LY367385通过抑制细胞外信号调节激酶途径减轻小鼠脑血管痉挛后神经细胞凋亡

目的: 探讨代谢型谷氨酸受体1(mGluR1)选择性拮抗剂LY367385对脑血管痉挛(CVS)后神经细胞凋亡的影响及细胞外信号调节激酶1/2(ERKl/2)途径的作用。方法: 采用非开颅血管内线栓法制备小鼠蛛网膜下腔出血(SAH)并CVS模型,随机分为3组:假手术组、模型对照组和mGluR1拮抗剂LY367385组,于SAH后10 min侧脑室注射生理盐水或LY367385(500 nmol)5 μL,术后行神经功能评分。分别在SAH后6、24和48 h 3个时点取右侧脑组织标本,在光镜和电镜下观察脑组织病理变化,采用逆转录-聚合酶链式反应检测各组mGluR1 mRNA的表达变化;蛋白免疫印迹法检测mGluR1和p-ERK1/2蛋白的表达;用TUNEL法检测神经细胞凋亡情况。结果: 与假手术组比较,模型组小鼠神经功能评分显著降低,随CVS时间延长,各组小鼠mGluR1 mRNA、mGluR1和p-ERK1/2蛋白均有不同程度增强,凋亡细胞增多,神经细胞出现变性坏死、神经轴索变性断裂。与模型组比较,拮抗剂组小鼠神经功能评分增加,mGluR1 mRNA、mGluR1和p-ERK1/2蛋白表达均有不同程度下调,神经细胞凋亡数目减少,脑组织形态学和超微结构损伤减轻。结论: (1)CVS后mGluR1的表达增强可通过激活ERK信号途径诱导神经细胞凋亡。(2)mGluR1选择性拮抗剂LY367385对CVS损伤具有拮抗作用。     

小鼠蛛网膜下腔出血后海马CA1区mGluR1和ERK1/2的表达变化

目的:探讨小鼠蛛网膜下腔出血(subarachnoid hemorrhage,SAH)后代谢型谷氨酸受体1(metabotropicglutamate receptor 1,mGluR1)及细胞外信号调节激酶1/2(ERK1/2)调控神经细胞凋亡的分子机制。方法:采用非开颅血管内穿线法制备小鼠SAH模型,随机分为3组:假手术组、SAH+生理盐水(SAH+NS)组、SAH+LY367385(mGluR1抑制剂,SAH+LY367385)组,于SAH后10 min侧脑室注射生理盐水或LY367385(500 nmol/L)5μl,术后行神经功能评分。分别在SAH后6、24、48 h 3个时间点取右侧脑组织标本,逆转录-聚合酶链式反应(RT-PCR)检测各组mGluR1的表达变化,免疫印迹法(Western Blot)检测p-ERK1/2蛋白的表达,TUNEL法检测右侧海马CA1区神经细胞的凋亡。结果:与假手术组比较,SAH+NS组小鼠神经功能评分均显著降低(P<0.05),随SAH时间延长,各组小鼠mGluR1、p-ERK1/2蛋白均有不同程度增强,凋亡细胞增多(P<0.05)。与SAH+NS组比较,SAH+LY367385组小鼠神经功能评分增加,mGluR1、p-ERK1/2蛋白表达均有不同程度下调,神经细胞凋亡数目有所减少。SAH后6～48 h,mGluR1的表达与p-ERK1/2呈正相关。结论:mGluR1和ERK在SAH的发病机制中发挥了重要作用,SAH后海马内mGluR1的表达增强可通过激活ERK信号途径诱导神经细胞的凋亡。     

载脂蛋白E拟肽在脑血管痉挛中的保护机制

目的:研究血管内皮生长因子(VEGF)-细胞外信号调节激酶(ERK)信号转导通路在脑血管痉挛后早期脑损伤中的作用,探讨载脂蛋白E(apoE)拟肽通过此途径发挥的保护机制.方法:采用非开颅血管内穿线法制备小鼠蛛网膜下腔出血(SAH)和脑血管痉挛模型,给予apoE-1410肽,术后脑血管铸型检测大脑中动脉(MCA)直径变化,分别在术后12、 24、 48h 3个时相点取右侧大脑动脉标本,应用免疫印迹法检测各组VEGF、 p-ERK1/2蛋白表达,TUNEL法检测MCA内皮细胞凋亡.结果:模型组MCA出现严重的血管痉挛,管腔直径减小,随脑血管痉挛时间延长,各组小鼠VEGF、 p-ERK1/2蛋白均有不同程度上调,凋亡细胞增多.与模型组比较,治疗组MCA管腔直径增加,各时相点3项指标的表达均不同程度下调.蛛网膜下腔出血12～48h VEGF表达与p-ERK1/2呈正相关;p-ERK1/2与TUNEL呈正相关.结论:脑血管痉挛后VEGF表达增强可通过激活ERK信号途径诱导大脑动脉内皮细胞凋亡;拟apoE-1410肽对脑血管痉挛具有一定的治疗作用,其机制之一可能是通过部分阻抑VEGF/ERK通路活化减少凋亡实现的.     

外源性拟载脂蛋白E肽对脑血管痉挛后血管内皮生长因子介导的脑动脉内皮细胞凋亡的影响

目的:探讨外源性拟载脂蛋白E(apoE)肽对脑动脉痉挛后内皮细胞凋亡的调节作用及对血管内皮生长因子(VEGF)表达的影响.方法:采用非开颅血管内穿线法制备小鼠蛛网膜下腔出血模型并给予apoE-1410肽,术后行神经功能评分并检测大脑中动脉直径变化,应用原位杂交法、免疫印迹法检测各组右侧大脑动脉VEGF表达,TUNEL法检测大脑中动脉内皮细胞凋亡.结果:出血组小鼠神经功能评分均降低,大脑中动脉直径减小,VEGF mRNA及VEGF蛋白显著升高,TUNEL显色阳性.治疗组小鼠神经功能评分及大脑中动脉管径增加,VEGF表达降低,内皮细胞凋亡数目减少.VEGF mRNA表达与TUNEL呈正相关.结论:蛛网膜下腔出血后痉挛脑动脉内皮细胞凋亡与VEGF高表达有关;拟apoE-1410肽对脑血管痉挛具有一定的治疗作用,其机制之一可能是通过抑制VEGF活化,减少大脑中动脉内皮细胞凋亡.     

ApoE肽段预处理通过调节c-jun氨基端激酶阻抑小鼠脑缺血再灌后神经细胞的凋亡

目的:为探讨外源性载脂蛋白E(apoE)肽段对局灶性脑缺血再灌(I/R)的保护机制,观察apoE-1410拟肽对I/R小鼠脑组织磷酸化c-jun氨基端激酶(JNK)表达变化的影响。方法:实验选用160只雄性ICR小鼠,随机分为3组:假手术对照组(sham组),模型组(I/R组),apoE治疗组(apoE组),采用可逆性大脑中动脉栓塞(MCAO)模型,术后行神经功能评分。I/R3、6、12、24、48h行HE染色观察皮质区神经细胞的形态变化,并计算存活神经元的数目。利用WesternBlot法、免疫组织化学法检测p-c-jun的表达,行TUNEL法检测凋亡细胞数。结果:与对照组比较,模型组小鼠神经功能评分均降低,I/R12h后存活神经元数目明显减少;p-c-jun阳性反应术后3、6、12、24h显著增高(P0.05);随缺血时间延长,凋亡细胞增多,并于48h达高峰(P0.05)。与模型组比较,治疗组小鼠神经功能评分增加,各时相点p-c-jun和TUNEL表达均不同程度下调(P0.05)。I/R3h至48h皮质区p-c-jun表达与TUNEL呈正相关(r=0.716,P0.01)。结论:缺血侧皮质区p-c-jun表达的增强可诱导I/R后神经细胞凋亡;拟apoE-1410肽对I/R具有一定的治疗作用,其机制之一可能是通过抑制JNK活化实现的。     

Changes of extracellular signal regulated kinase and caspase-8 expression in arteriae cerebri of mice with cerebral vasospasm model

目的:研究细胞外信号调节激酶(ERK)、半胱氨酸天冬氨酸特异性蛋白酶-8(caspase-8)在脑血管痉挛(CVS)后早期脑损伤中的作用,并探讨ERK特异性抑制剂U0126通过此途径发挥作用的保护机制.方法:采用非开颅血管内穿线法制备小鼠蛛网膜下腔出血(SAH)并脑血管痉挛模型,给予U0126,术后行神经功能评分,分别在术后12、24、48 h3个时相点取右侧大脑动脉标本,应用免疫印迹检测各组磷酸化ERK1/2( p-ERK1/2)、caspase-8蛋白表达,TUNEL法检测大脑中动脉内皮细胞凋亡.结果:模型组小鼠神经功能评分降低,大脑中动脉出现严重脑血管痉挛,随脑血管痉挛时间延长,各组小鼠p-ERK1/2、caspase-8蛋白均有不同程度增强,凋亡细胞增多.与模型组比较,治疗组小鼠神经功能评分增加,各时相点3项指标的表达均不同程度下调.脑血管痉挛12～48 h p-ERK1/2与caspase-8呈正相关.结论:脑血管痉挛后,ERK表达增强可通过激活caspase-8信号途径诱导大脑动脉内皮细胞凋亡;U0126对脑血管痉挛具有一定的治疗作用,其机制之一可能是通过部分阻抑ERK通路活化,减少凋亡实现的.     

依达拉奉通过抑制细胞外信号调节激酶1/2信号通路减轻大鼠弥漫性脑创伤后神经细胞凋亡

目的:探讨依达拉奉对脑创伤后神经细胞凋亡的影响及其机制.方法:雄性SD大鼠随机分为对照组、创伤组、依达拉奉组,Marmarou's法建立弥漫性脑创伤模型.H-E染色观察皮质区神经细胞组织形态变化;免疫组织化学法和免疫印迹法检测磷酸化ERK1/2及Bax的表达;原位缺口末端标记法(TUNEL)检测神经细胞的凋亡,并对大鼠综合运动功能进行评定.结果:与对照组比较,创伤组中皮质区部分神经细胞出现变性坏死和凋亡的改变,磷酸化 ERK1/2(1、 6、 24、 48h)和Bax(6、 24、 48、 72h)表达水平增高;神经细胞凋亡数目(6、 24、 48、 72h)增多;大鼠综合运动能力评分下降.与创伤组比较,依达拉奉组中脑组织形态结构损伤程度、磷酸化ERK1/2和Bax表达、神经细胞凋亡数目显著下降;大鼠的运动功能评分升高.结论:依达拉奉通过抑制ERK1/2信号途径活化,进而抑制促凋亡蛋白Bax表达,减少神经细胞凋亡,发挥对弥漫性脑创伤的保护作用.     

大鼠重型弥漫性脑创伤后ERK1/2信号途径的改变及意义

目的:探讨重型弥漫性脑创伤后细胞外信号调节激酶1/2(ERK1/2)信号的改变及意义。方法:SD大鼠分为正常对照组、创伤组、ERK1/2抑制剂U0126高、低剂量组。Marmarou等法制作弥漫性脑创伤模型。伤后30min、1、6、24、48、72h光镜和电镜下观察伤后脑组织形态变化;Western blotting法检测海马区磷酸化ERK1/2和c-Fos蛋白表达;TUNEL法检测海马区凋亡细胞;伤后3-7d水迷宫法测试动物学习记忆功能。结果:伤后,创伤组脑组织损伤严重,神经细胞出现变性坏死和凋亡改变、神经轴索变性断裂;磷酸化ERK1/2蛋白表达在伤后30min明显增高,6h达高峰,高表达状态持续至伤后24h;c-Fos在伤后30min明显增高,6h达高峰,24h接近正常水平;神经细胞凋亡数目伤后6h明显增多,72h达高峰;大鼠搜索安全岛时间延长。U0126干预后,脑组织形态损伤程度、磷酸化ERK1/2和c-Fos表达、神经细胞凋亡数目回降;大鼠搜索安全岛时间缩短;上述变化在U0126高剂量组中更为显著。结论:ERK1/2信号途径参与大鼠重型弥漫性脑创伤病理损伤过程,并在伤后神经细胞凋亡进程中发挥关键作用。     

利多卡因对大鼠SAH后神经细胞凋亡和迟发性脑血管痉挛的保护作用

目的:探讨利多卡因对大鼠蛛网膜下腔出血(SAH)后神经细胞凋亡和迟发性脑血管痉挛的保护作用。方法:将60只成年大鼠分为假手术组(sham)、SAH组(SAH)和lidocaine处理组(lidocaine)。采用枕大池二次注血法制备动物模型。lidocaine组注射盐酸利多卡因。分别于不同时间灌注后取脑。取用部分基底动脉行苏木精-伊红染色法(HE)观察形态改变和测量基底动脉的管径和管壁的厚度,使用脱氧核糖核酸(DNA)断裂的原位末端标记(TUNEL)法测定海马CA1区神经细胞的凋亡情况。结果:HE染色显示,利多卡因组从第3 d开始内径周长增加,管壁厚度缩小。TUNEL检测显示:SAH组凋亡细胞随时间逐渐增加,而利多卡因组的凋亡细胞逐渐减少。结论:利多卡因能明显减轻大鼠SAH后迟发性的脑血管痉挛及减少海马CA1区神经细胞的凋亡,具有一定的保护作用。     

mGluR5特异性激动剂CHPG促进人NSCs增殖

目的:研究代谢型谷氨酶受体mGluR5特异性激动剂CHPG对人胚胎皮质神经干细胞(NSCs)增殖的影响以及分子机制。方法:采用MTT比色法和神经球直径测量分析CHPG对人NSCs增殖的影响;流式细胞仪分析对细胞周期和细胞凋亡的影响;采用免疫蛋白印迹法,观察磷酸化ERK,JNK和p38 MAPKs表达水平的变化。结果:mGluR5激动剂CHPG促进人胚胎皮质NSCs增殖;CHPG组与对照组相比,S期与G2/M期细胞数量显著增加(P0.05),G1/G0期细胞数量显著减少(P0.05);CHPG组早期凋亡和晚期凋亡细胞显著减少(P0.05);CHPG组与对照组相比,ERK1/2和JNK2的磷酸化水平显著增高,但是p38 MAPKs的磷酸化水平显著降低(P0.01)。结论:mGluR5激动剂CHPG促进人胚胎皮质NSCs增殖,同时伴随着ERK和JNK的磷酸化激活。     

U0126对蛛网膜下腔出血小鼠细胞外信号调节激酶途径的调节作用

目的:研究细胞外信号调节激酶(ERK)、天冬氨酸特异性半胱氨酸蛋白酶-8(caspase-8)在蛛网膜下腔出血(SAH)后早期脑损伤中的作用,并探讨ERK特异性抑制剂U0126通过此途径发挥作用的保护机制。方法:采用稳定的非开颅血管内穿线法制备小鼠SAH模型,并于术前30 min经尾静脉给予U0126(0.1 mg/kg),分别在术后12、24、48 h 3个时相点取右侧大脑动脉标本,HE染色观察大脑动脉的形态改变,并检测大脑中动脉(MCA)的直径变化;应用免疫印迹法检测各组p-ERK1/2、caspase-8蛋白表达,TUNEL法检测MCA内皮细胞凋亡。结果:模型组小鼠MCA出现严重血管痉挛,直径减小,p-ERK1/2、caspase-8蛋白均有不同程度增强,凋亡细胞增多。与模型组比较,治疗组小鼠各时相点上述3项指标的表达均呈不同程度下调,MCA管径增加,脑血管痉挛缓解。SAH 12～48 h时p-ERK1/2与caspase-8的表达呈正相关。结论:SAH后ERK表达增强可通过激活caspase-8信号途径诱导大脑动脉内皮细胞凋亡;U0126可减少大脑动脉内皮细胞凋亡,其机制之一可能是通过阻抑ERK通路活化实现的。     

An apolipoprotein E-based therapeutic improves outcome and reduces Alzheimer's disease pathology following closed head injury: evidence of pharmacogenomic interaction

Apolipoprotein E (apoE) modifies glial activation and the CNS inflammatory response in an isoform-specific manner. Peptides derived from the receptor-binding region of apoE have been demonstrated to maintain the functional activity of the intact protein, and to improve histological and functional deficits after closed head injury. In the current study, APOE2, APOE3, and APOE4 targeted replacement (TR) mice expressing the human apoE protein isoforms (apoE2, apoE3 and apoE4) were used in a clinically relevant model of closed head injury to assess the interaction between the humanized apoE background and the therapeutic apoE mimetic peptide, apoE(133-149). Treatment with the apoE-mimetic peptide reduced microglial activation and early inflammatory events in all of the targeted replacement animals and was associated with histological and functional improvement in the APOE2TR and APOE3TR animals. Similarly, brain beta amyloid protein (Abeta)(1-42) levels were increased as a function of head injury in all of the targeted replacement mice, while treatment with apoE peptide suppressed Abeta(1-42) levels in the APOE2TR and APOE3TR animals. These results suggest a pharmacogenomic interaction between the therapeutic effects of the apoE mimetic peptide and the human apoE protein isoforms. Furthermore, they suggest that administration of apoE-mimetic peptides may serve as a novel therapeutic strategy for the treatment of acute and chronic neurological disease.     

p53在蛛网膜下腔出血小鼠脑皮质的表达及其与PI3K/Akt信号通路的关系

目的 探讨p53蛋白在实验性蛛网膜下腔出血小鼠脑皮质的表达及其与PI3K/Akt信号通路的关系.方法 将72只小鼠随机平均分为3组,即假手术组(Sham组),蛛网膜下腔出血组(SAH组),抑制剂LY294002组(LY组).采用线栓法制备小鼠蛛网膜下腔出血模型,术后观测6个时相点:0h、3h、6h、9h、12h、24h...     

Regulation of main olfactory bulb mitral cell excitability by metabotropic glutamate receptor mGluR1

In the rodent main olfactory bulb (MOB), mitral cells (MCs) express high levels of the group I metabotropic glutamate receptor (mGluR) subtype, mGluR1. The significance of this receptor in modulating MC excitability is unknown. We investigated the physiological role of mGluR1 in regulating MC activity in rat and mouse MOB slices. The selective group I agonist (RS)-3,5-dihydroxyphenylglycine (DHPG), but not group II or III agonists, induced potent, dose-dependent, and reversible depolarization and increased firing of MCs. These effects persisted in the presence of blockers of fast synaptic transmission, indicating that they are due to direct activation of mGluRs on MCs. Voltage-clamp recordings showed that DHPG elicited a voltage-dependent inward current consisting of multiple components sensitive to potassium and calcium channel blockade and intracellular calcium chelation. MC excitatory responses to DHPG were absent in mGluR1 knockout mice but persisted in mGluR5 knockout mice. Broad-spectrum LY341495, MCPG, as well as preferential mGluR1 LY367385 antagonists blocked the excitatory effects of DHPG and also potently modulated MC spontaneous and olfactory nerve-evoked excitability. mGluR antagonists altered spontaneous membrane potential bistability, increasing the duration of the up and down states. mGluR antagonists also substantially attenuated MC responses to sensory input, decreasing the probability and increasing the latency of olfactory nerve-evoked spikes. These findings suggest that endogenous glutamate tonically modulates MC excitability and responsiveness to olfactory nerve input, and hence the operation of the MOB circuitry, via activation of mGluR1.     

Differential roles for mGluR1 and mGluR5 in the persistent prolongation of epileptiform bursts.

Transient activation of group I metabotropic glutamate receptors (mGluRs) with the selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) produces persistent prolongation of epileptiform bursts in guinea pig hippocampal slices, the maintenance of which can be reversibly suppressed with group I mGluR antagonists. To determine the relative roles of mGluR1 and mGluR5 in these group I mGluR-dependent induction and maintenance processes, subtype-selective antagonists were utilized. In the presence of picrotoxin, DHPG (50 microM, 20-45 min) converted interictal bursts into 1- to 3-s discharges that persisted for hours following washout of the mGluR agonist. 2-methyl-6-(phenylethynyl)-pyridine (MPEP, an mGluR5 antagonist; 25 microM) and (+)-2-methyl-4-carboxyphenylglycine (LY367385, an mGluR1 antagonist; 20-25 microM) each significantly suppressed the ongoing expression of the mGluR-induced prolonged bursts. However, LY367385 was more effective, reducing the burst prolongation by nearly 90%; MPEP only produced a 64% reduction in burst prolongation. Nevertheless, MPEP was more effective at preventing the induction of the burst prolongation; all 10 slices tested failed to express prolonged bursts both during and after co-application of DHPG with MPEP. Co-application of DHPG with LY367385, in contrast, resulted in significant burst prolongation (in 68% of slices tested) that was revealed on washout of the two agents. These results suggest that while both receptor subtypes participate in both the induction and maintenance of mGluR-mediated burst prolongation, mGluR1 activation plays a greater role in sustaining the expression of prolonged bursts, whereas mGluR5 activation may be a more critical contributor to the induction process underlying this type of epileptogenesis.     

Olfactory nerve-evoked, metabotropic glutamate receptor-mediated synaptic responses in rat olfactory bulb mitral cells

The group I metabotropic glutamate receptor (mGluR) subtype, mGluR1, is highly expressed on the apical dendrites of olfactory bulb mitral cells and thus may be activated by glutamate released from olfactory nerve (ON) terminals. Previous studies have shown that mGluR1 agonists directly excite mitral cells. In the present study, we investigated the involvement of mGluR1 in ON-evoked responses in mitral cells in rat olfactory bulb slices using patch-clamp electrophysiology. In voltage-clamp recordings, the average EPSC evoked by single ON shocks or brief trains of ON stimulation (six pulses at 50 Hz) in normal physiological conditions were not significantly affected by the nonselective mGluR antagonist LY341495 (50-100 microM) or the mGluR1-specific antagonist LY367385 (100 microM); ON-evoked responses were attenuated, however, in a subset (36%) of cells. In the presence of blockers of ionotropic glutamate and GABA receptors, application of the glutamate uptake inhibitors THA (300 microM) and TBOA (100 microM) revealed large-amplitude, long-duration responses to ON stimulation, whereas responses elicited by antidromic activation of mitral/tufted cells were unaffected. Magnitudes of the ON-evoked responses elicited in the presence of THA-TBOA were dependent on stimulation intensity and frequency, and were maximal during high-frequency (50-Hz) bursts of ON spikes, which occur during odor stimulation. ON-evoked responses elicited in the presence of THA-TBOA were significantly reduced or completely blocked by LY341495 or LY367385 (100 microM). These results demonstrate that glutamate transporters tightly regulate access of synaptically evoked glutamate from ON terminals to postsynaptic mGluR1s on mitral cell apical dendrites. Taken together with other findings, the present results suggest that mGluR1s may not play a major role in phasic responses to ON input, but instead may play an important role in shaping slow oscillatory activity in mitral cells and/or activity-dependent regulation of plasticity at ON-mitral cell synapses.