红藻氨酸致癫痫持续状态大鼠海马neuropilin-2 mRNA及其蛋白表达的研究

目的研究轴索导向分子NPN-2mRNA及其蛋白对癫痫持续状态(SE)后大鼠海马内神经纤维外向性生长和突触重建中的调控作用。方法采用侧脑室内注射红藻氨酸(KA)制作TLE大鼠模型,用Nissl染色、原位杂交和免疫组织化学的方法,分别检测致SE后1d、1w、2w、3w、4w大鼠海马齿状回(DG)、CA1区、CA3区、门区神经元丢失程度以及NPN-2mRNA及其蛋白的表达。结果 KA致SE后1d开始出现神经元丢失,至4w神经元丢失明显增多。KA致SE后1d,NPN-2mRNA及其蛋白在DG和CA1区表达明显下降,持续至3w(P0.01),4w恢复至正常(P0.05);NPN-2mRNA及其蛋白在门区、CA3区表达实验组与对照组无明显差别(P0.05)。结论 KA致SE后,海马DG及CA1区神经元下调NPN-2mRNA及其蛋白的表达,促进DG及CA1区神经纤维外向性生长和突触的重建。     

红藻氨酸致颞叶癫痫大鼠海马内Semaphorin3C、Semaphorin3F基因表达的研究

目的研究神经轴索导向分子Sem aphorin3C(Sem a3C),Sem aphorin3F(Sem a3F)mRNA对颞叶癫痫(TLE)大鼠海马神经轴索环路重建的调控作用。方法采用侧脑室内注射红藻氨酸(KA)制作TLE大鼠模型,用N issl染色及原位杂交的方法,分别检测致痫后1d、1w、2w、3w、4w大鼠海马的齿状回(DG),CA1区、CA3区神经细胞丢失程度以及Sem a3C、Sem a3F mRNA的表达。结果KA致痫后1d始出现神经元丢失,至4w神经元丢失明显增多。KA致痫后1w,Sem a3C、Sem a3F mRNA在海马的CA1区、Sem a3F mRNA在海马的CA3区表达明显下降,持续至3w(P<0.01),4w时恢复至正常(P>0.05);Sem a3C、Sem a3F mRNA在DG的表达,Sem a3C在CA3区的表达,实验组与对照组均无明显差别(P>0.05)。结论KA致痫后海马CA1区神经元下调Sem a3C、Sem a3F mRNA的表达,CA3区神经元下调Sem a3F mRNA的表达,可能促进TLE大鼠海马神经轴索环路重建。     

海人酸致痫大鼠海马IL-1β、TNF-α的表达

目的 立体定向手术建立海人酸(KA)颞叶癫痫大鼠模型,检测海马内IL-1β、TNF-α蛋白及其mRNA的表达.方法 大鼠随机分为空白对照组、生理盐水对照组和模型组.模型组大鼠一侧海马CA3区注射KA (生理盐水组注射生理盐水),观察其行为学特征,HE染色和Nissl染色以及电镜观察其病理学改变,免疫组化法检测大鼠海马内IL-1β、TNF-α蛋白的表达,原位杂交法检测TNF-α mRNA的动态表达.结果 大鼠注射KA后出现湿狗样抖动、头面部肌阵挛、肢体阵挛及全面强直阵挛发作等,病理结果显示海马神经元变性、缺失及胶质细胞增生,模型组IL-1β在致痫后3 、6 h表达水平明显增加并于12 h达高峰,之后逐渐下降,7 d后与对照组相比差异无统计学意义(P＞0.05);TNF-α蛋白与mRNA表达时程基本一致,3 h出现,12 h达高峰,而后逐渐下降,7 d后回归至对照组表达水平,15 、30 d又高于对照组(P＜0.05).结论 (1)大鼠一侧海马注射KA是人类颞叶癫痫理想的动物模型;(2)内源性IL-1β、TNF-α参与了癫痫发病机制.     

低频重复经颅磁刺激对颞叶癫痫大鼠海马NF-κB及COX-2表达的影响

目的 研究低频重复经颅磁刺激(rTMS)对颞叶癫痫模型大鼠海马核转录因子κB(NF-κB)和环争化酶2(COX-2)表达的影响,进而从炎症反应角度探讨rTMS治疗癫痫的可能机制.方法 30只雄性SD大鼠随机分为颞叶癫痫刺激组(TIE+rTMS)、颢叶癫痫假刺激组(TLE+s-rTMS)和生理盐水对照组(NS),每组10只.利用立体定位仪向大鼠海马CA,区微量注射海人酸(KA)制备颞叶癫痫模型,对照组于同部位注射等量生理盐水.刺激组连续接受rTMS治疗10d.采用免疫组织化学染色、蛋白质印迹法(western blotting)研究大鼠海马NF-κBp65和COX-2表达情况.结果 与生理盐水对照组大鼠比较,造模后大鼠海马组织中NF-κBp65和COX-2表达明显增强NF-κBp65核移位增多,差异均有统计学意义(P＜0.05),TLE+rTMS组大鼠海马组织中NF-κBp65和COX-2表达较TLE+s-rTMS组明显降低,NF-κBp65核移位减少,差异均有统计学意义(P＜0.05).结论 rTMS可能通过降低癫痫大鼠海马NF-κB和COX-2的表达,阻止NF-κB核移位,从而抑制炎症反应发挥抗癫痫作用.     

α-细辛醚对KA癫痫大鼠海马组织中Bax、Bcl-2的影响

目的 探讨不同浓度的α-细辛醚对KA癫痫大鼠海马组织中细胞凋亡调节基因Bax、Bcl-2的mRNA及其蛋白表达的影响.方法 红藻氨酸(kainic acid,KA)侧脑室注入SD大鼠制备癫痫模型,随机分为模型对照组(KA组)、α-细辛醚低浓度组(6mg/kg)、中浓度组(12mg/kg)和高浓度组(24mg/kg),另设假手术组为正常对照组,每组10只.α-细辛醚组经腹腔注射连续给药10d后检测大鼠海马组织中Bax、Bcl-2的mRNA及其蛋白的表达.结果 α-细辛醚用药后,大鼠表现为明显的镇静、抗惊厥作用;与正常对照组相比,KA组、α-细辛醚低、中浓度组大鼠海马区Bax的mRNA及蛋白表达升高,差异有统计学意义(P均＜0.05),α-细辛醚高浓度组Bax的mRNA及蛋白表达下降(P＜0.05);与正常对照组相比,Bcl-2的mRNA及蛋白表达KA组显著降低,α-田辛醚中、高浓度组升高(P均＜0.05),低浓度组与对照组相比差异不明显(P＞0.05).结论 KA诱导的SD癫痫大鼠神经元存在Bax、Bcl-2的异常表达,α-细辛醚可能通过降低Bax和提升Bcl-2基因的表达从而减少SD癫痫大鼠神经元凋亡.     

难治性颞叶癫痫脑组织IL-6、IL-6受体及sIL-6R、 sgp130的表达及意义

目的 探讨IL-6、IL-6膜受体(IL-6R)及其下游信号转导相关分子在颞叶癫痫(TLE)发生中的作用. 方法 选择自2010年1月至2010年12月在第三军医大学新桥医院神经外科行手术治疗的TLE患者40例(TLE组),同期行手术治疗的外伤、高血压脑出血等患者40例(对照组);收集其手术过程中切除的颞叶组织.利用RT-PCR、ELISA等方法分析IL-6、IL-6R、sIL-6R以及sgp130的mRNA或蛋白水平的表达变化情况. 结果 TLE组和对照组中均可检测到IL-6和IL-6R mRNA的表达,但TLE组致病灶中IL-6和IL-6R mRNA水平均显著高于对照组,差异有统计学意义(P＜0.05).ELISA结果提示,sIL-6R在TLE组致痫灶中的表达量与对照组比较明显增高,差异有统计学意义(P＜0.05);sgpl30的含量略高于对照组,差异没有统计学意义(P＞0.05). 结论 局部高浓度的IL-6通过经典信号转导或者跨信号转导机制作用于TLE病灶细胞,影响这些细胞的生物学功能,参与癫痫发生过程.     

雷公藤内酯醇对癫痫大鼠电压门控钾通道kv1.1表达的影响

目的 探讨雷公藤内酯醇(TL)对癫痫大鼠神经的保护作用及其机制.方法 60只SD大鼠分成对照组、模型组、雷公藤组,每组各20只.雷公藤组大鼠腹腔注射TL(每日15μg/kg),模型组大鼠腹腔注射等量生理盐水,注射7d后,雷公藤组与模型组通过颈内皮下注射海人酸(KA)致痫,对照组则颈内皮下注射等量的生理盐水.用免疫组化和Western blot方法检测大鼠海马CA3区瞬时外向钾离子通道kv1.1蛋白表达.结果 模型组大鼠海马CA3区kv1.1蛋白表达水平低于对照组(P＜0.05);雷公藤组海马CA3区kv1.1蛋白表达高于模型组(P＜0.05);雷公藤组与对照组大鼠kv1.1蛋白表达水平无明显差异(P＞0.05).结论 TL对KA致痫大鼠神经元有保护作用,其作用的发挥可能与TL可增加海马CA3区神经元kv1.1的表达有关.     

KA致痫大鼠脑组织中N-cadherin mRNA的表达及意义

目的探讨癫痫鼠脑组织中N-cadherin mRNA的表达及意义。方法通过原位杂交方法对KA致痫鼠脑组织中N-cadherin mRNA的表达进行检测。结果对照组、手术对照组颞叶及海马均可见明显的N-cadherin mRNA的表达，但海马区明显高于颞叶。在充分点燃后12h，N-cadherin mRNA的表达明显下降，CA3区下降的最早且明显，在点燃后7d逐渐恢复，在14d时接近正常值，在28d高于对照组、手术对照组。结论N-cadherin是突触重建的重要的调控因子，是新的突触形成的介质，在癫痫的发生、发展中起着重要的调控作用。     

海人酸致痫大鼠海马IL-1β、TNF-α仪的表达

目的立体定向手术建立海人酸（KA）颞叶癫痫大鼠模型，检测海马内IL-1β、TNF-α蛋白及其mRNA的表达。方法大鼠随机分为空白对照组、生理盐水对照组和模型组。模型组大鼠-侧海马CA3区注射KA（生理盐水组注射生理盐水），观察其行为学特征，HE染色和Nissl染色以及电镜观察其病理学改变，免疫组化法检测大鼠海马内IL-1β、TNF-α蛋白的表达，原位杂交法检测TNF-α mRNA的动态表达。结果大鼠注射KA后出现湿狗样抖动、头面部肌阵挛、肢体阵挛及全面强直阵挛发作等，病理结果显示海马神经元变性、缺失及胶质细胞增生，模型组IL-1β在致痫后3、6h表达水平明显增加并于12h达高峰，之后逐渐下降，7d后与对照组相比差异无统计学意义（P〉0.05）；TNF-α蛋白与mRNA表达时程基本一致，3h出现，12h达高峰，而后逐渐下降，7d后回归至对照组表达水平，15、30d又高于对照组（P％0.05）。结论（1）大鼠-侧海马注射KA是人类颞叶癫痫理想的动物模型；（2）内源性IL-1β、TNF-α参与了癫痫发病机制。     

自发性癫痫大鼠脑内神经肽Y受体Y2R和Y5R的表达及分布

目的研究自发性癫痫大鼠(tremor rat,TRM)海马和颞叶皮质中神经肽Y(neuropeptide Y,NPY)Y2和Y5受体(Y2R和Y5R)的表达和分布。方法以TRM大鼠作为癫痫组,正常Wistar大鼠作为对照组,每组7只,以RT-PCR法检测Y2R和Y5R mRNA水平表达,Western Blot法检测其蛋白水平表达,免疫荧光法分析癫痫组和对照组大鼠海马CA1、CA3和齿状回(DG)区以及颞叶皮质中Y2R与Y5R的分布和定位。结果RT-PCR和Western Blot结果显示,与对照组大鼠相比较,癫痫组海马和颞叶皮质中Y2R mRNA相对表达水平(相对灰度值为海马:0.75±0.06 vs.0.51±0.07;颞叶皮质:0.70±0.05 vs.0.55±0.03)及蛋白相对表达水平(相对灰度值为海马:0.79±0.08 vs.0.42±0.05;颞叶皮质:0.72±0.05 vs.0.51±0.07)均显著上调(均P0.01),Y5R mRNA(相对灰度值为海马:0.52±0.10 vs.0.54±0.06;颞叶皮质:0.46±0.03 vs.0.42±0.04)及蛋白(相对灰度值为海马:0.28±0.06 vs.0.27±0.03;颞叶皮质:0.31±0.05 vs.0.27±0.07)表达均没有明显变化(均P0.05)。免疫荧光分析发现Y2R与Y5R在癫痫组大鼠海马CA1、CA3区神经元和DG区颗粒细胞以及颞叶皮质神经元中分布广泛且主要定位在细胞膜上。结论在TRM中Y2R表达在海马和颞叶皮质中均表达上调,但是Y5R的表达没有明显改变。     

青年脑卒中268例病因分析

目的 探讨青年脑卒中的病因,提高预防意识.方法 回顾性分析268例青年脑卒中患者的病因,其中67例患者接受了数字减影血管造影（DSA）检查.结果 本组268例青年脑卒中占我院同期全部住院脑卒中病例的9.92%（268/2 701）,青年脑卒中的发病率男性比例明显高于女性.268例中有明确病因者200例（74.63%）,病因不明者68例（25.37%）.在268例青年脑卒中患者中,缺血性脑卒中129例,占48.13%.其中有明确病因者101例,占77.51%,包括动脉粥样硬化65例,占50.38%;栓塞性脑血管病18例,占13.95%;非动脉硬化性血管病10例,占7.75%;凝血机制异常4例,占3.1%;疑似遗传性脑动脉病1例,占1.55%;偏头痛性脑梗死3例,占2.32%;出血性脑卒中139例,占51.86%.其中有明确病因者99例,占71.22%,包括高血压病53例,占38.12%;颅内血管发育异常（动脉瘤、动静脉畸形、烟雾病、海绵状血管瘤、脑膜动静脉瘘）37例,占26.62%;其他病因（如颅内肿瘤等）9例,占6.48%,病因不明40例,占28.78%.结论 青年缺血性脑卒中最主要的病因是动脉粥样硬化,其次是栓塞性脑血管病,其他病因如血管炎、烟雾病等较少见.青年出血性脑卒中最主要的病因是高血压病,其次是颅内血管发育异常.     

Basal expression of c-Fos and Zif268 in the rat basal ganglia: immunohistochemical characterization of striatal Zif268-positive neurons

Basal expression of the protein products of the inducible immediate early genes (IEGs), c-Fos and Zif268, was investigated in five regions of the rat basal ganglia using immunohistochemistry. In particular, high basal levels of Zif268 but very low levels of c-Fos were seen in the caudate-putamen (CPu). Double immunostaining revealed that many of the constitutively expressed Zif268-positive neurons were GABAergic but very few were cholinergic or neuronal nitric oxide synthase (nNOS)-positive, and some of the Zif268-positive neurons were also immunopositive for a glutamate NMDA receptor subunit NR1 or NR2A. No regional difference between the medial and lateral parts of the CPu was observed in the cellular phenotypes of Zif268-positive neurons. Almost no basal levels of Zif268 were seen in the other four regions: the globus pallidus, entopeduncular nucleus, subthalamic nucleus and substantia nigra pars reticulata. As in the CPu, negligible levels of c-Fos were seen in these four regions. Differential expression of these two IEGs may suggest gene-specific and region-specific functions of c-Fos and Zif268 in the basal ganglia. Constitutive expression of Zif268 existing mainly in the GABAergic neurons in the CPu may at least in part be maintained by glutamatergic afferents.     

Experience-dependent regulation of zif268 gene expression and spatial learning

Environmental enrichment (EE) is known to enhance the cognitive ability of rodents. To translate EE to the human condition, it is important to understand the parameters of its efficacy. In this study, we examine if the cognitive enhancement associated with EE is permanent and whether a developmental window exists for its efficacy. Rats were housed in continuous isolation (ISO), continuous enrichment (EE), enrichment from postnatal day (PN) 21-50, and then isolation from PN50-79 (PM), or isolation from PN21-50 and then enriched from PN50-79 (CW). Spatial learning ability and basal expression of the immediate-early genes zif268 and Arc as well as the NR1 subunit of the NMDA receptor were assessed. Rats housed in an enriched environment at the time of testing (EE and CW) performed significantly better in the spatial learning task than rats housed in an isolated environment at the time of testing (ISO and PM). Enhanced performance in the spatial learning task was associated with a higher expression of zif268 only in the CA3/CA4 region of the hippocampus. Our study further defines parameters that make environmental enrichment effective in enhancing learning performance and the findings may be helpful in the translation of this intervention to the human condition.     

Swim stress increases hippocampal Zif268 expression in the spontaneously hypertensive rat

The spontaneously hypertensive rat (SHR), which is used as an animal model of ADHD, displays numerous behavioural differences on learning and memory tasks. This study characterises differences in neural Zif268 expression in male SHR, Wistar Kyoto (WKY) and Sprague–Dawley (SD) rats after a 10-min forced swim. Swim stress increased Zif268 expression in the hippocampus of SHR only. In addition, SHR had increased expression in the prefrontal cortex, dorsal striatum and decreased expression in the nucleus accumbens shell in comparison to WKY and SD; and increased expression in the amygdala compared to SD. These findings: (i) support previous research indicating that SHR have altered neurobiological response to stressors, (ii) extends the characterisation of multiple memory systems in SHR to include differences in Zif268 expression in brain regions underlying their altered behaviour and (iii) supports previous findings that SHR may have a specific deficit within the shell of the nucleus accumbens.     

Increased expression of zif268 mRNA in rat retrosplenial cortex following administration of phencyclidine

Phencyclidine (PCP) has been shown to cause neurotoxicity in rat retrosplenial cortex following a single administration, although the precise mechanism underlying PCP-induced neurotoxicity is unclear. Using in situ hybridization and immunohistochemistry, we studied the effects of PCP on expression of immediate early gene zif268 mRNA and zif268 protein in the rat brain. High constitutive levels of zif268 mRNA and zif268 immunoreactivity were observed in the brain of control rats. Administration of PCP (12.5, 25 or 50 mg/kg, i.p., 6 h) caused marked induction of zif268 mRNA in the rat retrosplenial cortex, in a dose-dependent manner. However, the basal levels of zif268 mRNA in the other regions of cerebral cortex were decreased by administration of PCP. Emulsion-autoradiographical study suggested that marked expression of zif268 mRNA was observed in the layers III and IV of retrosplenial cortex where the neurotoxicity of PCP was detected. Furthermore, zif268 immunoreactivity in the layer IV of retrosplenial cortex was not changed by administration of PCP (25 mg/kg, i.p., 5 h), but that in the other layers of retrosplenial cortex was reduced by PCP. These results suggest that immediate early gene zif268 may, in part, play a role in the neurotoxicity of NMDA receptor antagonists such as PCP.     

Light-induced zif268 expression is dependent on noradrenergic input in rat visual cortex

In the present paper we investigated the role of the noradrenergic projection from the locus coeruleus on the expression of the immediate early gene zif268 in the visual cortex of rats exposed to ambient light stimulation. Local administrations of 6-hydroxydopamine (6-OHDA), a specific toxin directed against the catecholaminergic system, were performed in the locus coeruleus prior to visual stimulation. Animals were stimulated for 2 h by ambient light, after a 2-week dark adaptation period. Sham-operated controls displayed a massive increase in the number of zif268 positive cells after light stimulation. To the contrary, lesioned animals demonstrated a dramatic reduction in the number of zif268 positive nuclei across all cortical layers. A few scattered immunopositive nuclei were identified in cortical layer IV, however, this region also underwent a significant reduction in the number of zif268 immunopositive nuclei. Our results indicate that the noradrenergic system plays an important role in the expression of zif268 in the visual cortex of rats exposed to ambient light after dark isolation.     

Local and descending circuits regulate long-term potentiation and zif268 expression in spinal neurons

Long-term potentiation (LTP), a use dependent long-lasting modification of synaptic strength, was first discovered in the hippocampus and later shown to occur in sensory areas of the spinal cord. Here we demonstrate that spinal LTP requires the activation of a subset of superficial spinal dorsal horn neurons expressing the neurokinin-1 receptor (NK1-R) that have previously been shown to mediate certain forms of hyperalgesia. These neurons participate in local spinal sensory processing, but are also the origin of a spino-bulbo-spinal loop driving a 5-hydroxytryptamine 3 receptor (5HT3-R)- mediated descending facilitation of spinal pain processing. Using a saporin-substance P conjugate to produce site-specific neuronal ablation, we demonstrate that NK1-R expressing cells in the superficial dorsal horn are crucial for the generation of LTP-like changes in neuronal excitability in deep dorsal horn neurons and this is modulated by descending 5HT3-R-mediated facilitatory controls. Hippocampal LTP is associated with early expression of the immediate-early gene zif268 and knockout of the gene leads to deficits in long-term LTP and learning and memory. We found that spinal LTP is also correlated with increased neuronal expression of zif268 in the superficial dorsal horn and that zif268 antisense treatment resulted in deficits in the long-term maintenance of inflammatory hyperalgesia. Our results support the suggestion that the generation of LTP in dorsal horn neurons following peripheral injury may be one mechanism whereby acute pain can be transformed into a long-term pain state.