匹罗卡品致(癎)大鼠海马γ-氨基丁酸能中间神经元生长抑素和微清蛋白mRNA表达研究

目的 观察匹罗卡品致(癎)大鼠海马γ-氨基丁酸能中间神经元生长抑素(SS)mRNA和微清蛋白(PV)mRNA表达水平变化,拟从基因水平探讨其表达阳性γ-氮基丁酸能中间神经元在颞叶癫(癎)发生发展中的作用.方法 建立匹罗卡品致(癎)大鼠模型,采用原位杂交法检测各观察时间点海马SSmRNA和PVmRNA表达阳性神经元数目.结果 模型组大鼠海马各区γ-氨基丁酸能中间神经元SSmRNA表达水平均于出现癫(癎)持续状态后3d降低最为显著(均P=0.000),随后逐渐升高;至发病后60d,海马CA3区SSmRNA表达水平高于对照组(t=1.021,P=0.005),海马门区(t=3.211,P=0.009)和CA1区(t=1.902,P=0.048)则仍低于对照组.模型组大鼠海马门区γ-氨基丁酸能中间神经元PVmRNA表达水平于出现癫(癎)持续状态后6h开始降低,至发病后60d降低最为显著(均P=0.000);海马CA1区PVmRNA表达水平于发病后3d降低最为显著(均P=0.000),随后逐渐升高但仍低于对照组(江2.216,P=0.048);癫(癎)持续状态早期,海马CA3区PVmRNA表达水平无明显变化,至发病后7d逐渐升高且高于对照组(t=1.021,P=0.005).结论 γ-氨基丁酸能中间神经元SSmRNA和PVmRNA表达水平的下调可能在颞叶癫(癎)的发生中起重要作用,至慢性期γ-氨基丁酸能中间神经元SSmRNA和PVmRNA表达水平的恢复或上调可能与颞叶癫(癎)的发展或修复有关.γ-氨基丁酸能中间神经元数目的 变化,部分是由于其标志物mRNA表达水平的调节所致,并非神经元数目变化的唯一因素.     

雌激素致癎作用部位的研究

目的了解雌激素在癫癎大鼠中枢神经系统的作用部位.方法利用海人酸致癎和6-氟二乙酯致癎的两种不同机制的癫癎模型(单纯致癎组),应用免疫组化法检测单纯致癎及给予雌激素后再致癎大鼠海马、大脑皮质、纹状体的FOS表达.结果两种模型单纯致癎组海马、皮质、纹状体FOS表达较正常组显著增高(均P＜0.01).给予雌二醇(E2)后再致癎,海人酸模型中海马、皮质FOS表达较单纯致癎组增加(P＜0.01,P＜0.05);而6-氟二乙酯模型中无变化.结论在两种不同致癎机制的癫癎模型中,雌二醇对鼠脑FOS表达的影响不同.     

锂-毛果芸香碱致痫大鼠早期大脑NG2和O4表达及意义

目的探讨氯化锂-毛果芸香碱(匹罗卡品)致疒0 间大鼠早期大脑少突胶质前体细胞变化及意义.方法对雄性成年SD大鼠先后腹腔注射氯化锂、毛果芸香碱,制成癫癎持续状态动物模型;用免疫荧光组织化学法检测癎性发作后早期大鼠大脑皮质和海马CA1区NG2和O4阳性细胞数量.结果和对照组相比,除癫癎后1 d组外其余各组大鼠脑皮质内NG2和O4阳性细胞都有明显的增加;癫癎后1 d组海马CA1区的阳性细胞数明显减少;癫癎后7 d组皮质和海马CA1区NG2和O4阳性细胞数最多.结论氯化锂-毛果芸香碱致癎大鼠早期大脑NG2和O4表达增加,少突胶质前体细胞增多,并且和观测时间相关.     

匹罗卡品致癎大鼠海马γ-氨基丁酸能中间神经元生长抑素和微清蛋白mRNA表达研究

目的观察匹罗卡品致癎大鼠海马γ-氨基丁酸能中间神经元生长抑素（SS）mRNA和微清蛋白（PV）mRNA表达水平变化,拟从基因水平探讨其表达阳性叮一氨基丁酸能中间神经元在颞叶癫癎发生发展中的作用。方法建立匹罗卡品致癎大鼠模型,采用原位杂交法检测各观察时间点海马SSmRNA和PVmRNA表达阳性神经元数目。结果模型组大鼠海马各区吖．氨基丁酸能中间神经元SSmRNA表达水平均于出现癫癎持续状态后3d降低最为显著（均P=0．000）,随后逐渐升高;至发病后60d,海马CA3区SSmRNA表达水平高于对照组（t=1．021,P=0．005）,海马门区（t=3．211,P=0．009）和CA1区（t=1．902,JP=0．048）则仍低于对照组。模型组大鼠海马门区γ-氨基丁酸能中间神经元PVmRNA表达水平于出现癫癎持续状态后6h开始降低,至发病后60d降低最为显著（均P：0．000）;海马CA1区PVmRNA表达水平于发病后3d降低最为显著（均p=0．000）,随后逐渐升高但仍低于对照组（t=2．216,尸：0．048）;癫癎持续状态早期,海马CA3区PVmRNA表达水平无明显变化,至发病后7d逐渐升高且高于对照组（t=1．021,P=0．005）。结论γ-氨基丁酸能中间神经元SSmRNA和PVmRNA表达水平的下调可能在颞叶癫癎的发生中起重要作用,至慢性期γ-氨基丁酸能中间神经元SSmRNA和PVmRNA表达水平的恢复或上调可能与颞叶癫癎的发展或修复有关。γ-氨基丁酸能中间神经元数目的变化,部分是由于其标志物mRNA表达水平的调节所致,并非神经元数目变化的唯一因素。     

颞叶癫大鼠海马轴突导向分子Sema3F及其受体Np2表达的变化

目的 研究颞叶癫(癎)大鼠海马轴突导向分子Sema3F及其受体Np2表达的变化.方法 给SD大鼠腹腔注射匹罗卡品、氯化锂制作颞叶癫(癎)模型.用免疫组化法和原位杂交技术对致(癎)后不同时间点大鼠海马CA1区、CA3区、齿状回的Sema3F mRNA、Np2 mRNA和蛋白表达进行检测,并与正常对照组比较.结果 颞叶癫(癎)大鼠致(癎)后7 d、15 d,海马CA1区、CA3区Sema3F mRNA、Np2 mRNA和蛋白的表达明显低于正常对照组(P＜0.05～0.01), 致(癎)后30 d、60 d表达与正常对照组差异无统计学意义;而齿状回Sema3F mRNA、Np2 mRNA和蛋白的表达与正常对照组的差异无统计学意义.结论 颞叶癫(癎)大鼠海马CA1区、CA3区Sema3F、Np2表达在致(癎)后早期明显下调,而在慢性期恢复正常.     

托吡酯对戊四氮致癫癎大鼠海马AQP4表达水平的影响

目的探讨托吡酯对戊四氮致癫癎大鼠海马AQP4表达水平的影响。方法将30只Wistar大鼠随机分为戊四氮致癫癎组、托吡酯干预组和正常对照组，每组各10只；癫癎模型点燃后在不同时相点灌注取材，通过HE染色观察大鼠海马神经元的变化，并应用免疫组化法检测大鼠海马AQP4表达水平。结果HE染色显示托吡酯干预组神经元变性和坏死较戊四氮致癫癎组明显减轻；免疫组化显示戊四氮致癫癎组在致癫癎后12hAQP4的表达显著增强，致癫癎后24h达高峰，托吡酯干预组在致癫癎后12h～36h各时相点AQP4表达水平均分别低于戊四氮致癫癎组相应时间点（P〈0．05）。结论托吡酯通过下调大鼠海马AQP4的表达可能参与了对大鼠海马神经元的保护过程。     

Akt1在颞叶癫痫大鼠海马中的表达变化

目的研究颞叶癫癎模型海马区神经元Akt1表达变化,探讨其在癫癎发生发展中的作用。方法采用氯化锂-匹罗卡品方法制备颞叶癫癎大鼠模型,Western blotting检测海马区总蛋白、Quantity one软件行灰度值分析;免疫组织化学染色观察海马各区Akt1蛋白表达变化,计数不同处理组阳性神经元数目。结果 Western blotting检测结果显示,与正常对照组相比,癫癎模型组大鼠于癫癎持续状态发作即刻海马区Akt1蛋白表达升高(t=2.445,P=0.034),并于第30天时达峰值水平(t=1.214,P=0.002),发作后24 h表达水平迅速降低,并低于正常值范围(t=4.294,P=0.000),其余各测量时间点表达无明显改变;与氯化锂组相比,癫癎模型组大鼠于癫癎持续状态后1h海马区Akt1蛋白表达开始降低,24 h降至最低水平(t=4.134,P=0.000),至发作48 h后开始逐渐升高(t=2.481,P=0.002),并于发作第7天时升至氯化锂组水平。免疫组织化学染色显示,癫癎持续状态发作后海马CA3区Akt1蛋白表达阳性神经元数目立即增加,12h达高峰(t=16.586,P=0.000),48 h减少并降至正常值水平(t=0.357,P=0.089),发作后第10天再次增加(t=3.123,P=0.000),于第30天时阳性神经元数目再次达峰值水平(t=18.339,P=0.000),第50天开始恢复至正常值水平(t=3.219,P=0.000);氯化锂组仅海马CA3区Akt1蛋白表达于实验初始(0 h)升高并高于正常对照组(P<0.05),海马CA1和CA2区Akt1蛋白表达变化组间差异均无统计学意义(P>0.05)。结论海马及海马CA3区Akt1蛋白表达均呈现癫癎持续状态后升高、降低、再升高的动态过程,提示可能存在神经元保护作用,对抗细胞凋亡、促进细胞存活。     

普瑞巴林对慢性颞叶癫癎大鼠海马凋亡调控基因的影响

目的通过观察普瑞巴林对匹罗卡品慢性癫癎大鼠海马区Bcl-2和Bax表达的影响,探讨普瑞巴林治疗癫癎的药理学机制及对大鼠海马神经元的抗凋亡作用。方法采用氯化锂-匹罗卡品化学诱导方法建立慢性颞叶癫癎模型。经腹腔注射普瑞巴林40mg(/kg·d)连续治疗3周,免疫组织化学染色和Western blotting法检测不同处理组大鼠海马区Bcl-2和Bax表达变化。结果与生理盐水对照组比较,模型组大鼠海马区Bcl-2和Bax表达水平显著升高(均P=0.000);与模型组比较,普瑞巴林治疗组大鼠海马区Bcl-2表达水平升高、Bax表达水平降低,组间差异具有统计学意义(均P=0.000)。结论新型抗癫癎药物普瑞巴林可通过降低慢性颞叶癫癎大鼠海马区Bax表达、上调Bcl-2表达而抑制细胞凋亡,发挥神经元保护作用。     

生长抑素受体1在皮质发育障碍大鼠海马的异常表达

目的通过研究生长抑素受体1(sstr-1)在皮质发育障碍模型大鼠海马区中的表达,探讨皮质发育障碍模型大鼠海马形态改变的发生机制及致癫机制。方法制作皮质发育障碍大鼠模型,选取P60 d仔鼠,利用免疫组化、Western Blot检测sstr-1在皮质发育障碍模型大鼠海马区的变化。结果与正常对照组比较,sstr-1在皮质发育障碍模型大鼠海马区水平表达与正常对照组相比明显降低(0.59±0.15 vs 1.02±0.14,P<0.052)。结论 sstr-1在皮质发育障碍大鼠模型海马神经元的迁移紊乱及致癫机制中具有重要作用。     

癫(癎)大鼠海马凋亡诱导因子水平的改变及聚腺苷二磷酸核糖多聚酶抑制剂对其的影响

目的 探讨癫(癎)大鼠海马凋亡诱导因子(AIF)水平的改变及聚腺苷二磷酸核糖多聚酶(PARP)抑制剂3-氨基苯甲酰胺(3-AB)对其的影响.方法 (1)30只Wistar大鼠随机分为对照组及癫(癎)发作后3 h(Ep 3 h组)、8 h(Ep 8 h组)、24 h(Ep24 h组)和72 h(Ep 72 h组)组,每组6只.应用氯化锂-匹鲁卡品腹腔注射建立癫(癎)模型.分别于相应时间点处死大鼠取脑,应用免疫印记法(Western blot)检测大鼠海马线粒体和细胞核AIF的水平.(2)18只Wistar大鼠随机分为对照组、匹鲁卡品致(癎)组、3-AB干预组,每组6只.3-AB干预组大鼠于致(癎)前30 min腹腔注射3-AB 40 mg/kg.于癫(癎)发作24 h后处死并应用Western blot检测大鼠海马线粒体和细胞核AIF的水平.结果 与对照组比较,Ep 24 h组和Ep 72 h组大鼠海马线粒体AIF水平明显减低,而细胞核AIF水平显著增高(均P<0.05);Ep 3 h组、Ep 8 h组与对照组大鼠海马线粒体及细胞核AIF表达水平比较,差异无统计学意义.与匹鲁卡品致(癎)组比较,3-AB干预组大鼠海马线粒体AIF水平显著增高,而细胞核AIF水平显著降低(均P<0.05);匹鲁卡品致(癎)组与对照组大鼠海马线粒体及细胞核AIF表达水平比较,差异无统计学意义.结论 癫(癎)发作使海马线粒体AIF水平降低,细胞核AIF水平增高,3-AB能明显抑制这一变化.     

血脑屏障内皮细胞与细胞间粘附分子-1在鼠脑缺血性脑水肿发生机制中的作用

目的研究缺血性脑水肿病理过程中血脑屏障内皮细胞及其细胞间粘附分子-1(intercellularadhesionmolecule-1,ICAM-1)的表达与血脑屏障通透性的关系.方法用电镜技术和免疫组织化学方法观察脑缺血过程中大鼠血脑屏障内皮细胞超微结构及其ICAM-1的表达和对镧离子的通透性改变.结果脑缺血后1h,缺血区脑组织血脑屏障内皮细胞未见ICAM-1表达,但电镜下可见内皮细胞轻度肿胀.3h,ICAM-1开始表达,缺血区脑组织轻度水肿,内皮细胞肿胀.至12h,ICAM-1表达达到高峰,脑组织严重水肿,内皮细胞、神经细胞及胶质细胞明显肿胀;内皮细胞胞饮小泡包含有被吞噬的镧离子,有些镧离子进入紧密连接间隙.72h,ICAM-1表达已明显减少,脑组织水肿仍较重,内皮细胞严重破坏,大量镧离子漏出血管外,进入脑实质.结论脑缺血后血脑屏障的破坏与ICAM-1的表达密切相关,提示ICAM-1参与血脑屏障内皮细胞的破坏过程,而且这些改变在缺血性脑水肿的病理过程中起重要作用.     

戊四氮所致慢性癫痫发病过程中谷氨酸脱羧酶基因表达的研究

目的：观察谷氨酸脱羧酶（GAD）67mRNA表达在慢性癫痫演变过程的变化,并探讨其作用。方法：采用寡核苷酸探针原位组织杂交技术检测慢性癫痫演变过程中各期皮层,海马GAD67mRNA的表达水平。结果：慢性癫痫演变过程中GAD67mRNA阳性细胞数略有减少,但与对照组比较无显著性差异。而继续存活的GAD67mRNA阳性细胞平均吸光度明显增加,与对照组比较有显著性差异（P＜0．01）,结论：继续存活的GAD67m RNA阳性细胞GAD67mRNA表达水平的增高可能是机体的一种内源性抗病表现。     

Expression of the multidrug transporter P-glycoprotein in brain capillary endothelial cells and brain parenchyma of amygdala-kindled rats

Summary: Purpose: Based on data from brain biopsy samples of patients with pharmacoresistant partial epilepsy, overexpression of the multidrug transporter P‐glycoprotein (PGP) in brain capillary endothelium has recently been proposed as a potential mechanism of resistance to antiepileptic drugs (AEDs). We examined whether PGP is overexpressed in brain regions of amygdala‐kindled rats, a widely used model of temporal lobe epilepsy (TLE), which is often resistant to AEDs. Methods: Rats were kindled by stimulation of the basolateral amygdala (BLA); electrode‐implanted but nonkindled rats and naive (not implanted) rats served as controls. PGP was determined by immunohistochemistry either 1 or 2 weeks after the last kindled seizure, by using a monoclonal anti‐PGP antibody. Six brain regions were examined ipsi‐ and contralateral to the BLA electrode: the BLA, the hippocampal formation, the piriform cortex, the substantia nigra, the frontal and parietal cortex, and the cerebellum. Results: In both kindled rats and controls, PGP staining was observed mainly in microvessel endothelial cells and, to a much lesser extent, in parenchymal cells. The distribution of PGP expression across brain regions was not homogeneous, but significant differences were found in both the endothelial and parenchymal expression of this protein. In kindled rats, ipsilateral PGP expression tended to be higher than contralateral expression in several brain regions, which was statistically significant in the piriform cortex and parietal cortex. However, compared with controls, no significant overexpression of PGP in capillary endothelial cells or brain parenchyma of kindled rats was seen in any ipsilateral brain region, including the BLA. For comparison with kindled rats, kainate‐treated rats were used as positive controls. As reported previously, kainate‐induced seizures significantly increased PGP expression in the hippocampus and other limbic brain regions. Conclusions: Amygdala‐kindling does not induce any lasting overexpression of PGP in several brain regions previously involved in the kindling process. In view of the many pathophysiologic and pharmacologic similarities between the kindling model and TLE, these data may indicate that PGP overexpression in pharmacoresistant patients with TLE is a result of uncontrolled seizures but not of the processes underlying epilepsy. It remains to be determined whether transient PGP overexpression is present in kindled rats shortly after a seizure, and whether pharmacoresistant subgroups of kindled rats exhibit an increased expression of PGP. Furthermore, other multidrug transporters, such as multidrug resistance–associated protein, might be involved in the resistance of kindled rats to AEDs.     

氯化锂-毛果芸香碱致(痫)大鼠海马髓鞘及少突胶质细胞系变化初步研究

目的 研究氯化锂-毛果芸香碱致(痫)大鼠不同时期海马髓鞘损伤及少突胶质细胞系变化.方法 健康成年雄性SD大鼠建立氯化锂-毛果芸香碱慢性癫(痫)模型,分为对照组、急性期组(致(痫)后24 h内)、潜伏期组(致(痫)后2周内)、慢性期组(致(痫)后2个月),各组(均n=4).采用伊文思蓝法检测各组大鼠海马血脑屏障(BBB)通透性改变;免疫荧光染色法检测海马神经元数目、髓鞘碱性蛋白(MBP)表达水平、2',3'-环核苷酸3'-磷酸二酯酶(CNPase)阳性成熟少突胶质细胞数目、未成熟少突胶质细胞标记物(04)表达水平、硫酸软骨素蛋白多糖(NG2)阳性少突胶质祖细胞数目.结果 大鼠致(痫)后,急性期组、潜伏期组和慢性期组大鼠海马各区域BBB通透性均较对照组增加(P＜0.05),以急性期组增加最为显著.各组大鼠海马不同区域NeuN阳性神经元数目均较对照组显著下降(P＜0.05).潜伏期组和慢性期组海马各区域MBP表达水平及CNPase阳性细胞数目均显著低于对照组(P＜0.05);各组海马CA1、Hilus区O4表达水平及NG2阳性细胞数目均较对照组显著增加(P＜0.05),以潜伏期组增加最为明显.结论 氯化锂-毛果芸香碱致(痫)大鼠海马区BBB通透性增加,未成熟少突胶质细胞和少突胶质祖细胞免疫荧光表达上调,髓鞘及成熟少突胶质细胞存在一定程度损伤.     

癫癎大鼠海马区白细胞介素1受体1免疫反应阳性细胞的观察

目的:探讨白细胞介素1受体1(IL-1R1)在海马区的表达与癫(疒间)发病的关系。方法:用免疫细胞化学法,观察成年和美解眠致(疒间)大鼠海马区IL-1R1免疫反应(IL-1R1-IR)阳性细胞分布。结果:成年大鼠海马区存在IL-1R1-IR阳性细胞。美解眠致(疒间)后,海马区该阳性细胞数显著增多(P＜0.01);致(疒间)前后给予尼莫地平,海马区该阳性细胞均较癫(疒间)大鼠显著减少(P＜0.01),与正常比较无显著差异(P＞0.05)。结论:海马区IL-1R1参与癫(疒间)的病理过程,其机制可能与其介导该区细胞内信号传导有关。尼莫地平不仅可以阻止癫(疒间)发作,而且可以减轻其继发的脑损伤。     

Increased expression of the multidrug transporter P-glycoprotein in limbic brain regions after amygdala-kindled seizures in rats

Increased expression of the multidrug transporter P-glycoprotein (Pgp; ABCB1) has previously been found in epileptogenic brain tissue from patients with pharmacoresistant temporal lobe epilepsy (TLE) as well as in the hippocampus and other limbic brain regions in the rat kainate model of TLE. Approaches to the quantification of Pgp expression have mainly been based on subjective visual estimation of the level of Pgp immunoreactivity in brain sections. In the present study, computer-assisted image analysis based on optical density (OD) measurements was used to examine immunohistochemical expression of Pgp in the kindling model of TLE. Sections from kainate-treated rats were used for comparison. Using diaminobenzidine as chromogen, Pgp was exclusively located in brain capillary endothelial cells, which was confirmed by double-labeling with an antibody against the endothelial glucose transporter (GLUT-1). After kainate-induced seizures, the intensity of endothelial Pgp staining significantly increased by 70-80% in the dentate gyrus. A significant, albeit less marked increase in Pgp expression in this area was also seen after amygdala-kindled seizures. Furthermore, Pgp was upregulated after kindling in the hilus of the dentate gyrus, the CA1 and CA3 sectors of the hippocampus, and the piriform and cerebral cortex. In kindled rats, most Pgp alterations occurred ipsilateral to the electrode in the basolateral amygdala. The data demonstrate that computer-assisted image analysis using OD is an accurate and rapid method to determine the relative amount of Pgp protein in brain sections and the effects of seizures on this multidrug transporter. The fact that Pgp overexpression in brain capillary endothelial cells occurs in two established models of difficult-to-treat TLE substantiates the notion that seizure-induced upregulation of Pgp contributes to multidrug resistance (MDR) in epilepsy.     

Blood-brain barrier is impaired in the hippocampus of young adult spontaneously hypertensive rats

A causative role of blood-brain barrier (BBB) impairment is suggested in the pathogenesis of vascular dementia with leakage of serum components from small vessels leading to neuronal and glial damage. We examined the BBB function of young adult spontaneously hypertensive rats (SHR) in order to determine earlier changes in the BBB in chronic hypertension. SHR and stroke-prone SHR (SHRSP) were injected with horseradish peroxidase (HRP) as an indicator of BBB function and compared with Wistar Kyoto rats (WKY). The brain tissues were further examined with cationized ferritin, a marker for evaluating glycocalyx. The staining for HRP was distributed around the vessels in the hippocampal fissure of SHR and SHRSP, but not in WKY. With electron microscopy, the extravasated reaction product of HRP appeared in abluminal pits of the endothelial cells of arterioles and within the basal lamina in the hippocampus, but not the cerebral cortex, of SHR and SHRSP. On the contrary, the reaction product of HRP was never seen in the abluminal pits of the endothelial cells or the basal lamina of vessels in WKY. The number of cationized ferritin particles binding to the endothelial cells of capillaries was decreased in the hippocampus of SHR and SHRSP, while the number decreased in the cerebral cortex of SHRSP compared with those in WKY. However, the cationized ferritin binding was preserved in the endothelial cells of the arterioles with an increased vascular permeability. These findings suggest that the chronic hypertensive state induces BBB dysfunction in the hippocampus at an early stage.     

Upregulation of intercellular adhesion molecule-1 expression on human endothelial cells by tumour necrosis factor-α in an in vitro model of the blood–brain barrier

Adhesion molecules on the endothelial surface of the blood–brain barrier (BBB) play an important role in the pathogenesis of many encephalopathies, including multiple sclerosis (MS) and cerebral malaria (CM). The expression of four surface molecules of relevance to MS and CM on the immortalized human umbilical vein endothelial cell line, ECV304, was investigated using immunofluorescence flow cytometry. We found that ECV304 cells express intercellular adhesion molecule-1 (ICAM-1) and low levels of CD36, but not vascular cell adhesion molecule-1 (VCAM-1) or E-selectin. This expression pattern was unaltered on ECV304 cells which were co-cultured with C6 glioma cells; conditions under which the endothelial cells display enhanced barrier formation. Tumour necrosis factor-α (TNF-α), which is elevated in MS and CM, decreased the integrity of the barrier in co-cultured endothelial cells and upregulated the expression of ICAM-1 nine-fold. The significance of elevated ICAM-1 expression in relation to the binding of parasitised erythrocytes at the BBB in CM is discussed.     

N-methyl-D-aspartate receptor subunit 1 protein expression in the hippocampus and temporal cortex of kainic acid-induced epilepsy rats

BACKGROUND: The N-methyl-D-aspartate receptor subunit 1 (NMDAR1) contributes to the incidence of epilepsy. However, the relationship between epilepsy-induced brain injury and NMDAR1 remains poorly understood.OBJECTIVE: To investigate changes in NMDAR1 protein expression in the hippocampus and temporal cortex of kainic acid-induced epilepsy rats.DESIGN, TIME AND SETrlNG: A randomized, controlled, animal experiment was performed at the Department of Physiology and Department of Pathology, Basic Medical College of Jilin University from March 2002 to March 2003.MATERIALS: Rabbit anti-NMDAR1 antibody was purchased from Wuhan Boster Biological Technology, China.METHODS: A total of 80 healthy, male, Wistar rats, aged 22 weeks, were randomly assigned to sham-surgery (n = 10) and model (n = 70) groups. Epilepsy models were established by injecting kainic acid (1 μL) into the right amygdala, and rats were sacrificed at 2, 6, 24, 72 hours, and 7, 15, 30 days after surgery, with 10 animals at each time point. The rats in the sham-surgery group were injected with 1 μL phosphate buffered saline into the right amygdala.MAIN OUTCOME MEASURES: NMDAR1 protein expression in the hippocampus and temporal cortex at 2, 6, 24, 72 hours and 7, 15, 30 days after epilepsy was detected using immunohistochemistry and flow cytometry analysis.RESULTS: In the sham-surgery group, a few NMDARl-positive cells were distributed in the hippocampus and temporal cortex. In the model group, NMDARl-positive cells were increased in the hippocampus and temporal cortex at 2 hours following kainic acid-induced epilepsy. They were significantly increased at 6 hours, and slightly decreased at 7 days (CA3 region and temporal cortex), but remained greater than the sham-surgery group. This continued until day 30 (P < 0.01). In addition, there were more NMDAR1 positive cells in the hippocampal CA3 and dentate gyrus than the temporal cortex (P < 0.01).CONCLUSION: In epilepsy model rats, NMDAR1 protein expression was upregulated in the hippocampus and temporal cortex, and in particular in the hippocampal CA3 and dentate gyrus. NMDAR1 may participate in epilepsy and the excitation process of the epileptic brain.     

Changes in the Permeability and Expression of Markers of the Structural and Functional Integrity of the Blood–Brain Barrier under Early Postnatal Hypoxia in vivo

Pathologies associated with perinatal exposure of the CNS to damaging factors, including hypoxia, are a serious problem. However, the mechanisms by which they influence the development of brain damage have been insufficiently studied. The purpose of this study is to analyze the BBB permeability and expression of markers of its structural and functional integrity in animals with hypoxia (rats subjected to hypoxia at the age of P7) at the early (1 hour after the hypoxia) and delayed (P28) periods of their development. In sections of the rat brains, we immunohistochemically evaluated the expression of HIF-1 and Rac1; the Evans blue dye content was measured by the photometric method in the brain homogenates. In animals subjected to hypoxia, BBB permeability increased, CD31 expression was reduced, RAC1 expression increased, HIF-1-positive cells were retained in the hippocampus mainly at the early stage of development; CD31 and RAC1 expression was suppressed during the delayed period of development. The most-pronounced brain damage at the age of P7 corresponds to changes in the structural and functional integrity and permeability of the BBB; the recovery of a neurological deficit and the permeability of the BBB (at the age of P28) under damage to the brain corresponds to the period of reparative angiogenesis, as well as manifestations of HIF-1 effects in endothelial cells and astrocytes in the cortex and limbic system.