人参皂甙Rb_1对大鼠海马脑片缺血损伤的保护作用

目的　研究人参皂甙 Rb1 对脑缺血损的保护作用及其机制。方法　实验利用离体海马脑片模型 ,结合电生理学技术进行研究 :(1)观察大鼠海马脑片在缺血条件下顺向群峰电位 (orthodrom ic population spike,OPS)的变化及人参皂甙 Rb1 对它的影响。 (2 )观察谷氨酸对海马脑片 OPS的影响及人参皂甙 Rb1 抗谷氨酸毒性作用。结果　 (1)使用人参皂甙 Rb1 (6 0 0 μm ol/ L)后 ,可使缺氧损伤电位 (hypoxic injury potential,HIP)出现率明显减少 ,OPS恢复率和 OPS恢复程度与对照组比较有显著性差异。 (2 )使用人参皂甙 Rb1 (6 0 0 μm ol/ L)后 ,OPS恢复率和OPS恢复程度与对照组比较有显著性差异。结论　人参皂甙 Rb1 有明显的抗脑缺血损伤作用 ,其作用机制与抗谷氨酸的神经毒性作用有关。     

人参皂甙Rb1对大鼠海马脑片缺血损伤的保护作用

目的 研究人参皂甙Rb1对脑缺血损的保护作用及其机制。方法 实验利用离体海马脑片模型，结合电生理学技术进行研究：(1)观察大鼠海马脑片在缺血条件下顺向群峰电位(orthodromic population spike，OPS)的变化及人参皂甙Rb1对它的影响。(2)观察谷氨酸对海马脑片OPS的影响及人参皂甙Rb1抗谷氨酸毒性作用。结果 (1)使用人参皂甙Rbl(600μmol／L)后，可使缺氧损伤电位(hypoxic injury potential，HIP)出现率明显减少，OPS恢复率和OPS恢复程度与对照组比较有显著性差异。(2)使用人参皂甙Rb1(600μmol／L)后，OPS恢复率和OPS恢复程度与对照组比较有显著性差异。结论 人参皂甙Rb1有明显的抗脑缺血损伤作用，其作用机制与抗谷氨酸的神经毒性作用有关。     

低温对大鼠海马脑片缺氧无糖损伤的保护作用及其机制

目的利用离体海马脑片孵育液和电生理学技术研究低温对脑缺氧/无糖损伤的保护作用及其机制.方法 (1)观察大鼠海马脑片在缺氧/无糖条件下顺向群峰电位(orthodromic population spike,OPS)的变化及温度对它的影响;(2)用高效液相(high-performance liquid chromatography,HPLC)荧光法检测温度对缺氧/无糖导致的海马细胞外兴奋性/抑制性氨基酸(excitabilitory and inhibitory amino acid,EAA/IAA)水平变化的影响.结果 (1)37℃组海马脑片缺氧/无糖时缺氧损伤电位(hypoxic injury potential,HIP)出现率为87.5%(7/8),复氧/供糖1h后OPS恢复率为12.5%(1/8).低温组(32℃、25℃)OPS消失时间和HIP出现时间明显减少(P<0.01),复氧/供糖1h后OPS恢复程度(41%±34%、79%±33%)与37℃组(7.5%±21.2%)比较有显著性差异(P<0.05),而这3项指标25℃优于32℃;(2)37 ℃组缺氧/无糖时,EAA和IAA比对照组均显著增加(P<0.01),兴奋性毒性指数(excitory toxicity index,ETI)显著上升(P<0.01).复氧后各氨基酸水平均有所下降.而低温组缺氧/无糖导致的谷氨酸(Glu)、天门冬氨酸(Asp)和甘氨酸(Gly)升高显著减少(P<0.01),GABA却随着复氧时间的延长逐渐增加(P<0.01),ETI明显降低(P<0.01).这种效应25℃优于32℃.结论低温有明显的抗海马脑片缺氧/无糖损伤作用,其作用机制可能与低温维持了EAA和IAA的动态平衡,降低了EAA的兴奋性毒性作用有关.而其作用又以25℃优于32℃.     

N-乙酰半胱氨酸抗谷氨酸诱导海马神经元损伤的研究

目的　观察N-乙酰半胱氨酸(N- acetylcysteine,NAC)对不同浓度谷氨酸(Glutamte,Glu)诱导海马神经元损伤的影响,探讨其作用机制,评价毒性作用。方法　采用台盼蓝活细胞拒染与TUNEL法比较不同浓度NAC预处理3d给药与细胞毒性暴露后快速给药对10 0μmol/ L、5 0 0μm ol/ L Glu诱导体外培养海马神经元损伤的影响,并与MK- 80 1比较;利用激光扫描共聚焦显微镜(L SCM)观测细胞内Ca2 浓度变化;采用台盼蓝活细胞拒染、原子力显微镜(AFM)及细胞内酯酶活性判定方法评价NAC毒性。结果　NAC可降低10 0μmol/ L Glu诱导的海马神经元死亡率,以预处理组为佳,1m mol/ L NAC预处理保护效果类似于10μmol/ L MK - 80 1;NAC对5 0 0μm ol/ L Glu诱导的海马神经元损伤无保护作用。1mm ol/ L NAC预处理抑制Glu诱导的神经元Ca2 内流。经10 0 mmol/ L NAC作用的细胞虽然形态完整,但台盼蓝染色蓝染,失去对Glu毒性的反应性;AFM扫描见神经元细胞膜皱缩;培养基Ca2 经Fluo- 3(AM)标记后L SCM下无激发荧光。结论　NAC对轻度Glu细胞毒性损伤有保护作用,预防性用药效果更优越。认为抑制Glu诱导的神经元Ca2 内流为其保护机制之一。高浓度NAC具有固定作用     

谷胱甘肽与N-乙酰半胱氨酸对谷氨酸诱导海马神经元损伤的影响

目的观察还原型谷胱甘肽(reducedglutathione,GSH)与N-乙酰半胱氨酸(N-Acetylcysteine,N-NAC)对不同浓度谷氨酸(glutamate,Glu)诱导的海马神经元损害的影响。方法选用新生Wistar大鼠原代培养海马神经元谷氨酸细胞毒性模型,采用台盼蓝活细胞拒染及TUNEL细胞凋亡原位检测等方法比较GSH与NAC对100μmol/L及500μmol/LGlu细胞毒性损伤的影响,并与MK-801比较。结果GSH与NAC能够降低100μmol/LGlu作用下神经元死亡率与凋亡率,NAC组细胞存活率高于同条件下GSH组,其中1mmol/LNAC组神经元存活率达到90.4%±5.2%,与10μmol/LMK-801组相比,差异无统计学意义;在500μmol/LGlu作用下,GSH与NAC则不能增加神经元的存活率,但1mmol/LNAC抗500μmol/LGlu诱导调亡的作用与MK-801相比无明显差异。结论GSH及NAC对轻度Glu细胞毒性神经损伤有保护作用。     

nNOS抑制剂在大鼠海马缺氧损伤的保护作用

目的为进一步证实缺血、缺氧时神经原性ＮＯ的神经毒性作用。方法在大鼠离体海马脑片上,应用微电极记录技术,研究ｎＮＯＳ抑制剂７－ＮＩ对缺氧所致神经元损害的保护作用。结果ｎＮＯＳ抑制剂７－ＮＩ对海马脑片缺氧后群峰电位（ＰＳ）变化较对照组显著减小。结论７－ＮＩ对海马脑片缺氧损伤有明确保护作用。作用机理可能是７－ＮＩ抑制ｎＮＯＳ活性降低缺氧后海马神经原性ＮＯ的过多形成,从而提高海马脑片抗缺氧损伤的能力,更加明确了缺氧早期神经原性ＮＯ的神经毒性作用。     

应激性高血压大鼠海马CA1区在体外缺血的条件下形态结构和GM1的变化

本实验观察了应激性高血压大鼠海马脑片在体外人工缺血条件下30,60,120min海马CAl区神经元形态结构的变化。同时观察了神经节苷脂GMl对其形态结构变化的影响。发现缺血30min神经元胞体变小,胞内小空泡形成,细胞器减少,少量的线粒体和内质网肿胀。核不规则,染色质分布不均,出现边集。随缺血时间的延长胞内大量细胞器破碎,残存细胞器结构不清,胞核固缩,部分裂解。神经节苷脂GMl(0.01％)具有明显的抗损害作用,减轻缺血所造成的上述形态学上的改变。     

刺五加皂甙对谷氨酸毒性神经元凋亡的保护作用

目的观察神经元在谷氨酸毒性损伤时一氧化氮(NO)的动态变化及其与凋亡的关系,探讨刺五加皂甙(ASS)的有效保护浓度。方法采用谷氨酸(Glu)诱导的皮质神经元凋亡模型。随机分成Glu组、正常对照组及ASS3组;用流式细胞仪检测神经元凋亡率,用硝酸还原酶法测定细胞培养上清液中NO的含量,用MTT法测定神经元存活率并在电镜下观察细胞形态学变化。结果(1)Glu呈剂量和时间依赖性增加神经元培养液中NO含量,ASS能不同程度地减少NO含量;(2)与Glu共培养的神经元,其存活率呈剂量和时间依赖性下降,ASS能增加神经元存活率;(3)经Glu处理的神经元发生凋亡,细胞超微结构呈现凋亡样改变,其凋亡率与正常对照组比较有显著性差异(P<0.01)。ASS能减少Glu毒性神经元凋亡。结论NO介导了Glu毒性神经元凋亡,ASS可能通过抑制NO的释放及其神经毒性作用,拮抗Glu引起的神经元凋亡。     

神经节苷脂GM_1拮抗剂对海马CA_1区缺血神经元形态结构变化影响的离体研究

实验观察了神经节苷脂ＧＭ１拮抗剂——霍乱毒素对应激性高血压大鼠海马脑片体外人工缺血３０、６０、１２０ｍｉｎＣＡ１区神经元形态结构的影响。缺血３０ｍｉｎ时胞体变小，胞内小空泡形成，细胞器减少，少量的线粒体和内质网肿胀。核不规则，染色质分布不均，出现边集。随缺血时间的延长胞内细胞器破碎，残存细胞器结构不清，胞核固缩，部分裂解。而霍乱毒素则加重缺血３０、６０、１２０ｍｉｎＣＡ１区神经元形态学上的改变，具有明显的神经元损害作用。     

β-细辛醚对谷氨酸诱导损伤的脑皮层神经元凋亡线粒体膜电位和超微结构的影响

目的探讨β-细辛醚对谷氨酸(Glu)诱导损伤的脑皮层神经元凋亡、线粒体膜电位(mitochon-drial membrane potential,MMP)和超微结构的影响。方法体外培养乳鼠脑皮层神经元,β-细辛醚(7.5、15、30μg/ml)预给药4h,荡洗后加入终浓度为40mmol/L的Glu继续培养16h,采用流式细胞仪检测细胞凋亡率、MMP,并用透射电镜观察超微结构的变化。结果40mmol/L Glu组凋亡率明显增加(P<0.01),7.5、15、30μg/mlβ-细辛醚均能不同程度地抑制皮层神经元凋亡,30μg/mlβ-细辛醚抑制神经元凋亡的作用较7.5、15μg/mlβ-细辛醚明显(P<0.01),10μmol/L尼莫地平与30μg/mlβ-细辛醚抑制凋亡的作用相当(P>0.05);40mmol/L Glu组MMP明显低于正常对照组(P<0.01),10μmol/L尼莫地平组和30μg/mlβ-细辛醚组MMP明显高于40mmol/LGlu组(P<0.05,P<0.01),而与正常对照组比较无明显差别(P>0.05);电镜下40mmol/LGlu组的脑皮层神经元见染色质边聚、线粒体肿胀、部分内质网扩张,而30μg/mlβ-细辛醚组神经元则仅见少量内质网扩张、线粒体轻度肿胀。结论β-细辛醚可抑制Glu诱导的脑皮层神经元凋元,其作用机制可能与稳定细胞线粒体膜电位有关。     

An analog of thyrotropin-releasing hormone (TRH) is neuroprotective against glutamate-induced toxicity in fetal rat hippocampal neurons in vitro

TRH has been found to be efficacious in treating certain neurodegenerative disorders such as epilepsy, Alzheimer's disease, neurotrauma and depression, however, its mechanism of action is poorly understood. Since glutamate (Glu) toxicity has been implicated in these disorders, we utilized primary enriched cultures of rat fetal (E 17) hippocampal neurons to test the hypothesis that an analog of TRH, 3-Methyl-Histidine TRH (3Me-H TRH), given concurrently with Glu would protect such neurons against cell damage and cell death. Cell viability was assessed via Trypan Blue exclusion cell counts, and neuronal damage was determined by assaying lactic acid dehydrogenase (LDH) released in the conditioned media. Fetal hippocampal neurons were cultured in neurobasal media for 7 days. On day 7, neurons (10(6)/well) were treated with: control media, 10 microM 3Me-H TRH, 500 microM Glu or 500 microM Glu with either 10, 1, 0.1, 0.01 or 0.001 microM 3Me-H TRH. Both media and neurons were harvested 16 h after treatment. Prolonged exposure to 10 microM 3Me-H TRH was not toxic to the cells, whereas neurons exposed to 500 microM Glu resulted in maximal cell death. Notably, 10, 1 and 0.1 microM 3Me-H TRH, when co-treated with 500 microM Glu, protected fetal neurons against cell death in a concentration-dependent manner. These results provide support for an important neuroprotective effect of TRH/analogs against glutamate toxicity in primary hippocampal neuronal culture and implicate a potentially beneficial role of TRH/analogs in neurodegenerative diseases.     

刺五加多糖对H_2O_2损伤的海马神经元表达NF-κB的影响

目的研究刺五加多糖(ASPS)对氧化应激损伤的海马神经元表达核转录因子-κB(NF-κB)的影响,进一步探讨ASPS对海马神经元的保护作用。方法从新生大鼠脑分离海马,其神经元经原代培养后分为阴性对照组、损伤模型组(1 mmol/LH_2O_2诱导)、ASPS干预组(该组按ASPS10、5、2.5μg/ml的终浓度又分为3个亚组)。采用H_2O_2复制海马神经元氧化应激损伤模型。用免疫组化法和逆转录PCR法检测NF-κB的表达。同时观察各组海马神经元的形态学变化。结果与损伤模型组比较,ASPS干预组神经元损伤程度明显减轻。NF-κB在10μg/ml和5μg/ml ASPS干预组中的表达水平明显降低。结论 ASPS能明显减少受损海马神经元NF-κB的表达,对海马神经元有明显的保护作用。     

Ion Dependence and Receptor Mediation of Glutamate Toxicity in the Immature Rat Hippocampal Slice

Glutamate (glu) is a major excitatory transmitter and a toxin in the brain. In the present study, the immature rat hippocampal slice was used to determine the morphology, topography, ionic mediation and receptor specificity of glu toxicity. Slices were exposed to glu for 30 min, and the damage was evaluated after 3 h of recovery in regular medium. The effects on glu toxicity of changes of [Ca2+], [Cl-] and [Na+] were determined. The receptor preference of glu was assessed by using the N-methyl-D-aspartate (NMDA) antagonist MK-801 and the kainate (KA)/quisqualate (QA) antagonist DNQX, alone or in combination. Further, to see whether glu produces cytotoxicity via osmolysis, the effects of hyperosmolal sucrose on glu toxicity were studied. Glu toxicity was similar to the previously described NMDA toxicity with regard to cytopathology, but differed in some aspects from that caused by KA and QA. The severity of the lesion was determined by the proximity of neurons to the incubation fluid, probably as a consequence of cellular accumulation of the amino acid. Omission of Ca2+ abolished glu toxicity in all neurons except the granule cells of the outer blade. This population was completely protected when Ca2+ was omitted and [Cl-] was reduced. Elevation of [Ca2+] markedly aggravated the lesion caused by glu. Substitution of isethionate for Cl- worsened the glu-induced damage, whilst the amino acid produced qualitatively different neuropathology when choline substituted for Na+. Apparently glu did not damage hippocampal nerve cells through an osmolytic mechanism as medium supplemented with 100 mM sucrose increased the toxicity of glu. Since the lesion produced by glu was more widespread in the presence of high [Ca2+], the effects of receptor antagonists were studied under this condition. MK-801 inhibited glu toxicity whereas DNQX had no effect. Combination of MK-801 and DNQX did not offer better protection than did MK-801 alone. The results suggest that Ca2+ is the main (but not single) determinant of glu toxicity in the immature hippocampal slice. The ionic requirements of glu neurotoxicity are identical to those of NMDA, but differ from those of KA and QA. The notion that glu is a selective NMDA agonist in the present model was confirmed by the protection of MK-801, and by the lack of an effect of DNQX. This is the first report demonstrating that the toxicity of glu is mediated by NMDA receptors in brain tissue which has developed normally. The findings indicate that specific blockade of NMDA receptors may be the most rational strategy in the prevention of glu-related neuronal death occurring in certain neurological anomalies.     

A novel mouse brain slice preparation of the hippocampo-accumbens pathway

The nucleus accumbens (NAc) is an important component of circuitry that underlies reward related behaviors and the rewarding properties of drugs of abuse. Glutamatergic afferents to the nucleus are critical for its normal function and for behaviors related to drug addiction. An angled, sagittal mouse brain slice preparation has been designed to facilitate concurrent stimulation of two major glutamatergic afferent pathways to the nucleus accumbens. Medium spiny neurons at the medial core/shell boundary of the accumbens were depolarized by stimulation of either hippocampal or limbic cortical afferents through activation of AMPA-type glutamate receptors. High frequency but not low frequency stimulation of hippocampal afferents depolarized medium spiny neurons to a membrane potential that resembled the up state observed upon high frequency stimulation in vivo. The magnitude of the membrane depolarization was positively correlated with the amplitude of the stimulus-evoked EPSP. Concurrent stimulation of hippocampal and limbic cortical afferents at theta frequency selectively induced a long-term depression (LTD) in the magnitude of stimulus-evoked EPSPs on the hippocampal afferent only. These data suggest that this brain slice preparation can be used to study mechanisms underlying synaptic plasticity at two of the critical glutamatergic afferent synapses in the nucleus accumbens as well as characterizing potential interactions between afferents. Additionally, LTD at hippocampo-accumbens synapses can be induced at a stimulus frequency known to support reinstatement of drug seeking behavior.     

孕酮对原代培养海马神经细胞谷氨酸损伤的保护作用

目的观察孕酮对原代培养海马神经细胞谷氨酸损伤的保护作用。方法取新生大鼠海马神经细胞进行体外原代培养,建立谷氨酸对培养海马神经细胞兴奋毒性损伤模型,在相差显微镜下观察孕酮处理前后神经细胞形态变化,乳酸脱氢酶活性测定试剂盒检测细胞培养上清乳酸脱氢酶漏出量。结果孕酮组神经细胞部分形态保持较好,生长情况明显好于谷氨酸组,乳酸脱氢酶漏出量较谷氨酸组减少。结论孕酮对体外培养的海马神经细胞谷氨酸兴奋毒性损伤具有保护作用。     

内质网应激因子ATF6在hSOD1~(G93A)转基因小鼠脊髓中的表达

目的应用目前国际公认的肌萎缩侧索硬化(ALS)发病机制及临床前药物研究的动物模型hSOD1~(G93A)转基因小鼠,初步研究hSOD1~(G93A)小鼠脊髓中是否存在内质网应激中活性转录激活因子(ATF6)信号通路的激活,进而探究内质网应激在ALS发病机制中的作用。方法①hSOD1~(G93A)转基因小鼠及其同窝对照组小鼠的获得;②应用免疫组织化学方法观察转基因小鼠组和对照组显示脊髓前角运动神经元ATF6阳性细胞数量。结果症状早期和终末期转基因小鼠(分别为105~115d和120~130d),较对照组和无症状组转基因小鼠(60d)ATF6进入细胞核的运动神经元数量明显增多,差异有统计学意义(P0.05);症状早期和终末期相比较差异无统计学意义(P0.05);症状早期和终末期的一些细胞形态发生改变,出现皱缩,终末期运动神经元的数目减少。结论在无症状组时没有发现内质网应激因子ATF6的改变,但在疾病症状早期出现明显ATF6的改变,并随着疾病的进展而加重,说明内质网应激参与了ALS的发病,同时终末期脊髓前角运动神经元数量大幅减少。     

Induction of cellular stress and chaperone activation in organotypic slice cultures of hippocampus

Neurodegenerative diseases are often associated with the occurrence of misfolded proteins preceding neuronal cell death. Accumulation of misfolded proteins in the endoplasmic reticulum induces ER stress, which in consequence enhances chaperone expression to restore protein homeostasis. Here we used organotypic hippocampal slice cultures to analyze the time course of chaperone expression and neuronal death after induction of ER stress by tunicamycin treatment. Shortly after explantation many cells stain positive for Fluoro Jade B demonstrating neuronal cell death. While in control cultures the number of Fluoro Jade B labeled cells remarkably decrease over the total period of cultivation, neuronal death remains elevated in ER-stressed slice cultures. Caspase-3 staining revealed that neuronal death is primarily due to apoptosis in tunicamycin-treated slice cultures. The chaperone GRP78/BiP is expressed at low levels in control sections. Its expression is largely restricted to hippocampal neurons. Tunicamycin treatment resulted in upregulation of GRP78/BiP in the neuronal cells. Double-immunolabeling for GFAP shows a concomitant de novo expression of GRP78/BiP in astrocytes. The astrocytic GRP78/BiP upregulation might reflect an early, neuroprotective response. The increase of GRP78/BiP in neurons and astrocytes show successful induction of the ER stress response. The hippocampal slice cultures are, thus, a useful tool to examine the process of neurodegeneration and to investigate neuroprotective devices in an ER stress paradigm.     

Immunohistochemical detection of glutamate in rat vagal sensory neurons

Vagal primary afferent neurons have their cell bodies located in the nodose (inferior) and jugular (superior) vagal ganglia and send terminals into the nucleus tractus solitarii (NTS) which lies in the dorsomedial medulla. The presence of glutamate (Glu)-containing neurons in the rat nodose ganglion was investigated using immunohistochemistry. Glu-immunoreactivity on nodose sections was found in neuronal perikarya and nerve fibers, but not in non-neuronal elements such as Schwann cells and satellite cells. Both immunoreactive and non-immunoreactive ganglion cells were observed. The immunoreactive ganglion cells amounted to about 60% of the nodose population. No specific intraganglionic localization was observed for the non-immunoreactive cells. Immunoreactive perikarya were slightly smaller than the non-immunoreactive ones, but no relationship was found between size and staining intensities of immunoreactive neurons. The present data indicate that immunodetectable Glu is present in a large population of vagal afferent neurons. They therefore add to a growing body of evidence suggesting that Glu may be the main neurotransmitter released by vagal afferent terminals within the nucleus tractus solitarii.     

Cortical projection of giant neostriatal neurons in the cat. Light and electron microscopic horseradish peroxidase study

Following voluminous injections of horseradish peroxidase (HRP) in various neocortical fields, a small number of labeled large neurons are observed ipsilaterally in the putamen, striatal ponticuli, caudate nucleus, and nucleus accumbens septi. The bulk of the corticopetal cells are found in the putamen and in the striatal ponticuli. A more significant number of labeled neurons is encountered following injections in auditory and sensorimotor cortex, followed by the prefrontal and premotor cortex; very few cells project to the visual cortex. Ultrastructurally, the large HRP-labeled neurons display an eccentrically located, indented nucleus, abundant granular endoplasmic reticulum forming Nissl bodies, well developed Golgi zones, and numerous dense bodies. The simultaneous demonstration of retrogradely transported HRP and acetylcholinesterase (AChE) suggest that the large neurons are presumably cholinergic. These results provide evidence that at least some of the giant striatal neurons are efferent cells. The coincidence of cytological, histochemical, and hodological criteria invite the speculation that the giant corticopetal neostriatal neurons might be related to the magnocellular cholinergic groups of the basal forebrain (especially the Ch4 group).