NMDA受体拮抗剂剂量与脑缺血再灌注大鼠海马内源性神经干细胞的增殖

背景:脑缺血再灌注后,过度释放的兴奋性氨基酸可通过NMDA受体激活内源性神经干细胞,促使其增殖、分化,修复神经细胞,但同时也导致细胞内钙离子超载,引起神经细胞的损伤。通过控制NMDA受体活化的程度,刺激内源性神经干细胞激活的同时把损伤减到最小。目的:观察NMDA受体拮抗剂MK-801质量浓度对脑缺血再灌注大鼠海马内源性神经干细胞增殖的影响。方法:健康雄性SD大鼠54只随机数字表法分为对照组(不进行任何处理)、手术组(缺血模型制作)及不同剂量MK-801组。采用大鼠四条血管阻断方法(Pulsinelli-4VO法)制备大鼠全脑缺血再灌注模型。不同浓度MK-801组在模型制作前30min按照0.2,0.4,0.6,0.8,1.0,1.2mg/kg侧脑室注射MK-801。Western-blot、RT-PCR技术检测各组nestin蛋白及mRNA水平及表达。结果与结论:MK-801剂量在0.8mg/kg以下时,nestin mRNA及蛋白的表达差异无显著性意义(P0.05),呈现高表达;当剂量为0.8mg/kg时,可明显抑制内源性神经干细胞增殖;在0.8mg/kg以上,对神经干细胞的抑制作用随着剂量的升高呈递增趋势;在1.2mg/kg时,大鼠有躁动、共济失调等严重不良反应。nestin mRNA表达结果与蛋白表达趋势相吻合。说明MK-801的剂量与脑梗死后神经功能的修复有一定的关系,实验中0.6mg/kg是一个比较合适的实验应用剂量,在此剂量下既可使MK-801减少兴奋性氨基酸对神经元的毒性作用,保护神经细胞,又使内源性神经干细胞的增殖不受较大影响。     

MK-801的浓度与局灶性脑缺血大鼠内源性神经干细胞增殖的关系

目的 研究兴奋性氨基酸（NMDA）受体拮抗剂MK-801的浓度与局灶性脑缺血大鼠神经干细胞增殖的关系。方法 将60只大鼠随机分为正常对照组（6只）、假手术对照组（6只）、手术对照组（12只）和MK-801不同荆量组（0．2mg／kg、0．4mg／kg、0．6mg／kg、0．8mg／kg、1．0mg／kg、1．2mg／kg）（36只）。除正常对照组外，其它组均需制作大鼠大脑中动脉缺血（middle cerebral artery occlusion，MCAO）动物模型，在模型制作前30分钟按照不同剂量组腹腔注射MK一801，假手术组和手术对照组腹腔注射等量的生理盐水。在模型建立后第7天，通过免疫组化技术标记大鼠梗塞灶附近大脑皮质的Nestin阳性细胞数目。结果 MK-801浓度为0．4mg／kg时，可以明显抑制内源性神经干细胞的增殖，0．8mg／kg剂量以上有显著的抑制作用，并且随着浓度升高抑制作用呈递增趋势，在1．2mg／kg以上有明显的全身副作用。结论 NMDA受体拮抗剂MK-801在局灶性脑缺血后，对大鼠内源性神经干细胞的增殖有抑制作用，并且随浓度的增加抑制作用也随之加强。     

MK-801对皮质酮诱导 抑郁样行为大鼠海马mBDNF和proBDNF表达的影响

目的 N-甲基-D-天冬氨酸(NMDA)受体拮抗剂通过脑源性神经营养因子(BDNF)发挥抗抑郁作用。然而,NMDA受体拮抗剂调控BDNF表达的机制尚不清楚。方法口服皮质酮(CORT)诱导大鼠抑郁样行为。采用高架十字迷宫实验、蔗糖偏好实验、旷场实验和强迫游泳实验观察动物的抑郁样行为,采用蛋白免疫印迹方法检测动物背侧和腹侧海马DG脑区成熟BDNF(mBDNF)和前体BDNF(proBDNF)的表达。结果慢性给予CORT诱发大鼠抑郁样行为。NMDA受体拮抗剂地佐环平(MK-801)可减轻动物抑郁样行为,增加大鼠背侧和腹侧海马DG脑区中mBDNF的表达,同时降低proBDNF的表达(P0.05)。组蛋白去乙酰化酶拮抗剂辛二酰苯胺异羟肟酸(SAHA)能够减轻动物抑郁样行为,增加海马脑区的mBDNF表达,降低proBDNF的表达(P0.05)。然而,预先给予MK-801后,SAHA对大鼠抑郁样行为、海马DG脑区的mBDNF和proBDNF表达都无显著的影响。结论 MK-801可通过组蛋白乙酰化改变大鼠海马DG区mBDNF和proBDNF的表达,发挥抗抑郁作用,这将为抗抑郁治疗提供新靶点。     

芍药苷激活2型大麻素受体保护脑缺血再灌注大鼠海马神经元

目的探究芍药苷通过激活2型大麻素受体(CBR2)对脑缺血再灌注大鼠神经的保护作用。方法 144只雄性SD大鼠随机分为假手术组、模型组、溶媒组、10mg/kg芍药苷组、40mg/kg芍药苷组、CBR2选择性拮抗剂3mg/kg AM630组、40mg/kg芍药苷联合3mg/kg AM630组、CBR2选择性激动剂3mg/kg HU308处理组。线栓法制作大鼠局灶性脑缺血/再灌注模型。观察芍药苷对神经功能缺损评分、梗塞体积、脑水肿的影响;采用HE染色观察脑组织病理形态学改变;采用免疫组织化学染色法观察芍药苷对海马区环氧合酶2(COX-2)和caspase-3表达的影响。结果芍药苷能显著改善脑缺血大鼠神经功能评分,降低梗塞体积及缺血侧脑含水量,减轻脑组织病变,抑制海马区caspase-3和COX-2的表达;但预先注射AM630可显著拮抗芍药苷的脑神经保护作用。结论 CBR2可能参与芍药苷对脑缺血再灌注大鼠海马神经元的保护作用。     

海马NDMA受体与胃肌间神经丛NOS神经元分布在慢性应激性胃运动变化中的关系

 目的 探讨慢性应激对大鼠胃功能和胃肠神经系统的影响,并分析其海马谷氨酸（Glu）离子型受体机制。方法 通过建造慢性应激性抑郁模型大鼠,结合脑立体定位及微量注射Glu和N-甲基-D-天冬氨酸（NMDA）受体阻断剂MK-801,对实验鼠进行糖水偏爱等行为学检测、胃内压记录及胃内在神经丛的一氧化氮合酶(Nitric oxide synthase,NOS)阳性神经元表达的组织化学检测。结果 慢性不可预见性温和应激（CUMS）动物表现出抑郁样行为,且胃运动减弱;海马注射NMDA受体阻断剂MK-801,可以反转CUMS的效应;海马注射Glu,能增加游泳不动时间,但对胃运动无影响。CUMS使胃肌间神经丛NOS阳性神经元数量减少（73.74±16.38/LPF,P<0.05）,神经节数量减少（4.25±1.34/LPF, P<0.05）,但每个神经节内神经元数量明显增加（6.55±2.37,P<0.05）;海马注射MK-801,能改善CUMS引起的神经节数量减少的现象。结论 慢性应激诱发的抑郁样行为与海马Glu 及其NMDA受体有关,而胃活动的减弱可能与海马NMDA受体变化影响胃肌间神经丛NOS神经元分布格局有关     

间歇性低压缺氧预处理对大鼠全脑缺血/再灌注海马CA1区Ngb和Bcl-2蛋白表达的影响

 目的：探讨间歇性低压缺氧预处理对大鼠全脑缺血/再灌注海马CA1区脑红蛋白（Ngb）和Bcl-2蛋白表达的影响。方法：将Wistar大鼠随机分为假手术组、低压缺氧预处理对照组、全脑缺血/再灌注组、低压缺氧预处理+全脑缺血/再灌注组4组,将间歇暴露于低压缺氧环境的大鼠作为低压缺氧预处理对照组。采用Pulsinelli四血管闭塞法复制大鼠全脑缺血/再灌注模型,夹闭颈总动脉造成全脑缺血8 min后再灌注。用硫堇染色法和免疫组织化学方法分别观察大鼠海马组织学改变和海马组织Ngb、Bcl-2蛋白表达变化。结果：低压缺氧预处理+全脑缺血/再灌注组大鼠海马CA1区存活细胞数较全脑缺血/再灌注组明显增加,其海马组织Ngb和Bcl-2蛋白表达量较全脑缺血/再灌注组显著升高。结论：间歇性低压缺氧预处理可能通过上调海马组织Ngb和Bcl-2蛋白的表达,减少全脑缺血再灌注后海马神经元的死亡而发挥神经保护作用。     

基质细胞衍生因子1对内源性神经干细胞的趋化迁移作用

背景：目前研究表明,基质细胞衍生因子1在参与趋化迁移内源性神经干细胞中起着非常重要的作用,但其具体迁移机制尚不明确。目的：观察外源性基质细胞衍生因子1 对大鼠内源性神经干细胞的趋化迁移作用及海马区神经干细胞的激活增殖情况。方法：通过向SD大鼠海马区上大脑皮质内注射外源性基质细胞衍生因子1(注射量为5 μL,质量浓度为500 μg/L)建立动物模型,于3,7,14,21 d后灌注取脑,通过石蜡切片免疫组织化学检测大鼠皮质内注射区及海马区nestin阳性细胞表达情况,并设实验对照组与空白对照组。结果与结论：石蜡切片免疫组织化学显示：实验组注射区周围及海马区nestin表达阳性细胞的数量随时间推移逐渐增多,3,7 d时注射区及海马区nestin表达阳性细胞少量表达,14 d时注射区及海马区nestin表达阳性细胞进一步增多,并向注射区形成明显的迁移趋势,21 d时注射区及海马区nestin表达阳性细胞更多。实验对照组及空白实验组未见上述表现。结果表明外源性基质细胞衍生因子1 可能诱导海马区神经干细胞的增殖分化,参与内源性神经干细胞的趋化迁移过程。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

MK-801对新生大鼠脑外伤后神经元凋亡的影响

目的：探讨N-甲基-D-天冬氨酸(NMDA)受体拮抗剂MK-801对新生大鼠创伤性脑外伤(traumatic brain injury,TBI)后神经元凋亡的影响。方法：建立新生7 d大鼠顶叶皮质挫伤模型,在TBI前30 min、TBI后即刻、TBI后30 min分别给予腹腔注射MK-8011 mg/kg,在TBI后24 h取脑,连续切片,行H-E染色和Caspase-3免疫组化染色,检测脑神经元细胞的损伤和凋亡。结果：MK-801三组不同时间用药组与TBI组相比,在创伤同侧的扣带皮质、顶叶皮质和丘脑神经元凋亡细胞数减少,有显著性差异。其中TBI后即刻用MK-801治疗效果最好。结论：MK-80l能明显减少TBI后神经元的凋亡。     

Relationships between hippocampal NMDA receptor and NOS positive neurons in myenteric nerve plexus in stomach of CUMS rats

目的 探讨慢性应激对大鼠胃功能和胃肠神经系统的影响,并分析其海马谷氨酸(Glu)离子型受体机制.方法 通过建造慢性应激性抑郁模型大鼠,结合脑立体定位及微量注射Glu和N-甲基-D-天冬氨酸(NMDA)受体阻断剂MK-801,对实验鼠进行糖水偏爱等行为学检测、胃内压记录及胃内在神经丛的一氧化氮合酶(NOS)阳性神经元表达的组织化学检测.结果 慢性不可预见性温和应激(CUMS)动物表现出抑郁样行为,且胃运动减弱；海马注射NMDA受体阻断剂MK-801,可以反转CUMS的效应；海马注射Glu,能增加游泳不动时间,但对胃运动无影响.CUMS使胃肌间神经丛NOS阳性神经元数量减少[(73.74±16.38 )/LPF,P＜0.05],神经节数量减少[(4.25±1.34)/LPF,P＜0.05],但每个神经节内神经元数量明显增加(6.55±2.37,P＜0.05)；海马注射MK-801能改善CUMS引起的神经节数量减少的现象.结论 慢性应激诱发的抑郁样行为与海马Glu及其NMDA受体有关,而胃活动的减弱可能与海马NMDA受体变化影响胃肌间神经丛NOS神经元分布格局有关.     

Ro25-6981对脑缺血再灌注模型大鼠海马齿状回颗粒下层nestin和BrdU表达的影响

目的:研究Ro25-6981对成年大鼠短暂性脑缺血再灌注海马齿状回颗粒下层(subgranular zone,SGZ)神经干细胞增殖的影响。方法:健康成年雄性SD大鼠在进行侧脑室置管7 d后,随机分为正常组、假手术组和脑缺血再灌注组,每组又分别再分为Ro25-6981组和生理盐水组。缺血再灌注组大鼠采用四动脉阻断前脑缺血再灌注模型,即缺血15 min再灌注1、3、7、14、21、28 d和35 d,缺血前15 min经侧脑室套管注射Ro25-6981或生理盐水。各组大鼠均采用免疫组织化学显示SGZ区Brd U、nestin阳性细胞的表达情况,采用免疫荧光双重标记法检测再灌7 d时SGZ区Brd U/nestin双标阳性细胞数量的改变。结果:脑缺血再灌注生理盐水组SGZ区nestin阳性细胞表达再灌1 d时开始增多,7 d时达到峰值,14 d时急剧下降,至35 d时降至正常水平;缺血前给予Ro25-6981,各时间点的nestin阳性细胞表达均有所下降,其中在3、7 d和14 d时减少明显,差异有统计学意义(P0.05)。脑缺血再灌注生理盐水组SGZ区Brd U阳性细胞再灌1 d开始增殖,7 d达到峰值,35 d降至正常水平;缺血前给予Ro25-6981,各时间点的Brd U阳性细胞表达均有所下降,其中在3、7、14 d和21 d时减少明显,差异有统计学意义(P0.05)。与脑缺血再灌注生理盐水组相比,Ro25-6981可以降低脑缺血再灌7 d海马SGZ区的Brd U/nestin双标阳性细胞密度,差异有统计学意义(P0.05)。结论:Ro25-6981可以抑制前脑缺血再灌注后海马SGZ区神经干细胞的增殖,提示NR2B参与并促进了前脑缺血再灌注引起的神经干细胞的增殖。     

N-methyl-D-aspartate receptor antagonist reduces nitrotyrosine formation in caudate-putamen in rat focal cerebral ischemia-reperfusion

The aim of this study is to determine experimentally whether N-methyl-D-aspartate (NMDA) receptor is involved in nitrotyrosine formation in rat brain subjected to focal ischemia-reperfusion, by using the NMDA receptor antagonist MK-801. Halothane-anesthetized and artificially ventilated rats were given MK-801 (5 mg/kg, i.p.) or vehicle prior to 2 h of focal cerebral ischemia followed by 0.5 h of reperfusion. The brain was then removed and divided into four sections, cortical ischemic core, peri-ischemic cortex, lateral caudate-putamen and non-ischemic cortex. Tissue nitrotyrosine was measured by means of hydrolysis/HPLC. MK-801 significantly attenuated nitrotyrosine formation in the lateral caudate-putamen. We conclude that nitrotyrosine formation required activation of NMDA receptors, at least in part.     

Ro25-6981对缺血再灌注大鼠脑室下区神经干细胞增殖的影响

目的:探讨N-甲基-D-天冬氨酸(NMDA)受体亚单位NR2B特异性拮抗剂Ro25-6981对缺血再灌注大鼠脑室下区神经干细胞增殖的影响及可能的机制。方法:线栓法制作大鼠大脑中动脉栓塞2 h再灌注模型,随机分成假手术组、缺血再灌注对照组和Ro25-6981干预组。免疫组织化学显色观察脑室下区巢蛋白和增殖细胞核抗原(PCNA)阳性细胞数,行免疫印迹对缺血侧脑组织脑源性神经营养因子(BDNF)蛋白表达水平进行定量分析。结果:缺血再灌注后脑室下区巢蛋白和PCNA阳性细胞较假手术组增加,而Ro25-6981干预组巢蛋白和PCNA阳性细胞较缺血再灌注对照组减少。缺血再灌注损伤促进了缺血侧脑组织BDNF蛋白的表达,Ro25-6981干预下调了缺血侧脑组织BDNF蛋白的表达。结论:NMDA受体亚单位NR2B能促进脑室下区神经干细胞的增殖,可能与调节BDNF表达有关。     

Effect of NMDA receptor antagonist on proliferation of neurospheres from embryonic brain

The N-methyl-D-aspartate (NMDA) receptor, a subtype of ionotropic glutamate receptors, plays an important role in the regulation of neuronal development, learning and memory, and neurodegenerative diseases. NMDA receptor blockade enhances neurogenesis in the hippocampal dentate gyrus in vivo. The effect of NMDA receptor antagonist on proliferation of neural progenitor cells, however, remains to be determined. We investigated changes in the diameter and number of neurospheres derived from the embryonic rat brain after NMDA receptor blockade. Cortical progenitor cells were isolated from gestational day 18 fetal rats according to the Percoll density gradient method. Cultured spheres expressed neural progenitor markers, musashi-1 and nestin. Immunohistochemical analysis demonstrated that cells in Dulbecco's modified Eagle medium/F12 containing 1% fetal bovine serum on day 8 differentiated to MAP-2-positive neurons and GFAP-positive astrocytes. The expression of NR1 and NR2B subunits of the NMDA receptor in neurospheres was detected. Neither brief nor sustained exposure to NMDA altered the diameter and number of neurospheres. Brief exposure to 30 μM MK-801, an NMDA receptor antagonist, decreased the diameter of neurospheres. Sustained exposure to 30 μM MK-801 decreased the diameter and number of neurospheres. Our results provide evidence that MK-801 directly decreased proliferation of neural progenitor cells.     

MK-801对新生鼠脑外伤性神经变性的超微结构的影响

目的:研究非竞争性N-甲基-D-天门冬氨酸受体拮抗剂MK-801对新生鼠创伤性颅脑损伤(TBI)后同侧顶叶皮质和海马神经元超微结构的影响.方法:新生7 d SD大鼠,被随机分成正常对照组和实验组(实验组给予MK-801 1 mg/kg,并且细分为创伤前30 min给药、创伤即刻给药和创伤后30 min给药3个亚组).造模24 h取材,透射电镜下观察神经细胞超微结构的变化.结果:创伤后30 min给药的神经细胞出现胞质和核染色质的浓缩深染,胞质内充满大小不等的空泡;创伤即刻组的神经细胞胞质内线粒体有肿胀,但胞核的变化不明显.结论:适时和适量运用MK-180能延迟神经细胞核染色质和粒线体的变性损伤,使之停留在损伤早期,为临床联合其他药物治疗赢得时间.     

Contribution of peripheral N-methyl-D-aspartate receptors to c-fos expression in the trigeminal spinal nucleus following acute masseteric inflammation

In this study, we examined the contribution of N-methyl-D-aspartate (NMDA) receptors on c-fos expression in the trigeminal brainstem nuclei following acute muscle inflammation. Mustard oil (MO; 20%, 30 microL) injected into the masseter muscle induced extensive peripheral edema and Fos-like immunoreactivity (Fos-LI) in several trigeminal brainstem areas including the subnucleus caudalis of the trigeminal spinal nucleus (Vc), the ventral and dorsal regions of the Vc/subnucleus interpolaris transition zone, and the paratrigeminal nucleus. In order to assess the effect of antagonizing NMDA receptors on MO-induced Fos-LI, rats were pre-treated with two different doses of i.v. MK-801 (0.3 mg/kg, 3 mg/kg), a non-competitive NMDA receptor antagonist, 30 min prior to MO injection. Additional groups of rats received MK-801 (0.3 mg/kg) directly in the masseter muscle or in the biceps muscle 5 min prior to MO injection. A higher dose of i.v. MK-801 (3 mg/kg) and MK-801 given locally into the masseter muscle (0.3 mg/kg) produced a significant reduction in total number of MO-induced Fos-LI. Further analyses revealed that pre-treatment with MK-801 (3 mg/kg i.v.) significantly reduced the Fos-LI all throughout the Vc. Only at the caudal Vc, there was a dose-dependent reduction of MO induced Fos-LI. Pre-treatment with masseteric MK-801 also significantly reduced the Fos-LI in the caudal Vc, with the effect greater than that produced by the same dose of MK-801 given intravenously. These results suggest that peripheral NMDA receptors contribute to nociceptive processing from craniofacial muscles.     

永久性脑缺血神经干细胞迁移和细胞分裂、增殖的研究

目的　探讨永久性脑缺血神经干细胞 (NSC)迁移是否伴随自身的分裂与增殖。方法　以永久性脑缺血大鼠为模型 ,采用免疫组化染色方法 ,观察脑缺血 1,3,7,14和 2 8dNSC迁移及增殖细胞核抗原(PCNA)的表达。结果　室管膜下区 (SEZ)NSC和PCNA+ 细胞在缺血后沿胼胝体腹侧向缺血区方向发生了时空一致性的迁移模式 ;缺血梗死灶周围均有较多巢蛋白和PCNA阳性细胞的表达。结论　永久性脑缺血后NSC可能通过自身分裂和增殖向外迁移 ,巢蛋白和PCNA在缺血区周围表达可作为NSC迁移和增殖的标志物。     

MK-801和L-NA对成年仓鼠视网膜节细胞轴突损伤后存活与再生的影响

目的 研究NMDA受体阻断剂MK-801和一氧化氮合酶抑制剂L-NA对成年仓鼠视网膜节细胞(下称节细胞)轴突切断后存活和再生的影响。方法 切断动物左侧视神经后分两组：存活组存活2、7或14d；再生组视神经眶侧断端同一段坐骨神经吻合后存活28d。所有实验动物自视神经切断前1d开始，每日接受腹腔注射MK-801和／或LNA直至处死。结果存活节细胞均数在MK-801组与对照组间无显著性差异，但L-NA组在术后2和7d节细胞数较对照组显著增加，合用MK-801／LNA较单用MK-801或L-NA使更多节细胞存活。而MK-801或L-NA对节细胞轴突在周围神经移植物内的再生均无明显作用。结论 lmg／kg剂量的MK-801对节细胞的存活无明显作用，但同时阻断NMDA受体和抑制一氧化氮合酶比单纯抑制一氧化氮合酶对节细胞有更强的神经保护作用。1．0mg／kgMK-801或4．5mg／kgL-NA对节细胞轴突的再生无明显促进作用。     

Bivalent effects of MK-801 on ethanol-induced sensitization do not parallel its effects on ethanol-induced tolerance

The relationship between the effects of the N-methyl-D-aspartate (NMDA) antagonist MK-801 on acute responses to ethanol and its ability to block ethanol sensitization and tolerance was examined in DBA/2J mice. Cross-sensitization between these drugs was also studied. Repeated administration of 0.1 mg/kg MK-801 with ethanol potentiated, whereas 0.25 mg/kg attenuated, sensitization to ethanol's locomotor stimulant effects; rearing was similarly affected. There was evidence for cross-sensitization between ethanol and 0.25 mg/kg MK-801. MK-801 potentiated ethanol's ataxic effects in the grid test, but had no effect on tolerance to this effect. MK-801's effects on ethanol sensitization appeared to be related to its own behavioral effects, rather than NMDA receptor blockade per se. Further, these studies demonstrate dissociation between ethanol sensitization and tolerance.     

N-methyl-D-aspartate-induced excitotoxicity in adult rat retina is antagonized by single systemic injection of MK-801

Intravitreal injection of N-methyl-D-aspartate (NMDA) produced a substantial damage to the adult rat retina that was largely restricted to inner retinal layers, including the ganglion cell layer (GCL), inner nuclear layer (INL), inner, and outer plexiform layers. This retinal damage was significantly reduced by a systemic injection of a low dose of MK-801 (0.5 mg/kg), a potent NMDA-receptor antagonist. This neuroprotection was dose dependent and was most effective when the antagonist was given 1 h before NMDA insult. An intraperitoneal injection of 0.5 mg/kg MK-801 provided a virtually complete protection to the retina to the NMDA-induced toxicity, as indicated quantitatively by the number of DiI-filled retinal ganglion cells, the number of cells in the GCL and INL that undergo DNA fragmentation, and the edematous changes in retinal thickness. A post-lesion administration of MK-801 was still able to provide an effective neuroprotective effect to the retina, but this protection was lost when MK-801 was given 4 h after NMDA exposure. The current results indicate a therapeutic potential of systemic application of MK-801 in protecting the adult rat retina from neurologic disorders related to excessive activation of NMDA receptors.