抗氧化剂TA9901抑制加速老化鼠脑淀粉样颗粒的沉积

目的　观察抗氧化剂TA990 1对加速老化鼠P 8(SAM P 8)脑中淀粉样颗粒 (MSG)沉积的影响 ,为临床治疗阿尔采默病 (AD)提供科学依据。　方法　 2 5月龄雄性SAM P 8小鼠随机分为对照组、TA1组、TA2组和VE组 ,分别给予正常饮食、0 0 5 %TA990 1、0 5 %TA990 1和特制VitaminE(VE)饲料 (5 0 0mgVE kg)喂养 3 5个月。用Go mori六胺银法显示淀粉样颗粒 ,对海马CA1区的银染颗粒 (MSG)的体视学参数和分布进行图像分析。　结果　MSG为圆形和卵圆形 ,主要分布于海马、皮质深层和小脑 ;海马内MSG主要成群分布于各层。各组海马CA1区MSG颗粒数比较 ,差异均无显著性 (P >0 0 5 ) ,但TA1组、TA2组和VE组的MSG颗粒直径和面积均较对照组明显减小 (P <0 0 5 ) ,以TA2和VE组更为显著 (P <0 0 1) ;各实验组MSG的平均灰度均较对照组降低 (P <0 0 1) ;对照、TA1、TA2和VE组的低密度颗粒区分别占 12 0 5 %、36 5 2 %、4 7 91%和 4 4 6 5 % ,实验组在低密度区的颗粒数明显增多。　结论　TA990 1减少了SAM P 8脑中淀粉样颗粒的大小和密度 ,显示TA990 1对脑内淀粉样颗粒沉积有抑制作用     

抗氧化剂TA9901对加速老化鼠(SAM-P/8)行为学的影响

目的　观察抗氧化剂TA990 1对实验动物学习记忆功能的影响 ,为其临床应用提供实验依据。方法　用加速老化鼠 (SAM) P/ 8小鼠 2 0只随机分为对照组、TA1组 (0 0 5 %TA990 1)、TA2组 (0 5 %TA990 1)、和VE组 (5 0 0mgVE/kg饲料 ) ,分别进行Morris水迷宫行为学测试 ,观察TA990 1对SAM P/ 8小鼠的作用。结果　对照组平均逃避潜伏期是 (6 2 78± 8 75 )sec ,TA1组、TA2组和VE组的逃避潜伏期分别为 (5 3 94± 5 79)、(36 38± 4 77)和 (40 2 2± 5 5 1)sec。与对照组相比 ,TA2组和VE组逃避潜伏期减少均具有显著性意义 (P <0 0 5 ) ,TA1组和TA2组比较也有显著性差异 (P <0 0 5 )。空间探索试验结果以平台象限的游泳距离占总的游泳距离的百分比判断动物的空间学习记忆能力 ,对照组、TA1组、TA2组和VE组小鼠的平台象限游泳距离所占的百分比分别为 (11 33± 2 5 1)、(15 0 9± 4 0 2 )、(2 4 35± 7 89)和 (2 0 93± 5 0 2 ) % ,对照组与TA2组和VE组相比 ,差异均具有显著性 (P <0 0 5 ) ,TA1组与TA2组之间差异也有显著性 (P <0 0 5 )。结论　TA990 1能显著改善SAM P/ 8的学习记忆能力 ,初步显示TA990 1有改善学习记忆功能及抗痴呆的药理作用。     

银杏叶提取物改善AD模型鼠学习记忆功能的作用

目的 :研究银杏叶提取物 (EGb)对早老性痴呆 (AD)模型鼠学习记忆等认知功能的影响和对基底前脑隔区胆碱能神经元的保护作用。方法 :大鼠随机分成预防组、治疗组和对照组。各组切割右侧海马伞后分别用EGb(预防组及治疗组 )和蒸馏水 (对照组 )灌胃连续 6周。其中预防组在手术前先用EGb灌胃 2周。各组在术后次日及灌胃 6周后进行行为学检测。脑切片经Nissl、NOS组织化学和ChAT免疫组织化学染色 ,观察损伤侧内侧隔核和斜角带垂直支中NOS和ChAT阳性神经元的变化 ,并对ChAT阳性神经元作图像分析。数据分别用方差分析、SNK检验及 χ2 检验作统计学处理。结果 :行为学检测证实 ,EGb用药组AD模式鼠的学习记忆等认知功能较对照组明显改善 ,其中预防组效果又较治疗组为好 ;形态学检测证实 ,EGb用药组AD模式鼠损伤侧内侧隔核和斜角带垂直支中NOS、ChAT阳性神经元数目及ChAT阳性神经元的面积和周径等均优于对照组 ,预防组结果又优于治疗组。结论 :EGb对AD模型鼠学习记忆等认知功能障碍有一定的改善作用 ,这一作用是通过保护基底前脑隔区胆碱能神经元而实现的 ,其中预防效果比治疗效果更优     

雌激素对去卵巢大鼠基底前脑胆碱能神经元及一氧化氮合酶阳性神经元的影响

目的　探讨雌激素补充治疗对去卵巢大鼠胆碱能神经元及一氧化氮合酶 (NOS)阳性神经元表达的影响及剂量效应关系。方法　 2 0只去卵巢SD大鼠分成 4个不同剂量组 :0 μg(对照组 )、2 0 μg(0 0 8mg/kg)、5 0 μg(0 2 0mg/kg)、10 0 μg(0 4 0mg/kg)组 ;1周后 ,用乙酰胆碱转移酶 (ChAT)免疫化学方法及尼克酰胺腺嘌呤二核苷酸黄递酶 (NADPH d)组织化学方法研究。结果　NOS阳性神经元在内侧隔核 (MS) ,其数目在各组间无明显差异 (P >0 0 5 )。在斜角带垂直支 (VDB) ,5 0 μg剂量组与对照组相比有明显差异 (P <0 0 5 ) ;ChAT阳性神经元在内侧隔核 (MS) ,其 2 0 μg、5 0 μg剂量组数目出现剂量递增效应 ,与对照组比较有明显差异 (P <0 0 5 ) ,但 10 0 μg剂量组与对照组比较没有明显差异 (P >0 0 5 ) ,同时 5 0 μg与 10 0 μg剂量组比较有明显差异 (P <0 0 5 )。ChAT阳性神经元在斜角带水平支 (HDB) ,各组间均无明显差异 (P >0 0 5 )。结论　雌激素补充治疗能选择性影响基底前脑各亚区NOS和胆碱能神经元 ,并有可能影响学习和记忆能力。     

天然抗氧化剂减少神经根撕脱诱导的一氧化氮合酶表达和运动神经元死亡

为了研究抗老年性痴呆 990 1号和 TA990 1+银杏叶标准提取物合剂对臂丛神经根撕脱后前角运动神经元的神经元型一氧化氮合酶 ( n NOS)表达和存活的影响 ,用成年 SD雌性大鼠进行右侧臂丛 C5～ T1 神经根撕脱术后 ,随机分为撕脱组、撕脱 +TA990 1组以及撕脱 +TA990 1和 EGb761合剂组。对此三组每天分别进行腹腔注射 1ml生理盐水、0 .5 % TA990 1、0 .5 %TA990 1和 EGb761合剂。治疗 5 d、1、2、4和 6周后处死动物并进行 NADPH-d染色及中性红复染。定量比较各组 n NOS阳性和存活的运动神经元数量。结果证明 ,神经根撕脱后 5 d受损运动神经元开始表达 n NOS,2周时达高峰 ,随后下降至 6周。受损运动神经元的死亡从 2周开始 ,4～ 6周最明显。抗氧化剂治疗组 n NOS表达规律与对照组相似 ,但是阳性程度和阳性细胞数量均少于对照组 ,存活的运动神经元数量则明显多于对照组。撕脱后 4～ 6周 ,TA990 1治疗组抑制 n NOS表达和提高运动神经元存活的效果均明显优于 TA990 1和 EGb761合剂组。结论 :天然抗氧化剂可有效地减少臂丛神经根撕脱后 n NOS表达 ,提高受损运动神经元的存活。 TA990 1抑制 n NOS表达的作用强于 EGb761。     

γ-干扰素对大鼠胚胎基底前脑及隔区核团胆碱能神经元分化的影响

为了研究γ-干扰素(IFNγ)对大鼠胚胎基底前脑及隔区核团胆碱能神经元分化的作用,采用免疫组织化学方法对胆碱能神经元的特异性标记酶-胆碱乙酰基转移酶(ChAT)进行染色,ChAT阳性细胞的数量反映了胆碱能神经元的数量,并用14C-乙酰CoA作底物来检测ChAT活性。结果显示,IFNγ处理过的实验组,ChAT阳性细胞数量显著增加,ChAT活性也增加,这种增加被大鼠抗小鼠IFNγ单克隆抗体(Ab-IFNγ)完全拮抗。采用流式细胞术对细胞周期进行分析,细胞周期及细胞百分率无明显改变。用MAP2标记神经细胞,神经细胞数基本未增加。以上结果提示:IFNγ不能促进基底前脑和隔区神经元增殖,胆碱能神经元表达增加不是因为神经元数目增加而是分化的结果。     

艾司西酞普兰对APP/PS1小鼠海马神经元突触功能及对BDNF-TrkB-CREB信号通路的影响

目的探讨艾司西酞普兰对APP/PS-1小鼠海马神经元突触功能及与BDNF-TrkB-CREB信号通路的关系。方法将7月龄APP/PS-1转基因小鼠随机分为模型组及艾司西酞普兰组,每组10只;另取10只同龄C57BL/6小鼠作为阴性对照组。给予10 mg/kg艾司西酞普兰灌胃治疗,连续8周,每天一次;对照组及模型组给予等体积生理盐水。Morris水迷宫检测各组小鼠学习记忆能力;尼式染色检测各组小鼠海马DG区尼氏体变化;酶联免疫吸附实验(ELISA)检测小鼠海马区乙酰胆碱酯酶(AChE)及胆碱乙酰转移酶(ChAT);Western blot检测小鼠海马区SYN、PSD95、GAP43、BDNF、TrkB、p-TrkB、CREB及p-CREB蛋白表达。结果艾司西酞普兰组改善APP/PS-1小鼠认知功能,小鼠逃避潜伏期减少,穿越平台次数增加,在目标象限停留时间延长,小鼠海马DG区神经元排列整齐且尼氏体数量增加,下调AChE,上调ChAT表达,且能增加SYN、PSD95及GAP43蛋白表达;此外,艾司西酞普兰还能显著增加小鼠海马区BDNF、p-TrkB及p-CREB蛋白表达,与模型组相比,差异有统计学意义(P0.05)。结论艾司西酞普兰改善小鼠海马神经元突触功能,提高APP/PS-1小鼠的学习记忆能力,可能与调控BDNF-TrkBCREB信号通路相关蛋白表达有关。     

NGF/GDNF基因修饰神经干细胞移植对AD模型鼠前脑胆碱能神经元的保护作用

目的　观察NGF GDNF基因修饰神经干细胞 (NSC)单独和联合移植对穹窿海马伞 (FF)切断后胆碱能神经元的保护作用。　方法　将两种基因修饰的NSC单独和联合移植入FF切断的大鼠侧脑室。移植后 3周取脑行ChAT免疫组织化学染色 ,用Student NewmanKaels方法对内侧隔核 (MS)和斜角带垂直支 (VDB)的ChAT阳性细胞数 (损伤侧与正常侧的百分比 )进行计数分析。　结果　NGF组伤侧MS的ChAT阳性神经元数为 81% ,明显高于损伤组 (34% )、NSC组 (36 % )和GDNF组 (5 0 % ) ,P <0 0 1;NGF GDNF组为 6 8% ,明显高于损伤组和NSC组 (P <0 0 1) ,亦高于GDNF组 (P <0 0 5 ) ;GDNF组与损伤组和NSC组相比 ,有统计学意义 (P <0 0 5 )。NGF组和NGF GD NF组伤侧VDB的ChAT阳性神经元数分别为 80 %和 72 % ,明显高于损伤组 (5 6 % )和NSC组 (5 2 % ) ,P <0 0 1,亦高于GDNF组 (5 9% ) ,P <0 0 5 ;GDNF组与损害组和NSC组相比无明显差异 (P >0 0 5 )。　结论　NGF GDNF基因修饰NSC单独和联合移植均能不同程度地保护损伤的ChAT阳性神经元 ,NGF组和NGF GDNF组作用较大 ,GDNF组作用较小。     

神经生长因子诱导神经干细胞向胆碱能神经元的分化

为了探讨神经生长因子(NGF)是否具有诱导神经干细胞(NSCs)向胆碱能神经元分化的能力,利用无血清培养技术从胎鼠脑内获得神经干细胞,传代纯化后利用免疫细胞化学技术,观察不同剂量NGF作用后神经干细胞向胆碱乙酰转移酶(ChAT)阳性细胞分化的情况。结果发现:50、100、200ng/ml NGF组ChAT阳性细胞数明显比对照组增加,且以100ng/ml组最为明显;各NGF组,分化的细胞状态较好,且突起明显比对照组增粗增长,以200ng/ml组最为明显。此结果证明NGF具有诱导NSCs向胆碱能神经元分化的趋势,且能促进分化细胞突起的生长。     

大鼠多重脑震荡后多巴胺能与胆碱能神经元的变化

目的:观察大鼠多重脑震荡(MCC)后中枢神经系统内多巴胺能神经纤维与胆碱能神经元的变化。方法:用金属单摆打击装置复制MCC大鼠模型,随机分为对照组和伤后1、2、4、8、16、24 d多个损伤组,用免疫组织化学双标技术检测大鼠脑内多巴胺能神经纤维中酪氨酸羟化酶(TH)和胆碱能神经元中胆碱乙酰转移酶(ChAT)的表达变化。结果:TH阳性表达在伤后1 d开始上升,8 d达高峰,24 d组基本恢复;ChAT阳性表达在MCC后均呈现下调改变。结论:多重脑震荡后多巴胺能神经纤维和胆碱能神经元的变化可能与中枢神经系统的认知功能障碍有关。     

圆二色谱分析抗氧化剂EGb761配伍TA9901对老化Aβ_(1-42)二级结构改变的影响

目的　研究抗氧化剂EGb76 1、TA990 1及EGb76 1配伍TA990 1抑制Aβ1-42 聚集作用的分子机制。方法　运用圆二色谱 (CD)分析Aβ1-42 老化后以及其与EGb76 1、TA990 1和二者配伍物共同孵育后二级结构变化。结果　Aβ1-42 孵育 30min和 2 4h及用EGb76 1、TA990 1和其配伍物干预后 ,Aβ1-42 的CD图形发生了明显改变 ,30min以不规则卷曲和α 螺旋为主 ,其含量分别为 4 0 5 %和 2 5 4 % ;孵育 2 4h后以 β 折叠为主 ,占 4 0 3% ;TA990 1、EGb76 1和配伍物分别与Aβ1-42 共同作用后 ,则α 螺旋结构增多 ,分别为 37 6 %、4 5 3%和 10 0 % ,β 折叠消失。 结论　EGb76 1配伍TA990 1及其成分均能促使老化的Aβ1-42 由 β 折叠向α 螺旋转化 ,以配伍物的作用最强     

TA9901配伍EGb761对神经根撕脱后脊髓运动神经元的影响

目的　观察植物抗氧化剂TA990 1配伍EGb76 1对臂丛神经根撕脱后C7前角运动神经元c jun、NOS表达和存活的影响。方法　成年SD雌性大鼠 12 0只 ,随机分为撕脱组和治疗组。行C5～T1神经根撕脱术后 ,两组动物每天分别给予腹腔注射 1ml生理盐水或 0 5 % (TA990 1配伍EGb76 1)溶液。治疗后 4h、12h、1d、3d、5d、1周、2周、4周和 6周处死动物 ,行c jun免疫细胞化学、NADPH d组织化学和中性红复染。比较两组动物c jun阳性、NOS阳性和存活的运动神经元的数目。结果　臂丛神经根撒脱 4h后c jun开始表达 ,1d达高峰 ,随后下降至 6周。nNOS表达从 5d开始 ,2周时达高峰 ,以后逐渐下降至 6周。运动神经元的死亡开始于 2周 ,以 4周和 6周最明显。TA990 1配伍EGb76 1提高c jun表达的效果随治疗时间延长而增强、其抑制NOS表达作用则以 2周点最强。结论　TA990 1配伍EGb76 1能增强神经根撕脱后运动神经元c jun的反应 ,抑制NOS的表达 ,提高受损运动神经元的再生能力     

Effects of Ginkgo biloba extract (EGb 761) on hydroxyl radical-induced thymocyte apoptosis and on age-related thymic atrophy and peripheral immune dysfunctions in mice

In this study, the effect of EGb 761, a standard extract of Ginkgo biloba leaf, on thymocyte apoptosis and age-related thymic atrophy and on peripheral immune dysfunctions was investigated in mice. When primary culture of thymocytes was preincubated with 100 microg/ml EGb 761 before their exposure to hydroxyl radicals (*OH) generated by Fe(2+)-mediated Fenton reaction, apoptotic cell death induced by *OH was distinctly prevented as determined by DNA laddering, the TUNEL assay and flow cytometric analysis. Furthermore, oral EGb 761 administration (about 1.5 mg/day/mouse) for 60 consecutive days led to a significant thymic regrowth in 22-month-old mice as revealed by the increment of thymus weight and total numbers of thymocytes. Partial recovery of peripheral immune capacities such as mitogen responsiveness and NK cell activity were also found in the old mice after 60 days of EGb 761 supplementation. Taken together, our study indicates that in addition to its protective and rescuing abilities on neurodegenerative disorders and cardiovascular diseases, EGb 761 was also found active in the rejuvenation of degenerated thymus and accordingly the strengthening of the immune system. These beneficial effects of EGb 761 on immune system are based on its antioxidant properties as well as the cell proliferation-stimulating effect.     

Age-related spatial cognitive impairment is correlated with a decrease in ChAT in the cerebral cortex,hippocampus and forebrain of SAMP8 mice

At present, the mechanisms underlying cognitive disorders remain unclear. The senescence-accelerated mice (SAM) prone/8 (P8) has been proposed as a useful model for the study of aging, and SAM resistant/1 (R1) is its control as a normal aging strain. The purpose of this study was to investigate choline acetyltransferase (ChAT) expression in SAM brain. The age-related decline of learning and memory ability in P8 mice (4, 8 and 12 months old, n = 10 for each group) was proved in Morris water maze test (MWM). After the behavioral test, protein and mRNA levels of ChAT were determined in the cerebral cortex, hippocampus and forebrain by means of immunostaining, Western blotting, and real time quantitative PCR (QPCR). Comparing with 4-month-old P8 and R1, 8- and 12-month-old P8 showed age-related cognitive impairment in MWM test. The latencies of the 4-month-old P8 in a hidden platform trial were significantly shorter, and the retention time was significantly longer than that of the older P8 groups. In addition, significantly low level of ChAT protein was observed in older P8 groups. Comparing with the 4-month-old P8, ChAT mRNA in the 12-month-old P8 declined significantly in all three regions of P8 brain. Pearson correlation test showed that the latencies in the MWM were positively correlated with the level of ChAT in P8. Such phenomenon could not be detected in normal aging R1 mice. These findings suggest that the decrease of ChAT in P8 mice was responsible for the age-related learning and memory impairments in some sense.     

银杏叶提取物对拟血管性痴呆大鼠海马CA1区的保护作用

为了观察银杏叶提取物(EGb761)对拟血管性痴呆(vascular dementia,VD)大鼠海马CA1区神经细胞的保护作用,本研究通过反复夹闭再通大鼠双侧颈总动脉,同时腹膜腔内注射硝普钠制作拟血管性痴呆大鼠模型,选出造模成功者随机分为模型组及用药组,各为30只。另以条件匹配的30只大鼠为假手术组。采用Morris水迷宫和尼氏染色方法分别观察大鼠空间学习记忆能力及海马CA1区细胞形态学改变和细胞数目。结果显示:模型组大鼠在1、2和4月不同时间点测得的Morris水迷宫逃避潜伏期(EL)均较假手术组明显延长(P<0.01),药物组EL均显著短于模型组,但仍长于假手术组(P<0.05或P<0.01);各时间点模型组海马CA1区锥体细胞及其树突丢失,但药物组锥体细胞数多于模型组。上述结果说明EGb761对海马CA1区的保护作用可能是其改善VD大鼠学习记忆障碍的重要机制。     

不同年龄大鼠斜角带核水平支神经元内TrkA和ChAT的表达——免疫组织化学研究

目的 了解大鼠基底前脑斜角带核水平支神经元内酪氨酸激酶Ａ（ｔｙｒｏｓｉｎｅ ｋｉｎａｓｅ,Ａ,ＴｒｋＡ）、胆碱乙酰化转移酶（ｃｈｏｌｉｎｅ ａｃｅｔｙｌｔｒａｎｓｆｅｒａｓｅ,ＣｈＡＴ）样阳性神经元的生后发育规律及两乾的相互关系。方法 用免疫组织化学方法结合图像分析仪检测大鼠基底前脑斜角带核水平支ＴｒｋＡ、ＣｈＡＴ样阳性神经元的数量、面积和灰度值。结果 ＴｒｋＡ、ＣｈＡＴ分布于基底前脑神经元。生后１ｄ可见ＴｒｋＡ表达,但生后５ｄ才出现ＣｈＡＴ表达。生后２０ｄＴｒｋＡ、ＣｈＡＴ表达至高峰;生后３０ｄ下调,成年时维持相对较高水平。老年鼠ＴｒｋＡ、ＣｈＡＴ样阳性神经元出现萎缩、数量也分别减少３９．７％、３３．３％;胞体平均面积分别减少１５．７％、１２．８％;平均灰度值分别减少２９．９％、９．９％。同时,不同年龄大鼠Ｔ     

Distribution and role of Kv3.1b in neurons in the medial septum diagonal band complex

The medial septum diagonal band complex (MS/DB) projects via cholinergic and GABAergic pathways to the hippocampus and plays a key role in the hippocampal theta rhythm. In the MS/DB we have previously described a population of fast spiking GABAergic neurons that contain parvalbumin and mediate theta frequency activity in vitro. The Kv3.1 potassium channel is a delayed rectifier channel that plays a major role in fast spiking neurons in the CNS, and has previously been localized in the MS/DB. To determine which cell types in the MS/DB express the Kv3.1b ion channel subunit, transgenic mice in which the expression of GABAergic and glutamate markers are associated with the expression of green fluorescent protein (GFP; GAD67-GFP and VGluT2-GFP mice, respectively) were used for immunofluorescence and axonal tract tracing. Electrophysiological studies were also carried out on rat MS/DB slices to examine the role of the Kv3.1 channel in theta frequency oscillations. The results for the MS/DB were as follows: (1) cholinergic cells did not express GFP in either GAD67-GFP or VGluT2-GFP mice, and there was GAD67 immunoreactivity in GFP-positive neurons in GAD67-GFP mice and in a small proportion (6%) of GFP-positive neurons in VGluT2-GFP mice. (2) Kv3.1b immunofluorescence was associated with the somata of GABAergic neurons, especially those that contained parvalbumin, and with a minority of glutamatergic neurons, but not with cholinergic neurons, and with GABAergic axonal terminal-like processes around certain GABAergic neurons. (3) Both Kv3.1b-positive and -negative GABAergic neurons were septo-hippocampal, and there was a minor projection to hippocampus from VGluT2-GFP neurons. (4) Kainate-induced theta oscillations in the MS/DB slice were potentiated rather than inhibited by the Kv3.1 blocker 4-aminopyridine, and this agent on its own produced theta frequency oscillations in MS/DB slices that were reduced by ionotropic glutamate and GABA receptor antagonists and abolished by low extracellular calcium. These studies confirm the presence of heterogeneous populations of septo-hippocampal neurons in the MS/DB, and suggest that presence of Kv3.1 in the GABAergic neurons does not contribute to theta activity through fast spiking properties, but possibly by the regulation of transmitter release from axonal terminals.