Neuronal expression of the drug efflux transporter P-glycoprotein in the rat hippocampus after limbic seizures

In the brain, the efflux transporter P-glycoprotein (Pgp) is predominantly located on the luminal membrane of endothelial cells lining brain microvessels and forming the blood-brain barrier. Many lipophilic drugs, including antiepileptic drugs, are potential substrates for Pgp. Overexpression of Pgp in endothelial cells of the blood-brain barrier has been determined in patients with drug resistant forms of epilepsy such as temporal lobe epilepsy and rodent models of temporal lobe epilepsy and suggested to lead to reduced penetration of antiepileptic drugs into the brain. Expression of Pgp after seizures has also been described in astrocytes, whereas it is not clear whether neurons can express Pgp. In the present study, Pgp expression was studied by immunohistochemistry in rats 24 h after a status epilepticus induced by either pilocarpine or kainate, widely used models of temporal lobe epilepsy. Unexpectedly, in addition to endothelial Pgp staining, intense Pgp staining was found in neurons in the CA3c/CA4 sectors and hilus of the hippocampus formation, but not in other brain regions examined. The neuronal Pgp staining was confirmed by two different Pgp antibodies. Double immunolabeling and confocal microscopy showed that Pgp was colocalized with the neuronal marker neuronal nuclear antigen, but not with the glial marker glial fibrillary acidic protein. No neuronal Pgp staining was seen in control rats. The expression of Pgp in neurons after limbic seizures was substantiated by determining Pgp encoding genes (mdr1a, mdr1b) in neurons by real time quantitative RT-PCR. Increased Pgp expression in hippocampal neurons is likely to affect the action of drugs with intraneuronal targets and, in view of recent evidence from other cell types, could be associated with prevention of apoptosis which is involved in neuronal damage developing after seizures such as produced by pilocarpine.     

大鼠创伤性脑损伤后海马齿状回中脑脂结合蛋白的表达变化

Objective To investigate the change of brain lipid binding protein(BLBP) expression in hippocampus dentate gyrus(DG) at different time points after traumatic brain injury (TBI). Methods Seventy-two rats were divided into the injured group, sham group and control group randomly, and then were subjected to a lateral fluid percussion injury (FPI). Western blotting was used to detect BLBP expression. Brains were sectioned for immunofluorescence staining of BLBP and Vimentin at the time points of 1, 3, 7, 14 days after TBI.Results The results of Western blotting showed that the BLBP expression was lower than that of control group at 1 day post injury（EM>P/EM><0.01） and reached the peak compared with the other groups at 7 days after injury（EM>P/EM><0.01）, then descended at 14 days compared with control group after injury（EM>P/EM><0.01）. The changes of BLBP and Vimentin double-label positive cells were consistent with the results of Western blotting. The BLBP and Vimentin double-label positive cells were found mainly at the subgranular zone of ipsilateral injured hippocampus DG, and most of them were radial glia like cells; BLBP and Vimentin double-labelled positive cells were found at the hilus of DG at 7days after injury, and most of them looked like reactive astrocytes. Conclusion The expression of BLBP in DG after TBI decreased firstly, then increased and reached peak at 7 days after injury, decreased dramatically again at last. The     

Distribution of neuropeptides in the limbic system of the rat: the hippocampus

The distribution of several neuropeptides (vasoactive intestinal polypeptide, cholecystokinin octapeptide, substance P, neurotensin, methionine-enkephalin and somatostatin) in the hippocampal formation has been studied with immunocytochemical techniques. Numerous vasoactive intestinal polypeptide, cholecystokinin-octapeptide and somatostatin-positive cell bodies were found within the hippocampus and subiculum. Neurotensin-positive cell bodies were found within the subiculum, but no substance P or methionine-enkephalin-containing cell bodies were seen in either hippocampus proper or subiculum. Vasoactive intestinal polypeptide and cholecystokinin-octapeptide-positive cell bodies were predominantly located in the stratum moleculare and stratum radiatum of CA 1-2 regions and dentate gyrus, whilst somatostatin-containing cell bodies were found mainly in the stratum oriens. Nerve fibres containing each of the six peptides were found within the hippocampus. Large numbers of vasoactive intestinal polypeptide, cholecystokinin-octapeptide and somatostatin fibres innervated the pyramidal and granule cell layers, with smaller numbers in the stratum radiatum and fewer still in the stratum moleculare and stratum oriens. Other than a moderately dense neurotensin-positive fibre plexus in the dorsal subiculum, fewer neurotensin, substance P and methionine-enkephalin fibres were present. However, when present, these three peptides had a distribution restricted to a region close to the pyramidal layer in the CA 2/3 region and to the stratum moleculare of the CA 1 region. Peptide-containing fibres were identified entering or leaving the hippocampus in three ways, via (i) the fornix (all six peptides), (ii) the dorsal subiculum (neurotensin-positive fibres projecting to the cingulate cortex: somatostatin, vasoactive intestinal polypeptide, and cholecystokinin-octapeptide present in fibres running between the dorsal subiculum and occipito-parietal cortex) and (iii) the ventral subiculum (vasoactive intestinal polypeptide, cholecystokinin-octapeptide and somatostatin in fibres running between entorhinal cortex and hippocampus, and all six peptides in fibres crossing the amygdalo-hippocampal border). These findings indicate a major distinction between those peptides (vasoactive intestinal polypeptide, cholecystokinin-octapeptide, somatostatin, neurotensin) which are found in cell bodies intrinsic to the hippocampal formation and those peptides (substance P, methionine-enkephalin) which are found only in hippocampal afferents.(ABSTRACT TRUNCATED AT 400 WORDS)     

Statistical shape analysis of the rat hippocampus in epilepsy

In this study, we aimed to (1) propose landmarks for the hippocampus in a rat brain using an experimental study and (2) investigate hippocampus shape changes in a rat brain with epilepsy using the statistical shape analysis method. We have used the statistical shape analysis method to illustrate hippocampal shape deformation due to epilepsy. Statistical shape analysis is of increasing interest to the neuroimaging community because of its potential for locating morphological changes. Nineteen rat brains (ten healthy and nine epileptic) with hematoxylin and eosin images of the hippocampus were used. The results strongly indicated that the normalized hippocampal shape of the epileptic group was different from the nonepileptic group; deformation was seen most significantly in the medial regions of the cornu ammonis (CA1 and CA3) of the hippocampus. In conclusion, our landmark-based methodology detected regional differences in the hippocampus in epilepsy. This study may serve as an initial reference for future studies on shape alteration of the hippocampus associated with certain medical conditions.     

孤独症大鼠海马自噬相关蛋白的表达

目的 探讨细胞自噬在孤独症发病中的作用。 方法 健康繁殖期Wistar雌鼠20只(250～260g),雄鼠10只(280～290g),按2∶1比例合笼过夜,次日早上阴道涂片发现精子计为孕1d。在其孕12.5d时,10只雌鼠腹腔注射丙戊酸钠,其子代为孤独症模型组;10只注射同等剂量生理盐水,其子代为正常组。通过比较自梳理实验、三箱实验验证孤独症模型是否成功;Western blotting方法对比正常组与孤独症模型组大鼠生后42d（P42）海马自噬相关蛋白LC3-Ⅱ、Beclin-1和P62表达的变化。 结果 1.成功建立孤独症动物模型,与正常组比较,自梳理实验显示, 模型组理毛时间较正常显著增加（P<0.05）,三箱实验结果显示,模型组大鼠交互能力障碍、缺乏对新鲜事物的偏好性;2.与正常组相比较,模型组大鼠P42d海马自噬相关蛋白LC3-Ⅱ表达显著降低（P<0.05）,Beclin 1表达显著降低（P<0.05）,P62表达显著升高（P<0.05）。 结论 孤独症大鼠P42d海马自噬活性降低,提示增强自噬可能是一个潜在的治疗策略。     

单纯性脑震荡大鼠海马mPGES-1的表达

目的:观察单纯性脑震荡(pure cerebral concussion,PCC)大鼠脑内海马区膜结合型前列素E合酶1(membrane-associated prostaglandin E synthase-1,mPGES-1)的表达变化,探讨mPGES-1是否通过前列腺素代谢途径参与PCC的病理变化及其变化规律。方法:采用清醒状态下自制金属单摆闭合式脑损伤打击装置制备PCC模型,致伤后随机分为3 h、12 h、1 d、2 d、3 d、7 d组(n=5),另设正常对照组(n=5)。采用mPGES-1多克隆抗体进行免疫组织化学法和Western blot技术,检测PCC组和正常组大鼠海马CA1-4区mPGES-1的定位及定量情况。结果:正常状态下,海马CA1-4区mPGES-1阳性细胞极少,轮廓不清,致伤后3 h海马各区的mPGES-1表达水平开始增高,至3 d达高峰,与正常组比较有显著性差异(P<0.05),3 d后逐渐下降,至7 d接近正常水平。mPGES-1蛋白的变化规律和免疫组化结果相似。结论:PCC损伤早期海马区出现mPGES-1表达增强,提示mPGES-1通过前列腺素代谢途径参与PCC致伤后的病理变化。     

大鼠海马tau蛋白过度磷酸化时细胞周期蛋白cyclin D1的表达

目的:探讨大鼠海马tau蛋白过度磷酸化时细胞周期蛋白cyclin D1的表达.方法:用cAMP-依赖性蛋白激酶 A (PKA) 的激动剂Forskolin注射大鼠侧脑室,免疫印迹和免疫组织化学技术检测大鼠海马tau蛋白的磷酸化水平,同时采用免疫印迹、免疫组织化学和RT-PCR方法检测海马cyclin D1和cyclin D1 mRNA.结果:侧脑室注射Forskolin 48h,大鼠海马tau蛋白Ser214、Ser396和Ser202/Thr205位点的磷酸化水平升高,同时检测到细胞周期蛋白cyclin D1和cyclin D1 mRNA.结论:侧脑室注射Forskolin诱导大鼠海马 tau蛋白过度磷酸化的同时,大鼠海马出现细胞周期蛋白cyclin D1的表达,提示了tau蛋白过度磷酸化与细胞周期相关蛋白cyclin D1之间可能的内在联系.     

边缘系统相关膜蛋白在大鼠脑纹状体边缘区表达的Western blot研究

目的　研究边缘区与边缘系统是否存在共同的分子特征。方法　用Westernblot方法检测边缘区及有关脑区中边缘系统标志分子———边缘系统相关膜蛋白的表达。结果　边缘区及边缘系统脑区边缘系统相关膜蛋白有强表达 ,而非边缘系统脑区甚少或无表达。结论　边缘区与边缘系统具有共同的分子特征 ,边缘区可能是边缘系统的一个组成部分     

Caspase 6 expression in the rat hippocampus during epileptogenesis and epilepsy

The molecular basis of neuronal circuit reorganization during epileptogenesis is poorly understood. Such data are, however, critical for the search of new targets for the prevention of epileptogenesis. Here, we extended our previous studies on caspases in epileptogenesis by investigating the expression and activity of caspase 6 at different phases of the epileptic process in rats. Epileptogenesis was triggered by kainate-induced status epilepticus (SE) under video-electroencephalography-monitoring. Caspase 6 activity was measured fluorometrically in the hippocampus 8 h, 24 h, 48 h, 1 week, and 4 weeks after SE. Caspase 6 expression was examined using Western blot and immunohistochemistry. Our data demonstrated that the SE-induced increase in the expression of cleaved caspase 6 and its intraneuronal localization were dependent on the time delay from SE induction. Double-labeling with a neuronal marker, NeuN, indicated that within the first 48 h, caspase 6 immunoreactivity was present both in the hippocampal pyramidal cells and hilar neurons, some of which were also terminal transferase dUTP-end labeling-positive. This was coincident with a transient 18-fold increase in caspase 6 enzymatic activity. At the 1-week and 4-week time points, elevated caspase 6 immunoreactivity was detected in the dendritic processes and neuropil. These findings indicate that caspase 6 expression remains elevated long after the occurrence of acute cell death during epileptogenesis and epilepsy. Further, caspase 6 protein is not exclusively located in the somata of neurons, but also in dendrites. These data suggest that caspase 6 has functions other than execution of programmed cell death in epileptogenic hippocampus.     

单纯性脑震荡大鼠海马OX-42阳性小胶质细胞的表达

目的:观察单纯性脑震荡(pure cerebral concussion,PCC)大鼠脑内海马小胶质细胞(microglia,MG)的反应和变化,探讨小胶质细胞是否参与脑损伤后的病理变化以及变化规律。方法:采用清醒状态下自制金属单摆闭合式脑损伤打击装置制备PCC模型,致伤后随机分为3 h、12 h、1 d、2 d、3 d、7 d组(n=5),另设正常对照组(n=5)。采用小鼠抗鼠OX-42单克隆抗体(MG特异性标记物)进行免疫组织化学SP法(streptavidin-peroxidase,SP)和免疫荧光染色,观察PCC组和正常组大鼠海马CA1～4区和上、下齿状回OX-42的表达变化。结果:正常状态下,OX-42表达很少甚至很难发现,PCC组大鼠海马CA1～4区和上、下齿状回的OX-42的表达呈现伤后逐渐增高趋势,与正常组比较有显著性差异(P<0.05),7 d后有下降趋势,但仍高于正常组。结论:PCC损伤早期海马MG出现激活后的形态和数量的变化,提示MG参与PCC致伤后的病理变化。     

丙戊酸钠处理对于癫痫大鼠模型海马内细胞自噬的影响

目的:研究丙戊酸钠(VPA)对癫痫模型大鼠海马内细胞自噬、动物脑电图和行为学的影响。方法:制作癫痫大鼠模型并随机分为癫痫组、3甲基腺嘌呤(3MA)组、VPA组及VPA联合3MA组四组,设正常大鼠为对照组。观察各组行为学及脑电图变化,HE及Nissl染色显示神经元的损伤情况,免疫组织化学染色、Western Blot、Real-time PCR检测自噬标记物微管相关蛋白1轻链3(LC3)表达。结果:行为学观察及脑电图检测显示,丙戊酸钠组及丙戊酸钠联合3MA组均可降低癫痫发作等级,3MA组无抗癫痫效果。HE及Nissl染色显示,对照组未见明显异常,癫痫组神经元损伤严重,丙戊酸钠组与癫痫组相比,神经元损伤明显减轻;3MA组与癫痫组相比,神经元损伤明显减轻;丙戊酸钠联合3MA组与丙戊酸钠组相比,神经元损伤明显减轻。免疫组织化学染色、Western Blot、Real-time PCR检测显示,对照组LC3呈低表达,癫痫组LC3表达明显高于对照组;丙戊酸钠组LC3表达明显高于癫痫组;3MA组LC3表达明显低于癫痫组;丙戊酸钠联合3MA组LC3表达明显低于丙戊酸钠组。结论:VPA处理能够诱导癫痫大鼠海马内细胞自噬增加和导致神经元损伤。     

Expression of nitric oxide synthase in the developing rat hippocampus

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry was used to study the development of neurons synthesizing nitric oxide (NO) in the postnatal rat hippocampus. We show that NADPH-d reactive somata and processes are present from the day of birth until adulthood in the Ammon's horn. The dentate gyrus, however, has a more delayed period of nitric oxide synthase (NOS) expression with the staining appearing only by the end of the first postnatal week. Our results suggest that the time course of NOS expression correlates with the developmental sequence of events described in the hippocampus and that NO could be involved in the development of connections in this structure.     

颞叶癫痫大鼠海马差异表达基因和蛋白质的筛选研究

目的：寻找颞叶癫痫大鼠海马组织的差异表达基因和蛋白质，以期为进一步探讨颞叶癫痫的发病机制，寻找新的治疗靶点和研发新的治疗手段奠定基础。方法:运用cDNA微阵列、二维电泳和MALDI-TOF-MS技术，分析氯化锂-匹罗卡品（LiCl-PILO）致痫大鼠模型海马组织的基因表达谱和蛋白质表达谱，并对发现的差异表达基因和差异表达蛋白质进行分析和鉴定结果和。结论：发现LiCl-PILO致痫大鼠海马组织中192个基因差异表达，159条可在GenBank中登陆，其中表达上调的基因84条，表达下调的基因75条；筛选到78个差异表达蛋白质斑点，其中31个在癫痫组表达下调，47个在癫痫组表达上调。有5个蛋白质最终鉴定确认。本研究结果为运用蛋白质组学方法寻找癫痫治疗新靶点研究提供实验依据。     

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.     

Caspase-9在生后小鼠海马发育中的表达

目的观察小鼠海马发育过程中凋亡相关基因caspase-9的表达变化。方法取出生后1d、3d、7d、14d、21d和28d的小鼠海马,应用免疫组织化学技术、免疫印迹技术及图像分析技术检测caspase-9的表达变化。结果 P1 d海马CA区锥体细胞层和齿状回颗粒细胞层caspase-9阳性表达产物平均光密度最高,P3 d~P21 d caspase-9阳性表达产物平均光密度逐渐降低,P28表达水平最低。结论凋亡相关基因caspase-9在生后小鼠海马发育过程表达水平呈逐渐降低的趋势。     

Differential expression of small heat shock protein 27 in the rat hippocampus and septum after fimbria-fornix lesion

Preceding or immediately following fear-conditioning rats were exposed for 30 min to either a sham field, one of two symmetrical (sine-wave 7, 20 Hz) magnetic fields or to one of two complex magnetic fields whose waveforms were modeled after salient electrophysiological patterns within either the hippocampal formation (theta-burst) or the amygdaloid complex (burst-firing). The magnetic fields were presented in successive 2s intervals through each of the three spatial planes and then simultaneously within all three planes. Field strengths ranged between 0.5 and 1 microTesla. Only the group exposed after the conditioning to the theta-burst (hippocampal) magnetic fields displayed evidence of forgetting, as inferred by their marked attenuation of freezing behavior, during contextual extinction 24h later. This powerful treatment explained 75% of the variance in the extinction scores. Behavioral responses to the discrete conditioned stimulus were not affected. These findings are consistent with the involvement of the hippocampus in learned fear to contextual stimuli but not to discrete auditory stimuli and suggest that physiologically relevant stimuli may be delivered to the brain by weak, complex magnetic fields whose intensities are ubiquitous within modern environments.     

大鼠海马γ-氨基丁酸-A受体在生后发育中的表达

目的:研究大鼠生后发育过程中海马组织γ-氨基丁酸-A(GABA-A)受体的表达规律。方法:用免疫组织化学和PCR技术,检测不同年龄大鼠海马组织GABA-A受体及编码该受体的mRNA表达,并用图像分析方法进行定量研究。结果:大鼠海马组织在生后3 d已经出现GABA-A受体免疫反应,以后逐渐增强,到生后30 d达到最高值,海马各区GABA-A受体免疫反应强度没有显著的差别;编码GABA-A受体的mRNA表达也有类似的增龄性增多,但其表达高峰值提前到14 d。结论:生后大鼠海马GABA-A受体表达在一定时期内呈增龄性表达增强的趋势。     

Caspase-3在出生后大鼠海马中的表达

目的 探讨Wistar大鼠海马生后发育过程中,活化的Caspase-3与凋亡之间的关系. 方法 应用免疫荧光方法观测活化的Caspase-3和赫斯特荧光染料33342(Hoechst 33342)在生后不同时期大鼠海马CA1、CA3区和齿状回(DG)中的表达情况. 结果 在CA1区,活化的Caspase-3的表达在生后7d(P7)达到高峰;在CA3区,P2达到高峰,然后逐渐减弱.在DG,P7后又有所增强,到P14达到高峰,并在所观测的其余时段维持此水平.观测的3个区的凋亡细胞数目都在P7达到高峰,然后逐渐减少. 结论 在大鼠海马生后发育过程中,活化的Caspase-3的表达存在特定的时空格局.活化的Caspase-3在CA1区与CA3区有丝分裂后期细胞和DG神经前体细胞中的作用和机制不同.     

Expression of the neuronal calcium sensor visinin-like protein-1 in the rat hippocampus

Visinin-like protein-1 (VILIP-1) belongs to the neuronal calcium sensor (NCS) family of EF-hand Ca(2+)-binding proteins which are involved in a variety of Ca(2+)-dependent signal transduction processes in neurons. VILIP-1 has been implicated in the pathology of CNS disorders including Alzheimer's disease and schizophrenia, but its expression has also been found to be regulated following induction of hippocampal synaptic plasticity underlying learning and memory processes. VILIP-1 is strongly expressed in different populations of principal and non-principal neurons in the rat hippocampus. VILIP-1-containing interneurons are morphologically and neurochemically heterogeneous. On the basis of co-localizing markers, VILIP-1 is rarely present in perisomatic inhibitory parvalbumin containing cells. However, VILIP-1 is frequently expressed in mid-proximal dendritic inhibitory cells characterized by calbindin immunoreactivity, and most strongly co-expressed in calretinin-positive disinhibitory interneurons. Partial co-localization of the metabotropic glutamate receptor mGluR1alpha with VILIP-1 was often found in interneurons located in the stratum oriens of the hippocampal CA1 region and in hilar interneurons. Partial co-localization of alpha4beta2 nicotinic acetylcholine receptor with VILIP-1 was seen in stratum oriens interneurons and particularly at the border of the hilus in the dentate gyrus, where VILIP-1 also strongly co-localized with calretinin. We speculate that depending on the regulation of the expression of VILIP-1 in hippocampal pyramidal cells or defined types of interneurons, it may have different effects on hippocampal synaptic plasticity and network activity in health and disease.