缺血缺氧性新生大鼠皮层神经元胞浆Smac/Diablo、caspase-9的表达及参附注射液的干预作用

目的 了解缺血缺氧性脑损伤(HIBD)新生大鼠神经元胞浆Smac/Diablo、caspase-9的变化及其相互关系,同时观察参附注射液的干预效果.方法 HIBD模型制作参照Rice等方法.新生7dSD大鼠按照完全随机数字表法分为假手术组、生理盐水对照组与参附治疗组,再按照造模后不同观察时间点又分为3 h、6 h、12 h、24 h、3 d、7 d6个亚组,每亚组8只.参附治疗组于造模后即刻腹腔注射参附注射液10 mL/kg,1次/d,连续用药3 d.生理盐水对照组用生理盐水替代参附注射液,用法用量相同;假手术组仅切开颈正中皮肤和分离右侧颈总动脉,不结扎,不缺氧.Western blotting检测各时间点各组大鼠神经元胞浆中Smac/Diablo、活化caspase-9蛋白的表达.结果 假手术组Smac/Diablo、caspase-9蛋白表达微弱.生理盐水对照组与参附治疗组2种蛋白造模后3 h即明显增加,与假手术组比较,差异有统计学意义(P＜0.05);24 h时2种蛋白表达达到峰值,3 d后明显下降,7 d时接近假手术组.其中参附治疗组Smac/Diablo蛋白表达量12 h、24 h、3 d、7 d时较生理盐水对照组明显下降,差异有统计学意义(P＜0.05);参附治疗组caspase-9蛋白12 h、24 h、3 d时较生理盐水对照组明显下降,差异有统计学意义(P＜0.05).结论 (1)HIBD新生大鼠脑皮层神经元存在Smac/Diablo蛋白胞浆转位,同时伴有胞浆活化caspase-9表达增加:参附可减少HIBD大鼠Smac/Diablo蛋白的胞浆转位,减少caspase-9的活化.(2)Smac/Diablo、caspase-9参与了HIBD新生大鼠迟发性神经元损伤的病理机制.

Abstract：

Objective To investigate the expressions of Smac/Diablo and caspase-9 and their corelations in the cytoplasm of cerebral cortex of neonatal rats with hypoxic-ischemic brain damage (HIBD), and the possible interference effects of Shenfu injection on them. Methods Postnatal 7-d SD rats were randomly divided into Shenfu treatment group (group SF), normal saline group (group C) and sham-operated group (group S). Rat models of HIBD were established according to Rice's method.Based on the observing times after the success of model making, each group (n=48) was equally divided into 6 subgroups (3, 6, 12 and 24 h, and 3 and 7 d observing groups). Rats in the group S were not performed ligation of the right common carotid artery and not underwent hypoxia but only performed the separation of right common carotid artery. Shenfu injection was administered by intraperitioneal injection right after H/I insult and then once daily at a dosage of 10 mL/kg for 3 d into the rats of group SF. Saline was also administered into the rats of group C by the same methods and at the same dosage. Cytoplasm proteins were extracted from the cerebral cortex tissue, and then, Western blotting was employed to detect the protein levels of Smac/Diablo and activated caspase-9 in cytoplasm. Results Western blotting showed that the expression levels of Smac/Diablo and activated caspase-9 in cytoplasm of group S were very low; the expression levels of Smac/Diablo and activated caspase-9 in cytoplasm of group SF and group C were increased markedly since the 3rd h of the success of model making as compared with those in group S (P＜0.05): these in the 2 groups reached their peak levels 24 h after H/I insult, gradually decreased 3 d after H/I insult, and almost returned to the original levels 7 d after H/I insult. The expressions of Smac/Diablo in cytoplasm of group SF 12, 24 h and 3 d after H/I insult was significantly lower than that of group C (P＜0.05); the expression of activated caspase-9 in cytoplasm of group SF 12,24 h and 3 d after H/I insult was significantly decreased as compared with that of group C (P＜0.05).Conclusion Smac/Diablo cytoplasm translocation and increased expression level of caspase-9 appear in the cytoplasm of cerebral cortex cells after H/I injury; Shenfu injection can inhibit the expressions of Smac/Diablo and caspase-9; Smac/Diablo and caspase-9 involve in the pathophysiological mechanisms of neuronal damage after H/I.     

参附注射液对缺氧缺血性脑损伤新生大鼠脑保护机制研究

目的探讨参附注射液对缺氧缺血性脑损伤(HIBD)新生大鼠皮质区钙离子相关蛋白钙网蛋白(CRT)的表达、细胞内游离Ca2+浓度以及神经元凋亡的影响,探讨其可能的脑保护机制。方法将7日龄新生SD大鼠随机分为对照组、缺氧缺血组和参附干预组,各组进一步分为缺氧缺血后3、6、12、24、72h5个亚组,每组均10只。参照Rice等法制备HIBD模型。参附干预组于造模成功后立即腹腔注射参附注射液10mL/(kg·d),连用3d。缺氧缺血组和对照组注射等量生理盐水。HE染色观察大鼠病变侧脑组织病理变化;RT-PCR及Western-blot法检测各组CRT的表达变化;荧光显微镜法检测脑细胞中游离Ca2+浓度;流式细胞术检测神经元凋亡率。结果缺氧缺血组和参附干预组各时间点CRT mRNA及蛋白表达量均较对照组升高(P0.05),且参附干预组较缺氧缺血组升高(P0.05);缺氧缺血组各时间点细胞内游离Ca2+浓度均较对照组明显升高(P0.05),参附干预组细胞内游离Ca2+浓度较缺氧缺血组降低(P0.05),但仍高于对照组(P0.05);参附干预组各时间点的神经元凋亡率均明显低于缺氧缺血组(P0.05),但较对照组仍升高(P0.05)。结论参附注射液可能通过上调CRT的表达,降低细胞内钙超载来减轻新生大鼠缺氧缺血性脑损伤。     

大鼠脑缺血区皮层的Smac／Diablo变化

目的观察大鼠缺血区皮层Smac／Diablo的动态变化，并探讨其在缺血后神经元凋亡过程中的可能作用。方法采用RT-PCR，Westernblot方法分别测定大鼠脑缺血后不同时间点在缺血区皮层Smac／DiablomRNA和蛋白水平的表达水平。结果Smac／Diablo基因转录水平和蛋白表达的改变呈时间依赖性；RT-PCR显示模型组大鼠在脑缺血3h就有明显的Smac／DiablomRNA表达上调，6h达高峰，持续至24h后逐渐下降，48h时恢复至缺血前水平；western blot显示在脑缺血后3h有Smac／Diablo蛋白表达的增加，6h时Smac／Diablo蛋白表达至高峰（P〈0．01），48h基本恢复到缺血前水平。结论Smac／Diablo在脑缺血损伤后的病理生理过程中具有重要作用。     

亚低温对脑缺血后Smac表达和释放的影响

目的 观察脑缺血模型第2个线粒体源胱天酶激活剂(Smac)在脑缺血后不同时间点的表达和释放情况,探讨亚低温治疗与Smac表达的可能相关机制.方法 将大鼠随机分为常温假手术组、亚低温假手术组、常温缺血组和亚低温缺血组,用线栓法制作局部脑缺血模型.亚低温治疗3 h后,在脑缺血3、6、12、24 h及3、7 d后进行神经功能评分,并用免疫组化法、蛋白免疫印迹法检测Smac表达,实时定量PCR法测定Smac mRNA表达.结果 亚低温各组神经功能评分均较常温组降低(P＜0.05或P＜0.01).常温组Smac释放、蛋白及mRNA表达均表现为随时间延长而增高,约24 h达峰值,经亚低温治疗后各组数值均较相应常温组显著降低(P＜0.05或P＜0.01).结论 亚低温可能通过抑制Smac转录、表达和释放减轻细胞凋亡水平,从而发挥脑保护作用.     

Omi/HtrA2在无镁外液致痫海马神经元凋亡中的作用机制及其特异性抑制剂UCF-101的神经元保护作用

目的观察线粒体丝氨酸蛋白酶Omi/Htra2在体外颞叶癫痫模型中神经元凋亡方面发挥的作用及其机制,以及其特异性抑制剂UCF-101对痫性损伤神经元的保护作用。方法原代培养新生24 h内的SD大鼠海马神经元至10 d,随机分成对照组、无镁细胞外液处理后3 h组、8 h组以及24 h组,观察细胞形态学特征,采用免疫蛋白印迹法观察海马神经元中Omi/Htra2蛋白的表达变化;再随机将神经元分为对照组、无镁细胞外液处理后24 h组、UCF-101处理组以及DMSO组,采用TUNEL法检测各组神经元凋亡,免疫蛋白印迹法观察神经元XIAP、caspase3、HAX-1蛋白的表达变化。结果随着无镁外液作用时间的延长,Omi/Htra2在线粒体中的含量逐渐减少,胞浆中含量逐渐增多。与正常对照组相比,致痫24 h后神经元凋亡数目以及caspase3蛋白表达显著增多(P<0.05),且HAX-1蛋白表达下降(P<0.05),而XIAP蛋白的表达正常对照组与模型组相比无统计学差异。与致痫24 h组相比,UCF-101处理组神经元凋亡细胞数和caspase3的表达均明显减少(P<0.05),XIAP及HAX-1蛋白表达增多(P<0.05),DMSO组神经元凋亡细胞数、上述蛋白表达方面与致痫24 h组无明显差异(P>0.05)。结论神经元遭到无镁细胞外液作用后,Omi/Htra2从线粒体移位到了胞浆,其特异性抑制剂UCF-101能有效抑制神经元凋亡,下调神经元caspase3蛋白的表达,上调XIAP、HAX-1蛋白的表达,发挥神经元保护作用。     

针刺抑制幼鼠HIBD脑组织神经细胞凋亡的实验研究

目的探讨针刺对新生大鼠缺血缺氧性脑损伤(HIBD)脑神经元调亡的保护作用及其机制。方法 (1)选用85只7 d龄清洁级SD大鼠,将大鼠结扎左侧颈总动脉后恢复2 h,置于透明密闭容器中,并入37℃恒温水中以1 L/min的速度通入低氧气体,2.5 h后将动物取出,存活者继续作行为测定,获成功模型72只,随机分为3组,A组为HIBD+针刺Ⅰ组,B组为HIBD+针刺Ⅱ组,C组为HIBD对照组;另设假手术组D组,每组24只。(2)选用幼鼠百会、患侧颞Ⅰ针、内关、曲池、足三里、涌泉,A组于造模后24 h开始针刺,前7 d仅针四肢部穴位,第8天开始加针头部穴位;B组造模后第8 d开始针刺,头、体针同步。针刺均为1次/d,A组连续针刺20 d,B组连续针刺13 d,假手术组干预措施与A组相同,至HIBD后第21天处死取材。采用TUNEL法测定针刺对HIBD大鼠额叶与海马区组织神经元凋亡,用图像分析仪在光镜下计算神经细胞凋亡数目。结果 4组左侧海马CA1区神经元及左侧大脑额叶皮质神经元凋亡数差异有统计学意义(P<0.05),其中D相似文献   

新生大鼠缺氧缺血性脑损伤后CIRP表达与意义

目的观察新生大鼠缺氧缺血性脑损伤(HIBD)后冷诱导RNA结合蛋白(CIRP)表达的变化情况。方法将40只7日龄新生SD大鼠随机分为假手术组和HIBD组,分别采用real-time PCR及免疫组织化学方法检测假手术组和HIBD后不同时间点(6、12、24和48h)CIRP在大鼠脑皮质与海马表达的变化。结果利用免疫组化和real-time PCR检测发现,大鼠脑皮质的CIRP蛋白和CIRP mRNA表达呈持续降低趋势,HIBD后6、12 h开始减少;24~48h下降更为明显。海马CIRP蛋白和CIRP mRNA表达则表现为6 h先升48h后降。结论CIRP参与了新生大鼠脑缺氧缺血的应激过程,可能与缺氧缺血性脑损伤后的脑水肿及神经元凋亡相关。     

Omi/HtrA2在缺氧缺血性脑损伤新生大鼠顶叶皮质中的表达变化

目的 观察缺氧缺血性脑损伤(HIBD)新生大鼠顶叶皮质中Omi/HtrA2的表达变化,进而探讨其在HIBD中的可能作用. 方法 将新生7d龄SD大鼠按完全随机数字表法分为假手术组和模型组(制作新生大鼠HIBD模型),并按术后观察时间点不同进一步分为6h、12h、24 h、72 h4个亚组.分别于术前及缺氧后1h对大鼠进行行为学检查；不同时间点处死后观察每只大鼠的脑组织大体形态,并采用免疫组化染色和Western blotting检测病变侧脑组织中Omi/HtrA2蛋白的表达变化. 结果 (1)行为学检查:术前每只大鼠翻身能力、夹尾右旋测定均正常.术后假手术组测定结果同前.模型组大鼠中18只不能翻身,20只出现夹尾右旋,还有14只表现为不能翻身和夹尾右旋同时存在.(2)脑大体形态观察；模型组大鼠结扎侧半球脑组织受损严重,24h时苍白、水肿明显,体积明显大于对侧.假手术组大鼠大脑半球左右对称,未见任何病理变化.(3)Western blotting 结果:模型组Omi/HtrA2蛋白的表达水平在各时间点均较假手术组明显升高,差异均有统计学意义(P＜0.05).Omi/HtrA2在HIBD后6h开始表达,24 h达高峰,随后开始下降.(4)免疫组化染色结果:模型组病变侧大脑皮层Omi/HtrA2阳性细胞数在HIBD后24 h达到高峰,随后降低,与假手术组在各时间点比较差异均有统计学意义(P＜0.05). 结论 新生大鼠HIBD后病变侧大脑皮层Omi/HtrA2的表达水平明显升高,并具有时间差异性,表明其参与了HIBD的病理过程,可能在HIBD诱导的细胞凋亡中发挥重要作用.     

下调lncRNA-MALAT1表达对新生大鼠缺氧缺血性脑损伤的保护作用

目的 探讨小图lncRNA-MALAT1表达对新生大鼠缺氧缺血性脑损伤（HIBD）的影响。方法 取SPF级7 d龄SD大鼠40只,随机分为假手术组、模型组、lncRNA对照组、silncMALAT1组,每组10只。Rice-Vannucci法建立新生大鼠HIBD模型,造模后2 h,侧脑室分别注射生理盐水、生理盐水、空载体重组腺病毒液、silncMALAT1各5 μl。造模后7 d,Morris水迷宫试验评估空间学习和记忆能力,TUNEL染色检测海马组织神经元凋亡,PCR和免疫印迹法检测海马组织BDNF/TrkB信号通路mRNA和蛋白表达变化。结果 模型组造模失败2只,lncRNA组失败2只,silncMALAT1组失败1只,造模成功率为83.33%。下调lncRNA-MALAT1表达,明显增加新生大鼠学习和记忆能力（P<0.05）,明显减少海马组织神经元凋亡率（P<0.05）,明显下调BDNF/TrkB mRNA和蛋白表达水平（P<0.05）。结论 下调lncRNA-MALAT1表达可减轻新生大鼠HIBD,其机制可能与抑制BDNF/TrkB信号通路、减轻海马组织神经元凋亡有关。     

大鼠癫(癎)持续状态后海马内X-连锁凋亡抑制蛋白及其负性调控因子的表达

目的 研究大鼠癫(癎)持续状态(SE)后海马组织中X-连锁凋亡抑制蛋白(XIAP)及其负性调控因子Smac、HtrA2、XAF1的表达变化.方法 建立氯化锂-匹罗卡品致(癎)大鼠SE模型,应用免疫组织化学染色和Western blot方法检测大鼠SE后各时点海马CA3区XIAP、Smac、HtrA2、XAF1及半胱氨酸蛋白酶(caspase)-3蛋白的表达.结果 SE后大鼠海马CA3区XIAP蛋白呈弥散性分布于整个神经元内,SE后2 h(0.5503±0.0172)起逐渐增高(t=115.87),8 h(0.6221±0.0238)达高峰(t=136.69).与对照组(0.1507±0.0165)比较,差异有统计学意义(P<0.01).Same、HtrA2及XAF1蛋白在对照组弱表达,在SE后呈弥散性分布于整个神经元内.2～72 h增高.海马CA3区caspase-3活性蛋白在对照组呈阴性,在SE后4～72 h明显增高.Western blot发现,SE组各时间点XIAP蛋白表达量与对照组比较差异无统计学意义(P>0.05),Smac、HtrA2及caspese-3蛋白在对照组很少表达,在SE后2～72 h增高(P<0.01).结论 XIAP及其负性调控因子Smac、HtrA2、XAF1涉及了SE后神经元凋亡的调节,参与了SE后神经元损伤.     

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.     

Summary of legislation of 1935 of interest to the department of Mental Hygiene

Psychiatric Quarterly -