蒺藜皂苷对缺氧/复氧诱导大鼠皮层神经元凋亡及细胞内钙变化的作用

目的： 观察蒺藜皂苷(TTLS)对缺氧/复氧诱导的大鼠皮层神经元凋亡及细胞内游离钙离子浓度变化的影响。方法： 原代培养大鼠皮层神经元，建立缺氧/复氧损伤的皮层神经元凋亡模型。比色法测定细胞乳酸脱氢酶（LDH）释放漏出率；Annexin V-FITC/PI双染，流式细胞仪定量分析细胞凋亡百分率；Fluo-3荧光染色，激光扫描共聚焦显微镜观察细胞内钙平均荧光值变化。结果： 缺氧3 h复氧12 h可诱导出皮层神经元凋亡，引起细胞内钙离子浓度增高,高于对照组（P<0.01）。蒺藜皂苷(0.025 g/L)能明显降低缺氧/复氧诱导的神经元凋亡，并可降低细胞内钙浓度,与模型组比较有显著差异（P<0.01）。结论： 蒺藜皂苷可降低由缺氧/复氧诱导的皮层神经元的凋亡，减轻细胞损伤，这种作用机制可能与其抑制神经细胞内钙超载有关。     

丹参酮ⅡA定向诱导骨髓间质干细胞分化为神经元样细胞的研究

目的:丹参酮ⅡA体外定向诱导成人骨髓间质干细胞(MSC)分化为神经元样细胞。方法:采用Ficoll-Paque液离心和贴壁法分离成人MSC,体外扩增,流式细胞仪检测MSC表面抗原表达,FGF-2对MSC增殖和分化的影响。采用含丹参酮ⅡA的无血清达乐伯克改良必需基本培养基(DMEM)诱导MSC分化为神经元样细胞,免疫组化鉴定神经元特异性烯醇化酶(NSE)、神经丝蛋白(NF-M)、胶质纤维酸性蛋白(GFAP)、巢蛋白(nestin)的表达。结果:成人骨髓间质干细胞在体外扩增原代可获得0.5×10⁶,15代可获得(2-3)×10¹²个细胞,流式细胞仪检测结果显示CD29、CD44、CD90、CD105、CD166表达阳性,CD11a、CD14、CD34、CD38、CD45、CD80、CD86为阴性。FGF-2促进hMSC生长,并且对MSC的分化能力基本没有影响。丹参酮ⅡA诱导后,MSC胞体收缩,突起伸出;免疫组化显示诱导出的神经元样细胞NSE、NF-M、nestin表达阳性,GFAP阴性。结论:丹参酮ⅡA可以在体外诱导成人骨髓间质干细胞分化为神经元样细胞。     

局灶性脑梗死后半暗带区皮层血流量与神经细胞凋亡的实验研究

目的：研究大鼠局灶性脑梗死后半暗带区皮层血流量及神经细胞凋亡。 方法： 光化学法诱导大鼠局灶性脑梗死模型，应用激光多普勒微循环测量仪检测局灶性脑梗死后各时间点坏死区和半暗带皮层血流量；TdT-介导dUTP-生物缺口末端标记（TUNEL）法检测凋亡细胞。 结果： 脑梗死后坏死区皮层血流量呈波浪样改变，3 h降至最低，为正常血流量的9.36%±1.72%，6h后回升，12 h达高峰，为正常31.18%±1.44%，24-48 h血流量再度下降，与3 h相比无显著差异（P＞0.05）。半暗带皮层血流量6 h降至最低，为正常皮层血流量的30.88%±7.11%，9-48 h上升，至48 h血流恢复至正常91.90%±4.56%，与其它组差异显著 (P<0.05)。TUNEL结果显示，局灶性脑梗死组 6 h 开始出现凋亡细胞，主要集中于半暗带区，呈半球状向四周放射样扩展，随梗死时间的延长凋亡细胞逐渐增多，梗死48 h后凋亡细胞达峰值。 结论： 局灶性脑梗死后半暗带区皮层血流量与神经细胞凋亡具有明显的关系，其血流量的恢复引起的再灌注损伤最终导致了迟发性神经细胞凋亡。     

Connectionist networks learn to transmit chaos

Evidence presented in the preceding paper indicates that the activity of some neurons during the generation of coordinated motor patterns may be attributable to chaos. Because even "simple" biological systems are difficult to control, we have used connectionist networks in order to inquire into the question of whether a chaotic signal originating in one part of the nervous system can be learned and transmitted by another. We have examined a number of different architectures, and report here the findings for a simple network consisting of one input unit, four hidden units, and one output unit. During training sessions, the input of the circuit was given analog values of either the 3.60 or 3.95 logistic equation, or of one variable of the three-variable R?ssler attractor. The backpropagated error in the learning algorithm was a function of the difference between the input value and the output at each iteration. Iterations involving small changes in analog value resulted in good similarity between the input and output signals, but little learning occurred because of the small error propagated back to the synapses. With larger differences in the analog values (and larger feedback error) at each iteration, we found that networks learned to transmit different chaotic attractors. Once the network learned one input, it could transmit another without changing the synapses. Increasing the number of hidden units increased the rate of learning.(ABSTRACT TRUNCATED AT 250 WORDS)     

丹参注射液椎管内局部灌注对急性脊髓损伤的保护作用

目的 探讨丹参对急性脊髓损伤的防治作用。方法 以改良Allen's法造成兔不完全性脊髓损伤的模型,硬膜下插管。随机分成丹参治疗组和对照组。术后按每天0.3ml/kg体重的总量分4次从硬膜下导管推入丹参注射液,对照组推入生理盐水。损伤后8、72h对脊髓损伤区进行过氧化物歧化酶(SOD)、丙二醛(MDA)、组织形态学观察、神经元凋亡、bcl-2等进行评价。结果 丹参组SOD含量高于对照组(P<0.01),MDA含量低于对照组(P<0.01)。细胞凋亡数目TUNEL法丹参组低于对照组(P<0.01),流式细胞术检测凋亡丹参组低于对照组(P<0.05)。bcl-2的表达丹参组高于对照组(P<0.05)。神经元及神经纤维变性、坏死轻于对照组。结论 丹参能改善损伤脊髓微循环,抑制和减轻脊髓损伤后的两种死亡方式坏死和凋亡。     

依达拉奉对过氧化氢刺激神经细胞的保护作用及机制研究

目的 探讨依达拉奉对神经元的保护作用及其保护机制.方法 提取胎龄16 ～ 18 d的孕大鼠皮层组织,消化、离心后培养,鉴定神经元细胞纯度90％以上.实验共分为3组:空白对照组、过氧化氢(H2O2)处理组(300 μmol/L刺激0.5、2、6及12 h),依达拉奉预处理组(刺激前预防性应用500 μmol/L保护30 min,分组同H2O2处理组).电镜观察线粒体形态的变化,流式细胞仪检测凋亡细胞的比率,免疫印迹及免疫沉淀检测凋亡关键蛋白的活性及蛋白间的关系.结果 相比依达拉奉预处理组,H2O2处理组相同时间点的线粒体明显呈水泡样肿胀变及空泡样变.H2O2处理组刺激后0.5、2、6及12h细胞凋亡比率分别为14.40％±1.23％、45.50％±2.81％、56.40％±3.53％、62.50％±4.23％;与空白对照组比较神经元凋亡明显增加(F=274.8,P＜0.01).依达拉奉预处理组相应时间点的细胞凋亡比率分别为0.90％±0.07％、1.10％±0.08％、3.50％±1.90％、12.60％±1.10％;与H2 02处理组比较明显降低(F=362.7,P＜0.01).刺激0.5h后,磷酸化c-Jun氨基端激酶(p-JNK)、细胞质中细胞色素C、线粒体膜上B细胞白血病-淋巴瘤-2相关X蛋白(BAX)表达水平开始明显升高,直至12 h时达到顶峰;与此同时,细胞质中B细胞白血病-淋巴瘤-2关联死亡蛋白(BAD)、磷酸化BAD、BAX和14-3-3水平表现为持续性降低,而这些改变在依达拉奉预处理组均被抑制.结论 作为一种自由基清除剂,依达拉奉可能通过抑制JNK的活性、抑制BAD与14-3-3分离及抑制BAX选择性地由细胞质向线粒体移位来保护神经细胞.     

银杏叶提取物对周围神经损伤后运动神经元的保护作用

目的　研究银杏叶提取物在大鼠坐骨神经损伤后对运动神经元的保护作用。方法　SD大鼠 3 6只 ,按手术先后随机平分为银杏叶提取物 (EGb 761)组和生理盐水 (SAL)组。大鼠均制成坐骨神经部分切除后结扎断端的模型 ,术后每天经腹腔分别注射EGb 761(10 0mg/kg-1·d-1)和同体积的生理盐水直到取材。术后 2、4、6周取材 ,大鼠处死后取L4～ 6节段脊髓 ,经酶组化染色后观察乙酰胆碱脂酶 (AChE)及酸性磷酸酶 (ACP)的活性变化 ;冰冻切片硫槿染色后对前角大型运动神经元计数 ;电镜观察脊髓前角运动神经元超微结构的变化。结果　与对照组相比 ,银杏叶提取物组的乙酰胆碱脂酶、酸性磷酸酶活性、损伤侧前角运动神经元数目及前角运动神经元超微结构 ,均有明显的改善。结论　银杏叶提取物对周围神经损伤后的脊髓前角运动神经元损害有保护作用     

不同浓度七氟醚预处理对大鼠海马神经元缺氧复氧时细胞凋亡的影响及线粒体ATP敏感型钾通道在其中的作用

目的 探讨不同浓度七氟醚预处理对大鼠海马神经元缺氧复氧时细胞凋亡的影响及线粒体ATP敏感型钾通道(mito-KATP通道)在其中的作用.方法 新生(出生＜24 h)SD大鼠,雌雄不拘,体重5～6 g,原代培养海马神经元,接种于培养孔或培养皿中,采用随机数字表法,将其随机分为7组,每组48孔和12皿,正常对照组(C组):不予任何处理;缺氧复氧组(HR组):缺氧4 h复氧24 h;6%七氟醚预处理组(S1 组)、4%七氟醚预处理组(S2 组)、2%七氟醚预处理组(S3 组):分别经6%、4%、2%七氟醚预处理后行缺氧复氧;5-羟葵酸100 μmol/L预处理组(5-HD组):经mito-KATP通道阻断剂5-羟葵酸(终浓度100 μmol/L)预处理后进行缺氧复氧;5-羟葵酸100 μmol/L+6%七氟醚预处理组(5-HD+S组):同时行5-羟葵酸和6%七氟醚预处理后进行缺氧复氧.各组以上处理结束后,测定神经元活力、凋亡率、Bcl-2和Bax蛋白的表达水平.结果 与C组比较,其余6组海马神经元活力降低,细胞凋亡率升高,Bcl-2和Bax蛋白表达上调(P＜0.01);与HR组比较,S1组～S3组海马神经元活力增强,细胞凋亡率降低,Bcl-2蛋白表达上调,Bax蛋白表达下调(P＜0.01),5-HD组和5-HD+S组上述指标比较差异无统计学意义(P＞0.05);与S1组比较,S2组、S3组和5-HD+S组海马神经元活力降低,细胞凋亡率升高,Bcl-2蛋白表达下调,Bax蛋白表达上调(P＜0.01);与S2组比较,S3组海马神经元活力降低,细胞凋亡率升高,Bcl-2蛋白表达下调,Bax蛋白表达上调(P＜0.01).结论 七氟醚预处理可抑制大鼠海马神经元缺氧复氧时细胞凋亡,从而减轻神经元损伤,且呈浓度依赖性,机制可能与开放神经元mito-KATP通道,上调Bcl-2蛋白表达,下调Bax蛋白表达有关.

Abstract：

Objective To investigate the effect of preconditioning with different concentrations of sevoflurane on hypoxia-reoxygenation(H/R)-induced apoptosis in rat hippocampal neurons and the role of mitochondrial KATP(mito-KATP)channels.Methods Primary cultured hippocampal neurons isolated from newborn SD rats(＜24h)of both sexes,weighing 5-6 g,were randomly divided into 7 groups with 48 wells and 12 dishes in each one:control group(C group),H/R group,preconditioning with 6%,4%and 2% sevoflurane groups(S1-3 groups),5-hydroxydecanoate(5-HD,mito-KATP channel blocker)100 μmol/L preconditioning group(5-HD group)and preconditioning with 5-HD 100 μmol/L+6% sevoflurane group(5-HD+S group).The neurons were exposed to 4 h hypoxia followed by 24 h reoxygenation. In S1-3 groups, preconditioning was performed with 6% , 4% and 2% sevoflurane respectively before H/R. In 5-HD group, preconditioning was performed with 5-HD (final concentration 100 μmol/L) before H/R. In 5-HD + S group, preconditioning was performed with 5-HD 100 μmol/L and 6% sevoflurane before H/R. The neuronal viability, apoptosis rate and expression of Bcl-2 and Bax were determined after 24 h reoxygenation.Results The neuronal viability was significantly lower,while the apoptosis rate and expression of Bcl-2 and Bax were significantly higher in the other 6 groups than in group C(P＜0.01).The neuronal viability and expression of Bcl-2 were significantly higher,while the apoptosis rate and Bax expression were lower in S1-3 groups than in group H/R. There was no significant difference in the parameters mentioned above between 5-HD and 5-HD + S groups(P＞0.05).The neuronal viability and expression of Bcl-2 were significantly lower, while the apoptosis rate and Bax expression were higher in S2, S3 and 5-HD + S groups than in group S1, and in group S3 than in group S2(P＜0.0l) .Conclusion Sevoflurane preconditioning can inhibit H/R-induced apoptosis in rat hippocampal neurons and reduce the injury to neurons in a concentration-dependent manner, and the underlying mechanism may be related to activation of mito-KATP channels, up-regulation of Bcl-2 expression and down-regulation of Bax expression.     

氯胺酮对海马神经细胞大电导钙激活钾通道的作用

目的:研究氯胺酮对海马神经细胞大电导钙激活钾(BK)通道的作用。方法:将培养的神经元置于对称性高钾溶液中,应用膜片钳技术,记录其单通道电流。用循环灌流的方法,观察了不同浓度氯胺酮对BK通道的作用。结果:(1)应用内面向外式膜片,该通道电导为170pS左右,随着胞内钙离子浓度的增加,通道开放概率明显增加(P<0.05),当膜内侧加入钾通道阻断剂(TEA)后,通道活动被阻断;(2)应用细胞贴附式膜片,当浴液内氯胺酮浓度为0.01～0.20mmol/L时,通道开放概率及电流幅值均降低(P<0.05);当其浓度为0.50～1.00mmol/L时,通道开放概率增加(P<0.05),而电流幅值与加药前相比无明显变化(P<0.05)。结论:氯胺酮对BK通道具有双向作用,其抑制作用可能是发挥麻醉效用时产生不良反应的原因之一,而其激活作用可能与全麻作用的分子机制有关。     

Magnetic resonance microscopy of mammalian neurons

Magnetic resonance imaging (MRI) is now a leading diagnostic technique. As technology has improved, so has the spatial resolution achievable. In 1986 MR microscopy (MRM) was demonstrated with resolutions in the tens of micrometers, and is now an established subset of MRI with broad utility in biological and non-biological applications. To date, only large cells from plants or aquatic animals have been imaged with MRM limiting its applicability. Using newly developed microsurface coils and an improved slice preparation technique for correlative histology, we report here for the first time direct visualization of single neurons in the mammalian central nervous system (CNS) using native MR signal at a resolution of 4–8 μm. Thus MRM has matured into a viable complementary cellular imaging technique in mammalian tissues.