电刺激小脑顶核对大鼠脑缺血/再灌注后氧化性DNA损伤的保护作用

目的预电刺激小脑顶核对大鼠脑缺血/再灌注后DNA氧化性损伤的保护作用, 以了解电刺激小脑顶核对实验性脑缺血及再灌注后神经保护的分子机制.方法健康雄性Wistar大鼠106只, 体重(250±30)g, 随机分为4组: (1) 单纯造模组; (2) 预刺激组; (3) 毁损小脑顶核组; (4) 假手术组.毁损小脑顶核组大鼠用鹅膏氨酸毁损两侧小脑顶核, 预刺激组、毁损小脑顶核组大鼠均以电刺激器刺激左侧小脑顶核, 前3组大鼠用线栓法成功制作可复流的MCAO模型2 h后再灌注.在再灌注后6、24、48 h将大鼠断头取脑, 取第3片提取DNA或RNA.DNA样品经酶解后上高效液相-电化学检测器检测8-ohdG.RNA样品通过RT-PCR的方法探测rOOG1 mRNA的表达.结果 (1) 大鼠脑缺血/再灌注后缺血区8-ohdG堆积.预刺激组再灌注各时点的8-ohdG含量均较单纯造模组及毁损小脑顶核组减少(P＜0.01); (2) 单纯造模组及毁损小脑顶核组脑缺血/再灌注后rOGG1的转录水平相似, 再灌注后6 h其rOGG1 mRNA几乎检测不到, 随时间的延长其转录水平有所增加, 但仍较假手术组及预刺激组低(P＜0.01).预刺激组再灌注后6 h其rOGG1 mRNA的表达量与假手术组无显著性差异, 但再灌注后24及48 h其rOGG1 mRNA的表达量均较假手术组增加(P＜0.01).结论 (1) 大鼠脑缺血/再灌注后脑缺血区存在DNA氧化性损伤; (2) 预电刺激小脑顶核减少缺血区神经元凋亡可能与DNA修复酶活性上调有关.     

电刺激小脑顶核对大鼠局灶性脑缺血／再灌注后Ku70表达影响的研究

目的:研究预电刺激小脑顶核对大鼠脑缺血再灌注后Ku70 mRNA的表达及其神经保护的分子机制。方法:Wistar大鼠通过原位杂交的方法及末端标记法检测Ku70 mRNA的表达及Tunel阳性细胞数。结果:①单纯造模组及毁损小脑顶核组缺血/再灌注后各时点Ku70 mRNA的表达无显著性差异,均较预刺激组及假手术组明显减少(P<0.01),预刺激组除缺血/再灌注后6h Ku70mRNAR的表达较假手术组减少(P<0.01)外,余时点与假手术组Ku70 mRNA表达无明显差异;②预刺激组Tunel阳性细胞数较未给预电刺激的两组明显减少(P<0.01)。结论:①预电刺激小脑顶核能减少缺血区神经元凋亡可能与DNA修复酶Ku70活性上调有关;②毁损小脑顶核后电刺激对脑缺血/再灌注引起的氧化性DNA损伤无保护作用。     

端粒酶逆转录酶在预电刺激小脑顶核后大鼠局灶性脑缺血再灌注损伤中的作用

目的 探讨预电刺激小脑顶核对大鼠脑缺血再灌注后端粒酶逆转录酶(TERT)蛋白表达水平的影响及其抗细胞凋亡作用的可能机制. 方法 将Wistar雄性大鼠150只采用完全随机数字表法分为单纯造模组及预电刺激组(即电刺激小脑顶核1h,24 h后制作右侧局灶性脑缺血模型).2组均缺血2h后行再灌注,并按再灌注时间不同分为24h、48h、72 h亚组(各25只).分别用TTC染色测定脑梗死体积,Western blotting检测TERT与Bax蛋白的表达,TUNEL组化染色检测凋亡细胞数,透射电镜观察线粒体超微结构的变化. 结果 单纯造模组再灌注24h、48 h、72h各亚组的脑梗死体积均明显大于预电刺激组相应亚组,差异均有统计学意义(P＜0.05).预电刺激组再灌注24 h、48 h、72 h各亚组TERT蛋白的相对表达值(0.87±0.51、0.91±0.40、0.80±0.24)较单纯造模组相应亚组(0.73±0.37、0.80±0.51、0.64±0.33)明显增加,TUNEL阳性细胞数(53.60±5.18、64.00±2.37、49.83±4.26)较单纯造模组相应亚组(63.57±3.74、75.40±5.55、60.00±2.37)明显减少,差异均有统计学意义(P＜0.05).预电刺激组再灌注24 h、48 h、72 h各亚组Bax蛋白水平与单纯造模组相应亚组比较差异无统计学意义(P＞0.05).预电刺激组再灌注24 h、48 h、72 h各亚组线粒体损伤级别(1.50±0.41、1.75±0.52、1.33±0.52)均较单纯造模组相应亚组(2.50±0.63、3.08±0.58、2.33±0.41)明显减轻,差异有统计学意义(P＜0.05). 结论 电刺激小脑顶核可以促进TERT蛋白表达的增加,增加的TERT可能通过保护线粒体来减少缺血区神经元的凋亡.     

脑缺血再灌注损伤时 c-fos、c-jun 的表达和细胞凋亡

目的　研究脑缺血再灌注大鼠神经细胞凋亡和原癌基因c fos、c jun表达。 方法　 8周龄健康雄性Wistar大鼠 2 4只 ,随机分为缺血再灌注组、假手术组和对照组 ,每组各 8只。制作大鼠大脑中动脉栓塞 (MCAO)再灌注模型 ,缺血 4h再灌注 2h后断头处死 ,TUNEL法检测神经细胞凋亡 ,免疫组织化学法检测神经细胞c fos、c jun蛋白的表达。结果　缺血再灌注组细胞凋亡率、平均吸光度及c fos、c jun阳性细胞率、平均吸光度均高于假手术组和对照组 (P <0 .0 5 )。结论　脑缺血再灌注损伤可诱导c fos、c jun蛋白的表达和细胞凋亡 ;脑缺血再灌注大鼠神经功能评分与c fos、c jun蛋白的表达和细胞凋亡呈正相关。     

移植神经干细胞的存活与迁移：电刺激小脑顶核可提高移植效率吗？**

背景：缺血早期半暗带中神经元和内皮细胞的坏死和凋亡无法得到缓解,加之移植后的存活率低,向功能细胞的分化率低,是单纯神经干细胞移植的缺陷。课题组提出整体干预理念,期望给移植细胞提供优化的整体环境。 目的：观察神经干细胞移植与电刺激小脑顶核相结合整体干预对移植神经干细胞存活与迁移的影响。 方法：体外分离、培养新生Wistar大鼠海马表皮生长因子反应性神经干细胞,Brdu标记,并用胎牛血清诱导,观察其多向分化潜能;80只Wistar大鼠分为4组：顶核刺激+神经干细胞移植组(n=32)：左侧小脑顶核刺激24 h后行右侧大脑中动脉梗死,再24 h后将神经干细胞立体定向注入右侧侧脑室内;顶核刺激组(n=8)：PBS代替神经干细胞,其余同顶核刺激+神经干细胞移植组;单纯神经干细胞移植组(n=32)：同心圆电极插入小脑顶核,不通电其余同顶核刺激+神经干细胞移植组;对照组(n=8)：同心圆电极插入小脑顶核后不通电,其余同顶核刺激组。记录梗死后6,24 h,3,7,14,28 d大鼠的神经功能;分别在移植后3,7,14,28 d处死大鼠,以梗死灶为中心切片,Brdu免疫组织化学染色,观察移植神经干细胞的存活与迁移。 结果与结论：分离培养的表皮生长因子反应性细胞表达nestin抗原,并有自我更新及多向分化潜能,在胎牛血清诱导下可分化为神经胶质细胞和神经元。经Brdu标记后,85%以上神经干细胞表达Brdu抗原。移植28 d内顶核刺激+神经干细胞移植组功能评分明显优于与其他3组(P < 0.05~0.01)。顶核刺激+神经干细胞移植组在移植14,28 d存活细胞数明显多于单纯神经干细胞移植组;提示电刺激小脑顶核可显著提高移植神经干细胞的存活率及大脑中动脉梗死大鼠的神经功能评分;优化的整体环境可提高移植神经干细胞对局灶性脑缺血损伤细胞的替代作用。     

大鼠脑缺血再灌注后内质网应激相关因子表达的改变

目的观察内质网应激相关因子GRP78和CHOP在大鼠局灶性脑缺血再灌注后的表达,探讨内质网应激在脑缺血再灌注损伤中的作用。方法采用大鼠大脑中动脉线栓模型,应用免疫组化及RT-PCR方法检测脑缺血再灌注后不同时相缺血周围区GRP78和CHOP的表达;缺口末端标记法检测细胞凋亡。结果模型组GRP78和CHOP表达均高于假手术组,分别于再灌注12h和24h达高峰,CHOP表达与神经细胞凋亡时间趋势一致。结论脑缺血再灌注诱导GRP78和GADD153表达上调,与神经细胞凋亡有关,内质网应激机制参与了脑缺血再灌注后的神经细胞凋亡。     

iASPP在大鼠脑缺血再灌注中的表达及其意义

目的 观察p53凋亡刺激蛋白（apoptosis stimulating protein of p53,ASPP）家族的抑制因子（inhibitorymember of the ASPP family,iASPP）在大鼠脑缺血再灌注后的表达及其意义。方法 48只清洁级、成年健康雄性SD大鼠随机分为假手术组（8只）和脑缺血再灌注组（40只）。脑缺血再灌注组又分为缺血2h再灌注4h、12h、24h、48h和72h 5个时间点,其中每个时间点8只大鼠。苏木素-伊红染色测定脑梗死体积;原位末端转移酶标记技术检测半暗带细胞凋亡情况;免疫印迹法测定半暗带iASPP的表达变化。结果 脑缺血再灌注4h组和24h组的凋亡细胞阳性率与假手术组存在统计学差异（P <0.01）。脑缺血再灌注组的脑梗死体积百分比与假手术组相比具有统计学意义（P <0.05）。与假手术组相比,在脑缺血再灌注12h时iASPP表达开始下降（P <0.01）,再灌注24h降至最低（P <0.01）,再灌注48h开始回升（P <0.01）。结论 在脑缺血再灌注后,缺血半暗带凋亡细胞显著增多,iASPP表达下降,提示在脑缺血再灌注中iASPP的表达下降可能在细胞凋亡的发生中发挥重要作用,其表达上调可能具有神经保护作用。     

西洛他唑对大鼠脑缺血再灌注损伤后PARP/AIF介导的细胞凋亡途径的影响

目的 研究大鼠局灶性脑缺血再灌注损伤后多聚腺苷二磷酸核糖聚合酶(PARP)和凋亡诱导因子(AIF)在海马CAI区的表达,探讨西洛他唑预处理能否通过PARP/AIF途径发挥脑保护作用. 方法 将135只雄性SD大鼠按随机数字表法分为3组:假手术组、模型组、西洛他唑组,每组45只.采用线栓法阻塞大鼠大脑中动脉制作局灶性脑缺血再灌注损伤模型.西洛他唑组造模前灌胃给予30 mg/kg剂量西洛他唑(2次).每组根据再灌注时间点不同(6 h、24 h、72 h)分为3个亚组,每亚组15只.应用TUNEL法检测神经细胞凋亡变化,Western blotting法检测AIF、腺苷二磷酸核糖(PAR)在不同时间点的变化,RT-PCR法检测AIF mRNA的表达变化. 结果 大鼠局灶性脑缺血再灌注损伤后出现AIF核移位.与假手术组比较,模型组凋亡细胞数明显增加,AIF、PAR含量及AIF mRNA表达明显增加,24 h时最显著,差异均有统计学意义(P＜0.05).西洛他唑组再灌注6h、24 h、72 h各亚组凋亡细胞数较模型组中各亚组明显减少,AIF、PAR含量较模型组各亚组明显降低,AIF mRNA表达亦明显减少,24h时最显著,差异有统计学意义(P＜0.05). 结论 西洛他唑对大鼠脑缺血再灌注损伤有一定保护作用,其抗神经细胞凋亡的机制之一可能是通过抑制脑缺血损伤引起的PARP的过度活化及AIF的易位而实现的.     

电刺激小脑顶核对脑卒中大鼠的治疗作用与机制

研究电刺激小脑顶核对脑卒中大鼠的治疗作用及其机制。方法采用线栓法制备大鼠脑缺血模型,于２４ｈ测定脑血流量、脑含水量、髓过氧化物酶活性、梗塞体积;胶原酶法制备脑出血模型,于３０ｈ测定血肿周围组织含水量、脑血流量和血肿体积。结果电刺激脑缺血大鼠小脑顶核后,缺血组织的脑血流量上升,脑含水量和髓过氧化物酶活性下降,梗塞体积缩小;电刺激脑出血大鼠小脑顶核后,血肿周围组织血流量增加,含水量下降。电刺激后大鼠的生理参数和血肿体积无改变。结论电刺激小脑顶核可改善脑卒中大鼠的局部血供,缩小梗塞体积,减轻组织水肿。电刺激脑卒中大鼠小脑顶校无明显副作用。     

局灶性脑缺血再灌注大鼠海马CA1区神经元凋亡研究

目的 观察大鼠局灶性脑缺血再灌注(ischemia reperfusion,I/R)损伤后海马CA1区神经元凋亡、TUNEL阳性细胞变化,以及凋亡相关蛋白Bcl-2与Bax蛋白的表达情况.方法 将健康雄性SD (Sprague-Dawley)大鼠随机分为假手术组和I/R组,每组再分为缺血再灌注后3、6、12、24、48、72 h亚组.应用免疫组化方法检测再灌注后不同时间点大鼠海马CA1区神经元凋亡基因Bcl-2和Bax蛋白的表达及Bcl-2/Bax比值变化,采用原位细胞凋亡检测(TUNEL)技术检测凋亡阳性细胞数.结果 各组非缺血侧相应区域神经元胞质中Bcl-2均有微弱表达.I/R组缺血侧海马CA1区于再灌注3 h开始出现Bcl-2和Bax蛋白微弱表达,随再灌注时间延长神经元内Bcl-2表达逐渐增强,再灌注24 h后Bcl-2表达达高峰,假手术组与I/R组比较差异有统计学意义(P<0.05).结论 I/R损伤后海马CA1区神经元不仅存在变性坏死,还存在明显的细胞凋亡且细胞凋亡在大鼠I/R损伤中发挥重要作用;I/R可诱导海马CA1区细胞凋亡基因Bcl-2和Bax蛋白表达,且其表达呈一定规律.     

Denervation study of synapses of organ of corti of old world monkeys

Fine structural characteristics of synapses in the spiral organ of Corti were examined, with reference to differences between inner and outer haircell systems, and to location of neurons of origin of efferent axons. Surgical interruption of crossed olivocochlear bundle, of vestibular nerve, of facial nerve, and excision of superior cervical ganglia were used to determine the pathways of efferent axons. Interruption of the vestibular nerve near the brainstem results in degeneration of all efferent terminals on outer hair cells. Mid-line lesions at, and caudal to, the facial colliculus result in degeneration of about half of these efferent terminals. Efferent synaptic bulbs to the inner hair-cell system are small, of the order of one micron, and form type 2 junctions with afferent dendrites. They tend to have more large dense-core vesicles (about 80 nm) than the large efferent terminals of the outer hair-cell system, and appear to be the terminals of axons in the habenula perforata, which exhibit varicosities laden with large dense core vesicles. The varicosities are unaffected by excision of the superior cervical ganglia. So far as our material can reveal, it appears that the varicosities in the habenula perforata do not survive vestibular root interruption, nor do the efferent processes in the internal spiral bundle or at the base of inner hair cells. Most interestingly, the afferent processes of the inner hair-cell system, as identified for example by their relation to pre-synaptic bodies in the inner hair cells, are subject to a trans-synaptic reaction after severance of the vestibular root. They undergo a dramatic cytological transformation, characterized by increase of volume, engorgement with microtubules, microfilaments, microvesicles of various sizes, and clusters of lysosomes. Thus, both the efferent and afferent terminals of the inner hair-cell system show marked cytological differences from the corresponding terminals of the outer hair cell system.     

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.