氟西汀预处理对大鼠脑缺血再灌注海马区BDNF和bcl-2表达的影响

目的探讨氟西汀对大鼠缺血后再灌注海马的影响及可能机制。方法将40只Wistar大鼠随机等分成4组：①单纯用药组（氟西汀灌胃20d）；②正常对照组（氯化钠灌胃20d）：③术前用药组（氟西汀灌胃20d制作脑缺血再灌注模型）；④单纯手术组（氯化钠灌胃20d制作脑缺血再灌注模型）．于实验终点进行苏木精-伊红染色及免疫组化检查．检测bcl-2和脑源性神经营养因子（BDNF）表达情况。结果与正常对照组比较，单纯用药组bcl-2检测指标无显著性差异（P〉0．05），BDNF表达显著性增多（P〈0．05）。术前用药组与单纯手术组比较，bcl-2表达无显著性差异（P〉0．05），BDNF表达显著性增高（P〈0．05）。结论氟西汀可以诱导增加BDNF表达．但不增加bcl-2的表达．从而减轻缺血后的再灌注损伤．产生神经保护作用。     

脑梗死后认知功能障碍患者血清BDNF水平及氟西汀的治疗作用

目的探讨脑梗死后认知功能障碍与血清BDNF的关系,观察氟西汀对血清BDNF和认知功能的影响。方法应用ELISA法检测血清BDNF水平,比较脑梗死后认知功能障碍组患者和认知功能正常组患者血清BDNF水平。将脑梗死后认知功能障碍组患者按照有无接受氟西汀治疗随机分为对照组和氟西汀组,观察两组治疗前后血清BDNF水平及MMSE分值的变化。分析血清BDNF与MMSE分值的相关性。结果脑梗死后认知功能障碍组患者血清BDNF水平低于认知功能正常组;对照组治疗前、后MMSE分值及血清BDNF水平无明显变化;氟西汀治疗组治疗后MMSE分值及血清BDNF水平均较治疗前升高;氟西汀治疗组治疗后MMSE分值及血清BDNF水平均较对照组治疗后升高;脑梗死后认知功能障碍患者MMSE分值与血清BDNF水平呈正相关。结论脑梗死后认知功能障碍的发生和程度与血清BDNF水平有关,氟西汀能改善脑梗死后认知功能患者的认知功能,并可能部分是通过增加血清BDNF来实现的。     

脑源性神经营养因子对海马神经元NMDA受体功能下调的逆转作用

应用全细胞膜片钳技术研究BDNF对培养养海马神经元NMDA受体的调控作用。结果发现,培养18d的海马神经元NMDA诱发电流小,BDNF可快速、可逆地增加NMDA诱发电流,而培养10,14d的海马神经元NMDA诱发电流大,BDNF增强NMDA诱发电流不明显。本文结果提示BDNF对功能低下的海马神经元NMDA受体具有上调作用。     

脑源性神经营养因子对海马神经元NMDA受体功能下调的逆转作用

应用全细胞膜片钳技术研究BDNF对培养海马神经元NMDA受体的调控作用.结果发现,培养18d的海马神经元NMDA诱发电流小,BDNF可快速、可逆地增加NMDA诱发电流,而培养10,14d的海马神经元NMDA诱发电流大,BDNF增强NMDA诱发电流不明显.本文结果提示BDNF对功能低下的海马神经元NMDA受体具有上调作用.     

电抽搐与氟西汀对大鼠部分脑区脑源性神经营养因子及其受体表达的影响

目的 观察两种抗抑郁措施对大鼠部分脑区脑源性神经营养因子(BDNF)及其受体酪氨酸激酶受体B(Trk B)表达的影响.方法 根据电抽搐(ECS)与氟西汀(Flu)处理持续时间分为短期/长期(10 d/28 d)组,分别采用免疫组织化学、定量逆转录聚合酶链反应(RT-PCR)检测实验大鼠6个目标脑区BDNF/TrkB蛋白和信使核酸的表达水平.结果 ①与对照组相比,短期ECS与Flu组丘脑(Tha)、海马齿状回(DG)BDNF免疫反应性(IR)均明显增强(P＜0.05);短期ECS组海马阿蒙角(CA)和下丘脑(HI)BDNF/TrkB-IR也增强(P＜0.05).长期氟西汀组所有目标脑区的BDNF-IR以及Amy、Ht、CA、DG的TrkB-IR均较对照有明显增强,而长期ECS组与对照比较,只DG的BDNF/TrkB-IR均无变化,余目标脑区的BDNF-IR增强,而Ht和CA区的TrkB-IR也增强(P均小于0.05).②短期ECS组只Hc区BDNFmRNA较对照组有明显上调(P＜0.05),两长期组HC区BDNF/TrkBmRNA表达以及Amy区BDNFmRNA表达水平较相应对照组均明显上调(P＜0.05).而长期ECS组Ht区域BDNFmRNA则明显下调(P＜0.05).结论 电休克与氟西汀治疗这两种抗抑郁措施可影响脑内不同区域BDNF/TrkB转录表达和蛋白合成,BDNF可能参与抗抑郁治疗效应的发生.     

慢性应激与氟西汀对大鼠海马结构与杏仁核神经营养因子蛋白与核酸表达的影响

目的观察慢性温和未预知应激抑郁模型及氟西汀对大鼠海马与杏仁核神经元BDNF／Trk与NT-3／TrkC表达的影响。方法将32只SD雄性大鼠随机均分为正常组、慢性应激模型组、氟西汀组与生理盐水组。选用慢性轻度不可预见性应激加孤养造模,分别采用免疫组织化学、定量逆转录聚合酶链反应检测慢性应激与氟西汀处理的大鼠海马与杏仁核BDNF／TrkB蛋白和信使核酸的表达。结果与其相应的对照组相比,两干预组海马海马回区（CA）与齿状核（DG）BDNF表达呈现相反的变化,而杏仁核的表达则相似。慢性应激组与氟西汀组NT-3在海马与齿状核受调节的方向迥异。TrkB与TrkC表达水平与其配体调节方向略有差别。结论慢性温和未预知应激与氟西汀对海马与杏仁核BDNF／Trk与NT-3／TrkC表达的不同调节方向,可能反映慢性应激抑郁模型动物的病理过程与氟西汀治疗效应之间的差异。     

氟西汀对大鼠星形胶质细胞分泌的胶质源性神经营养因子的影响

目的 探讨氟西汀对大鼠星形胶质细胞分泌的胶质源性神经营养因子(GDNF)的影响.方法 以氟西汀干预体外培养的大鼠海马星形胶质细胞,通过四甲基偶氮唑盐法(MTT)检测不同浓度氟西汀对细胞活力的影响;采用酶联免疫吸附测定法(ELISA)检测细胞培养液GDNF浓度及Real-time PCR法检测GDNFmRNA的表达.结果 (1)氟西汀浓度超过35 μmol/L浓度时,可降低细胞活性,差异有统计学意义(P<0.01或P<0.05);(2)10 μmol/L氟西汀干预星形胶质细胞不同时间后,48 h组细胞培养液GDNF浓度[(68±13)fg/L]高于0 h组[(32±11)fg/L]、6 h组[(34±12)fg/L]、12 h组[(41±17)fg/L]、24 h组[(45±13)fg/L],差异均有统计学意义(P均<0.01);(3)不同浓度氟西汀作用星形胶质细胞48 h后,10 μmol/L浓度组的细胞培养液GDNF浓度[(64±17)fg/L]高于0 μmol/L[(39±15)fg/L]和1 μmoVL浓度组[(39±18)fg/L],差异均有统计学意义(P均<0.05);(4)氟西汀作用星形胶质细胞48 h后,撤离氟西汀24 h后星形胶质细胞仍明显分泌GDNF,差异有统计学意义(P<0.01或P<0.05);(5)不同浓度氟西汀作用星形胶质细胞24 h后,10 μmol/L和20 μmol/L浓度组细胞GDNFmRNA表达量[分别为(0.008 1±0.001 1)和(0.006 3±0.000 3)]高于0 μmol/L、1 μmol/L及5 μmol/L浓度组[分别为(0.003 1±0.000 7)、(0.003 9±0.000 3)和(0.004 1±0.000 2)],差异均有统计学意义(P均<0.01).结论 氟西汀可能通过促进星形胶质细胞GDNF的分泌来发挥其神经保护作用.     

Notch1信号系统在氟西汀上调大鼠海马神经再生中的作用

目的 了解慢性氟西汀干预正常大鼠所导致的海马神经再生上调与Notch1信号系统功能改变的关系.方法 应用大鼠腹腔注射氟西汀建立在体模型,分为对照组、14 d干预组、28 d干预组(n=12),采用免疫组化、real time PCR和Western blot,测定大鼠海马神经干细胞的增殖、存活和分化以及Notch1信号通路各个因子(NICD、Hes1、Hes5、Jag1)的基因及蛋白表达水平的改变.结果 ①与对照组(2919.50±188.80)比较,14 d氟西汀干预组(3706.50±228.04)、28 d氟西汀干预组(4334.33±217.48)海马齿状回神经干细胞增殖明显增加(P<0.001);与对照组(2404.50±148.77)相比,Flu干预28 d组(3273.16±156.68)海马齿状回神经干细胞存活明显增加(P<0.001);与对照组比较,氟西汀干预组NeuN/BrdU、GFAP/BrdU比例无明显差异(P＞0.05).②与对照组[NICDmRNA (0.30±0.03),Hes1mRNA (0.53±0.03),Hes5mRNA (0.21±0.02),Jag1mRNA(1.04±0.07)]比较,氟西汀(Flu)干预14d组[NICDmRNA (0.45±0.05),Hes1mRNA (0.65±0.06),Hes5mRNA (0.31±0.06),Jag1mRNA(2.46±0.39)]和Flu干预28 d组[NICDmRNA (0.42±0.03),Hes1mRNA (0.85±0.06),Hes5mRNA (0.39±0.02),Jag1mRNA(3.21±0.34)]Notch1信号通路各因子基因水平均明显升高(P<0.01或P<0.001).③与对照组[NICD(2.36±0.17),Hes1(1.09±0.25),Jag1(2.33±0.31)]比较,Flu干预14 d组[NICD(3.20±0.25),Jag1(2.86±0.25)]和Flu干预28 d组[NICD(3.40±0.19),Hes1(1.43±0.13),Jag1(3.35±0.14)]NICD、Hes1、Jag1蛋白水平明显升高,差异有统计学意义(P<0.01或P<0.001).与对照组Hes5比较,Flu干预14 d组Hes5和Flu干预28 d Hes5蛋白水平无改变,差异无统计学意义(P＞0.05).结论 氟西汀促进大鼠海马齿状回神经干细胞的增殖和存活,但对分化无影响;同时,海马Notch信号功能激活,提示Notch1信号系统可能参与氟西汀介导的大鼠海马神经再生上调.     

电针及氟西汀对抑郁大鼠行为学及海马神经元凋亡的影响

目的 探究电针及氟西汀对慢性应激抑郁大鼠行为学及海马神经元凋亡的影响.方法 65只Sprague-Dawley雄性大鼠,按随机数字表随机分为空白组、空白电针组、模型组、电针组、氟西汀组,每组13只.慢性应激后进行行为学评价;采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶双染法检测海马神经元凋亡率;采用免疫组织化学方法检测海马bcl-2蛋白表达.结果 模型组大鼠旷场试验水平穿越格数[(1.6±1.3)格]、竖立次数[(0.4 ±0.2)次]、体质量增加量[(34±18)g]均明显低于空白组[分别为(51.1 ±22.3)格、(13.2±4.6)次、(128 ±21)g],P均＜0.05;海马神经元细胞凋亡率[(67±10)%]高于空白组[(53±13)%],P＜0.05;海马组织bcl-2蛋白阳性细胞计数[(28±10)个/mm<'2>]低于空白组[(78±22)个/mm<'2>],P＜0.05.电针组[分别为(39.3 ±14.3)格,(9.6 ±4.1)次,(81±43)g]、氟西汀组[分别为(37.2±15.1)格,(9.3±4.6)次,(80 ±35)g]上述指标均明显高于模型组(P＜0.05);海马神经元细胞凋亡率[电针组(30±9)%,氟西汀组(51±13)%]均低于模型组(P＜0.05);海马组织bcl-2蛋白阳性细胞计数[电针组(56±18)个/mm<'2>,氟西汀组(62±24)个/mm<'2>]均高于模型组(P＜0.05),而电针组与氟西汀组间的差异无统计学意义(P＞0.05).结论 电针和氟西汀可改善抑郁大鼠行为学症状,这种改善与海马细胞凋亡机制有一定相关性.     

氟西汀对缺血性脑卒中患者脑源性神经营养因子的影响

目的观察氟西汀对缺血性脑卒中患者血清脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)的影响。方法将60例缺血性脑卒中的患者随机分为氟西汀组和安慰剂组,两组患者均同时接受物理治疗。采用酶联免疫吸附法(ELISA)测定两组缺血性脑卒中患者治疗前后BDNF的变化,并与正常对照组比较;用改良Barthel指数(modified Barthel index,MBI)和简化Fugl-Meyer运动功能量表(Fugl-Meyer assessment,FMA)评定两组患者的日常生活活动能力(activties of daily living,ADL)和运动功能。结果治疗3m后,氟西汀组BDNF浓度显著高于治疗前及安慰剂组、对照组(P<0.05);治疗后两组MBI及FMA两项评分均有改善(P<0.05),且氟西汀治疗组疗效优于对照组(P<0.05)。结论缺血性脑卒中患者早期给氟西汀和物理治疗后可促进其运动功能的恢复,提高日常生活活动能力,这种效应可能是通过提高BDNF的浓度,促进神经元再生和对抗神经元损伤后凋亡而发挥作用。     

Neuroprotection against excitotoxicity by N-alkylglycines in rat hippocampal neurons

Excessive activation of glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype is considered a relevant initial step underlying different neurodegenerative diseases. Recently, with the approval of memantine to treat Alzheimer dementia, NMDA receptors have regained clinical interest. Accordingly, the development and validation of NMDA receptor antagonists is being reconsidered. We recently identified a family of trialkylglycines that act as channel blockers of the NMDA receptor. Their neuroprotective activity against excitotoxic insults remains elusive. To address this issue, we first characterized the contribution of glutamate receptor sub-types to hippocampal death in culture as a function of days in culture in vitro (DIV). Whereas at 7 DIV neither NMDA nor glutamate produced a significant neuronal death, at 14 and 21 DIV, NMDA produced the death of 40% of the neurons exposed to this receptor agonist that was fully protected by MK-801. Similar results were obtained for l-glutamate at 14 DIV. In contrast, when neurons at 21 DIV were used, glutamate killed 51.1±4.9% of the neuronal population. This neuronal death was only partially prevented by MK-801, and fully abrogated by a combination of MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Glucose deprivation injured 37.1±9.2% of the neurons through a mechanism sensitive to MK-801. The family of recently identified N-alkylglycines tested protected neurons against NMDA and glucose-deprivation toxicity, but not against glutamate toxicity. Noteworthy, N-alkylglicines with a moderate protection against NMDA-induced toxicity strongly protected from β-amyloid toxicity. Collectively, these findings imply both NMDA and non-NMDA receptors in excitotoxicity of hippocampal neurons, and suggest that blockade of NMDA receptors alone may not suffice to efficiently abrogate neurodegeneration.     

Agmatine protects against cell damage induced by NMDA and glutamate in cultured hippocampal neurons

Agmatine is a polyamine and has been considered as a novel neurotransmitter or neuromodulator in the central nervous system. In the present study, the neuroprotective effect of agmatine against cell damage caused by N-methyl-D-aspartate (NMDA) and glutamate was investigated in cultured rat hippocampal neurons. Lactate dehydrogenase (LDH) activity assay, beta-tubulin III immunocytochemical staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay were conducted to detect cell damage. Exposure of 12-day neuronal cultures of rat hippocampus to NMDA or glutamate for 1 h caused a concentration-dependent neurotoxicity, as indicated by the significant increase in released LDH activities. Addition of 100 microM agmatine into media ablated the neurotoxicity induced by NMDA or glutamate, an effect also produced by the specific NMDA receptor antagonist dizocilpine hydrogen maleate (MK801). Arcaine, an analog of agmatine with similar structure as agmatine, fully prevented the NMDA- or glutamate-induced neuronal damage. Spermine and putrescine, the endogenous polyamine and metabolic products of agmatine without the guanidine moiety of agmatine, failed to show this effect, indicating a structural relevance for this neuroprotection. Immunocytochemical staining and TUNEL assay confirmed the findings in the LDH measurement. That is, agmatine and MK801 markedly attenuated NMDA-induced neuronal death and significantly reduced TUNEL-positive cell numbers induced by exposure of cultured hippocampal neurons to NMDA. Taken together, these results demonstrate that agmatine can protect cultured hippocampal neurons from NMDA- or glutamate-induced excitotoxicity, through a possible blockade of the NMDA receptor channels or a potential anti-apoptotic property.     

Neuroprotection by pigment epithelial-derived factor against glutamate toxicity in developing primary hippocampal neurons.

Pigment epithelial-derived factor (PEDF) has been shown to be a survival factor for cerebellar granule neurons. Here we investigated the ability of PEDF to enhance the survival of hippocampal neurons in culture, and to protect these neurons against acute glutamate toxicity. Hippocampal neurons prepared from 1- to 3-day postnatal rat brain were cultured for either 7 or 14 days in vitro (DIV). At 14 DIV, neurons were only slightly protected (13% +/- 4%) against 50 microM glutamate toxicity when treated with 1 microg/ml of PEDF for 3 successive days before glutamate exposure as measured by lactate dehydrogenase (LDH) release. In comparison, basic fibroblast growth factor (bFGF) at 10 ng/ml for the same treatment period protected 58% +/- 8% of the neurons against glutamate. Using quantitative image analysis of digitized micrographs, we found that the average size of neurons in young, developing hippocampal cultures (7 DIV), was greatly decreased by treatment with 50 microM glutamate. Treatment for up to 5 successive days with 1 microg/ml of PEDF before glutamate addition dramatically increased the average hippocampal neuron soma size, compared to cells treated with glutamate alone. Thus, PEDF may promote the growth of hippocampal neurons, and, if added to developing hippocampal neurons, can also protect these cells from subsequent injury, such as the excitotoxicity of glutamate.     

BDNF-TrkB signaling pathway is involved in pentylenetetrazoleevoked progression of epileptiform activity in hippocampal neurons in anesthetized rats

Pentylenetetrazole（PTZ）is a widely-used convulsant used in studies of epilepsy;its subcutaneous injection generates an animal model with stable seizures.Here,we compared the ability of PTZ via the intravenous and subcutaneous routes to evoke progressive epileptiform activity in the hippocampal CA1 neurons of anesthetized rats.The involvement of the BDNF-TrkB pathway was then investigated.When PTZ was given intravenously,it induced epileptiform bursting activity at a short latency in a dose-dependent manner.However,when PTZ was given subcutaneously,it induced a slowly-developing pattern of epileptogenesis;first,generating multiple population-spike peaks,then spontaneous interictal discharge-like spike,leading to the final ictal discharge-like,highly synchronized bursting firing in the CA1 pyramidal layer of the hippocampus.K252a,a TrkB receptor antagonist,when given by intracerebroventricular injection,significantly reduced the probability of multiple population spike peaks induced by subcutaneous injection of PTZ,delayed the latency of spontaneous spikes,and reduced the burst frequency.Our results indicate that PTZ induces a progressive change of neuronal epileptiform activity in the hippocampus,and the BDNF-TrkB signaling pathway is mainly involved in the early phases of epileptogenesis,but not the synchronized neuronal burst activity associated with epileptic seizure in the PTZ animal model.These results provide basic insights into the changing pattern of hippocampal neuronal activity during the development of the PTZ seizure model,and establish an in vivo seizure model useful for future electrophysiological studies of epilepsy.     

去铁敏对体外培养的大鼠海马神经元谷氨酸毒性的保护作用

目的 探讨去铁敏对体外培养的海马神经元谷氨酸损伤的保护作用及可能机制.方法 建立体外培养的大鼠海马神经元谷氨酸毒性模型.细胞分为去铁敏组、对照组.采用形态学观察、Hoechst 33342染色、乳酸脱氢酶(LDH)检测评价细胞损伤情况.生化法检测羟自由基、丙二醛变化.用显微荧光测量技术监测神经元内钙信号的动态变化.结果 去铁敏组较对照组神经元形态保持良好.去铁敏组及对照组细胞核固缩率分别为14%±6%和58%±6%(t=8.98,P＜0.01),LDH分别为(36.42±8.99)U/L和(68.06±11.26)U/L(t=3.25,P＜0.05),羟自由基分别为(34.21±4.23)U/L和(47.06±8.79)U/L(t=3.11,P＜0.05),丙二醛分别为(12.26±2.78)nmol/mg和(28.86±5.19)nmol/mg(t=4.88,P＜0.01).结论 去铁敏能减轻谷氨酸导致的神经元损伤,其可能的机制与去铁敏减少谷氨酸导致的神经元内钙浓度的升高及自由基水平有关.     

Vasopressin-induced calcium signaling in cultured hippocampal neurons

We recently demonstrated that the neural peptide vasopressin (AVP) can act as a neurotrophic factor for hippocampal nerve cells in culture. Because the neurotrophic effect of vasopressin is mediated by the V₁ receptor [11], we investigated AVP activation of calcium signaling pathways in cultured hippocampal neurons. Results of this investigation demonstrate that exposure of cultured hippocampal neurons prelabeled with [³H]myo-inositol to vasopressin induced a significant accumulation of [³H]inositol-1-phosphate ([³H]IP₁). The selective V₁ vasopressin receptor agonist, [Phe², Orn²]vasotocin, induced a s significant accumulation of [³H]IP₁ whereas a selective V₂ vasopressin receptor agonist, [deamino¹, d-Arg⁸]-vasopressin, did not. Moreover, V₁ agonist-induced accumulation of [³H]IP₁ was blocked by the selective V₁ vasopressin receptor antagonist d(CH₂)₅[Tyr(Me)²]-vasopressin. V₁ agonist-induced accumulation of [³H]IP₁ was concentration dependent and exhibited a steep inverted U-shaped curve that included both stimulation and inhibition of [³H]IP₁ accumulation. Time course analysis of V₁ agonist-induced accumulation of [³H]IP₁ revealed significant increase by 20 min which continued to be significantly elevated for 60 min. Investigation of the effect of closely related peptides on [³H]IP₁ accumulation indicated that the vasopressin metabolite peptide AVP_4–9 and oxytocin significantly increased [³H]IP₁ accumulation whereas the vasopressin metabolite peptide AVP_4–8 did not. AVP_4–9 and oxytocin induced [³H]IP₁ accumulation were blocked by the V₁ vasopressin receptor antagonist d(CH₂)₅[Tyr(Me)²]-vasopressin. V₁ receptor activation was associated with a pronounced rise in intracellular calcium. Results of calcium fluorometry studies indicated that V₁ agonist exposure induced a marked and sustained rise in intracellular calcium that exhibited oscillations. Interestingly, absence of calcium in the extracellular medium abolished both the rise in intracellular calcium and the appearance of oscillations. The loss of the intracellular calcium signal is not due to a failure to induce PIP₂ hydrolysis since activation of the phosphatidylinositol pathway occurred in the absence of extracellular calcium. V₁ agonist (250 nM) induced a highly significant increase in⁴⁵Ca²⁺ uptake from the extracellular medium within 5 sec of exposure.⁴⁵Ca²⁺ uptake remained significantly greater than basal for 300 sec. The increase in⁴⁵Ca²⁺ uptake was followed by a significant inhibition of uptake by 20 min of exposure. These results indicate that in cultured hippocampal neurons, V₁ vasopressin receptor activation leads to activation of the phosphatidylinositol signaling pathway, uptake of calcium from the extracellular medium and induction of complex intracellular calcium signals. These data provide the first step in delineating the biochemical mechanism that underlies vasopressin-induced neutrophism.     

Neuroprotective effects of hibernation-regulating substances against low-temperature-induced cell death in cultured hamster hippocampal neurons

The neuroprotective effects of hibernation-regulating substances (HRS) such as adenosine (ADO), opioids, histamine and thyrotropin-releasing hormone (TRH) on low-temperature-induced cell death (LTCD) were examined using primary cultured hamster hippocampal neurons. LTCD was induced when cultures were maintained at <22 degrees C for 7 days. ADO (10-100 microM) protected cultured neurons from LTCD in a dose-dependent manner. The neuroprotective effects of ADO were reversed by both 8-cyclopenthyltheophilline (CPT; A(1) receptor antagonist) and 3,7-dimethyl-1-propargylxanthine (DMPX; A(2) receptor antagonist). Morphine (a non-selective opioid receptor agonist) was also effective in attenuating LTCD at an in vitro dose range of 10-100 muM. The neuroprotective effects of morphine were antagonized by naloxone (a non-selective opioid receptor antagonist). In addition, although [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]-enkephalin (DAMGO; mu-opioid receptor agonist), [D-Pen(2,5)]-enkephalin (DPDPE; delta-opioid receptor agonist) and U-69593 (kappa-opioid receptor agonist) were also effective, LTCD of cultured hippocampal neurons was not affected by TRH. Furthermore, histamine produced hypothermia in Syrian hamsters and protected hippocampal neurons in vitro at 100 microM. The neuroprotective effect of histamine was reversed by pyrilamine (H(1) receptor antagonist). Apoptosis was probably involved in LTCD. These results suggest that ADO protected hippocampal neurons in vitro via its agonistic actions on both A(1) and A(2) receptors, whereas morphine probably elicited its neuroprotective effects via agonistic effects on the mu-, delta- and kappa-opioid receptors. In addition, histamine also protected hippocampal neurons via its agonistic action on the H(1) receptor. Thus, HRS-like adenosine-, opioid- and histamine-like hypothermic actions would most likely induce neuroprotective effects against LTCD in vitro.     

Nicotine protects against the dexamethasone potentiation of kainic acid-induced neurotoxicity in cultured hippocampal neurons

To elucidate the neuroprotective effect of nicotine, we investigated whether nicotine may attenuate dexamethasone potentiation of kainic acid-induced neurotoxicity. Primary hippocampal culture was pre-treated with nicotin for 24 h followed by dexamethasone (10⁻⁴ M) for 24 h. Then, cultures were exposed with kainic acid (10⁻⁴ M) and cellular viability was determined by LDH effluxmetry. Nicotine pre-treatment (10⁻⁹−10⁻⁷ M) dose-dependently attenuated dexamethasone potentiation of kainic acid-induced neurotoxicity. These results may support the epidemiological data suggesting a neuroprotective effect of cigarette smoking on Alzheimer's disease or Parkinson's disease.     

Protective effects of paeonol on cultured rat hippocampal neurons against oxygen-glucose deprivation-induced injury

Mounting evidence has suggested that paeonol possesses plenty of pharmacologic actions. Our research is to determine if paeonol can protect cultured rat hippocampal neurons from oxygen-glucose deprivation(OGD)-induced injury and elucidate the underlying mechanism. We cultivated the rat hippocampal neurons as the object of study and then established the model of oxygen-glucose deprivation. Neuronal viability was measured by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), while intracellular Ca(2+) concentration was observed by fluorospectrophotometer. The binding force of N-methyl-D-aspartate (NMDA) receptor was evaluated by liquid scintillation counting. Compared with oxygen-glucose deprivation group, paeonol treatment obviously increased cell survival rate and reduced the activity of the binding force of NMDA receptors, reversing the overload of intracellular Ca(2+). These results demonstrate that paeonol protected rat neurons from oxygen-glucose deprivation-induced injury, resulting in alleviating the morphological damage and increasing neuron viability and suggest that paeonol may exhibit its protective effect against oxygen-glucose deprivation-induced injury by targeting on NMDA receptors.     

Development of N-methyl-D-aspartate excitotoxicity in cultured hippocampal neurons

Immature hippocampal neurons (E-18) were maintained in defined medium for up to 3 weeks and their susceptibility to N-methyl-D-aspartic acid (NMDA)-induced cell death was studied at various days in vitro. Upon acute exposure to NMDA (5 min), hippocampal neurons in vitro (8-12 days after plating) showed cell body swelling and dendritic degeneration that preceded cell death 24 h later. NMDA-induced neurodegeneration could be prevented by MK-801 treatment but not by tetrodotoxin. In contrast, immature (5-7 days old) neurons were unaltered by exposure to 500 microM NMDA for either 5 min or 24 h. One explanation for the resistance of immature neurons to glutamate neurotoxicity may be related to maturation of the NMDA receptor complex. Glutamate binding to the NMDA receptor in vivo increased from 14.6 +/- 1.6% (0 day) to 55.2 +/- 4.5% (day 7), 79 +/- 4.9% (day 14), 93.8 +/- 2.8% (day 21) until it reached the adult Sprague-Dawley value of 100 +/- 0.8% (day 90).