Contrasting patterns in the localization of glutamic acid decarboxylase and Ca2+/calmodulin protein kinase gene expression in the rat central nervous system.

The expression of the genes encoding the alpha subunit of type II calcium calmodulin-dependent protein kinase (CaM II kinase alpha) and the 67,000 mol. wt form of glutamic acid decarboxylase was examined throughout the rat central nervous system. In situ hybridization histochemistry, using cRNA probes, revealed a dense population of CaM II kinase alpha-expressing cells throughout the telencephalon and diencephalon. CaM II kinase alpha mRNA was also expressed in the midbrain, cerebellum and medulla oblongata, but at greatly reduced levels. No CaM II kinase alpha gene expression was detected in nuclei producing monoamines or acetylcholine. By contrast, the glutamic acid decarboxylase gene was moderately to highly expressed throughout the central nervous system. In several regions there was a complementarity in the distributions of cells expressing the glutamic acid decarboxylase or CaM II kinase alpha genes. Cells in certain nuclei such as the thalamic reticular nucleus or globus pallidus showed glutamic acid decarboxylase gene expression only; others such as the majority of the dorsal thalamic nuclei showed CaM II kinase alpha gene expression only. Several regions contained both glutamic acid decarboxylase and CaM II kinase alpha expressing cells. However, simultaneous immunostaining for both proteins revealed only two regions where CaM II kinase alpha and glutamic acid decarboxylase immunoreactivity were colocalized: the cerebellar Purkinje cells and the commissural nucleus of the stria terminalis. The results imply that CaM II kinase alpha is primarily expressed in non-GABAergic neurons. In several regions CaM II kinase alpha mRNA is concentrated in nuclei known to contain populations of neurons that use excitatory amino acid transmitters.     

Dendritic localization of type II calcium calmodulin-dependent protein kinase mRNA in normal and reinnervated rat hippocampus.

In situ hybridization histochemistry has revealed a diffuse distribution of the alpha subunit of type II calcium calmodulin-dependent protein kinase (CaM II kinase alpha) mRNA in the neuropil of regions containing CaM II kinase alpha-expressing cells and has led some to propose that it may be expressed in dendrites. In order to determine if CaM II kinase alpha mRNA is expressed in dendrites and if the gene encoding CaM II kinase alpha is regulated in response to synaptic reinnervation, we examined its expression in the hippocampus of normal rats, of rats that had received a unilateral injection of kainic acid and of rats with a unilateral entorhinal cortex lesion. The relatively specific elimination of the CA3 pyramidal cells by kainate lesions precisely correlated with the loss of CaM II kinase alpha cRNA hybridization in the stratum radiatum as well as the stratum pyramidale. Following entorhinal cortex lesions, during the period of new synapse formation in the dentate gyrus, there was no detectable change in the level of CaM II kinase alpha gene expression. These data suggest that CaM II kinase alpha mRNA is expressed in the dendrites of hippocampal pyramidal cells and, therefore, is likely to be expressed in dendrites in other regions of the central nervous system exhibiting CaM II kinase alpha cRNA labeling in the neuropil. However, changes in expression were not found to accompany new synapse formation.     

小鼠脑缺血时自噬相关基因5的抗损伤作用

 目的: 观察自噬相关基因5(Atg5)在小鼠脑缺血再灌注损伤中的抗损伤作用。方法: 将雄性BALB/c小鼠随机分为假手术(sham)组、缺血再灌注(I/R)组、Atg5 siRNA组和control siRNA组。I/R组采用大脑中动脉阻塞(MCAO)60 min后再灌注24 h。Atg5 siRNA组和control siRNA组将5 μL Atg5 siRNA或scrambled siRNA在MCAO前24 h侧脑室注射。实时荧光定量PCR和Western blot检测Atg5的表达;2,3,5-氯化三苯基四氮唑(TTC)染色法检测抑制Atg5对缺血再灌注损伤后脑梗死面积和水肿率的影响;神经行为学评分法检测抑制Atg5对缺血再灌注损伤后神经症状的影响。结果: MCAO后再灌24 h,缺血半影区Atg5 mRNA和蛋白水平显著增高(P<0.05);Atg5 siRNA明显降低缺血再灌后Atg5 mRNA和蛋白的表达(P<0.05);侧脑室给予Atg5 siRNA能显著增加脑梗死面积和水肿率,并加重神经行为学损伤(P<0.05)。结论: 沉默Atg5加重小鼠脑缺血再灌损伤,提示MCAO后诱导的 Atg5 可减轻小鼠局灶性脑缺血再灌注损伤。     

Ca2+/calmodulin-dependent protein kinase II immunoreactivity in the rat hippocampus after forebrain ischemia

The influence of transient forebrain ischemia on the temporal alteration of Ca2+/calmodulin-dependent kinase II (CaM kinase II) in the rat hippocampus was analysed by the immunohistochemical method using antigen-affinity purified polyclonal antibodies against CaM kinase II of rat brain. Six to twenty-four hours after ischemia, CA1 and CA3 pyramidal cells, and dentate granule cells lost CaM kinase II immunoreactivity in neuronal perikarya, although immunoreactivity in the dendritic fields was preserved. The recovery of immunoreactivity of the CA3 pyramidal cells and dentate granule cells was noted 3 days after recirculation. Seven days after ischemia, immunoreactivity in the CA1 subfield was greatly reduced. These results suggest that CaM kinase II molecules in the CA1 subfield are preferentially located on the CA1 pyramidal cells and that CaM kinase II plays a critical role in the reconstruction of neuronal cytoskeleton and neuronal networks damaged by ischemic insult.     

醒脑静注射液对全脑缺血再灌注大鼠血脑屏障ZO-1表达的影响

目的:探讨醒脑静(XNJ)注射液对大鼠全脑缺血再灌注后血脑屏障通透性及紧密连接蛋白1(ZO-1)表达的影响。方法:采用改良Pulsinelli四血管闭塞法建立大鼠全脑缺血再灌注模型。将雄性Wistar大鼠随机分为4组,即假手术组、全脑缺血再灌注模型组、溶剂对照组和XNJ组。每组均在缺血再灌注后24 h、48 h和72 h处理。用干湿重法测定脑组织中水含量,分光光度计法检测脑组织伊文思蓝(EB)含量,Western blot检测大脑皮层的ZO-1蛋白含量。结果:缺血再灌注后24 h,模型组、溶剂对照组和XNJ组的脑组织含水量均显著高于假手术组(P0.05),但在缺血再灌注后48 h和72 h,模型组和溶剂对照组脑组织含水量显著高于XNJ组和假手术组(P0.05)。缺血再灌注后24 h,模型组、溶剂对照组和XNJ组大鼠脑组织内EB含量均高于假手术组(P0.05),缺血再灌注后48 h和72 h,假手术组和XNJ组的EB含量显著低于模型组和溶剂对照组(P0.05)。缺血再灌注后24h,模型组、溶剂对照组和XNJ组大鼠脑皮层中的ZO-1蛋白表达水平显著低于假手术组(P0.05),同样缺血再灌注后48 h和72 h,假手术组和XNJ组皮层中ZO-1蛋白含量显著高于模型组和溶剂对照组(P0.05)。结论:在缺血再灌注后的48 h和72 h,醒脑静注射液对血脑屏障具有保护作用,可能与醒脑静注射液上调ZO-1蛋白的表达有关。     

Early increase in mRNA levels of pro-inflammatory cytokines and their interactions in the mouse hippocampus after transient global ischemia

There is convincing evidence that cytokines are involved in the inflammatory response following cerebral ischemia, but the interactions among the pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in the early stage of ischemic reperfusion are not yet completely understood. In this study, we examined the early mRNA expressions of pro-inflammatory cytokines in the ischemic hippocampus after 30 min of bilateral common carotid artery occlusion in C57BL/6J wild-type (WT) and TNF-alpha, IL-1alpha/beta or IL-6 gene knockout (KO) mice utilizing real-time polymerase chain reaction. The mRNA expressions of the pro-inflammatory cytokines TNF-alpha, IL-6 and IL-1beta were significantly induced in ischemic WT mice compared with in the sham-operated mice. These increases peaked at 3 to 24 h for TNF-alpha, at 12 h for IL-1beta, and at 6 to 24 h for IL-6 after ischemia. The pattern of temporal expression of the cytokine mRNAs in ischemic gene KO mice, however, differed from that in WT mice. The TNF-alpha mRNA expression showed a similar temporal expression pattern in IL-6 KO mice compared to in WT mice following ischemic reperfusion, and the levels at all time points were lower than in WT mice. The IL-1beta mRNA level was very low in ischemic TNF-alpha KO mice and IL-6 KO mice in spite of a small peak observed in both at 24 h. The IL-6 mRNA level was significantly upregulated at all time points in both ischemic WT and TNF-alpha KO mice; however, the peak was delayed by 12-h in IL-1alpha/beta KO mice. In conclusion, the present study indicates that the rapid increases in cytokine levels are interdependent, interactive, and possibly modulate each other in the mouse hippocampus after transient global ischemia.     

局灶性脑缺血再灌注大鼠 Nogo-AmRNA的表达

为观察 Nogo-A m RNA在脑缺血再灌注后的动态变化 ,探索其在大脑皮质及纹状体神经元表达的意义及作用 ,应用原位杂交方法在大鼠局灶性大脑中动脉阻塞动物模型上观察脑缺血再灌注时大鼠 Nogo-A m RNA的表达。结果显示 :脑缺血再灌注大鼠缺血侧大脑皮质 Nogo-A m RNA的表达与假手术组比较明显增加 ,在 12 h内达高峰 (P<0 .0 1) ,2 4h时下降 ,48h时又升高 ,出现第二个高峰 ,然后逐渐降低 ,7～ 14 d降至基础水平 ;大脑纹状体脑缺血 1h后再灌流 ,2 h时 Nogo-A m RNA的表达明显增加 ,之后逐渐下降 ,到 2 4h时接近基础水平 ,48h时又升高 ,以后表达水平下降 ,3～ 14 d时 ,接近基础水平。结果提示 ,脑缺血后内源性 Nogo-A m RNA的表达呈动态性变化 ,参与了脑缺血后神经元的再生过程的调节。     

大鼠肢体缺血再灌注致肺损伤时肺组织中血红素氧合酶的表达

目的:观察大鼠肢体缺血再灌注致肺损伤时肺组织中HO-1表达的变化。方法:采用夹闭大鼠腹主动脉下段造成双下肢缺血和再灌注性肺损伤模型,分别采集假手术组,缺血4h组及缺血4h再灌注4、8、16、24、48h组肺组织标本,以Northern印迹、Western印迹及免疫组化分析,观察HO-1表达的变化。结果:假手术组和单纯缺血组未见HO-1mRNA表达;再灌注4h可见其表达信号,且随着再灌注时间延长表达信号逐渐增强,到16h时达到高峰,之后渐减弱,48h时已消失。蛋白表达水平与mRNA表达一致。免疫组化研究显示,HO-1阳性信号主要出现在气道上皮细胞、血管平滑肌细胞和肺泡巨噬细胞,而假手术组和单纯缺血组均未见阳性信号。结论:假手术及肢体的单纯缺血未诱发肺组织HO-1的表达,而肢体缺血再灌注可诱发其表达,并具有时相变化。     

Induction of cyclooxygenase-2 mRNA and protein following transient focal ischemia in the rat brain

Expression of cyclooxygenase-2 (cox-2) mRNA and inducible heat-shock protein-70 (hsp-70) mRNA was studied with in situ hybridization techniques at 30 min and 4 h following 1 h transient middle cerebral artery (MCA) occlusion in the rat brain. In addition, immunoreactivity for cox-2 was studied after 8 h of reperfusion. Induction of hsp-70 and cox-2 mRNA was found in the brain side ipsilateral to MCA occlusion. Hsp-70 mRNA was induced in the parietal cortex and striatum within the territory of the occluded MCA. Induction of cox-2 mRNA was particularly seen in cortical layer II in the brain side ipsilateral to MCA occlusion. At 30 min of reperfusion, areas showing cox-2 mRNA induction included the cingulate and frontal cortices located perifocally to the areas showing hsp-70 mRNA induction, and the piriform cortex. At 4 h of reperfusion, induction of cox-2 mRNA was seen within the parietal cortex. At 8 h of reperfusion, immunoreactivity for cox-2 was mainly seen in the ipsilateral cortex. These results demonstrate that transient focal ischemia induces the expression of cox-2 mRNA and protein in discrete areas of the rat brain during reperfusion, which might lead to local increases of arachidonic acid metabolism     

Neuregulin attenuated cerebral ischemia-Creperfusion injury via inhibiting apoptosis and upregulating aquaporin-4

It has been demonstrated that neuregulin-1beta (NRG-1beta) plays a neuroprotective role in cerebral ischemic injury, however, its defined mechanisms and the perfect treatment window are still elusive. Therefore, we established the animal model of MCAO/R to evaluate cerebral damage. As a result, neurological deficit scores were increased, and a small infarction focus could be seen in ischemic cortex in the control group at ischemic 0.5h/reperfusion 24h. With the duration of ischemia time, deficit scores and infarction sizes obviously elevated in the control group. A large number of positive-apoptotic cells were widespread in the ischemic cortex. Simultaneously, the expression of AQP-4 mRNA and its protein increased. NRG-1beta significantly improved neurological function, decreased the infarction volume, and elevated the expression levels of AQP-4 compared with that in the control group. The therapeutic effect of NRG-1beta was notable, especially at the ischemic 1.0h. These results demonstrate that NRG-1beta might play a neuroprotective effect on cerebral ischemia and reperfusion by inhibiting mitochondrial apoptosis pathway and regulating the activation of AQP-4. The perfect treatment window is at ischemic 1.0h after MCAO.     

3-硝基丙酸预处理对脑缺血耐受模型GLUT1和GLUT3表达的影响

目的: 探讨3-硝基丙酸(3-NPA)预处理对大鼠局灶性脑缺血半暗带区不同再灌注时点GLUT1和GLUT3 mRNA及蛋白水平表达的影响。方法: 雄性SD大鼠40只,随机分为假手术组(sham组,n=4)、预处理对照组(3-NPA组,n=4)、大脑中动脉缺血组(M组,n=16)、3-NPA 预处理组(IPC组,n=16)。M组和IPC组按再灌注时间(4 h、12 h、24 h及48 h)不同又分为4个亚组,每组动物4只。将大鼠在相应时点断头取脑,取缺血侧(左侧)冠状面中间1/3皮质,分别采用RT-PCR和Western blotting方法检测GLUT1、GLUT3 mRNA和蛋白水平表达情况。结果: IPC组GLUT1 mRNA表达在缺血再灌注后4 h开始升高,48 h最大,显著高于sham组和M组相应时点。IPC组GLUT3 mRNA表达在24 h增高,48 h最高,与M组相应时点24 h、48 h及sham组比较显著增高。IPC组比M组的GLUT1蛋白、GLUT3蛋白表达增高,有显著差异(F=5.848,P<0.05;F=6.295,P<0.05),尤以缺血再灌注后48 h两者差异最明显。结论: 3-NPA预处理能诱导脑缺血耐受,其机制可能是上调GLUT1和GLUT3 mRNA及蛋白表达水平,维持脑组织的能量供给。     

NGF对局灶性脑缺血再灌注后大鼠海马CHOP mRNA及蛋白表达的影响

探讨NGF对局灶性脑缺血再灌注后大鼠海马神经元CHOPmRNA及蛋白表达的影响。用线栓法制作大鼠右侧大脑中动脉阻塞再灌注模型(MCAO),应用原位杂交方法检测CHOPmRNA的表达;应用免疫组织化学SABC法检测CHOP蛋白表达;应用显微图像分析系统进行分析。结果显示:假手术组大鼠海马CHOPmRNA及蛋白表达极少;缺血组较假手术组CHOP表达mRNA和蛋白均显著增加(P<0.01),缺血再灌注3h后CHOPmRNA表达明显增加,24h达高峰,48h开始显著下降,72h接近对照组水平,CHOP蛋白在缺血再灌注12h时明显表达增高,24h达到高峰,72h显著下降,但仍高于对照组水平(P<0.01);在缺血再灌注12、24、48h,NGF组CHOPmRNA及蛋白表达明显低于缺血组(P<0.01)。本研究表明,外源性NGF能显著抑制局灶性脑缺血再灌注后CHOPmRNA及蛋白表达,提示NGF可能对脑缺血再灌注损伤后的海马神经元有保护作用。     

黄芪注射液对脑缺血再灌注大鼠海马神经元凋亡及JNK3表达的影响

目的:观察黄芪注射液对脑缺血再灌注大鼠海马神经元凋亡及c-Jun N末端激酶3(JNK3)表达的影响。方法:四血管阻断法制备脑缺血再灌注大鼠模型。设假手术组、脑缺血再灌注模型组(模型组)、脑缺血再灌注模型+黄芪注射液组(黄芪注射液组)和脑缺血再灌注模型+黄芪注射液溶剂对照组(溶剂对照组)。除假手术组外其余3组根据再灌注时间不同又分为0 h、0.5 h、2 h、6 h、24 h、72 h和120 h 7个亚组。采用TUNEL法检测海马神经元凋亡,Western blotting法检测海马组织JNK3蛋白变化,real-time PCR法检测海马组织JNK3 mRNA 的表达变化。结果:与假手术组比,模型组大鼠各个时点凋亡细胞数均增多(P<0.05);与模型组比,黄芪注射液组各个时点的细胞凋亡数明显减少(P<0.05),而黄芪注射液溶剂对照组各个时点的细胞凋亡数无明显变化(P>0.05)。除120 h外,模型组各时点海马组织JNK3蛋白及mRNA表达均较假手术组增加(P<0.05);与模型组相比,黄芪注射液可减弱除120 h之外的各时点JNK3 蛋白及mRNA的表达(P<0.05),而黄芪注射液溶剂对照组则无明显变化(P>0.05)。结论:黄芪注射液可抑制脑缺血再灌注大鼠海马神经元凋亡,其抗凋亡机制可能与下调JNK3 mRNA及蛋白表达有关。     

Relationship between metabolic dysfunctions, gene responses and delayed cell death after mild focal cerebral ischemia in mice.

The evolution of brain injury was examined in mice subjected to focal cerebral ischemia as induced by 30 min of intraluminar thread occlusion of the middle cerebral artery, followed by 3 h to 3 days of reperfusion. Metabolic dysfunctions were studied by 3H-leucine autoradiography for the measurement of cerebral protein synthesis and by regional ATP bioluminescent imaging. Metabolic changes were compared with responses of the genes c-fos, c-jun, heat-shock protein gene (hsp)72, p53-activated gene (pag)608 and caspase-3, which were investigated by in situ hybridization histochemistry and immunocytochemistry, and correlated with the degree of DNA fragmentation, as assessed by the terminal TdT-mediated dUTP-biotin nick end labeling method. Intraluminar thread occlusion led to a reproducible reduction of cerebral laser Doppler flow to 20-30% of control. Thread withdrawal was followed by a short-lasting post-ischemic hyperperfusion to approximately 120%. In non-ischemic control animals, fractional protein synthesis values of 0.81+/-0.26 and 0.94+/-0.23 were obtained. Thread occlusion resulted in a suppression of protein synthesis throughout the territory of the middle cerebral artery after 3 h of reperfusion (0.04+/-0.08 in caudate-putamen and 0.14+/-0.19 in somatosensory cortex, P<0.05). Protein synthesis partly recovered in the cortex after 24 h and 3 days (0.71+/-0.40 and 0.63+/-0.26, respectively), but remained suppressed in the caudate-putamen (0.14+/-0.22 and 0.28+/-0.28). Regional ATP levels did not show any major disturbances at the reperfusion times examined. Thread occlusion resulted in a transient increase of c-fos mRNA levels in ischemic and non-ischemic parts of the cortex and caudate-putamen at 3 h after ischemia, which suggests that spreading depressions were elicited in the tissue. At the same time, c-jun and hsp72 mRNAs were elevated only in ischemic brain areas showing inhibition of protein synthesis. C-fos and c-jun responses completely disappeared within 24 h of reperfusion. Hsp72 mRNA levels remained elevated in the cortex after 24 h, but decreased to basal values in the caudate-putamen. Twenty-four hours after reperfusion, pag608 and caspase-3 mRNA levels increased in the caudate-putamen, where protein synthesis rates were still reduced, and remained elevated even after 3 days. However, pag608 and caspase-3 mRNA levels did not increase in the cortex, where protein synthesis recovered. After 24 h and 3 days, functionally active p20 fragment of caspase-3 was detected in the caudate-putamen, closely associated with the appearance of DNA fragmented cells. Neither activated caspase-3 nor DNA fragmentation were noticed in the cortex.In summary, the suppression of protein synthesis is reversible in the ischemia-resistant cortex following 30 min of thread occlusion in mice, but persists in the vulnerable caudate-putamen. In the caudate-putamen, apoptotic programs are induced, closely in parallel with the manifestation of delayed cell death. Thus, the recovery of protein synthesis may be a major factor influencing tissue survival after transient focal ischemia.     

大鼠脑局灶性缺血再灌注后的细胞凋亡及bcl-2、bax蛋白表达的改变和银杏叶提取物干预的研究

目的探讨大鼠大脑中动脉重度栓塞再灌注动物的脑细胞凋亡及其相关调控基因bcl鄄2、bax阳性蛋白质表达的变化和银杏叶提取物(EGB)的治疗作用。方法采用插线法制作大鼠大脑中动脉栓塞后再灌注模型。将大鼠随机分为对照组、栓塞3h组、栓塞3h再灌注24h组、栓塞3h再灌注48h组、栓塞3hEGB干预、再灌注24hEGB干预组、再灌注48hEGB干预组。观察脑缺血/再灌注不同时点大鼠脑细胞凋亡(TUNEL法)、凋亡相关基因bcl鄄2、bax(S鄄P免疫组化法)的阳性表达和EGB对其治疗作用。EGB干预方法:术后2min腹腔注射,8h/次。结果大鼠大脑中动脉栓塞3h、栓塞3h/再灌注24、48h时,bcl鄄2、bax表达增强,细胞凋亡逐步加重,损伤加重。EGB干预后,bcl鄄2表达增强、bax表达减弱,细胞凋亡明显减少。结论细胞凋亡与bcl鄄2/bax有关,银杏叶提取物(EGB761)可以明显抑制大鼠脑组织缺血/再灌注后细胞凋亡的发生。     

Ca2+/calmodulin-dependent protein kinase II in the rat cerebellum: an immunohistochemical study with monoclonal antibodies specific to either alpha or beta subunit.

Monoclonal antibodies specific to either alpha or beta subunit of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) of the rat brain were produced and the distribution of each subunit in the rat cerebellum was examined immunohistochemically. Each antibody detected solely the corresponding subunit in immunoblot analysis of crude homogenates of the rat forebrain and cerebellum, and purified CaM kinase II from the rat forebrain. Immunoreactivity for alpha subunit was present selectively in Purkinje cells: perikarya, dendrites with their spines, axons and their terminal-like structures in the cerebellar cortex, cerebellar nuclei and lateral vestibular nucleus. Many of these alpha subunit-immunoreactive axons from the cerebellum were traced only through the inferior cerebellar peduncle. beta Subunit was detected in perikarya and dendrites of a limited number of Purkinje cells, many granule cells and neurons in the cerebellar nuclei. Thus, different distributions of alpha and beta subunits of CaM kinase II in the cerebellum were demonstrated.     

亚低温对脑缺血-再灌注损伤后PLC-γ 1mRNA的影响

目的探讨脑缺血-再灌注(I/R)损伤后PLC-γ1mRNA的表达变化及亚低温对脑损伤的保护作用。方法 70只雄性SD大鼠随机分为正常组、脑缺血-再灌注组(I/R)、亚低温组(SHP组),I/R、SHP组又分为24h、36h、72h亚组,每组10只动物。采用RT–PCR技术检测各时间点脑皮质中PLC-γ1mRNA的表达。结果正常组、I/R组、SHP组脑皮质中均有PLC-γ1mRNA的阳性表达,I/R组的阳性表达水平随损伤时间的延长逐渐下调,SHP组各时间点脑皮质PLC-γ1mRNA表达水平均高于I/R组。结论亚低温的脑缺血-再灌注损伤的保护作用可能与上调PLC-γ1mRNA表达相关。