高血糖对大鼠局灶性脑缺血再灌注后神经细胞凋亡及caspase-9、caspase-3表达的影响

目的探讨高血糖对大鼠局灶性脑缺血再灌注后神经细胞凋亡及半胱氨酸天冬氨酸蛋白酶-9(caspase-9)、caspase-3表达的影响,进一步探讨其加重脑缺血损伤的作用机制。方法将30只雄性Sprague-Dawley(SD)大鼠随机分为3组:高血糖组、正常血糖组和假手术组。按体质量4g/kg给予大鼠尾静脉注射25%(质量浓度)的葡萄糖,造成大鼠高血糖状态;继而制作大脑中动脉阻塞脑缺血再灌注模型。于缺血2h再灌注24h进行神经功能评分,采用脱氧核糖核苷酸末端转移酶介导的dUTP缺口末端标记法(terminal-deoxy-nucleotidyl transferase mediated dUTP nick end labeling,TUNEL)检测大鼠脑组织细胞凋亡数,采用免疫组化染色检测大鼠脑组织caspase-9和caspase-3表达。结果 (1)高血糖组神经功能评分为(4.20±0.63)分,正常血糖组为(3.50±0.70)分,假手术组为0分。高血糖组及正常血糖组的神经功能评分较假手术组均显著增加(P<0.05),且高血糖组神经功能评分较正常血糖组增加(P<0.05)。(2)高血糖组凋亡细胞数为(16.30±3.30)个,正常血糖组为(14.60±3.27)个,假手术组为(0.50±0.53)个。高血糖组及正常血糖组的凋亡细胞数较假手术组增多(P<0.05),且高血糖组细胞凋亡数较正常血糖组增多(P<0.05)。(3)高血糖组caspase-9和caspase-3灰度值分别为114.30±3.83、111.50±3.17,正常血糖组分别为117.20±4.34、117.40±3.24,假手术组分别为146.20±1.98、146.80±0.74。高血糖组及正常血糖组的caspase-9和caspase-3表达水平较假手术组均增强(P<0.05),且高血糖组caspase-9和caspase-3表达水平较正常血糖组增强(P<0.05)。结论高血糖可增加缺血再灌注后脑神经细胞凋亡;caspase-9、caspase-3的激活及表达增强可能是高血糖加重脑缺血再灌注损伤的机制之一。     

替普瑞酮诱导阿尔茨海默病大鼠海马热休克蛋白70表达及抗凋亡机制

目的研究替普瑞酮(Geranylgeranylacetone,GGA)诱导阿尔茨海默病(Alzheimer's disease,AD)模型大鼠海马热休克蛋白70(heat shock protein 70,HSP70)的表达及其抗细胞凋亡机制。方法 36只SD大鼠随机分为对照组、模型组与GGA组(n=12)。双侧海马注射β淀粉样蛋白1-42(β-amyloid1-42,Aβ1-42)建立AD大鼠模型。GGA组大鼠予GGA800mg/(kg·d)灌胃,持续21d。Y迷宫测试大鼠行为学变化;HSP70-TUNEL免疫荧光双标染色观察大鼠海马细胞凋亡与HSP70表达变化、分布情况;Western-blot测定大鼠海马HSP70及凋亡相关因子caspase-9、cytochrome C、FADD、caspase-8蛋白表达。结果术后模型组大鼠获得记忆所需电击次数(14d:21.1±5.6;21d:27.4±5.2),对照组(14d:15.9±3.8;21d:14.7±3.9),GGA组(14d:17.3±2.7;21d:20.8±4.6)。术后21d海马HSP70表达:模型组(0.22±0.04),对照组(0.43±0.07),GGA组:(0.71±0.15);凋亡细胞:模型组(38.4±6.8),对照组(9.7±3.8);GGA组(16.7±4.3);caspase-9表达:模型组(0.69±0.09),对照组(0.14±0.04),GGA组(0.23±0.06)。FADD表达GGA组与模型组无统计学差异。结论 GGA诱导AD大鼠海马HSP70表达上调,可能通过抑制线粒体凋亡途径的激活及部分死亡受体凋亡途径的激活而发挥抗凋亡作用,减轻神经元损伤,从而改善AD大鼠的学习记忆能力。     

蛋白酶体抑制剂MG132对动脉粥样硬化的影响

目的观察蛋白酶体抑制剂MG132对动脉粥样硬化的影响。方法将新西兰白兔30只随机分成高脂组、MG132(1)组和MG132(2)组。3组兔普通饲料喂养1w后行颈动脉球囊损伤术,术后高脂饲料(含1%胆固醇、3%猪油和15%蛋黄)喂养;MG132(1)组在术后高脂饲料喂养的同时血管局部应用蛋白酶体抑制剂MG132;MG132(2)组在术后高脂饲料喂养4w后血管局部应用蛋白酶体抑制剂MG132;3组均喂养8w后取颈总动脉血管制成病理切片行HE染色。结果高脂组兔的右颈总动脉管壁呈动脉粥样硬化改变。MG132(1)组兔右颈动脉血管组织结构基本正常。MG132(2)组兔的右颈总动脉管腔介于二者之间。结论局部应用蛋白酶体抑制剂MG132能够抑制血管内膜增生及动脉粥样形成,同时可能具有稳定斑块的作用。     

细胞凋亡在热打击致大鼠大脑皮质神经元损害中的作用及机制

目的探讨热打击后大鼠大脑皮质神经元凋亡及caspase-3的表达变化。方法将Wistar大鼠随机分为正常对照组和热打击组,正常对照组大鼠始终置于(25±0.5)℃,相对湿度(35±5)%环境下;热打击组置于人工智能气候舱内100 min,舱内温度(40±0.5)℃,相对湿度(60±5)%。TUNEL染色观察大鼠大脑皮质神经元的凋亡情况;免疫组织化学方法及荧光定量PCR检测大鼠大脑皮质神经元caspase-3的表达。结果热打击1 d后大脑皮质神经元出现凋亡,第3天达高峰。caspase-3于热打击2 d后开始表达,于第3天表达最明显。荧光定量PCR检测结果显示,caspase-3在1 d、2 d、3 d和7 d各时间点含量与正常对照组比较差异均有统计学意义(P0.05),3 d时caspase-3含量最高。结论热打击可激活caspase-3表达,诱导皮质神经元凋亡。     

地佐辛后处理对大鼠局灶性脑缺血/再灌注损伤影响

目的评价地佐辛后处理对大鼠局灶性脑缺血/再灌注损伤的作用及可能机制。方法健康雄性SD大鼠72只,随机分为4组(n=18):假手术组(S组)、缺血/再灌注损伤组(IR组)、地佐辛后给药组(D组)和5-羟基葵酸钠组(5-HD组)。采用线栓法阻断大脑中动脉制备大鼠局灶性脑缺血/再灌注损伤模型。再灌注24 h末,行神经功能缺陷评分和脑梗死体积测定;取血测定血浆SOD活性与皮质醇水平;TUNEL法检测细胞的凋亡;Western blot测定缺血侧皮质caspase-3表达。结果与S组比较,D组、IR组和5-HD组皮质醇水平、神经功能缺陷评分升高,凋亡神经细胞、脑梗死体积增加,SOD活性下降,caspase-3表达上调(P<0.05);与IR组和5-HD组比较,D组神经功能缺陷评分、皮质醇水平降低,SOD活性升高,脑梗死体积、凋亡神经元减少(P<0.05),caspase-3表达下调;IR组与5-HD组比较无统计学意义(P>0.05)。结论地佐辛后给药可减少局灶性脑缺血/再灌注损伤大鼠神经元凋亡,机制可能与线粒体ATP敏感性钾通道激活及下调caspase-3表达相关。     

自噬和蛋白酶体降解途径在突变型α-突触核蛋白PC12细胞凋亡中的作用

目的 利用建立好的转染A53T突变型α-突触核蛋白的大鼠嗜铬细胞瘤(PC12)细胞株,探讨自噬和泛素-蛋白酶体通路在细胞凋亡途径中的具体作用.方法 选择特异性蛋白酶体抑制剂和大自噬抑制剂及诱导剂作用于稳定转染的A53T细胞株,四甲基偶氮唑盐法和流式细胞仪检测细胞活力和各组细胞凋亡率,电镜观察超微结构,并测定细胞培养液中NO的活力和热休克蛋白70(Hsp70)及Caspase-3蛋白表达.结果 蛋白酶体抑制剂环氧霉素(100 nmol/L)和(或)大自噬抑制剂3-MA(10 mmol/L)处理A53T细胞24 h后环氧霉素组、3-MA组和环氧霉素+3-MA组细胞活力(A值分别为0.23±0.01、0.19±0.01、0.17±0.01)较对照A53T细胞组(A值为0.32±0.06)明显下降(P<0.05);而大自噬诱导剂雷帕霉素(0.2 μg/ml)处理后细胞存活率(A值为0.44±0.08)显著高于其余用药组.与对照组(1.55%±1.15%)相比,3-MA、环氧霉素、环氧霉素+3-MA组作用24 h后A53T细胞的凋亡百分率(分别为4.74%±0.91%、4.59%±1.18%、5.40%±1.75%)显著增高(P<0.05),而大自噬诱导剂下调蛋白酶体抑制剂导致的凋亡;蛋白酶体抑制剂组NO和Hsp70蛋白含量也明显高于对照组.结论 大自噬和蛋白酶体途径障碍促进了凋亡发生,抑制或诱导自噬对Hsp70和NO的影响不如蛋白酶体途径对其影响大.     

烟酰胺单核苷酸腺苷酰转移酶1对慢性帕金森病小鼠模型黑质中酪氨酸羟化酶及半胱氨酸天冬氨酸蛋白水解酶3表达的影响

目的探讨烟酰胺单核苷酸腺苷酰转移酶1(NMNAT1)对慢性帕金森病(PD)小鼠模型(C57BL/6小鼠)黑质中酪氨酸羟化酶(TH)及半胱氨酸天冬氨酸蛋白水解酶3(caspase-3)表达的影响。方法利用重组慢病毒转染1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)制备慢性PD小鼠模型。依据不同处理方式分为3组:对照组[经立体定位仪向小鼠双侧黑质注射空白对照重组慢病毒(LV-GFP),1周后腹腔注射生理盐水];MPTP组(经立体定位仪向小鼠双侧黑质注射LVGFP,1周后建立MPTP慢性PD模型);NMNAT1组[经立体定位仪向小鼠双侧黑质注射NMNAT1基因的过表达重组慢病毒(LV-NMNAT1),1周后建立MPTP慢性PD模型]。采用Western blot法检测3组小鼠黑质中TH及caspase-3半定量表达水平。结果 NMNAT1呈高表达的NMNAT1组黑质中TH蛋白表达量较MPTP组高(t=7.985,P0.001),caspase-3蛋白表达量较MPTP组低(t=8.901,P0.001)。结论 NMNAT1能增加TH表达发挥一定的神经保护作用,并通过减少caspase-3表达水平参与抗细胞凋亡。     

凝血酶预处理对脑出血大鼠神经细胞凋亡及半胱氨酸蛋白酶-3表达的影响

目的 探讨凝血酶预处理(TPC)对脑出血(ICH)大鼠神经细胞凋亡及半胱氨酸蛋白酶-3(caspase-3)表达的影响.方法 60只成年雄性SD大鼠随机平均分为10组:假手术(SO)组、TPC 3 h组、TPC6 h组、TPC 12 h组、TPC 1 d组、TPC 3 d组、TPC 7 d组、TPC 14 d组、TPC 21 d组及ICH组.TPC各组大鼠与ICH组大鼠予以右侧尾状核注射凝血酶10 U诱发脑出血,SO组大鼠予以同部位注射生理盐水50μl,用dUTP缺口末端标记法(TUNEL)染色及免疫组化染色法观察神经细胞凋亡及caspase-3表达的情况.结果 ICH组与TPC各组凋亡细胞及cmpase-3表达均明显多于SO组(均P<0.01),TPC 12 h、TIC 1 d、TPC 3 d、TPC 7 d、TIC 14 d和TPC 21 d组细胞凋亡及caspase-3阳性表达明显少于ICH组(均P<0.05),其中TIC 7 d组凋亡细胞数及caspase-3阳性表达最少(P<0.05～0.01).结论 TPC可减少ICH所致的神经细胞凋亡及caspase-3表达,其中以TPC 7 d的作用最强.     

NADPH氧化酶在ATP7Btx-J小鼠脑损伤氧化应激过程中的作用

目的 探讨还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶是否通过干扰氧化应激过程参与ATP7Btx-J纯合型小鼠脑损伤. 方法 50只ATP7Btx-J小鼠分为3组:(1)20周野生型(WT)小鼠,共18只;(2)20周ATP7Btx-J纯合型小鼠,共18只;(3)NADPH氧化酶抑制剂干预小鼠(即apo-ATP7Btx-J纯合型小鼠),共14只.其中apo-ATP7Btx-J纯合型小鼠于16周开始灌胃给予apocynin,200 mg/(kg·d),至20周.其余2组给予等体积生理盐水.采用电感耦合等离子体质谱仪检测纹状体铜含量;比色法测定其纹状体NADPH氧化酶的活性;二氢乙啶(DHE)染色法检测超氧阴离子的含量;Western blotting检测凋亡蛋白活化caspase-3的表达;TUNEL法检测神经元凋亡情况. 结果 (1) 20周ATP7Btx-J纯合型小鼠脑组织纹状体中的铜含量显著高于20周WT小鼠,差异有统计学意义(P＜0.05).(2) ATP7Btx-J纯合型小鼠脑组织纹状体中的NADPH氧化酶活性和超氧阴离子水平高于同周龄同品系的WT小鼠及apo-ATP7Btx-J纯合型小鼠,差异均具有统计学意义(P＜0.05).(3) 20周ATP7Btx-J纯合型小鼠脑组织纹状体活化caspase-3蛋白表达水平和凋亡神经元数较WT小鼠及apo-ATP7Btx-J纯合型小鼠明显增高(多),差异均具有统计学意义(P＜0.05). 结论ATP7Btx-J纯合型小鼠纹状体神经元凋亡与NADPH氧化酶存在一定联系,apocynin可通过抑制NADPH氧化酶发挥神经保护作用.     

大鼠酒精中毒后大脑皮质、海马、小脑的病理学改变及caspase-3的异常表达

目的探讨大鼠慢性酒精中毒后大脑皮质、海马、小脑的病理学改变及caspase-3的异常表达。方法选用健康雄性Wistar大鼠随机分为两组,其中酒精中毒组30只;盐水对照组20只。酒精中毒组每日每只大鼠分别按8ml/kg灌胃2w,随后再按照10ml/kg灌胃1w,按12ml/kg灌胃1w,共灌胃4w。每日灌胃两次,其间隔均为6h,酒精浓度为50%。对照组用等量的生理盐水灌胃。并对两组大鼠进行体重、一般生物学特征、HE染色、TUNEL染色、免疫组化caspase-3的检测。结果造模成功后,两组大鼠的体重存在的统计学差异;HE染色后酒精组大鼠大脑皮质、海马、小脑锥体细胞数目减少,部分神经元变性、坏死;TUNEL法测定酒精组大鼠凋亡细胞数量明显多于对照组(P<0.05),酒精组大鼠大脑皮质、海马、小脑的caspase-3表达明显高于对照组(P<0.05)。结论慢性酒精中毒可引起大鼠大脑皮质、海马及小脑的病理学改变,出现神经细胞凋亡,引起与凋亡相对应部位caspase-3阳性表达,并参与大鼠酒精中毒后凋亡机制的发生、发展。     

Mechanism of action of tubocurarine on nicotinic cholinoreceptors of neurons of the sympathetic ganglia of rats

Tubocurarine (Tc) effect on membrane currents elicited by acetylcholine (ACh) was studied in isolated superior cervical ganglion neurons of rat using patch-clamp method in the whole-cell recording mode. The "use-dependent" block of ACh current by Tc was revealed in the experiments with ACh applications, indicating that Tc blocked the channels opened by ACh. Mean lifetime of Tc-open channel complex, tau, was found to be 9.8 +/- 0.5 s (n = 7) at -50 mV and 20-24 degrees C. tau exponentially increased with membrane hyperpolarization (e-fold change in tau corresponded to the membrane potential shift by 61 mV). Inhibition of the ACh-induced current by Tc (3-30 microM/1) was completely abolished by membrane depolarization to the level of 80-100 mV. Inhibition of ACh-induced current was augmented at increased ACh doses. It is concluded that the open channel block produced by Tc is likely to be the only mechanism for Tc action on nicotinic acetylcholine receptors in superior cervical ganglion neurons of rat.     

The effect of age of onset of PD on risk of dementia

Background Dementia occurs in the majority of patients with Parkinson’s disease (PD). Late onset of PD has been reported to be associated with a higher risk for dementia. However, age at onset (AAO) and age at baseline assessment are often correlated. The aim of this study was to explore whether AAO of PD symptoms is a risk factor for dementia independent of the general effect of age. Methods Two community-based studies of PD in New York (n = 281) and Rogaland county, Norway (n = 227) and two population-based groups of healthy elderly from New York (n = 180) and Odense, Denmark (n = 2414) were followed prospectively for 3–4 years and assessed for dementia according to DSM-IIIR. All PD and control cases underwent neurological examination and were followed with neurological and neuropsychological assessments. We used Cox proportional hazards regression based on three different time scales to explore the effect of AAO of PD on risk of dementia, adjusting for age at baseline and other demographic and clinical variables. Findings In both PD groups and in the pooled analyses, there was a significant effect of age at baseline assessment on the time to develop dementia, but there was no effect of AAO independent of age itself. Consistent with these results, there was no increased relative effect of age on the time to develop dementia in PD cases compared with controls. Interpretation This study shows that it is the general effect of age, rather than AAO that is associated with incident dementia in subjects with PD. Received in revised form: 22 December 2005     

Limits of deinstitutionalization--perspectives of relatives of psychiatric patients

After a hopeful beginning, the social process of the reintegration of those with severe mental illness has come to a standstill. I am led to wonder whether "the community" really wants to live together with people suffering from severe mental illness, and if so, how closely? As long as the medical treatment of mental illness provided by the general practitioners is fundamentally deficient, as they are not able to prescribe the necessary interventions--such as out-patient psychiatric nursing, and service providers in the out-patient sector are content with offering increasingly intensive forms of care for the less seriously ill at the cost of the Social Welfare System--the reintegration of those with serious mental illness remains an illusion--which is mainly to the benefit of providers of residential care in homes and hostels.     

Sense of coherence as a predictor of subjective state of health: results of 4 years of follow-up of adults

A number of cross-sectional population studies have shown that a strong sense of coherence (SOC) is associated with various aspects of good perceived health. The association does not seem to be entirely attributable to underlying associations of SOC with other variables, such as age or level of education. OBJECTIVE: The aim of the study reported here was to determine whether SOC predicted subjective state of health. METHODS: The study was carried out as a two-way panel mail survey of 1976 individuals with 4 years interval for two collections of data. The statistical method used was multivariate cumulative logistic modeling. Age, initial subjective state of health, initial occupational training level, and initial degree of social integration were included as potential explanatory variables. RESULTS: A strong SOC predicted good health in women and men. CONCLUSIONS: SOC can be interpreted as an autonomous internal resource contributing to a favorable development of subjective state of health. SOC data should, however, be regarded as complementary to and not a substitute for information already known to be associated with increased risk of future ill health.