异氟烷预处理对电磁脉冲辐射所致脑损伤的保护作用研究

目的:探讨异氟烷预处理对电磁脉冲辐射所致脑损伤的保护作用。方法:选取成年雄性SD大鼠48只,采用随机数字表法,将其随机分为4组(n=12),分别为:假辐照组(CON组)、电磁辐照组(EMP组)、异氟烷预处理组(IP组)和异氟烷预处理+电磁辐照组(IP+EMP组)。EMP组场强为400 KV/m,脉冲为200次,连续辐照3天;IP组吸入2.0%异氟醚2h;IP+EMP组吸入2.0%异氟醚2 h,24 h后制备EMP损伤模型。于辐照后24 h处死大鼠,每组随机抽取3只大鼠,取脑组织,采用ELISA法检测大鼠海马IL-6和TNF-α的表达变化;尼氏染色法观察大鼠海马区神经元的凋亡;采用Western blot法检测大鼠海马区BDNF蛋白的表达情况;采用免疫荧光法检测大鼠海马区BDNF细胞水平的表达。结果:与CON组比较,EMP组、IP组、IP+EMP组的IL-6和TNF-α的表达增高,尼氏小体减少,BDNF蛋白及细胞水平的表达均下调(P0.05);与EMP组比较,IP组和IP+EMP组IL-6和TNF-α的表达降低,尼氏小体增多,BDNF蛋白及细胞水平的表达上调(P0.05)。结论:异氟烷预处理可减轻电磁脉冲辐射所致脑损伤,其机制可能与减轻大鼠炎症反应有关。     

PTSD样大鼠海马神经元凋亡及其ACP变化的研究

目的研究PTSD（posttraumatic stress disorder创伤后应激障碍）样大鼠海马神经元凋亡及ACP（Acid phosphatase酸性磷酸酶）的变化。方法建立大鼠PTSD模型-SPS（single-prolonged stress）,于模型建立后的6h、12h、1d、7d、14d取材;同时取材正常组作为对照,应用Annexin V-F1TC／PI双标记流式细胞术、透射电镜、酶组化方法分别进行各组海马神经元凋亡及ACP表达变化的观察及定量检测。结果模型建立后的6h、12h海马神经元的凋亡细胞增加、ACP活性增强,1d时凋亡细胞增加更为明显、ACP活性更为显著,7d、14d时凋亡细胞逐渐减少、ACP活性减弱。结论PTSD样大鼠海马神经元出现凋亡,凋亡增加的同时ACP酶活性增强,说明ACP酶参与PTSD大鼠海马神经元的凋亡。     

电磁辐射对原代培养海马神经元的损伤效应及其机制探讨

研究X带高功率微波、S带高功率微波及电磁脉冲辐射对原代培养海马神经元的损伤效应并探讨其机制。通过体外培养原代海马神经元,建立电磁波辐照细胞模型。采用Annexin V-PI双标记、流式细胞术检测细胞凋亡与坏死,原子力显微镜检测细胞膜表面形态,Fluo-3-AM荧光探针负载、激光扫描共聚焦显微镜测定胞内[Ca2 ]i。结果表明,辐射后海马神经元凋亡与坏死均增加,其中坏死增加明显;细胞膜表面粗糙度加大,膜穿孔增多;胞内[Ca2 ]i明显升高。且以上变化均以X带高功率微波组最重,S带高功率微波组次之,电磁脉冲组最轻。提示细胞膜穿孔增多,膜通透性增加,导致胞外Ca2 内流增加,甚至胞内钙超载是辐射致海马神经元凋亡与坏死的机制之一;三种电磁辐射对海马神经元的损伤程度与照射频率呈正相关。     

大鼠脑缺血再灌注后神经元和星形胶质细胞凋亡的比较研究

目的比较研究大鼠局灶性脑缺血再灌注后神经元和星形胶质细胞的凋亡规律。方法建立大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)再灌注模型,在缺血再灌注后1、3、7、14d断头取脑,应用流式细胞分选技术和原位末端标记法分别检测各组MCAO后不同时期神经元和星形胶质细胞凋亡情况。结果局灶性脑缺血再灌注后,海马区星形胶质细胞凋亡数量超过神经元,其凋亡以再灌注3d最为显著,而神经元则以7d最为显著;而皮层区神经元凋亡数量超过星形胶质细胞,两种细胞凋亡均在再灌注后7d达高峰。结论脑缺血再灌注后,皮层和海马区的神经元及星形胶质细胞均可发生凋亡,海马区星形胶质细胞比皮层区更易凋亡,而皮层区神经元比海马区更易凋亡。     

竹叶黄酮缓解异氟醚引起的老年大鼠认知功能障碍

研究竹叶黄酮对异氟醚吸入诱发老年大鼠神经细胞凋亡及认知功能障碍的影响.分离培养原代海马神经细胞,暴露于体积分数为2%异氟醚6 h后,分别给予50、80及100 mg/L竹叶黄酮处理,MTT法检测细胞增殖能力,流式细胞仪检测细胞凋亡情况.大鼠经吸入1.4%体积的异氟醚2 h后,分别于1~5 d连续腹腔注射25、50、100 mg/kg竹叶黄酮,于第6天进行水迷宫实验检测大鼠空间学习记忆能力,ELISA法检测海马组织匀浆中的Aβ1-42、TNF-α、INF-γ含量.结果发现,竹叶黄酮可显著促进海马神经细胞的增殖,并且抑制异氟醚所致的海马神经细胞凋亡.与异氟醚单独处理组相比较,各浓度竹叶黄酮可显著降低大鼠逃避潜伏期并增加大鼠过台次数,ELISA结果显示竹叶黄酮可显著抑制海马组织中Aβ1-42、TNF-α及INF-γ的表达.表明竹叶黄酮可增强大鼠抗炎能力,抑制海马组织中Aβ1-42的产生,从而抑制异氟醚所致的老年大鼠神经损伤及认知功能障碍.     

过量皮质酮致原代培养的大鼠海马神经元死亡方式的研究

目的和方法：以体外原代培养的大鼠海马神经元为研究对象,采用原位染色的方法,对不同剂量的皮质酮（CORT）致海马神经元死亡的方式进行研究。结果：在CORT作用下,海马神经元不仅会发生快速的坏死,而且还会发生慢性的凋亡;并且,随着CORT剂量增大和作用时间延长,海马神经元坏死和凋亡的发生率会随之增高。结论：海马神经细胞坏死和凋亡的发生,可能与CORT抑制神经元能量代谢的程度和增高神经元对谷氨酸神经毒性的敏感性有关。     

HSP70 高表达保护低氧诱导的大鼠海马DG 区神经细胞凋亡

目的:研究诱导HSP70高表达对低氧引起的大鼠海马DG区神经细胞凋亡的保护作用。方法:大鼠海马DG区神经细胞分别在41℃温浴2h和加入砷酸钠诱导HSP70高表达。对照低氧而不预热,并用HSP70反义寡核苷酸链抑制HSP70合成,观察HSP70与低氧大鼠海马DG区神经细胞凋亡的关系。DNA碎片法检测细胞凋亡,Western Blotting检测HSP70。结果:预热和砷酸钠都可以诱导细胞HSP70高表达;HSP70高表达可以明显减少低氧诱导的细胞凋亡。在预热前导入HSP70反义核酸,可以降低HSP70抑制细胞凋亡的作用。结论:HSP70高表达可以保护细胞由于低氧引起的细胞凋亡。     

急性酒精中毒合并中度创伤性脑损伤大鼠海马GFAP的表达

目的:观察急性酒精中毒合并中度创伤性脑损伤后大鼠海马星形胶质细胞标记物胶质纤维酸性蛋白(GFAP)表达的变化.方法:健康成年雄性SD大鼠72只,随即机分为4组:假手术组(N组)、急性酒精中毒组(E组)、中度创伤性脑损伤组(T组)和急性酒精中毒合并中度创伤性脑损伤组(E T组).腹腔注射酒精(2.5g/kg)致使大鼠急性酒精中毒,2h后,按改进的Feeney's自由落体硬膜外撞击方法使其合并中度创伤性脑损伤(600g.cm).各组动物术后6h、24h和48h处死.中性红染色观察海马CA1区神经元形态学改变;用免疫组织化学的方法检测海马CA1区GFAP表达变化.结果:与N组和E组相比,T组和E T组GFAP表达显著增多(P＜0.01).术后6h和24h,T组GFAP表达显著高于E T组(P＜0.05);T组和E T组的海马CA1区神经元细胞出现胞体肿胀,排列散乱,但T组上述形态学改变较E T组明显.结论:急性酒精中毒合并中度创伤性脑损伤的早期可通过减少GFAP的表达,抑制星形胶质细胞激活,减少炎症反应发挥保护作用.     

大鼠脑缺血再灌注后海马神经细胞NOS表达与细胞凋亡及复方丹参的保护作用

目的探讨大鼠局灶性脑缺血再灌注后海马神经细胞一氧化氮合酶(NOS)的表达与神经细胞凋亡的关系及中药复方丹参的保护作用。方法采用大脑中动脉内栓线阻断法(MCAO)造成局灶性脑缺血再灌注模型。用原位细胞凋亡检测方法观察海马神经细胞凋亡;用免疫组织化学方法检测大鼠海马神经细胞(nNOS、iNOS)的表达并做图像分析。结果与假手术对照组比较,脑缺血再灌注2h后缺血侧海马CA1、CA3区神经细胞nNOS、iNOS表达升高,并出现神经细胞凋亡,随着再灌注时间的延长,神经细胞iNOS的表达明显增强,凋亡神经细胞数逐渐增多,至24h达高峰,但神经细胞nNOS的表达并未见明显增强。复方丹参保护组神经细胞nNOS、iNOS的表达和凋亡神经细胞数明显低于缺血再灌组(P<0.01)。结论脑缺血再灌注后缺血侧海马CA1、CA3区神经细胞nNOS的表达增强,iNOS的表达显著升高,使NO的形成增加,这可能是介导脑缺血再灌注后神经细胞凋亡的机制之一。复方丹参具有下调神经细胞nNOS、iNOS的表达,减少NO的生成,抑制细胞凋亡,减轻缺血再灌注对大鼠海马损伤的作用。     

Aβ25-35诱导大鼠记忆力障碍与海马细胞色素氧化酶活性及线粒体超微结构的变化

目的探讨Aβ诱导模拟人类Alzheimer's病(AD)大鼠模型中海马CA1区细胞色素氧化酶的表达和神经元线粒体超微结构的变化及其与老年性记忆力减退的关系,揭示Aβ对神经元的毒性机制.方法通过将Aβ25-35注射入海马建立阿尔茨海默病动物模型,使用Y形迷宫试验检测大鼠的学习记忆能力,运用酶组织化学方法测定大鼠海马CA1区细胞色素氧化酶活性,应用电镜观察大鼠海马CA1区神经细胞线粒体超微结构的变化.结果与对照组比较,接受Aβ注射的大鼠学习记忆能力降低(P<0.05),线粒体数量及形态发生了明显的变化,海马CA1区脑组织细胞的细胞色素氧化酶活性相对于对照组也有显著的下降(P<0.05).结论 Aβ在神经退行性变中的作用可能与细胞色素氧化酶表达下降及神经元线粒体超微结构的改变导致的细胞能量代谢障碍有关.     

LncRNA TUG1 inhibits neuronal apoptosis in status epilepticus rats via targeting the miR-421/mTOR axis

Status epilepticus (SE) induces apoptosis of hippocampal neurons. However, the underlying mechanism in SE is not fully understood. Recently, lncRNA TUG1 is reported as a significant mediator in neuronal development. In present study, we aimed to investigate whether lncRNA TUG1 induces apoptosis of hippocampal neurons in SE rat models. TUG1 expression in serum of normal volunteers and SE patients, SE rats and neurons with epileptiform discharge was detected. SE rat model was established and intervened with TUG1 to evaluate hippocampal neuronal apoptosis. The experiments in vitro were further performed in neurons with epileptiform discharge to verify the effects of TUG1 on neuronal apoptosis of SE rats. The downstream mechanism of TUG1 was predicted and verified. miR-421 was intervened to perform the rescue experiments. Levels of oxidative stress and inflammation-related factors and mTOR pathway-related proteins in SE rats and hippocampal neurons were detected. TUG1 was highly expressed in serum of SE patients, SE rats and neurons with epileptiform discharge. Inhibition of TUG1 relieved pathological injury, oxidative stress and inflammation and reduced neuronal apoptosis in SE rats, which were further verified in hippocampal neurons. TUG1 upregulated TIMP2 expression by targeting miR-421. Overexpressed miR-421 inhibited hippocampal neuronal apoptosis. TUG1 knockout inactivated the mTOR pathway via the miR-421/TIMP2 axis to relieve neuronal apoptosis, oxidative stress and inflammation in SE rats and hippocampal neurons. Taken together, these findings showed that downregulation of lncRNA TUG1 inhibited apoptosis of hippocampal neurons in SE rats, and attenuated oxidative stress and inflammation damage through regulating the miR-421/mTOR axis.     

Disruption of microRNA‐214 during general anaesthesia prevents brain injury and maintains mitochondrial fusion by promoting Mfn2 interaction with Pkm2

Duration of surgical general anaesthesia is associated with severe brain injury and neurological deficits. The specific mechanisms underlying post‐general anaesthesia brain injury, however, still remain to be elucidated. Herein, we explore the role of microRNA‐214 (miR‐214) in the occurrence of brain injury after general anaesthesia and its underlying mechanism. Hippocampal tissues and neurons were isolated from rats exposed to 2% sevoflurane. TUNEL stains reflect hippocampal neuron apoptosis. Cultured hippocampal neurons stained with JC‐1 and MitoTracker dyes were imaged by fluorescence microscope to visualize changes of mitochondrial membrane potential and mitochondrial fusion. Mitochondrial function was evaluated. Mitofusin 2 (Mfn2) binding to miR‐214 or pyruvate kinase M2 (Pkm2) was confirmed by co‐immunoprecipitation, immunofluorescence, dual luciferase reporter gene and RNA immunoprecipitation assays. After exposure to 2% sevoflurane, up‐regulated miR‐214 expression and impaired interaction between Mfn2 and Pkm2 were found in rat hippocampal tissues. Rats exposed to 2% sevoflurane also experienced neuronal injury, mitochondrial defects and deficits in the brain‐derived neurotrophic factor (Bdnf) signalling. miR‐214 was shown to target Mfn2 by impairing its binding with Pkm2. Inhibiting miR‐214 expression using its specific inhibitor improved mitochondrial membrane potential, enhanced mitochondrial fusion, maintained mitochondrial function, restored interaction between Mfn2 and Pkm2, and activated the Bdnf signalling in cultured hippocampal neurons. Adenovirus infection of miR‐214 inhibitor reduced neuron apoptosis and maintained mitochondrial function in the hippocampus of rats exposed to 2% sevoflurane. Taken together, the study demonstrates inhibition of miR‐214 is cerebral protective against brain injury following general anaesthesia.     

Astroglial trophic support and neuronal cell death: influence of cellular energy level on type of cell death induced by mitochondrial toxin in cultured rat cortical neurons

Previous in vivo and in vitro analyses have shown that both necrosis and apoptosis are involved in neuronal cell death induced by energy impairment caused by mitochondrial dysfunction. However, little is known about the key factors that determine whether the cells undergo necrosis or apoptosis. In the present study, we analyzed neuronal cell death induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II, in a primary culture system of rat cortical neurons. The neurons were maintained for a week in coculture with astroglial cells, and then they were treated with 3-NP in the presence or absence of astroglial cells. As judged from morphological (Hoechst 33258 staining) and biochemical (DNA fragmentation and caspase activation) analyses, the cortical neurons appeared to die through an apoptotic process after 3-NP treatment in the presence of astroglial cells. However, caspase inhibitors did not suppress the 3-NP-induced cell death, suggesting the involvement of a caspase-independent pathway of 3-NP-induced neuronal cell death in the presence of astroglial cells. On the other hand, 3-NP induced necrotic cell death within 1 day in the absence of astroglial cells, following a rapid decrease in intracellular ATP level. These changes were attenuated by the presence of astroglial cells or the addition of astroglial conditioned medium. These results suggest that astroglial trophic support influences the alteration of the intracellular energy state in 3-NP-treated neurons and consequently determines the type of neuronal cell death, apoptosis or necrosis.     

Viral interference with neuronal integrity: what can we learn from the Borna disease virus?

The neurotropic Borna disease virus (BDV) is unusual in that it can persistently infect neurons of the central nervous system (CNS) without causing general cell death, reflecting its favourable adaptation to the brain. The activity-dependent enhancement of neuronal network activity is however disturbed after BDV infection, possibly by its effect on the protein kinase C signalling pathway. The best model for studying BDV, which has a non-cytolytic replication strategy in primary neurons, is the rat. Infection of adult rats results in a fatal immune-mediated disease, whereas BDV establishes persistent infection of the brain in newborn rats resulting in progressive neuronal cell loss in defined regions of the CNS. Our recently developed system of BDV-infected hippocampal slice cultures has clearly shown that the onset of granule cell loss begins after the formation of the mossy fibre projection. Quantitative analysis has revealed a significant increase in synaptic density on identified remaining granule cell dendrites at 6?weeks after infection, followed by a decline. Granule cells are the major target of entorhinal afferents. However, despite an almost complete loss of dentate granule cells during BDV infection, entorhinal axons persist in their correct layer, both in vivo and in slice cultures, possibly exploiting rewiring capabilities and thereby allowing new synapse formation with available targets. These morphological observations, together with electrophysiological and biochemical data, indicate that BDV is a suitable model virus for studying virus-induced morphological and functional changes of neurons and connectivity patterns.     

远志皂苷对丙泊酚麻醉所致大鼠认知功能障碍的保护作用及机制

本研究旨在探讨远志皂苷对丙泊酚麻醉所致大鼠认知功能障碍的保护作用及机制。将SD大鼠分别应用远志皂苷(200 mL·kg^-1·d^-1)和/或丙泊酚(60 mL·kg^-1·d^-1)处理3周,通过Morris水迷宫实验来评价认知功能情况,通过苏木精伊红(hematoxylineosin, HE)染色评价组织病理改变。采用原位末端标记技术(TdTmediated dUTP nick and labeling, Tunel)检测大鼠海马神经细胞的凋亡情况。采用酶联免疫吸附法(enzymelinked immunosorbent assay, ELISA)检测氧化损伤指标。采用Western blotting检测Bcl-2和Caspase-3的表达。Morris水迷宫实验显示,远志皂苷显著降低了丙泊酚麻醉大鼠的逃避潜伏期,并提高了穿越平台次数(p<0.05)。苏木精伊红(HE)染色显示,远志皂苷可显著降低丙泊酚引起的大鼠海马组织病变程度。原位末端标记技术(Tunel)实验显示,远志皂苷抑制了丙泊酚引起的大鼠海马神经细胞凋亡(p<0.05)。Western blotting检测显示,远志皂苷抑制了丙泊酚对大鼠脑组织中Bcl-2蛋白的下调及Caspase-3的上调(p<0.05)。酶联免疫吸附检测显示,远志皂苷提高了大鼠脑组织中SOD和GSH的水平,并降低了MDA水平(p<0.05)。远志皂苷可显著改善丙泊酚麻醉大鼠的认知功能并降低海马组织病变程度,其机制与抑制海马神经细胞凋亡和减弱氧化损伤有关。     

The effects of α-lipoic acid on immature rats with traumatic brain injury

Abstract

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality during childhood. TBI enhances formation of reactive oxygen species that cause neuron damage and apoptosis. α-Lipoic acid (LA) is a free radical scavenger and biological antioxidant. We investigated the effects of LA treatment on the parietal and prefrontal cortex, and on the hippocampal regions of the brain in 7-day-old rat pups that had been subjected to contusion injury. Forty-two male rats were divided randomly into a control group, a TBI group and a TBI + LA treated group. LA was administered 30 min after TBI through an intragastric tube once daily for 2 days. Forty-eight hours after TBI, the animals were sacrificed and tissues were examined for apoptosis and density of neurons. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and active caspase-3 immunostaining were used to detect apoptosis. Glutathione peroxidase (GPx), superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels also were measured. Histological evaluation showed that LA treatment significantly reduced TBI-induced neuronal death in the hippocampus, prefrontal and parietal cortex; TUNEL- and caspase-3-positive cells also were decreased in the same regions. In addition, LA administration increased GPx and SOD activity in the prefrontal cortex. It appears that LA may be beneficial for TBI in rats.     

Post-Treatment with Voltage-Gated Na+ Channel Blocker Attenuates Kainic Acid-Induced Apoptosis in Rat Primary Hippocampal Neurons

Injection of rats with kainic acid (KA), a non-N-methyl-D-aspartate (NMDA) type glutamate receptor agonist, induces recurrent (delayed) convulsive seizures and subsequently hippocampal neurodegeneration, which is reminiscent of human epilepsy. The protective effect of anti-epileptic drugs on seizure-induced neuronal injury is well known; however, molecular basis of this protective effect has not yet been elucidated. In this study, we investigated the effect and signaling mediators of voltage-gated Na(+) channel blockers (Lamotrigine, Rufinamide, Oxcarbazepine, Valproic Acid, and Zonisamide) on KA-induced apoptosis in rat primary hippocampal neurons. Exposure of hippocampal neurons to 10 μM KA for 24 h caused significant increases in morphological and biochemical features of apoptosis, as determined by Wright staining and ApopTag assay, respectively. Analyses showed increases in expression and activity of cysteine proteases, production of reactive oxygen species (ROS), intracellular free [Ca(2+)], and Bax:Bcl-2 ratio during apoptosis. Cells exposed to KA for 15 min were then treated with Lamotrigine, Rufinamide, Oxcarbazepine, Valproic Acid, or Zonisamide. Post-treatment with one of these anti-epileptic drugs (500 nM) attenuated production of ROS and prevented apoptosis in hippocampal neurons. Lamotrigine, Rufinamide, and Oxcarbazepine appeared to be less protective when compared with Valproic Acid or Zonisamide. This difference may be due to blockade of T-type Ca(2+) channels also by Valproic Acid and Zonisamide. Our findings thus suggest that the anti-epileptic drugs that block both Na(+) channels and Ca(2+) channels are significantly more effective than agents that block only Na(+) channels for attenuating seizure-induced hippocampal neurodegeneration.     

Erythropoietin receptor-mediated inhibition of exocytotic glutamate release confers neuroprotection during chemical ischemia

Erythropoietin (EPO) reduced Ca(2+)-induced glutamate (Glu) release from cultured cerebellar granule neurons. Inhibition was also produced by EPO mimetic peptide 1 (EMP1), a small synthetic peptide agonist of EPO receptor (EPO-R), but not by iEMP1, an inactive analogue of EMP1. EPO and EMP1 induced autophosphorylation of Janus kinase 2 (JAK2), a tyrosine kinase that associates with EPO-R. Furthermore, genistein, but not genistin, antagonized both the phosphorylation of JAK2 and the suppression of Glu release induced by EPO and EMP1. During chemical ischemia, substantial amounts of Glu were released from cultured cerebellar and hippocampal neurons by at least two distinct mechanisms. In the early phase, Glu release occurred by exocytosis of synaptic vesicle contents, because it was abolished by botulinum type B neurotoxin (BoNT/B). In contrast, the later phase of Glu release mainly involved a BoNT/B-insensitive non-exocytotic pathway. EMP1 inhibited Glu release only during the early exocytotic phase. A 20-min exposure of hippocampal slices to chemical ischemia induced neuronal cell death, especially in the CA1 region and the dentate gyrus, which was suppressed by EMP1 but not iEMP1. However, EMP1 did not attenuate neuronal cell death induced by exogenously applied Glu. These results suggest that activation of EPO-R suppresses ischemic cell death by inhibiting the exocytosis of Glu.