小檗碱对大鼠海马CA_1区迟发性神经元坏死的影响

本文采用Ｐｕｌｓｉｎｅｌｌｉ－Ｂｒｉｅｒｌｅｙ４血管结扎致ＳＤ大鼠全脑缺血（１０ｍｉｎ）再灌流模型，分别观察了早期不同再灌流时间（１２、２４、４８ｈ）点上，大鼠海马ＣＡ１区神经元的超微结构以及再灌７ｄ时光镜结构变化，同时观察了小檗碱对ＣＡ１区迟发性神经元坏死的影响。结果显示脑缺血再灌流早期，ＣＡ１区神经元超微结构发生明显改变，７ｄ时光镜下绝大部分细胞脱失；而用药组大鼠海马ＣＡ１区神经元在相应时间点上超微结构变化相对较轻，７ｄ时仍有绝大多数（８２％）细胞存活，细胞密度为１７２±１２．２个／ｍｍ，显著高于缺血对照组２７±７．６个／ｍｍ，Ｐ＜０．００１。提示小檗碱对大鼠短暂脑缺血再灌流造成的海马ＣＡ１区迟发性神经元坏死具有显著的对抗作用。     

小檗碱对小鼠海马CA1区迟发性神经元坏死的影响

本文采用Ｐｕｌｓｉｎｅｌｌｉ－Ｂｒｉｅｒｌｅｙ４血管结扎致ＳＤ大鼠全脑缺血（１０ｍｉｎ）再灌流模型，分别观察了早期不同再灌流时间（１２、２４、４８ｈ）点上，大鼠海马ＣＡ１区神经元的超微结构以及早灌７ｄ时光镜结构变化，同时观察了小檗碱对ＣＡ１区迟发性神经元坏死的影响。结果显示脑缺血再灌流早期，ＣＡ１区神经元超微结构发生明显改变，７ｄ时光镜下绝大部分细胞脱失，而用药组大鼠海马ＣＡ１区神经元在相应时间点     

沙鼠脑缺血再灌注后海马中c—fos蛋白表达及纳洛酮 …

目的 观察沙鼠脑缺血再灌注后纳洛酮对海马神经元ｃ－ｆｏｓ蛋白表达及神经元形态改变的影响。方法 采用沙鼠急性全脑缺血模型,用免疫组织化学及组织化学方法观察海马ｃ－ｆｏｓ蛋白表达及细胞形态改变。结果 纳洛酮能明显加强急性全脑缺血沙鼠海马各区ｃ－ｆｏｓ蛋白的表达,ＣＡ１区尤为明显。同时纳洛酮能明显改善缺血后ＣＡ１区神经元细胞的变性坏死。结论 纳洛酮对缺血后海马神经元细胞的保护作用可能与加强ｃ－ｆｏｓ蛋白     

大鼠脑缺血再灌流后海马氨基酸水平变化与神经元损害的关系探讨

目的探讨脑缺血再灌流后海马氨基酸递质变化与神经元损害的关系。方法建立大鼠前脑缺血再灌流模型,测定海马ＣＡ１区和ＣＡ３／齿状回区游离氨基酸含量,观察阻断隔－海马通路对海马神经元损害和氨基酸水平的影响。结果（１）海马结构中仅ＣＡ１区神经元明显损害,但ＣＡ１区和ＣＡ３／齿状回区的Ｇｌｕ、Ａｓｐ和ＧＡＢＡ含量无差异。（２）阻断隔－海马通路可明显减轻海马神经元损害,但对海马氨基酸水平变化无影响。结论脑缺血再灌流后,氨基酸递质水平的异常变化不是海马ＣＡ１区神经元选择性易损的唯一决定因素,隔－海马通路末梢释放的神经递质也参与海马神经元损害过程。     

脑缺血再灌流大鼠脑胶质源性神经营养因子基因表达的变化

探讨脑缺血再灌流不同时程及不同程度缺血对海马及皮层胶质源性神经营养因子（ｇｌｉａｌｃｅｌｌｌｉｎｅ ｄｅｒｉｖｅｄ ｎｅｕｒｏｔｒｏｐｈｉｃ ｆａｃｔｏｒ, ＧＤＮＦ）基因表达的影响,以及Ｎ甲基Ｄ天冬氨酸（Ｎｍ ｅｔｈｙｌＤｓａｐａｒｔａｔｅ, ＮＭＤＡ）受体拮抗剂,钙离子通道阻断剂是否能调节缺血病态下ＧＤＮＦｍ ＲＮＡ的表达。参照Ｓｍ ｉｔｈ 等方法建立大鼠前脑缺血再灌流动物模型。用ＤＩＧＯｌｉｇｏｎｕｃｌｅｏｔｉｄｅ ３′ｅｎｄ ｌａｂｅｌｉｎｇ Ｋｉｔ,标记５１ ｍ ｅｒ的ＧＤＮＦ寡核苷酸探针在含有海马结构的冰冻组织切片上进行原位杂交检测ＧＤＮＦｍ ＲＮＡ的表达。１０ ｍ ｉｎ 缺血再灌流２ ｈ,齿状回ＧＤＮＦｍ ＲＮＡ表达上调。再灌流６ ｈ,ＣＡ１,ＣＡ３ 和皮层ＰＡＲ区ＧＤＮＦｍ ＲＮＡ表达亦见增多,２４ ｈ 达高峰。Ｋｅｔａｍ ｉｎｅ 可使ＧＤＮＦ的基因表达在海马结构及皮层ＰＡＲ区明显低于相应的缺血再灌流组,统计学差异显著（Ｐ＜ ００５）。脑缺血再灌流时ＧＤＮＦ基因表达增加,对缺血神经元可能起保护作用。Ｋｅｔａｍ ｉｎｅ可阻断缺血后ＧＤＮＦｍ ＲＮＡ 的表达增加,提示ＮＭＤＡ谷氨酸受体很可能参与介导了缺     

大鼠脑缺血再灌流后海马区一氧化氮合酶活性的变化

目的：观察鼠全脑缺血再灌流后海马区ＮＯＳ活性的变化。方法：采用大鼠４血管关闭方法制作全脑缺血再灌流模型。实验动物分为假手术组、缺血１０ｍｉｎ组、再灌注１、２、３ｄ组。测定脑缺血再灌流后海马区ＮＯＳ活性的变化。结果：全脑缺血曹澡注后海马组织ＮＯＳ活性被激活上调。结论：ＮＯ可能参与了海马ＣＡ１区迟发性神经元死亡（ＤＮＤ）的发生。     

急性脑缺血再灌流后脑组织钙依赖性中性蛋白酶的变化

采用大鼠全脑缺血模型，观测脑缺血再灌流脑组织钙依赖性中性蛋白酶（ｃａｌｃｉｕｍ－ａｃｔｉｚａｔｅｄｍｅｕｔｕａｌｐｒｏｔｅｉｍａｓｅ，ｃａｌｐａｉｎ）活性的变化及海马ＣＡ１区神经元损害改变。结果显示脑缺血再灌流脑组织ｃａｌｐａｉｎⅠ和ｃａｌｐａｉｎⅡ活性都明显升高（Ｐ＜０．０１），ＣＡ１区神经元密度相应下降，提示ｃａｌｐａｉｎｓ在脑缺血损害过程中可能起一定作用。     

保精增智液对大鼠迟发性神经元坏死海马CA_1区光镜和电镜改变的影响

本研究采用Ｗｉｓｔａｒ大鼠４血管关闭法制成全脑缺血１０ｍｉｎ再灌流动物模型造成迟发性神经元坏死（ＤＮＤ），分别观察了海马ＣＡ１区再灌流后３ｄ和５ｄ的普通病理和超微结构改变，同时观察了中药保精增智液对ＤＮＤ的保护作用，结果显示再灌流３ｄ时电镜下ＣＡ１区神经元内亚细胞结构改变明显，５ｄ时光镜下出现明显的神经元脱失，造模前８ｄ给药组可明显改善再灌流３ｄ时的亚细胞结构的改变，使５ｄ时神经细胞存活数上升（１８１．６±１５．１个／ｍｍ，对照组４１．４±４．０，Ｐ＜０．０１）。该药对大鼠短暂全脑缺血再灌流造成的ＤＮＤ有保护作用。     

用逆转录-多聚酶链反应检测局灶脑缺血及再灌注后c-fos和c-jun基因表达

本研究应用逆转录－多聚酶链反应（ＲＴ－ＰＣＲ）方法检测大鼠局灶脑缺血模型中即早基因ｃ－ｆｏｓ和ｃ－ｊｕｎ的表达。结果发现缺血１５ｍｉｎ时可见到ｃ－ｆｏｓ和ｃ－ｊｕｎｍＲＮＡ表达缺血３０ｍｉｎ时引起轻微左侧局灶脑缺血改变，可诱导左侧局灶脑缺血区ｃ－ｆｏｓ和ｃ－ｊｕｎｍＲＮＡ广泛的表达；缺血９０ｍｉｎ后，导致大面积局灶脑缺血改变，诱导上述两种基因在同侧缺血区与同侧非大脑中动脉（ＭＣＡ）供血区的海马中表达。后者有相当轻的缺血症状。再灌流６０ｍｉｎ后诱导两种基因的共同表达立即达高峰。我们采用标准化的大鼠局灶脑缺血及再灌注模型，在分子水平上动态观察缺血／再灌注后基因变化特征，为缺血性脑损害的防治提供实验依据。     

脑局部缺血后C—fos基因表达的意义及机理研究

目的：探讨脑局部缺血后神经元损伤的分子机制。方法：经颞骨开窗结扎大鼠大脑中动脉，制成脑局部缺血模型，采用地高辛标记Ｃ－ｆｏｓ探针原位杂交法，观察脑缺血后大脑皮层及海马等区域Ｃ－ｆｏｓ基因的表达状况，用Ｃ－ｆｏｓ阳性细胞密度作定量研究指标。结果：脑缺血组、氯胺酮组和对照组，在大脑皮层及海马区域Ｃ－ｆｏｓ阳性细胞分别呈高密度、低密度和散在分布，三组间相差显著（Ｐ＜０．０１）。在脑的其余部位，包括丘脑、脑干、小脑等区域，三组Ｃ－ｆｏｓ阳性细胞均呈散在分布，无组间差异。结论：脑局部缺血可诱发大脑皮层及海马Ｃ－ｆｏｓ基因表达，Ｃ－ｆｏｓ基因表达可能是缺血致神经元损伤的分子机制之一；氯胺酮对脑缺血后Ｃ－ｆｏｓ基因表达有部分抑制作用；ＮＭＤＡ受体激活可能参与了缺血后Ｃ－ｆｏｓ基因表达过程     

Selective proteasomal dysfunction in the hippocampal CA1 region after transient forebrain ischemia.

Delayed neuronal death in the hippocampal CA1 region after transient forebrain ischemia may share its underlying mechanism with neurodegeneration and other modes of neuronal death. The precise mechanism, however, remains unknown. In the postischemic hippocampus, conjugated ubiquitin accumulates and free ubiquitin is depleted, suggesting impaired proteasome function. The authors measured regional proteasome activity after transient forebrain ischemia in male Mongolian gerbils. At 30 minutes after ischemia, proteasome activity was 40% of normal in the frontal cortex and hippocampus. After 2 hours of reperfusion, it had returned to normal levels in the frontal cortex, CA3 region, and dentate gyrus, but remained low for up to 48 hours in the CA1 region. Thus, the 26S proteasome was globally impaired in the forebrain during transient ischemia and failed to recover only in the CA1 region after reperfusion. The authors also measured 20S and 26S proteasome activities directly after decapitation ischemia (at 5 and 20 minutes) by fractionating the extracts with glycerol gradient centrifugation. Without adenosine triphosphate (ATP), only 20S proteasome activity was detected in extracts from both the hippocampus and frontal cortex. When the extracts were incubated with ATP in an ATP-regenerating system, 26S proteasome activity recovered almost fully in the frontal cortex but only partially in the hippocampus. Thus, after transient forebrain ischemia, ATP-dependent reassociation of the 20S catalytic and PA700 regulatory subunits to form the active 26S proteasome is severely and specifically impaired in the hippocampus. The irreversible loss of proteasome function underlies the delayed neuronal death induced by transient forebrain ischemia in the hippocampal CA1 region.     

Changes in mint1, a novel synaptic protein, after transient global ischemia in mouse hippocampus.

Mints (munc18-interacting proteins) are novel multimodular adapter proteins in membrane transport and organization. Mint1, a neuronal isoform, is involved in synaptic vesicle exocytosis. Its potential effects on development of ischemic damage to neurons have not yet been evaluated. The authors examined changes in mint1 and other synaptic proteins by immunohistochemistry after transient global ischemia in mouse hippocampus. In sham-ischemic mice, immunoreactivity for mint1 was rich in fibers projecting from the entorhinal cortex to the hippocampus and in the mossy fibers linking the granule cells of the dentate gyrus to CA3 pyramidal neurons. Munc18-1, a binding partner of mint1, was distributed uniformly throughout the hippocampus, and synaptophysin 2, a synaptic vesicle protein, was localized mainly in mossy fibers. After transient global ischemia, mint1 immunoreactivity in mossy fibers was dramatically decreased at 1 day of reperfusion but actually showed enhancement at 3 days. However, munc18-1 and synaptophysin 2 were substantially expressed in the same region throughout the reperfusion period. These findings suggest that mint1 participates in neuronal transmission along the excitatory pathway linking the entorhinal cortex to CA3 in the hippocampus. Because mint1 was transiently decreased in the mossy fiber projection after ischemia, functional impairment of neuronal transmission in the projection from the dentate gyrus to CA3 pyramidal neurons might be involved in delayed neuronal death.     

Changes of neuronal transmission in the hippocampus after transient ischemia in spontaneously hypertensive rats and the protective effects of MK-801.

BACKGROUND AND PURPOSE: I studied the mechanism of postischemic neuronal degeneration in the hippocampus by an electrophysiological method. METHODS: Sequential changes of field potentials evoked by perforant path stimulation in the dentate gyrus and the CA1 region of the hippocampus were evaluated in spontaneously hypertensive rats up to 7 days after transient global ischemia induced by bilateral occlusion of the carotid arteries for 20 minutes after electrocauterization of the vertebral arteries. Animals were treated with vehicle or the excitotoxin antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10 amine (MK-801, 2 mg/kg or 5 mg/kg) intraperitoneally 30 minutes before ischemia. RESULTS: Complete recovery of the population spike was observed in the dentate gyrus within 24 hours after recirculation, followed by a gradual reduction of population spike amplitude. In contrast, population spike in the CA1 region showed partial recovery 24 hours after recirculation, and an abrupt reduction of population spike amplitude occurred on day 2. There was no significant enhancement of population spike amplitude in either region throughout the experiment. Interneuronal recurrent inhibition in the dentate gyrus was enhanced on day 4, and ischemic changes were apparent in the CA1 pyramidal cells on day 7. Pretreatment with 5 mg/kg MK-801 prevented field potential and pathological changes completely in the dentate gyrus and partially in the CA1 region. CONCLUSIONS: My results indicate that pathological changes of the CA1 pyramidal neurons after transient ischemia may not be the result of postischemic overstimulation. However, neuronal transmission in the CA1 region may be persistently impaired during or after transient ischemia.     

Activation of mitogen-activated protein kinases after transient forebrain ischemia in gerbil hippocampus.

We investigated the expression, activation, and distribution of c-Jun N-terminal kinases (JNKs), p38 mitogen-activated protein kinases (p38s) and extracellular signal-regulated kinases (ERKs) using Western blotting and immunohistochemistry in gerbil hippocampus after transient forebrain ischemia to clarify the role of these kinases in delayed neuronal death (DND) in the CA1 subfield. Immunoblot analysis demonstrated that activities of JNK, p38, and ERK in whole hippocampus were increased after 5 min of global ischemia. We used an immunohistochemical study to elucidate the temporal and spatial expression of these kinases after transient global ischemia. The immunohistochemical study showed that active JNK and p38 immunoreactivities were enhanced at 15 min of reperfusion and then gradually reduced and disappeared in the hippocampal CA1 region. On the other hand, in CA3 neurons, active JNK and p38 immunoreactivities were enhanced at 15 min of reperfusion and peaked at 6 hr of reperfusion and then gradually reduced but was continuously detected 72 hr after ischemia. Active ERK immunoreactivity was observed transiently in CA3 fibers and dentate gyrus. Pretreatment with SB203580, a p38 inhibitor, but not with PD98059, an ERK kinase 1/2 inhibitor, reduced ischemic cell death in the CA1 region after transient global ischemia by inhibiting the activity of p38. These findings indicate that the p38 pathway may play an important role in DND during brain ischemia in gerbil. Components of the pathway are important target molecules for clarifying the mechanism of neuronal death.     

Immunohistochemical distribution of protein kinase C isozymes is differentially altered in ischemic gerbil hippocampus

We used monoclonal antibodies to examine the immunohistochemical distribution of the three major Ca(2+)-dependent protein kinase C (PKC) isozymes (I, II, and III) in ischemic gerbil hippocampus. Groups of four animals were sacrificed at 15 min, 4 h, 1 day, 2 days, 3 days, and 7 days after a 10-min episode of global forebrain ischemia. In control animals, PKC-I immunoreactivity was greater in CA1 neurons than in CA3-4. Terminal-like staining was not evident. PKC-II immunoreactivity was observed in all CA fields and in the outer molecular layer of the dentate gyrus. PKC-III staining was present in the CA fields, the inner molecular layer of the dentate gyrus and the subiculum. Dentate granule cells and mossy fibers were not stained with any of the PKC antibodies. Fifteen minutes and 4 h after ischemia, PCK-I, -II and -III immunoreactivity were all increased in CA1 neurons and PKC-III immunoreactivity alone was visualized in granule cells and mossy fibers. Staining patterns returned to baseline one day after ischemia. PKC-II and -III terminal-like staining were preserved in the stratum lacunosum-moleculare for 3 days and 2 days after ischemia respectively and then disappeared. The altered patterns of PKC staining in the hippocampus may reflect activation and/or down-regulation of PKC isozymes. Ca(2+)-dependent PKC isozymes may, therefore, potentially play a role in the pathogenesis of delayed ischemic neuronal death.     

Hyperbaric oxygen treatment decreases post-ischemic neurotrophin-3 mRNA down-regulation in the rat hippocampus.

The therapeutic effect of hyperbaric oxygen (HBO) on ischemic injury was investigated using in situ hybridization to detect the mRNA expression of neurotrophin-3 (NT-3), which is thought to play a crucial role in protecting against neuronal death induced by brain ischemia. The rats under investigation were subjected to 10 min transient forebrain ischemia, and subsequently exposed to HBO (100% oxygen, 2.5 atm absolute) for 2 h. Levels of NT-3 mRNA in the CA1, CA2 and CA3 regions, and the dentate gyrus of the hippocampus were measured after various reperfusion periods. Neuronal death in the hippocampal CA1 region was also measured by Nissl staining, seven days post ischemia. The results demonstrated that HBO treatment significantly reduced the ischemia-induced down-regulation of the NT-3 mRNA level at 4 h post ischemia, and significantly increased cell survival 7 days after reperfusion. The findings suggest that an HBO treatment maintaining the NT-3 mRNA level in the hippocampus can be beneficial to the ischemic brain within a certain time frame.     

Ischemia-induced changes in α-tubulin and β-actin mRNA in the gerbil brain and effects of bifemelane hydrochloride

Using in situ hybridization histochemistry, we examined changes in the cytoskeletal protein α-tubulin and β-actin mRNAs in the gerbil brain 14 days after transient ischemia. In an attempt to identify the changes induced in the synthesis of cytoskeletal protein by schemia, we also evaluated the effects of post-ischemia administration of bifemelane on these cytoskeletal proteins. α-Tubulin and α-actin mRNAs were decreased in the CA1 region 14 days after transient ischemia. These decreases coincided with the loss of CA1 pyramidal cells, suggesting that they may have been related to delayed neuronal death. The β-actin mRNA level in ischemic controls was significantly increased in the dentate gyrus, habenular nucleus, and medial and lateral thalamic nuclei, where some afferent nerves project into the hippocampal pyramidal cells. The increased β-actin mRNA suggests that there may be a compensatory enhancement of actin synthesis in the afferent neurons that restores loosened synaptic connections with the ischemic cells in the CA1-4 fields. Administration of bifemelane just after recirculation prevented most of the ischemia-induced mRNA reductions in the CA1 field. Bifemelane's effect may be related to inhibition of Ca²⁺ influx and its radical scavenging activity. When bifemelane was administered to the ischemic group, α-tubulin mRNA levels significantly increased in the dentate gyrus and amygdaloid nucleus, and β-actin mRNAs showed a tendency to increase in the CA3 and CA4 fields, dentate gyrus, and medial and lateral thalamic nuclei. These findings suggest that bifemelane may enhance synthesis of cytoskeletal protein, especially in the ischemic brain, inducing axon outgrowth or synapse formation.     

脑缺血后脑内HSP70表达的实验研究

热休克蛋白(HSP)是细胞对缺血等应激反应的敏感标记.通过建立大鼠全脑缺血模型,采用HSP70单克隆抗体LSAB免疫组化技术对脑缺血再灌注后的神经元进行了检测.发现在海马CA_3区,齿状回,尾壳核及杏仁核等处的神经元中有HSP70过量表达,并于再灌注后48h达到高峰.初步研究了HSP70在不同神经元中的分布情况,基本肯定不同神经元中HSP70的表达与神经元对缺血的耐受性有关.