
41. La Porta CA, Gena P, Gritti A, Fascio U, Svelto M, Calamita G. Adult murine CNS stem cells express aquaporin channels. Biol Cell. 2006; 98(2): 89-94.
42. Cavazzin C, Ferrari D, Facchetti F, Russignan A, Vescovi AL, La Porta CA, et al. Unique expression and localization of aquaporin-4 and aquaporin-9 in murine and human neural stem cells and in their glial progeny. Glia. 2006; 53(2): 167-181.
43. Schwartz PH, Nethercott H, Kirov II, Ziaeian B, Young MJ, Klassen H. Expression of neurodevelopmental markers by cultured porcine neural precursor cells. Stem Cells. 2005; 23(9): 1286-1294.
44. Nicchia GP, Frigeri A, Liuzzi GM, Svelto M. Inhibition of aquaporin-4 expression in astrocytes by RNAi determines alteration in cell morphology, growth, and water transport and induces changes in ischemia-related genes. FASEB J. 2003; 17(11): 1508-1510.
45. Wen H, Nagelhus EA, Amiry-Moghaddam M, Agre P, Ottersen OP, Nielsen S. Ontogeny of water transport in rat brain: postnatal expression of the aquaporin-4 water channel. Eur J Neurosci. 1999; 11(3): 935-945.
46. Nagelhus EA, Mathiisen TM, Ottersen OP. Aquaporin-4 in the central nervous system: cellular and subcellular distribution and coexpression with KIR4.1. Neuroscience. 2004; 129(4): 905-913.
47. Verkman AS. Novel roles of aquaporins revealed by phenotype analysis of knockout mice. Rev Physiol Biochem Pharmacol. 2005; 155: 31-55.
48. Padmawar P, Yao X, Bloch O, Manley GT, Verkman AS. K~+ waves in brain cortex visualized using a long-wavelength K~+-sensing fluorescent indicator. Nat Methods. 2005; 2(11): 825-827.
49. Binder DK, Yao X, Zador Z, Sick TJ, Verkman AS, Manley GT. Increased seizure duration and slowed potassium kinetics in mice lacking aquaporin-4 water channels. Glia. 2006; 53(6): 631-636.
50. Saadoun S, Papadopoulos MC, Watanabe H, Yan D, Manley GT, Verkman AS. Involvement of aquaporin-4 in astroglial cell migration and glial scar formation. J CellSci. 2005; 118(Pt 24): 5691-5698.
51. Auguste KI, Jin S, Uchida K, Yan D, Manley GT, Papadopoulos MC, et al. Greatly impaired migration of implanted aquaporin-4-deficient astroglial cells in mouse brain toward a site of injury. FASEB J. 2007; 21(1): 108-116.
52. Nicchia GP, Srinivas M, Li W, Brosnan CF, Frigeri A, Spray DC. New possible roles for aquaporin-4 in astrocytes: cell cytoskeleton and functional relationship with connexin43.FASEB J. 2005; 19(12): 1674-1676.
53. Fan Y, Zhang J, Sun XL, Gao L, Zeng XN, Ding JH, et al. Sex- and region-specific alterations of basal amino acid and monoamine metabolism in the brain of aquaporin-4 knockout mice. J Neurosci Res. 2005; 82(4): 458-464.
54. Tomas-Camardiel M, Venero JL, de Pablos RM, Rite I, Machado A, Cano J. In vivo expression of aquaporin-4 by reactive microglia. J Neurochem. 2004; 91(4): 891-899.
55. Tomas-Camardiel M, Venero JL, Herrera AJ, De Pablos RM, Pintor-Toro JA, Machado A, et al. Blood-brain barrier disruption highly induces aquaporin-4 mRNA and protein in perivascular and parenchymal astrocytes: protective effect by estradiol treatment in ovariectomized animals. J Neurosci Res. 2005; 80(2): 235-246.
56. Zhou J, Kong H, Hua X, Xiao M, Ding J, Hu G. Altered blood-brain barrier integrity in adult aquaporin-4 knockout mice. Neuroreport. 2008; 19(1): 1-5.
57. Caplan LR. Stroke treatment: promising but still struggling. JAMA. 1998; 279(16): 1304-1306.
58. Fisher M, Bogousslavsky J. Further evolution toward effective therapy for acute ischemic stroke. JAMA. 1998; 279(16): 1298-1303.
59. Heiss WD, Thiel A, Grond M, Graf R. Which targets are relevant for therapy of acute ischemic stroke? Stroke. 1999; 30(7): 1486-1489.
60. Muir KW, Grosset DG. Neuroprotection for acute stroke: making clinical trials work. Stroke. 1999; 30(1): 180-182.
61. Hickenbottom SL, Barsan WG. Acute ischemic stroke therapy. Neurol Clin. 2000; 18(2): 379-397.
62. Manley GT, Fujimura M, Ma T, Noshita N, Filiz F, Bollen AW, et al. Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke. Nat Med. 2000; 6(2): 159-163.
63. Woitzik J, Schneider UC, Thome C, Schroeck H, Schilling L. Comparison of different intravascular thread occlusion models for experimental stroke in rats. J Neurosci Methods. 2006; 151(2): 224-231.
64. Ma T, Yang B, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS. Generation and phenotype of a transgenic knockout mouse lacking the mercurial-insensitive water channel aquaporin-4. J Clin Invest. 1997; 100(5): 957-962.
65. Koizumi J, Yoshida Y, Nakazawa T, Ooneda G. Experimental studies of ischemic brain edema: A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. Jpn Stroke J. 1986; 8: 1-8.
66. Ngai AC, Nguyen TS, Meno JR, Britz GW. Postischemic augmentation of conducted dilation in cerebral arterioles. Stroke. 2007; 38(1): 124-130.
67. Ribeiro Mde C, Hirt L, Bogousslavsky J, Regli L, Badaut J. Time course of aquaporin expression after transient focal cerebral ischemia in mice. J Neurosci Res. 2006; 83(7): 1231-1240.
68. Frydenlund DS, Bhardwaj A, Otsuka T, Mylonakou MN, Yasumura T, Davidson KG, et al. Temporary loss of perivascular aquaporin-4 in neocortex after transient middle cerebral artery occlusion in mice. Proc Natl Acad Sci USA. 2006; 103(36): 13532-13536.
69. Kleffner I, Bungeroth M, Schiffbauer H, Schabitz WR, Ringelstein EB, Kuhlenbaumer G. The role of aquaporin-4 polymorphisms in the development of brain edema after middle cerebral artery occlusion. Stroke. 2008; 39(4): 1333-1335.
70. Panickar KS, Norenberg MD. Astrocytes in cerebral ischemic injury: morphological and general considerations. Glia. 2005; 50(4): 287-298.
71. Lai AY, Todd KG. Microglia in cerebral ischemia: molecular actions and interactions. Can J Physiol Pharmacol. 2006; 84(1): 49-59.
72. Denes A, Vidyasagar R, Feng J, Narvainen J, McColl BW, Kauppinen RA, et al. Proliferating resident microglia after focal cerebral ischaemia in mice. J Cereb Blood Flow Metab. 2007; 27(12): 1941-1953.
73. Tanaka R, Komine-Kobayashi M, Mochizuki H, Yamada M, Furuya T, Migita M, et al. Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia. Neuroscience. 2003; 117(3): 531-539.
74. Schousboe A. Transport and metabolism of glutamate and GABA in neurons are glial cells. Int Rev Neurobiol. 1981; 22: 1-45.
75. Tanaka K, Watase K, Manabe T, Yamada K, Watanabe M, Takahashi K, et al. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science. 1997; 276(5319): 1699-1702.
76. Schousboe A, Divac I. Difference in glutamate uptake in astrocytes cultured from different brain regions. Brain Res. 1979; 177(2): 407-409.
77. Rosenberg PA, Amin S, Leitner M. Glutamate uptake disguises neurotoxic potency of glutamate agonists in cerebral cortex in dissociated cell culture. J Neurosci. 1992; 12(1): 56-61.
78. Frandsen A, Schousboe A. Development of excitatory amino acid induced cytotoxicity in cultured neurons. Int J Dev Neurosci. 1990; 8(2): 209-216.
79. Griffin S, Clark JB, Canevari L. Astrocyte-neurone communication following oxygen-glucose deprivation. J Neurochem. 2005; 95(4): 1015-1022.
80. Danbolt NC. Glutamate uptake. Prog Neurobiol. 2001; 65(1): 1-105.
81. Fiacco TA, McCarthy KD. Astrocyte calcium elevations: properties, propagation, and effects on brain signaling. Glia. 2006; 54(7): 676-690.
82. Araque A. Astrocyte-neuron signaling in the brain-implications for disease. Curr Opin Investig Drugs. 2006; 7(7): 619-624.
83. Benarroch EE. Neuron-astrocyte interactions: partnership for normal function and disease in the central nervous system. Mayo Clin Proc. 2005; 80(10): 1326-1338.
84. Bender AS, Schousboe A, Reichelt W, Norenberg MD. Ionic mechanisms in glutamate-induced astrocyte swelling: role of K~+ influx. J Neurosci Res. 1998; 52(3): 307-321.
85. Sass JB, Ang LC, Juurlink BH. Aluminum pretreatment impairs the ability of astrocytes to protect neurons from glutamate mediated toxicity. Brain Res. 1993; 621(2): 207-214.
86. Ozog MA, Siushansian R, Naus CC. Blocked gap junctional coupling increases glutamate-induced neurotoxicity in neuron-astrocyte co-cultures. J Neuropathol ExpNeurol. 2002; 61(2): 132-141.
87. Kosugi T, Kawahara K. Reversed actrocytic GLT-1 during ischemia is crucial to excitotoxic death of neurons, but contributes to the survival of astrocytes themselves. Neurochem Res. 2006; 31(7): 933-943.
88. Du Y, Chen CP, Tseng CY, Eisenberg Y, Firestein BL. Astroglia-mediated effects of uric acid to protect spinal cord neurons from glutamate toxicity. Glia. 2007; 55(5): 463-472.
89. Haugeto O, Ullensvang K, Levy LM, Chaudhry FA, Honore T, Nielsen M, et al. Brain glutamate transporter proteins form homomultimers. J Biol Chem. 1996; 271(44): 27715-27722.
90. Marcaggi P, Attwell D. Role of glial amino acid transporters in synaptic transmission and brain energetics. Glia. 2004; 47(3): 217-225.
91. Torres GE, Gainetdinov RR, Caron MG. Plasma membrane monoamine transporters: structure, regulation and function. Nat Rev Neurosci. 2003; 4(1): 13-25.
92. Munch C, Ebstein M, Seefried U, Zhu B, Stamm S, Landwehrmeyer GB, et al. Alternative splicing of the 5'-sequences of the mouse EAAT2 glutamate transporter and expression in a transgenic model for amyotrophic lateral sclerosis. J Neurochem. 2002; 82(3): 594-603.
93. Maragakis NJ, Dykes-Hoberg M, Rothstein JD. Altered expression of the glutamate transporter EAAT2b in neurological disease. Ann Neurol. 2004; 55(4): 469-477.
94. Ganel R, Ho T, Maragakis NJ, Jackson M, Steiner JP, Rothstein JD. Selective up-regulation of the glial Na~+-dependent glutamate transporter GLT1 by a neuroimmunophilin ligand results in neuroprotection. Neurobiol Dis. 2006; 21(3): 556-567.
95. Hoogland G, van Oort RJ, Proper EA, Jansen GH, van Rijen PC, van Veelen CW, et al. Alternative splicing of glutamate transporter EAAT2 RNA in neocortex and hippocampus of temporal lobe epilepsy patients. Epilepsy Res. 2004; 59(2-3): 75-82.
96. Matute C, Melone M, Vallejo-Illarramendi A, Conti F. Increased expression of the astrocytic glutamate transporter GLT-1 in the prefrontal cortex of schizophrenics. Glia. 2005; 49(3): 451-455.
97. Smith RE, Haroutunian V, Davis KL, Meador-Woodruff JH. Expression of excitatory amino acid transporter transcripts in the thalamus of subjects with schizophrenia. Am J Psychiatry. 2001; 158(9): 1393-1399.
98. Rothstein JD, Patel S, Regan MR, Haenggeli C, Huang YH, Bergles DE, et al. Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature. 2005; 433: 73-77.
99. Hurtado O, Pradillo JM, Fernandez-Lopez D, Morales JR, Sobrino T, Castillo J, et al. Delayed post-ischemic administration of CDP-choline increases EAAT2 association to lipid rafts and affords neuroprotection in experimental stroke. Neurobiol Dis. 2008; 29(1): 123-131.
100.Diamond JS, Jahr CE. Transporters buffer synaptically released glutamate on a submillisecond time scale. J Neurosci. 1997; 17(12): 4672-4687.
101.Takayasu Y, lino M, Shimamoto K, Tanaka K, Ozawa S. Glial glutamate transporters maintain one-to-one relationship at the climbing fiber-Purkinje cell synapse by preventing glutamate spillover. JNeurosci. 2006; 26(24): 6563-6572.
102.O'Shea RD. Roles and regulation of glutamate transporters in the central nervous system. Clin Exp Pharmacol Physiol. 2002; 29(11): 1018-1023.
103.Kanai Y, Hediger MA. The glutamate/neutral amino acid transporter family SLC1: molecular, physiological and pharmacological aspects. Pflugers Arch. 2004; 447(5): 469-479.
104.Rae C, Lawrance ML, Dias LS, Provis T, Bubb WA, Balcar VJ. Strategies for studies of neurotoxic mechanisms involving deficient transport of L-glutamate: antisense knockout in rat brain in vivo and changes in the neurotransmitter metabolism following inhibition of glutamate transport in guinea pig brain slices. Brain Res Bull. 2000; 53(4): 373-381.
105.Had-Aissouni L, Re DB, Nieoullon A, Kerkerian-Le Goff L. Importance of astrocytic Inactivation of synaptically released glutamate for cell survival in the central nervous system—are astrocytes vulnerable to low intracellular glutamate concentrations? J Physiol Paris. 2002; 96(3-4): 317-322.
106.Re DB, Boucraut J, Samuel D, Birman S, Kerkerian-Le Goff L, et al. Glutamate transport alteration triggers differentiation-state selective oxidative death of cultured astrocytes: a mechanism different from excitotoxicity depending on intracellular GSH contents. J Neurochem. 2003; 85(5): 1159-1170.
107.Aoyama K, Suh SW, Hamby AM, Liu J, Chan WY, Chen Y, et al. Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse. Nat Neurosci. 2006; 9(1): 119-126.
108.O'Shea RD, Lau CL, Farso MC, Diwakarla S, Zagami CJ, Svendsen BB, et al Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1. Neurochem Int. 2006; 48(6-7): 604-610.
109.Zhou J, Sutherland ML. Glutamate transporter cluster formation in astrocytic processes regulates glutamate uptake activity. J Neurosci 2004; 24(28): 6301-6306.
1. Fisher M, Brott TG. Emerging therapies for acute ischemic stroke: new therapies on trial. Stroke. 2003; 34(2): 359-361.
2. Kaur J, Zhao Z, Klein GM, Lo EH, Buchan AM. The neurotoxicity of tissue plasminogen activator? J Cereb Blood Flow Metab. 2004; 24(9): 945-963.
3. Gropen TI, Gagliano PJ, Blake CA, et al. Quality improvement in acute stroke: the New York State Stroke Center Designation Project. Neurology. 2006; 67(1): 88-93.
4. Nicole O, Docagne F, Ali C, Margaill I, Carmeliet P, MacKenzie ET, et al. The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling. Nat Med. 2001; 7(1): 59-64.
5. Liot G, Roussel BD, Lebeurrier N, Benchenane K, Lopez-Atalaya JP, Vivien D, et al. Tissue-type plasminogen activator rescues neurones from serum deprivation-induced apoptosis through a mechanism independent of its proteolytic activity. J Neurochem. 2006; 98(5): 1458-1464.
6. Zhuo M, Holtzman DM, Li Y, Osaka H, DeMaro J, Jacquin M, et al. Role of tissue plasminogen activator receptor LRP in hippocampal long-term potentiation. J Neurosci. 2000; 20(2): 542-549.
7. Siao CJ, Tsirka SE. Tissue plasminogen activator mediates microglial activation via its finger domain through annexin II. J Neurosci. 2002; 22(9): 3352-3358.
8. Benchenane K, Lopez-Atalaya JP, Fernandez-Monreal M, Touzani O, Vivien D. Equivocal roles of tissue-type plasminogen activator in stroke-induced injury. Trends Neurosci. 2004; 27(3): 155-160.
9. Lebeurrier N, Vivien D, Ali C. The complexity of tissue-type plasminogen activator: can serine protease inhibitors help in stroke management? Expert Opin Ther Targets. 2004; 8(4): 309-320.
10. Samson AL, Medcalf RL. Tissue-type plasminogen activator: a multifaceted modulator of neurotransmission and synaptic plasticity. Neuron. 2006; 50(5): 673-678.
11. Benchenane K, Berezowski V, Ali C, Fernandez-Monreal M, Lopez-Atalaya JP, Brillault J, et al. Tissue-type plasminogen activator crosses the intact blood-brain barrier by low-density lipoprotein receptor-related protein-mediated transcytosis. Circulation. 2005; 111(17): 2241-2249.
12. Fernandez-Monreal M, Lopez-Atalaya JP, Benchenane K, Cacquevel M, Dulin F, Le Caer JP, et al. Arginine 260 of the amino-terminal domain of NR1 subunit is critical for tissue-type plasminogen activator-mediated enhancement of N-methyl-D-aspartate receptor signaling. J Biol Chem. 2004; 279(49): 50850-50856.
13. Fernandez-Monreal M, Lopez-Atalaya JP, Benchenane K, Leveille F, Cacquevel M, Plawinski L, et al. Is tissue-type plasminogen activator a neuromodulator? Mol Cell Neurosci. 2004; 25(4): 594-601.
14. Benchenane K, Castel H, Boulouard M, Bluthe R, Fernandez-Monreal M, Roussel BD, et al. Anti-NR1 Nterminal-domain vaccination unmasks the crucial action of tPA on NMDA-receptor-mediated toxicity and spatial memory. J Cell Sci. 2007; 120(Pt 4): 578-585.
15. Liberatore GT, Samson A, Bladin C, Schleuning WD, Medcalf RL. Vampire bat salivary plasminogen activator (desmoteplase): a unique fibrinolytic enzyme that does not promote neurodegeneration. Stroke. 2003; 34(2): 537-543.
16. Lopez-Atalaya JP, Roussel BD, Ali C, Maubert E, Petersen KU, Berezowski V, et al. Recombinant Desmodus rotundus salivary plasminogen activator crosses the blood-brain barrier through a low-density lipoprotein receptor-related protein-dependent mechanism without exerting neurotoxic effects. Stroke. 2007; 38(2): 1036-1043.
17. Wahlgren NG, Ahmed N. Neuroprotection in cerebral ischaemia: facts and fancies-the need for new approaches. Cerebrovasc Dis. 2004; 17 Suppl 1: 153-166.
18. Gill R, Foster AC, Woodruff GN. MK-801 is neuroprotective in gerbils when administered during the post-ischaemic period. Neuroscience. 1988; 25(3): 847-855.
19. Rother J. Neuroprotection Does Not Work! Stroke. 2008; 39(2): 523-524.
20. Grotta J. Neuroprotection is unlikely to be effective in humans using current trial design. Stroke. 2002; 33(1): 306-307.
21. Faden AI. Neuroprotection and traumatic brain injury: theoretical option or realistic proposition. Curr OpinNeurol. 2002; 15(6): 707-712.
22. Faden AI, Stoica B. Neuroprotection: challenges and opportunities. Arch Neurol. 2007; 64(6): 794-800.
23. Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke. 1999; 30(12): 2752-2758.
24. Fisher M, Hanley DF, Howard G, Jauch EC, Warach S; STAIR Group. Recommendations from the STAIR V meeting on acute stroke trials, technology and outcomes. Stroke. 2007; 38(2): 245-248.
25. Shuaib A, Hussain MS. The past and future of neuroprotection in cerebral ischaemic stroke. Eur Neurol. 2008; 59(1-2): 4-14.
26. Edaravone Acute Infarction Study Group. Effect of a novel free radical scavenger, edaravone (MCI-186), on acute brain infarction. Randomized, placebo-controlled, double-blind study at multicenters. Cerebrovas Dis. 2003; 15(3): 222-229.
27. Noble M. Ethics in the trenches: a multifaceted analysis of the stem cell debate. Stem Cell Rev. 2005; 1(4): 345-376.
28. Tahashi K, Tanabe K, Ohruki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5): 861-872.
29. Meissner A, Wernig M, Jaenisch R. Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol. 2007; 25(10): 1177-1181.
30. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007; 318(5858): 1917-1920.
31. Kalra L, Ratan RR. Advances in Stroke Regenerative Medicine 2007. Stroke. 2008; 39(2): 273-275.
32. Ohab JJ, Carmichael ST. Post-stroke neurogenesis: Emerging priniciples of migration and localization of immature neurons. Neuroscientist. 2007. Epub ahead of print.
33. Ratan RR, Siddiq A, Smirnova N, Karpisheva K, Haskew-Layton R, McConoughey S, et al. Harnessing hypoxic adaptation to prevent, treat and repair stroke. J Mol Med. 2007; 85(12): 1331-1338.
34. Kleim JA, Chan S, Pringle E, Schallert K, Procaccio V, Jimenez R, et al. BDNF val66met polymorphism is associated with modified experience-dependent plasticity in human motor cortex. Nat Neurosci. 2006; 9(6): 735-737.
35. Young AR, Ali C, Duretete A, Vivien D. Neuroprotection and stroke: time for a compromise. J Neurochem. 2007; 103(4): 1302-1309.