过氧化氢诱发小脑皮质神经元凋亡

采用分离细胞培养的新生SD大鼠小脑皮质神经元，用Ara－c抑制非神经元细胞生长，研究了过氧化氢能否诱导种经元凋亡．用荧光染料Hoechst33258染色鉴别凋亡神经元，可见凋亡神经元的细胞核体积缩小至正常神经元的40％～50％，核形不规则，核内染色质块状深染．少量神经元可见到不规则的核碎片及凋亡小体。结果表明：（1）小剂量过氧化氢（50μmol／L）作用12h，可见1％～3％神经元凋亡，与对照组比较，无明显差异；24、36h凋亡神经元比对照组显著增多；48h凋亡神经元数量达高峰。（2）大剂量过氧化氢（70μmol/L）作用12h，凋亡神经元的出现明显增多，作用24、36h凋亡神经元增加均比小剂量组更显著，出现高峰更早、更高．（3）对照组随培养时间延长凋亡细胞的数量增长速度比较缓慢．以上结果提示过氧化氢能诱发小脑皮质神经元凋亡，尤以大剂量过氧化氢的诱发作用为更强．     

丙烯酰胺对原代培养大鼠大脑皮质神经元毒性作用的研究

目的:研究丙烯酰胺(acrylamide,ACR)对体外原代培养的大鼠大脑皮质神经元的毒性作用。方法:原代培养新生SD大鼠大脑皮质神经元,将培养至第7天的神经元分为空白对照组,丙烯酰胺不同浓度组。染毒后,四甲基偶氮唑盐比色法(MTT)检测各组皮质神经元的存活率,倒置显微镜观察各组皮质神经元的形态学变化,Hoechst33258染色法观察皮质神经元核形态的变化。结果:MTT结果显示各剂量ACR组的神经元存活率与对照组相比都显著降低,并且呈时间和剂量依赖性。ACR可造成皮质神经元突起萎缩,胞体肿胀,并可以诱导皮质神经元发生核固缩、碎裂,出现典型的凋亡特征,且随着浓度的升高,毒性作用加重。结论:ACR可对原代培养的大鼠大脑皮质神经元产生毒性损伤作用。它能抑制皮质神经元的存活,诱导神经元形态结构的改变、凋亡的发生。     

卵巢类固醇激素对雌性小鼠额叶皮质神经元超微结构的影响

电镜观察苯丙酸雌二醇和己酸孕酮对昆明雌小鼠额叶皮质锥体神经元细胞器，树突和树突朵微管超微结构的影响。根据雌鼠的动情周期，对卵巢切除的幼鼠每天注射１次８μｇ雌二醇或８μｇ雌二醇加２ｍｇ孕酮，治疗２个月。结果显示：卵巢切除用雌二醇治疗组，卵巢切除用雌二醇加孕酮治疗组和正常     

皮质丘脑投射神经元的超微结构及突触联系

采用ＨＲＰ逆行追踪技术与电镜相结合的方法，对猫大脑皮质体感Ⅰ区内皮质丘脑投射神经元超微结构及突触联系进行了研究。结果证明，皮质丘脑投射神经元超微结构的特点为锥体形的胞体，胞浆丰富，含有多量的粗面内质网，游离核糖体及线粒体。ＨＲＰ标记的皮质丘脑投射神经元作为突触后成份与轴突和树突分别形成轴－树突触，轴－体突触和树－体实触。这些结果提示：皮质丘脑投射神经元接受广泛的传入联系和皮质间的联系。     

槲皮素对小鼠前额叶皮质锥体神经元的放电抑制作用研究

目的:探讨槲皮素对小鼠前额叶皮质(prefrontal cortex,PFC)锥体神经元动作电位的效应。方法:成年C57小鼠或Thy1-GCaMP 3.0小鼠,制作含PFC的冠状脑片。分成正常脑脊液孵育组(对照组)和槲皮素孵育组(50μmol/L),给予高钾(5 mmoL/L KCl)人工脑脊液灌流两组脑片记录GCaMP荧光信号,全细胞膜片钳记录两组PFC锥体神经元放电,碘化丙啶(propidium iodide,PI)染色检测神经元活性。结果:5 mmol/L KCl人工脑脊液可以明显引起对照组GCaMP脑片PFC锥体神经元荧光信号增强,而对槲皮素组无明显作用。全细胞膜片钳记录显示槲皮素组神经元能够产生动作电位。但与对照组相比,产生放电所需刺激强度增加,连续放电能力降低。PI染色显示槲皮素组神经元死亡数目减少。结论:槲皮素孵育可以通过抑制小鼠前额叶皮质锥体神经元产生动作电位的能力,从而提高神经元在体外环境的生存能力。其具体的离子通道机制及细胞信号通路研究需进一步的深入研究。     

一种同时培养原代皮质及海马神经元的实验方法北大核心

背景:近年来原代神经元细胞模型在颅脑疾病中扮演着重要的角色,此类细胞模型特性更加贴近于疾病细胞,可用于模拟研究各类神经系统疾病。目的:建立一种便捷实用的可同时提取皮质及海马原代神经元的培养方法。方法:取新生24 h内的SD大鼠,麻醉后断脊法处死,采用体积分数为75%乙醇消毒,用镊子分离出颅骨及脑膜,解剖出完整大脑,剥离脑血管膜,游离出大脑皮质及海马组织,采用木瓜蛋白酶及适量DNA酶顺序消化方案,吹打、离心、过滤,然后接种于左旋多聚赖氨酸包被的6孔板和爬片内,以含体积分数10%胎牛血清的DMEM-F12培养基为种植液,6 h后换用含有B27的Neurobasal无血清专用培养基,持续培养7 d后,显微镜下观察细胞形态及生长状态。分别采用β-Tubulin免疫荧光法、Neun抗体免疫组化法鉴定皮质神经元与海马神经元,以及采用MAP2免疫荧光法鉴定其纯度。结果与结论:①接种24 h后细胞体积变清晰,呈不规则圆形、周围环绕光晕,少数细胞已有细小突起,均已贴壁生长;持续培养3 d后,胞体逐步增大,部分呈聚团性生长,突触延长,细胞间出现交联;持续培养7 d后,胞体成熟饱满,细胞质显著增多,光晕增强,形成更为密集的神经元网络;②经Neun抗体免疫组化法、β-Tubulin免疫荧光法鉴定为神经元,神经元标志物MAP2免疫荧光法鉴定皮质神经元和海马神经元纯度分别为(94.00±0.34)%和(91.00±0.26)%,可用于后期实验;③结果表明,采用同一批24 h内新生SD大鼠分离大脑皮质和海马组织,经木瓜蛋白酶与DNA酶顺序消化后,可提取到优质的海马神经元及皮质神经元。该方案得到的神经元纯度高,操作简化,可作为研究各类神经系统疾病细胞模型的基础。     

小鼠小脑皮质发育中的神经元凋亡

目的探讨小鼠小脑皮质发育中神经元凋亡的规律和机制。方法用激活型Caspase-3多抗免疫组织化学标记及Hoechst 33258染色液染色,检测从出生至成年小鼠小脑皮质中神经元的凋亡,用Western blotting方法对小脑组织中Caspase-3和Caspase-8的活化片段进行半定量测定。结果外颗粒层、普肯耶细胞层和内颗粒层凋亡细胞密度最高分别在出生后第8d(P8)、P5及P9,P20各层凋亡细胞密度都很低。Caspase-3活化片段的表达量在P5较高,P5以后逐渐降低,至P14消失;Caspase-8活化片段的表达量从P0到P10都较高,P10以后逐渐降低,至P30消失。结论P0至P14是小脑皮质神经元凋亡的重要时期,通过启动Caspase-8的活化进而激活效应性Caspase-3的细胞凋亡途径存在于小脑皮质的塑型成熟过程。     

鱼藤酮对多巴胺能神经元的损伤作用及机制

鱼藤酮等环境毒物的长期接触可能与帕金森病发病有关。鱼藤酮(Rotenone)是线粒体复合酶Ⅰ抑制剂，可以抑制多巴胺能神经元线粒体复合酶Ⅰ的活性、降低线粒体膜电位、诱导细胞内活性氧的产生，从而导致多巴胺能神经元α-突触核蛋白(α-synuclein)表达增加、路易小体(Lewy bodies)形成，DJ-1和Parkin的突变以及细胞微管破坏。     

N-copine基因反义核酸对原代培养的小鼠皮质神经元的影响

目的：通过研究N-copine基因反义核酸(ant isense)对体外培养的小鼠大脑皮质神经元的影响，在细胞学层面上探索N-copine基因的功 能。 方法： 原代培养小鼠大脑皮质神经元，用反义核酸抑制N-copine基 因的表达，分析抑制N-copine基因表达对神经元生长及死亡的影响。 结果：寡核苷酸处理72 h，antisense组神经元轴突长度短了而且胞体变小，而对照组神经元轴 突和胞体都正常生长；antisense组台盼蓝着色细胞明显多于对照组，培养液中LDH活力明显 高于 对照组。 结论： 抑制N-copine基因表达，能影响体外培养的皮质神经元 的生长，并导致神经元严重损伤和死亡。这提示N-copine可能是神经元的一种保护性蛋白， 可能具有防止神经元退变和促进再生的重要作用。     

aFGF基因在大鼠脑皮质损伤神经元的表达

目的：为aFGF对成年大鼠脑损伤后脑内神经元的作用提供理论依据。方法：应用原位杂交和免疫组织化学方法观察了大鼠脑损伤后aFGF基因mRNA和蛋白在皮质神经元的表达变化。结果：脑损伤后15min及30min伤侧大脑皮质神经元aFGF mRNA和蛋白表达增加,aFGF mRNA表达于伤手6h达高峰,持续至伤后24h,以后表达逐步降低,于伤后72h恢复至正常水平。神经元aFGF蛋白表达于伤后1h达高峰,持续至伤后3h,伤后24h表达恢复正常。结论：脑损伤后皮质神经元aFGF基因表达增加与神经元抗损伤修复机制有关。     

人参皂苷Rg1抑制叔丁基过氧化氢诱导的原代大鼠皮层神经元损伤

 目的：观察人参皂苷Rg1对叔丁基过氧化氢(t-BHP)诱导的原代大鼠皮层神经元损伤的改善作用,并探讨其可能机制。方法：将神经元随机分为正常对照组、10 μmol/L t-BHP组及10 μmol/L t-BHP+10 μmol/L人参皂苷Rg1组,培养24 h。采用MTT检测不同浓度t-BHP处理的神经元活性,神经元三维重建研究神经元平均总纤维长度及总突起数量,免疫荧光检测caspase-3表达水平及免疫印迹方法检测Bcl-2、caspase-3以及磷酸化糖原合成酶激酶3β (pGSK-3β)蛋白表达水平。结果：10 μmol/L人参皂苷Rg1能够对抗10 μmol/L t-BHP引起的原代大鼠皮层神经元活性水平的降低,并且上调Bcl-2及pGSK-3β蛋白表达量,降低caspase-3活化为cleaved caspase-3的水平(P<0.05)。结论:人参皂苷Rg1可能通过提高GSK-3β自身磷酸化从而增强神经元的抗t-BHP损伤能力。     

叶黄素抑制叔丁基过氧化氢诱导的视网膜神经节细胞凋亡

目的:观察叶黄素(lutein)对叔丁基过氧化氢(t-BHP)处理的视网膜神经节细胞(RGC-5细胞系)的保护效应并探讨其作用机制。方法:用免疫荧光染色检测视网膜神经节特异性蛋白Brn-3和神经微管结合蛋白MAP-2的表达来鉴定RGC-5细胞;将RGC-5细胞随机分为对照组、t-BHP处理组、t-BHP和lutein共同处理组、lutein处理组,培养24 h,MTT实验检测细胞活力;Annexin V-FITC/PI双染流式细胞术检测细胞凋亡;免疫细胞化学技术检测caspase-3蛋白的活化情况;Western blot检测Bcl-2/Bax、cleaved caspase-3、JNK和c-Jun蛋白的变化。结果:MTT实验和流式细胞检测结果显示,lutein能提高t-BHP处理的RGC-5细胞的活力,并降低t-BHP诱导的RGC-5细胞凋亡;免疫荧光结果显示lutein能抑制t-BHP诱导的caspase-3的活化;与对照组比较,t-BHP处理后RGC-5细胞抗凋亡蛋白Bcl-2表达下调(P0.05),Bax/Bcl-2比率升高,cleaved caspase-3表达上调(P0.05),JNK和c-Jun蛋白的磷酸化水平增加(P0.05),t-BHP的上述作用可被lutein部分逆转。结论:Lutein能够降低t-BHP诱导的RGC-5细胞凋亡,其机制与其上调Bcl-2的表达、抑制caspase-3的活化并降低JNK和c-Jun蛋白的磷酸化有关。     

Resveratrol protects against inorganic arsenic-induced oxidative damage and cytoarchitectural alterations in female mouse hippocampus

Prolonged inorganic arsenic (iAs) exposure is widely associated with brain damage particularly in the hippocampus via oxidative and apoptotic pathways. Resveratrol (RES) has gained considerable attention because of its benefits to human health. However, its neuroprotective potential against iAs-induced toxicity in CA1 region of hippocampus remains unexplored.Therefore, we investigated the neuroprotective efficacy of RES against arsenic trioxide (As₂O₃)-induced adverse effects on neuronal morphology, apoptotic markers and oxidative stress parameters in mouse CA1 region (hippocampus).Adult female Swiss albino mice of reproductive maturity were orally exposed to either As₂O₃ (2 and 4 mg/kg bw) alone or in combination with RES (40 mg/kg bw) for a period of 45 days. After animal sacrifice on day 46, the perfusion fixed brain samples were used for the observation of neuronal morphology and studying the morphometric features. While the freshly dissected hippocampi were processed for biochemical estimation of oxidative stress markers and western blotting of apoptosis-associated proteins.Chronic iAs exposure led to significant decrease in Stratum Pyramidale layer thickness along with reduction in cell density and area of Pyramidal neurons in contrast to the controls. Biochemical analysis showed reduced hippocampal GSH content but no change in total nitrite (NO) levels following iAs exposure. Western blotting showed apparent changes in the expression levels of Bax and Bcl-2 proteins following iAs exposure, however the change was statistically insignificant. Contrastingly, iAs +RES co-treatment exhibited substantial reversal in morphological and biochemical observations.Together, these findings provide preliminary evidence of neuroprotective role of RES on structural and biochemical alterations pertaining to mouse hippocampus following chronic iAs exposure.     

谷氨酸诱导新生小鼠皮层神经细胞损伤体外模型的建立

目的:本实验建立谷氨酸(Glu)诱导神经细胞的损伤模型,观察Glu对神经细胞的兴奋毒性作用,探索Glu诱导神经细胞损伤模型的最佳浓度,为进一步研究Glu与神经系统疾病之间的关系奠定基础。方法:原代培养新生小鼠皮层神经细胞,鉴定成功后,采用不同浓度的Glu诱导神经细胞损伤,酶标仪测定乳酸脱氢酶(LDH)漏出率,用流式细胞仪检测细胞凋亡率和死亡率,以获得Glu诱导神经细胞损伤模型的最佳浓度。结果:成功培养新生小鼠皮层神经细胞,Glu诱导神经细胞损伤呈浓度依赖性。实验中Glu浓度=100μmol/L,细胞凋亡率(%)为44.34±6.19而细胞死亡率仅为4.6±0.90说明在Glu浓度=100μmol/L诱导神经细胞,能得到较大的凋亡率和较小的死亡率。结论:成功建立Glu诱导的神经细胞损伤模型,验证Glu=100μmol/L为诱导神经凋亡的最佳浓度。     

白藜芦醇对氧化应激诱导的大鼠海马神经元损伤的影响

目的:研究不同剂量的白藜芦醇对大鼠海马神经元氧化损伤的影响。方法:通过体外原代培养获得大鼠海马神经元。实验分为对照(control,Con)组、三甲基氯化锡(trimethyltin chloride,TMT)组、溶剂对照(vehicle)组和白藜芦醇(resveratrol,Res)组。其中,Res设4个浓度:5μmol/L、10μmol/L、20μmol/L和40μmol/L;除对照组外,其它各组细胞均采用三甲基氯化锡(TMT)处理使细胞出现氧化应激状态。采用CCK-8法和荧光探针DCFH-DA法观察各组神经元的活力与细胞内活性氧(reactive oxygen species,ROS)含量的变化;TUNEL荧光染色法检测各组神经元的凋亡程度。结果:与TMT组相比,不同浓度的白藜芦醇均能提高神经元的活力,其中20μmol/L的作用最显著。20μmol/L白藜芦醇能抑制经TMT处理后神经元内的ROS水平以及凋亡程度的增加。结论:白藜芦醇具有抑制TMT诱导的大鼠海马神经元氧化损伤的作用。     

The protective effect of lycopene against oxidative kidney damage associated with combined use of isoniazid and rifampicin in rats

It is known that the combined use of antibiotics, such as isoniazid and rifampicin, in the treatment of tuberculosis causes oxidative kidney damage. The aim of this study was to biochemically and histopathologically investigate the effect of lycopene on oxidative kidney damage due to the administration of isoniazid and rifampicin in albino Wistar male rats. Lycopene at a dose of 5 mg/kg was orally administered to lycopene+isoniazid+rifampicin (LIR) rats, and normal sunflower oil (0.5 mL) was orally administered to isoniazid+rifampicin (IR) and healthy control (HG) rats as vehicle by gavage. One hour after the administration of lycopene and vehicle, 50 mg/kg isoniazid and rifampicin were given orally to the LIR and IR groups. This procedure was performed once a day for 28 days. Rats were sacrificed by a high dose of anesthesia at the end of this period, and oxidant-antioxidant parameters were measured in the removed kidney tissues. Creatinine and blood urea nitrogen (BUN) levels were measured in blood samples, and kidney tissues were also evaluated histopathologically. The combined administration of isoniazid and rifampicin changed the oxidant-antioxidant balance in favor of oxidants, and it increased blood urea nitrogen and creatinine levels, which are indicators of kidney function. Co-administration of isoniazid and rifampicin also caused oxidative kidney damage. Lycopene biochemically and histopathologically decreased oxidative kidney damage induced by isoniazid and rifampicin administration. These results suggested that lycopene may be beneficial in the treatment of nephrotoxicity due to isoniazid and rifampicin administration.     

Selective susceptibility of different populations of sympathetic neurons to diabetic neuropathy in vivo is reflected by increased vulnerability to oxidative stress in vitro

Diabetes is the major cause of autonomic neuropathy in humans. Sympathetic neurons from the celiac/superior mesenteric ganglia (CG/SMG) develop neuropathic changes in diabetes whereas sympathetic superior cervical ganglion (SCG) neurons do not. Glucose-induced oxidative stress is proposed as a major factor in the development of diabetic neuropathy. The aim of this study was to investigate whether sympathetic neurons that develop neuropathy in diabetes are more susceptible to oxidative stress. Explants of CG/SMG and SCG from control adult rats were cultured in media free of serum and NGF, exposed to menadione for 48 h to induce oxidative stress and assessed for neuronal viability, TUNEL-positive nuclei and tyrosine hydroxylase- (TH)-immunoreactivity. TH-immunoreactivity was also assessed in ganglia from control and 8 week streptozotocin-diabetic rats. Menadione caused a concentration-dependent loss of neuronal viability and increase in TUNEL staining in both ganglia. However, at low concentrations, menadione had a significantly greater effect (p<0.01) on CG/SMG neurons than SCG neurons. At 1 nM, menadione caused a significant increase (p<0.05) in the number of CG/SMG neurons containing intense TH-immunoreactivity without affecting SCG neurons. Similarly, 8 weeks streptozotocin-induced diabetes resulted in a significant increase (p<0.05) in intensely fluorescent TH-containing CG/SMG neurons but not SCG neurons. This is the first demonstration that oxidative stress in vitro causes the same accumulation of TH in CG/SMG neurons as is observed following streptozotocin-induced diabetes in vivo. Furthermore, the selective vulnerability of CG/SMG neurons to diabetes is reflected by increased sensitivity to oxidative stress.     

Heme oxygenase protects hippocampal neurons from ethanol-induced neurotoxicity

Ethanol has deleterious effects on neuronal cells both in vivo and in vitro, but the mechanisms are unknown. Here, treatment with increasing doses of ethanol (from 20 up to 600mM) decreased the viability of a mouse hippocampal neuroblastoma cell line, HT22. The glutathione concentration decreased and intracellular reactive oxygen species (ROS) increased in a dose-and time-dependent manner, suggesting that the neurotoxicity was due to oxidative stress. Expression of heme oxygenase (HO)-1, a redox regulator and heat shock protein, increased with time after ethanol treatment, but HO-2 was expressed constitutively. The addition of 5microM zinc protoporphyrin IX (ZnPP IX), a competitive HO inhibitor, with the ethanol further reduced cell viability and increased intracellular ROS, but these effects were reversed by co-treatment with 50nM bilirubin, a well-known antioxidant and a product of HO catalysis. These results suggest that HO has a protective role in hippocampal neurons as an intrinsic factor against ethanol-induced oxidative stress and the protection depends on the degree of oxidative stress.     

4-PBA通过抑制内质网应激减轻原代海马神经元的损伤

目的:探讨内质网应激后原代海马神经元树突棘密度及突触蛋白表达的变化,以及通过内质网应激分子伴侣4-苯基丁酸(4-phenylbutyric acid,4-PBA)抑制内质网应激对这种神经元损伤的抑制作用。方法:原代培养新生大鼠海马神经元,将表达增强型绿色荧光蛋白的质粒转染到原代培养5～7 d(DIV 5～7)的大鼠海马神经元内持续培养,DIV 20时分为对照组、衣霉素(tunicamycin,Tm)处理组和Tm+4-PBA预处理组(Tm处理前1 h给予4-PBA),采用Western blot法检测内质网应激标志蛋白Bi P和突触蛋白的表达水平,激光共聚焦显微镜下观察神经元,分析树突棘密度,采用MTT法分析细胞活力。结果:Tm处理后使Bi P蛋白水平明显升高,而4-PBA预处理使Bi P蛋白水平显著下降(P 0. 05)。Tm引起的原代海马神经元树突棘密度下降及突触蛋白的表达下降能够被4-PBA抑制。Tm引起的细胞活力下降可被4-PBA抑制。结论:Tm能够通过诱导内质网应激而引起原代海马神经元树突棘密度下降及突触蛋白表达下降,而提前给予4-PBA预处理可明显降低内质网应激反应,抑制树突棘密度下降及突触蛋白表达下降,从而减轻原代海马神经元的损伤。