缺血预处理诱导大鼠海马CA_1区神经元HSP_(70)提前表达

目的前脑缺血再灌注可引起大鼠海马迟发性神经元死亡。但若在致死性缺血前，预先给于短暂的非致死性缺血刺激，即缺血坝处理（ｉｓｃｈｅｍｉｃ　ｐｒｅｃｏｎｄｉｔｉｏｎｉｎｇ，ＩＰＣ），则可对其后的长时间致死性缺血产生耐受，此现象称为缺血耐受。ＨＳＰ_７０基因的转录被认为是细胞应激反应的基因标志。其基因产物ＨＳＰ_７０。具有细胞保护作用。本研究旨在探讨缺血预处理引起的缺血耐受中 ＨＳＰ_７０表达的变化。方法（１）建立动物模型：选用健康成年雄性Ｗｉｓｔａｒ大鼠，参照Ｐｕｌｓｉｎｅｌｌｉ的四血管阻断法建立前脑缺血模型。动物分三组：单纯缺血再灌注组（ＩＲ组），给予假手术３ｄ后行８ｍｉｎ致死性缺血；缺血预处理组（ＩＰＣ组），先给予３ｍｉｎ非致死性缺血．３ｄ后再给予８ｍｉｎ缺血：对照组，只给于假手术，不给予缺血刺激，（２）免疫投化检测；十缺血再灌注２ｈ，取脑制备冰冻切片，用ＳＡＢＣ 法检测各组海马ＣＡ_１区神经元ＨＳＰ_７０的表达。（３）流氏细胞术检测：于缺血再灌庄２４ｈ，迅速取而背侧新鲜海马组织，用流氏细胞仪检测海马ＣＡ_１区神经元ＨＳＰ_７０的阳性表达率。（４）病理学检查：再灌注７ｄ时取脑制备石蜡切片，行尼氏染色，光镜下观察海马ＣＡ     

针刺对小鼠腹腔巨噬细胞的热休克蛋白、诱导性一氧化氮合酶及其基因表达的效应

目的研究针刺对小鼠腹腔巨噬细胞的热休克蛋白（ＨＳＰ）、诱导性一氧化氮合酶（ｉＮＯＳ）及其ｍＲ－ＮＡ表达的效应。方法将２４只昆明小鼠腹腔注射无菌石蜡油后,随机分为３组：电针（ＥＡ）组、对照１（Ｃ１）组和对照２（Ｃ２）组。将３组收集的腹腔巨噬细胞制备成玻片和硝酸纤维素膜（ＮＣＭ）两种标本,应用原位杂交、免疫细胞化学、细胞化学及斑点印迹技术检测ＨＳＰ７０、ｉＮＯＳ及ｉＮＯＳｍＲＮＡ。结果ＨＳＰ７０定位于巨噬细胞的胞质和胞核;ｉＮＯＳｍＲＮＡ及ｉＮＯＳ均定位于巨噬细胞的胞质;３组的ｉＮＯＳｍＲＮＡ、ｉＮＯＳ、ＨＳＰ７０的斑点印迹的扫描数值均为ＥＡ组＞Ｃ２组＞Ｃ１组（Ｐ＜０．０１）。结论电针可显著提高小鼠腹腔巨噬细胞的ＨＳＰ７０、ｉＮＯＳ及ｉＮＯＳｍＲ－ＮＡ表达。     

大鼠全脑缺血再灌后海马区bcl—2和Bax表达的检测

目的：检测ｂｃｌ－２和Ｂａｘ在大鼠嵛脑缺血再灌后海马区的表达。方法：用原位杂交组织化学检测ｂｃｌ－２ｍＲＮＡ,用免疫组织化学检测Ｂａｘ蛋白。结果：ｂｃｌ－２主要在海马ＣＡ３区表达,再灌８小时出现,２４小时阳性达到高峰,７２小时明显下降;Ｂａｘ则主要主要在ＣＡ１区表达,再灌８小时出现,２４小时阳性信号达到高峰,和,ＣＡ１２、ＣＡＳ４、ＤＧ区也有表达,但变化不明显,高倍镜下可见紫蓝色的阳性信号和棕黄色     

热应激诱导兔脑热休克蛋白70的合成研究

目的探讨热应激与热休克诱导的脑组织不同细胞热休克蛋白（ＨＳＰ）７０合成。方法用免疫组织化学ＡＢＣ方法，对８只新西兰兔在不同温度热应激中诱导脑组织ＨＳＰ７０合成进行了观察。结果发现热刺激组对兔脑组织ＨＳＰ７０合成刺激较弱，而热休克组大量诱导ＨＳＰ７０的合成。在脑不同区域ＨＳＰ７０合成并不相同，在皮质ＨＳＰ７０主要分布于颗粒Ⅱ层，在海马ＣＡ１和ＣＡ２区阳性细胞较多，而下丘脑阳性神经元主要分布于室周区，阳性物质主要分布在胞浆中，少数细胞可见核深染。结论ＨＳＰ７０的合成与热刺激强度及脑组织热敏感区有关，并且ＨＳＰ７０的合成可能反应了体温调节中枢与神经内分泌功能的损伤     

尼莫地平对大鼠脑血管痉挛缺血性脑损害的防治作用

目的：探讨尼莫地平（ＮＤ）对蛛网膜下腔出血（ＳＡＨ）后脑血管痉挛（ＣＶＳ）缺血性脑损害的保护作用。方法：应用非开颅大鼠模型,观察ＳＡＨ组和ＮＤ处理组２４ｈ内微区脑血流量（ＣＢＦ）和海马组织Ｃａ含量动态变化及３天后海马ＣＡ１区形态学改变。结果：在ＳＡＨ后２４ｈ内,ＳＡＨ组ＣＢＦ明显而持续降低,海马组织Ｃａ含量逐渐增加,３天后海马ＣＡ１区神经元明显受损。ＮＤ使上述改变均减轻。结论：ＮＤ通过对微循环的改善增加ＳＡＨ后ＣＶＳ时ＣＢＦ,通过阻断脑缺血时的有害代谢环节而减轻ＣＶＳ缺血性神经元损伤。     

小鼠胸腺树突状细胞的形态学观察

目的 观察小鼠胸腺树突状细胞（ＴＤＣ）的形态。方法 密度梯度离心结合小鼠ＣＤ５单抗淘洗法（ｐａｎｎｉｎｇ），分离出小鼠ＴＤＣ并行光镜及电镜观察。结果 台盼蓝排斥试验示９０％以上细胞存活，光镜下发现初分离的ＴＤＣ常和周围的胸腺细胞形成紧密的细胞簇。Ｓ－１００蛋白染色见ＴＤＣ胞体和突起内有棕黄色反应产物，ＡＮＡＥ反应呈阴性或核附近有数个小颗粒反应。ＡＴＰ酶反应阴性。电镜见细胞表面有许多树状突起，核形不     

针刺对小鼠腹腔巨噬细胞的热休克蛋白诱导性一氧化氮合酶？…

目的 研究针刺对小鼠腹腔巨噬细胞的热休克蛋白（ＨＳＰ）,诱导性一氧化氮合酶（ｉＮＯＳ）及其ｍＲＮＡ表达的效应。方法 将２４只昆明小鼠腹腔注射无菌石蜡油后,随机分为３组;电针（ＥＡ）组,对照１（Ｃ１）组和对照２（Ｃ２）组。将３组收集的腹腔巨噬细胞制备成玻片和硝酸纤维素（ＮＣＭ）两种标本,应用原位杂交,免疫细胞化学,细胞化学及斑点印变技术检测ＨＳＰ７０,ｉＮＯＳ,及ｉＮＯＳ ｍＲＮＡ。     

超抗原致小鼠免疫细胞凋亡的形态学观察

目的：探讨超抗原（ＳＥＡ）对小鼠免疫细胞的影响。方法：采用免疫学及光镜和电镜等方法观察ＳＥＡ注射小鼠体内引起免疫细胞的改变。结果：ＳＥＡ在小鼠体内早期引起中枢及外周免疫器官细胞增殖，表现为３Ｈ－ＴｄＲ掺入量增多和淋巴细胞母细胞化；晚期则致免疫细胞凋亡，在光镜和电镜下可见细胞呈核固缩、核碎裂，其ＤＮＡ琼脂糖凝胶电泳呈“梯状”改变；作为刺激细胞进行混合淋巴细胞培养时，反应细胞无３Ｈ－ＴｄＲ掺入量增多（Ｐ＞０．０５）；细胞周期蛋白依赖抑制因子（ｐ１６）阳性细胞数明显多于正常对照组及早期阶段。结论：这些变化可能与细胞周期紊乱、信号传导障碍有关     

下丘脑室旁核神经元多重神经支配的电镜研究

为了探讨下丘脑神经内分泌的突触调控机制，本文用电镜细胞化学与免疫电镜双标技术相结合的方法，研究了大鼠下丘脑室旁核神经元的多重神经支配。即先用６－ＯＨＤＡ损毁ＣＡ能神经末梢，再于振动切片上用包埋前免疫电镜法，分别以ＤＡＢ和ＴＡＢ为呈色剂先后对肽能（ＯＴ或ＳＰ）神经元和ＧＡＢＡ神经元进行双重标记。电镜观察结果表明：在下丘脑室旁核内存在肽能（ＯＴ，ＳＰ）和氨基酸（ＧＡＢＡ）能神经元及ＣＡ神经末梢；ＯＴ神     

肿瘤免疫活化剂沙培林的研究

本文应用沙培林诱导人外周血单个核细胞（ＰＢＭＣ）对胃癌（ＳＧＣ－７９０）、肺癌（ＳＰＣＡ－１）以及白血病细胞（ＨＬ－６０）能进行有效的杀伤。个体不同，ＰＢＭＣ对沙培林的应答不同，如对胃癌有的Ｓ．Ｉ、值达４．８６，有的无应答。在实验浓度下，对肝癌（７７２１）的杀伤作用较差。将小鼠肝癌（ＨＡＣ）细胞接种于小鼠腹腔，经腹腔连续给药后于不同时间测定小鼠脾及腹水单个核细胞的杀伤活性。结果表明治疗组的杀伤活性明显升高，并能非特异地提高对Ｌ９２９细胞的杀伤。腹腔接种ＨＡＣ经腹腔给药，单个核细胞杀伤ＨＡＣ的效应更强。上述结果表明，国产沙培林在体内能有效地激活单个核细胞。     

Distribution of 70kDa heat shock protein in rabbit brains after heat stress and heat stroke

OBJECTIVE: Evaluation of the relationship between the induction of 70kDa heat shock protein in rabbit brains and heat stress. METHODS: HSP70 was detected using monoclonal antibody by ABC method in rabbit hypothalamus, hippocampus and cerberal cortex. RESULTS: Intense HSP70 staining was displayed in rabbit brains of the heat stroke group (rectal temperature 43 degrees C to death). Positive cells were distributed mainly in the CA1, CA2 regions of the hippocampus; granular cell layer I and pyramidal layer (II) of the cerebral cortex; and the periventricular area of hypothalamus. HSP70-psoitive substances were localized in the cytoplasm and neuronal processes, a few neurons exhibited dark staining nucle. Hosever, the rabbit brains of the general heat stress group (rectal temperature 42.0 degrees C, 30 minutes) had much weaker staining. CONCLUSION: Hyperthermia causes neuronal expression of HSP70, particularly under strong heat stress, and may be sustained till death.     

Induction of mature neuronal properties in immortalized neuronal precursor cells following grafting into the neonatal CNS

Summary RN33B, a conditionally-immortalized neuronal cell line, survives and differentiates following grafting into the neocortex and hippocampus of adult and neonatal rat hosts. We have previously shown that these cells assume shapes characteristic of endogenous neurons at the integration site and persist up to 24 weeks post-grafting. In the present study we use electron microscopy and immunohistochemistry to characterize such cells. Differentiated RN33B cells were identical in size to endogenous neurons and their sizes depended on the specific location of integration. RN33B cells in the granule cell layer of the dentate gyrus and CA3 and CA1 pyramidal layers were 9.0, 15.3, and 12.6 m in diameter, respectively. Grafted RN33B cells received synapses from fibres of host origin. Differentiated cells expressed neuronal markers, but not glial markers. Some differentiated cells expressed glutamate bothin vitro andin vivo whereas undifferentiated cells did not. Grafted RN33B cells that differentiated with morphologies similar to CA3 pyramidal neurons and pyramidal cortical neurons expressed Py antigen, a neuronal marker that is differentially expressed in endogenous large pyramidal neurons of the cerebral cortex and large pyramids of hippocampal field CA3. This Py immunoreactivity was region-specific and corresponded to the endogenous pattern of Py immunostaining. Collectively, these data indicate that RN33B cells are capable of region-specific differentiation and have the potential to integrate functionally into the host CNS.     

Alteration in the immunoreactivity of the calcineurin subunits after ischemic hippocampal damage.

Dephosphorylation processes of target proteins are critical to the reversible regulation of intracellular signal transduction systems. Further, brain damage such as ischemic insult induces marked changes in protein kinase activity. To study these changes more thoroughly, specific monoclonal antibodies of the A and B subunits of calcineurin (protein phosphatase 2B) were raised, and regional alterations in the immunoreactivity of calcineurin in the rat hippocampus were investigated after a transient forebrain ischemic insult causing selective and delayed hippocampal CA1 pyramidal cell damage. In normal rats it was found that both the calcineurin A and the B subunits showed high immunoreactivity in the dendritic fields of the hippocampal formation. The immunoreactivity of subunit A in the strata oriens, the radiatum of the CA1 subfield and in the stratum lucidum of the CA3 subfield was most intense, whereas the immunoreactivity in the other CA3 subfields and in the dentate gyrus was relatively low. In contrast, the dendritic fields of the hippocampal formation were equally immunoreactive to calcineurin subunit B, although the stratum lucidum of the CA3, where the mossy fibers from the dentate granule cells terminate, showed a very high immunoreactivity of the B subunit. After transient forebrain ischemia in the CA1 subfield, where selective pyramidal cell death occurred two days after this ischemia, a marked loss of immunoreactivity in both subunits was observed, along with morphological pyramidal cell damage. A recovery of the immunoreactivity of A and B subunits in the strata oriens and radiatum was later noted 30 days after ischemia. In the stratum lucidum of the CA3, the immunoreactivity of both the A and B subunits was transiently depressed from 6 to 24 h, followed by a marked immunoreactivity enhancement from four to 30 days after ischemia. Further, in the histologically intact dentate gyrus, both the immunoreactivity of the A and B subunits in the molecular layer were transiently enhanced from four to 14 days after ischemia, particularly in the supragranular layer. The results clearly indicate that the protein dephosphorylation systems were markedly altered in the whole hippocampal formation during the recirculation period following ischemia. Further, the transient depression in the calcineurin immunoreactivity seen in the mossy fiber terminals may reflect modulated synaptic activity of the dentate granule cells, which may play a pivotal role in the delayed and selective death of the CA1 pyramidal cells. Thus, calcineurin appears to be an excellent marker enzyme for the detection of neuronal activity and synaptic plasticity after brain damage, such as an ischemic insult.     

Transient enhancement of inhibitory synaptic transmission in hippocampal CA1 pyramidal neurons after cerebral ischemia

Pyramidal neurons in hippocampal CA1 regions are highly sensitive to cerebral ischemia. Alterations of excitatory and inhibitory synaptic transmission may contribute to the ischemia-induced neuronal degeneration. However, little is known about the changes of GABAergic synaptic transmission in the hippocampus following reperfusion. We examined the GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons 12 and 24 h after transient forebrain ischemia in rats. The amplitudes of evoked inhibitory postsynaptic currents (eIPSCs) were increased significantly 12 h after ischemia and returned to control levels 24 h following reperfusion. The potentiation of eIPSCs was accompanied by an increase of miniature inhibitory postsynaptic current (mIPSC) amplitude, and an enhanced response to exogenous application of GABA, indicating the involvement of postsynaptic mechanisms. Furthermore, there was no obvious change of the paired-pulse ratio (PPR) of eIPSCs and the frequency of mIPSCs, suggesting that the potentiation of eIPSCs might not be due to the increased presynaptic release. Blockade of adenosine A1 receptors led to a decrease of eIPSCs amplitude in post-ischemic neurons but not in control neurons, without affecting the frequency of mIPSCs and the PPR of eIPSCs. Thus, tonic activation of adenosine A1 receptors might, at least in part, contribute to the enhancement of inhibitory synaptic transmission in CA1 neurons after forebrain ischemia. The transient enhancement of inhibitory neurotransmission might temporarily protect CA1 pyramidal neurons, and delay the process of neuronal death after cerebral ischemia.     

Relationships of 5-hydroxytryptamine immunoreactive terminal-like varicosities to 5-hydroxytryptamine-2A receptor-immunoreactive neuronal processes in the rat forebrain

The distributions of 5-hydroxytryptamine (5-HT)-immunoreactive (IR) varicosities and 5-hydroxytryptamine-2A receptor (5-HT2A)-IR neuronal structures in the rat brain have previously been described individually. Using double labeling immunocytochemistry, the relationships between 5-HT2A-IR and 5-HT-IR elements in the forebrain of male rats has been studied at the light microscopic level. In neocortical regions (frontal, parietal and retrosplenial cortex), the strongest 5-HT2A-IR was found in the apical dendrites of pyramidal cells in layers III-V, while 5-HT-IR terminal-like varicosities were present in all layers but most prominently in the outer layers. In other forebrain regions, the olfactory bulb, the hippocampal formation, and the islands of Calleja and Calleja magna, localized discrepancies were present between the 5-HT2A-IR neuronal profiles and the 5-HT-IR terminal-like varicosities. Hardly any additional juxtapositions between the 5-HT2A-IR neuronal profiles and 5-HT-IR terminal-like varicosities were revealed when the intraneuronal level of 5-HT was increased by monoamine oxidase inhibitor pretreatment (nialamide, 250 mg/kg, 3 h). Thus, in most forebrain regions, there were overall few juxtapositions between 5-HT terminal-like varicosities and 5-HT2A-IR neuronal structures. This observation suggests that 5-HT2A receptor mediated 5-HT transmission in the rat forebrain is mainly a volume transmission process mediated via short distance diffusion in the extra-cellular space.     

Hyperexcitability of hippocampal CA1 neurons after fluid percussion injury of the rat cerebral cortex

Effects of fluid percussion injury (FPI) of the parietal cerebral cortex on the neuronal activity in the temporal region of the rat hippocampal CA1 area were investigated by using optical and extracellular recording techniques. Application of moderate impact (1.5-2.0 atm) to the parietal cerebral cortex enhanced the optical signal of the neuronal activity in the ipsilateral hippocampal CA1 area. The field potential evoked by the Schaffer collaterals had multiple population spikes in the ipsilateral hippocampal CA1 pyramidal cell layer. Bicuculline (15 microM) increased the amplitude and the number of population spikes of the field potential even after the brain injury. These results suggest that FPI produces hyperexcitability of hippocampal CA1 neurons, probably by increasing the activity of the Schaffer collaterals of hippocampal CA3 neurons.     

Impairment of protein ubiquitination may cause delayed neuronal death

The hippocampus is a brain structure specifically vulnerable to short periods of transient cerebral ischemia, and which displays delayed neuronal necrosis. Protein ubiquitination is a posttranslational modification of proteins and an important factor in heat shock response and a regulator of ATP-dependent protein degradation. Using affinity purified antibodies against ubiquitin and ubiquitin-protein conjugates we have found that the ubiquitin immunoreactivity (UIR), normally present in all neurons of the hippocampus, disappears in the early recirculation period following cerebral ischemia from all hippocampal cells except the interneurons. Later UIR reappears in the different hippocampal regions over a 72 h period in the following order: granule cells-CA3 pyramidal cells-CA2 pyramidal cells. This is the inverse order of sensitivity of these cells to ischemia. The UIR never recovers in the CA1 pyramidal neurons where a 95% neuronal necrosis is seen following three days of recovery. We propose that the loss of UIR in the pyramidal neurons in the CA1 region signifies a persistent impairment of protein ubiquitination, and thus a change in the turnover of structural and regulatory proteins, which could be an essential part of the mechanism of slow neuronal death following cerebral ischemia.     

葛根素对大鼠局灶性脑缺血再灌注损伤后炎症反应的抑制作用

目的： 观察葛根素对大鼠局灶性脑缺血再灌注损伤后炎症反应的抑制作用并探讨其作用机制。方法: 采用大鼠大脑中动脉内栓线阻断法（MCAO）复制大鼠脑缺血再灌注损伤模型，于缺血开始及再灌注即刻由尾静脉注射葛根素18 mg·kg-1，缺血 2 h 再灌注 24 h 后取缺血侧脑组织，HE染色观察海马存活锥体细胞数，比色法测定脑组织MPO活性观察脑组织中性粒细胞浸润程度，Western blotting及RT-PCR法测定脑组织中ICAM-1 蛋白及mRNA表达情况及NF-κB p65亚基核转位情况。结果: 葛根素组缺血侧脑组织海马存活锥体细胞数明显高于缺血再灌注损伤组（P<0.01）， MPO活性明显低于缺血再灌注损伤组（P<0.01），ICAM-1 蛋白及mRNA表达较缺血再灌注损伤组明显减少（P<0.01），NF-κB p65蛋白亚基核转位明显少于缺血再灌注损伤组（P<0.01）。结论: 葛根素可减轻大鼠脑缺血再灌注损伤后炎症反应，这可能是其发挥脑保护作用的机制之一。     

Hyperglycemia increases superoxide production in the CA1 pyramidal neurons after global cerebral ischemia

Transient global cerebral ischemia results in selective neuronal death in the vulnerable hippocampal CA1 pyramidal neurons in a delayed manner. Hyperglycemia accelerates and exacerbates neuronal damage in this region. The object of this study was to determine whether hyperglycemia-enhanced damage is associated with increased production of superoxide anion after ischemia. The results showed that hyperglycemic ischemia caused a significant increase of superoxide production in the hippocampal CA1 neurons compared to normoglycemic animals after 18 h of recirculation, suggesting that enhanced superoxide anion production may mediate the hyperglycemia-accelerated and -enhanced neuronal death in the hippocampal CA1 area after ischemia and reperfusion.     

Post-ischemic administration of progesterone in rats exerts neuroprotective effects on the hippocampus

Progesterone is neuroprotective in models of focal or global ischemia when treatment starts either before the insult or at the onset of reperfusion. In these cases the steroid may act during the occurrence of the early pathophysiological events triggered by ischemia or reperfusion. As opposed to this condition, the aim of the present study was to assess the effect of delayed, post-injury administration of progesterone on the preservation of pyramidal neurons of the hippocampus of rats 21 days after been exposed to global ischemia by the four vessel occlusion model. Progesterone (8 mg/kg, i.v.) or its vehicle, were administered at 20 min, 2, 6, and 24h after the end of ischemia. At histological examination, brains of the ischemic vehicle-treated rats showed a severe reduction of the population of pyramidal neurons in the CA1 and CA2 subfields (12% and 29% remaining neurons, respectively), and a less severe neuronal loss in the CA3 and CA4 subfields of the hippocampus (68% and 63% remaining neurons, respectively), as compared to rats exposed to sham procedures. They also showed a two-fold enlargement of the lateral ventricles and 33% shrinkage of the cerebral cortex as compared to the sham group. Progesterone treatment resulted in a significant preservation of pyramidal neurons in CA1 and CA2 (40% and 62% remaining neurons), with no ventricular dilation and only a mild (12%) cortical shrinkage. Results suggest that progesterone is able to interfere with some late pathophysiological mechanisms leading both to selective neuronal damage in the hippocampal CA1 and CA2 subfields, and to shrinkage of the cerebral cortex.