氯化锂-毛果芸香碱致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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模型,分为对照组、急性期组(致后24 h内)、潜伏期组(致后2周内)、慢性期组(致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(O4)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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(P0.05),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(P0.05)。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(P0.05);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(P0.05),以潜伏期组增加最为明显。结论氯化锂-毛果芸香碱致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。     

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.     

氯化锂-匹罗卡品致(疒间)大鼠脑髓鞘转录因子的表达及其意义

目的探讨氯化锂-匹罗卡品致疒间大鼠脑髓鞘转录因子1(MyT1)的表达及其意义.方法给SD大鼠先后腹腔注射氯化锂、匹罗卡品,制成癫疒间动物模型;用免疫荧光组化法检测癫疒间大鼠癫疒间发作后不同时间大脑皮质和海马CA1区MyT1阳性细胞数.结果与对照组相比,癫疒间后1 d组大鼠海马CA1区MyT1阳性细胞数显著减少(P＜0.05),癫疒间后其他各时间组大鼠脑皮质和海马CA1区MyT1阳性细胞数均有明显的增加,其中癫疒间后7 d组MyT1阳性细胞数最多(P＜0.01,P＜0.05).结论氯化锂-匹罗卡品致疒间大鼠早期大脑MyT1表达增加,并有时程性变化.     

NBQX对脑缺血再灌注后成年大鼠大脑少突胶质前体细胞的保护作用

目的 探讨脑缺血再灌注后成年大鼠大脑少突胶质前体细胞的变化及MK-801和NBQX对其保护作用.方法 以线栓法制作成年SD大鼠局灶性脑缺血再灌注模型(阻塞90min再灌注1d、7d和14d),分别向其大脑皮质缺血区立体定向注射5mmol/L 的MK-801和50 mmol/L的NBQX各1μl,用免疫荧光组织化学法检测脑缺血再灌注后成年大鼠早期大脑梗死中心区、梗死周边区和缺血对侧NG2和O4阳性细胞数量.结果 脑缺血再灌注后成年大鼠大脑皮质梗死中心区NG2 和O4阳性细胞逐渐减少, NBQX组大鼠NG2和O4阳性细胞数减少的幅度较少.脑缺血再灌注后梗死周边区NG2和O4阳性细胞数逐渐增加,NBQX组大鼠增加更明显,而MK-801组与生理盐水组无明显差别.结论 成年SD大鼠脑缺血再灌注后梗死周边区少突胶质前体细胞增多.NBQX对少突胶质前体细胞早期的缺血性损伤有保护作用.     

局灶性脑缺血再灌注大鼠大脑NG2和O4表达变化

目的:探讨脑缺血再灌注大鼠早期大脑少突胶质祖细胞及未成熟少突胶质细胞变化及意义。方法:以线栓法制作SD大鼠局灶性脑缺血再灌注模型(阻塞90min再灌注1d、3d和7d);用免疫荧光组织化学法检测脑缺血再灌注后早期大鼠大脑梗死中心区、梗塞周边区和缺血对侧NG2和O4阳性细胞数量。结果:脑缺血再灌注后各时间点梗死中心区NG2和O4阳性细胞数明显减少;梗塞周边区NG2和O4阳性细胞数随着再灌注时间延长而逐渐增加,再灌注3d和7d增加显著;脑梗死对侧区NG2和O4阳性细胞数无明显变化。结论:成年SD大鼠脑缺血再灌注后梗塞周边区少突胶质祖细胞及未成熟少突胶质细胞增多,可能参与缺血损伤的修复过程。     

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.     

皮质发育障碍模型的建立及其致痫敏感性的研究

目的:建立皮质发育障碍模型,探讨皮质发育障碍模型的敏感性。方法:在SD大鼠孕17d腹腔注入1,3-二氯乙烯-亚硝基脲(BCNU)制作皮质发育障碍模型;Nissl染色观察P60d仔鼠病理变化;选取P60d雄性仔鼠,腹腔注射氯化锂-毛果芸香碱,分别比较两组大鼠癫发生的潜伏期、持续状态时间和死亡率。结果:同龄仔鼠脑组织湿重实验组比对照组显著减轻(P<0.01);Nissl染色显示皮质变薄、皮质层次紊乱、海马区域异位细胞异常聚集;有皮质发育障碍的仔鼠注射氯化锂-毛果芸香碱后,癫发生的潜伏期显著缩短(P<0.01),癫持续状态时间延长(P<0.01),死亡率显著升高(P<0.05)。结论:BCNU致皮质发育障碍模型具有癫易感性。     

氨甲酰化促红细胞生成素对毛果芸香碱癫(癎)模型小鼠脑保护作用的研究

目的:研究氨甲酰化促红细胞生成素(CEPO)对毛果芸香碱诱导小鼠癫(癎)模型海马神经元的保护作用.方法:C57/B6小鼠随机分为正常未干预组、对照组和CEPO干预组.正常未干预组小鼠不作任何处理;对照组和CEPO干预组小鼠用毛果芸香碱350 mg·kg-1腹腔注射诱导癫(癎)发作,并于癫(癎)发作30 min后用地西泮...     

低频重复经颅磁刺激的抗作用及其对癫大鼠海马CA3区膜连蛋白A7表达的影响

目的:观察低频重复经颅磁刺激(rTMS)对大鼠性发作行为及海马CA3区膜连蛋白A7表达的影响。方法:取85只健康雄性SD大鼠,按预处理方式将其分成rTMS组(rTMS刺激+毛果芸香碱致)、对照组(假刺激+毛果芸香碱致)及生理盐水对照组(假刺激+生理盐水)。各组大鼠经相应处理后,rTMS组和对照组(各n=30)大鼠制作氯化锂-毛果芸香碱癫持续状态(SE)模型;生理盐水对照组(n=25)则腹腔注射生理盐水。观察各组大鼠行为表现及SE潜伏期,应用免疫组化法观察膜连蛋白A7表达的动态变化(6 h、24 h、1周、3周、6周)。结果:①rTMS组SE潜伏期为(41.37±5.45)min,与对照组(23.86±4.42)min比较明显延长(P<0.01);②海马CA3区膜连蛋白A7阳性细胞数在各时间点均为对照组最多,rTMS组次之,生理盐水对照组最少(均P<0.05)。但是膜连蛋白A7的表达随时间变化的趋势rTMS组与对照组明显不同。结论:低频rTMS有一定抗作用;低频rTMS可影响大鼠海马CA3区膜连蛋白A7表达并呈现独特的动态变化特点。     

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。     

氯化锂-匹罗卡品致痫大鼠脑髓鞘转录因子的表达及其意义

目的探讨氯化锂-匹罗卡品致     

锂-匹罗卡品致癫大鼠早期大脑皮质MyT1 mRNA表达变化

目的 探讨氟化锂-匹罗卡品致癫大鼠脑髓鞘转录因子1基因表达变化及其意义。方法 以氯化锂、匹罗卡品对雄性成年SD大鼠先后腹腔注射,制成癫痫持续状态动物模型;采用5′末端标记地高辛的寡核苷酸探针荧光原位核酸分子杂交检测癫性发作后早期大鼠大脑皮质MyT1 mRNA阳性细胞数量。结果 与对照组相比,癫痫后1d组大鼠脑皮质MyT1 mRNA阳性细胞数减少（P＜0.05）;其他各组大鼠脑皮质MyT1 mRNA阳性细胞数都有明显的增加,其中癫痫后7d和14d组MyT1mRNA阳性细胞数都有非常显著的增加（P＜0.05,P＜0.01）。结论 氯化锂-匹罗卡品致癫大鼠早期大脑MyT1 mRNA表达增加,并有时程性变化,提示与早期脑损伤修复有关。     

Activation of NG2-positive oligodendrocyte progenitor cells during post-ischemic reperfusion in the rat brain.

This study examines the alteration of oligodendrocyte progenitor cells which express membrane NG2 chondroitin sulfate proteoglycan after focal ischemia in the rat brain. Adult male Sprague-Dawley rats were subjected to 90 min occlusion of the middle cerebral artery, followed by reperfusion time of up to 2 weeks. The distribution and morphological changes in NG2-positive oligodendrocyte progenitor cells were immunohistochemically examined. Stellate-shaped NG2-positive cells with multiple branched processes were detected in both the gray and white matter of normal brain. After 2 weeks of reperfusion, NG2-positive cells in the area surrounding the infarction site (peri-infarct area) clearly showed enlarged cell bodies with hypertrophied processes. These stained strongly for NG2. Although the number of NG2-positive cells was increased significantly in the peri-infarct area, it decreased markedly in the infarct core compared to controls. Double immunostaining revealed that these NG2-positive cells were neither astrocytes nor microglia, but NG2-positive oligodendrocyte progenitor cells. These progenitor cells are known to differentiate into oligodendrocytes. As such, this upregulation of NG2 expression may be an adaptive mechanism attempting to remyelinate rat brain tissue after ischemic insult. Only further study will elucidate this hypothesis.     

Functional and morphological effects of NG2 proteoglycan deletion on hippocampal neurogenesis

NG2-expressing cells are the largest proliferating cell population in the adult central nervous system. The function of NG2 proteoglycan or NG2-expressing cells in the adult brain, however, is unknown. So far, NG2-positive cells are thought to be mainly oligodendrocyte precursor cells. This view was recently challenged when NG2+/CNP-EGFP-positive cells were identified as multipotent progenitor cells in the postnatal and adult CNS (e.g., [Belachew, S., Chittajallu, R., Aguirre, A.A., Yuan, X., Kirby, M., Anderson, S., Gallo, V., 2003. Postnatal NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional neurons. J. Cell Biol. 161, 169-186]). In addition, purified NG2-expressing progenitor cells, were shown to differentiate into neurons and astrocytes in vitro [Sellers, D.L., Horner, P.J., 2005. Instructive niches: environmental instructions that confound NG2 proteoglycan expression and the fate-restriction of CNS progenitors J. Anat. 207, 727-734]. In this study, we focus on the influence of NG2 ablation on neurogenesis in the hippocampus, where putative multipotent NG2-positive cells reside, and on hippocampus-dependent behavior using NG2 knockout mice. Using the thymidine analogue bromodeoxyuridine (BrdU) to label dividing cells in vivo we show that the number of BrdU-positive cells was unchanged in the hippocampus of NG2 knockout mice 1 day after a series of BrdU injections. This finding suggests that the proliferation rate of hippocampal progenitor cells is not influenced by NG2. A few BrdU-positive cells were found in deeper layers of the granule zone 1 day after a series of BrdU injections, which is different from the wild type. The presence and the phenotype of newborn hippocampal cells were studied 4 weeks after a series of BrdU injections. The survival and differentiation of BrdU-positive cells in NG2 knockout hippocampus did not significantly differ from wild-type mice. Concurrently, the water maze task did not reveal obvious differences compared to wild-type animals. These results suggest that the null mutation for NG2 does not influence adult hippocampal neurogenesis or hippocampal-dependent behavioral tasks.     

The role of piriform cortex adenosine A1 receptors on hippocampal kindling

INTRODUCTION: The hippocampus and piriform cortex have a critical role in seizure propagation. In this study, the role of adenosine A1 receptors of piriform cortex on CA1 hippocampal kindled seizures was studied in rats. METHODS: Animals were implanted with a tripolar electrode in the right hippocampal CA1 region and two guide cannulae in the left and right piriform cortex. They were kindled by daily electrical stimulation of hippocampus. In fully kindled rats, N6- cyclohexyladenosine (CHA; a selective adenosine A1 receptors agonist) and 1,3-dimethyl-8-cyclopenthylxanthine (CPT a selective adenosine A1 receptor antagonist) were microinfused into the piriform cortex. The animals were stimulated at 5, 15 and 90 minutes (min) after drug injection. RESULTS: Obtained data showed that CHA (10 and 100 microM) reduced afterdischarge duration, stage 5 seizure duration, and total seizure duration at 5 and 15 min after drug injection. There was no significant change in latency to stage 4 seizure. CPT at concentration of 20 microM increased afterdischarge duration, stage 5 seizure duration, and total seizure duration and decreased latency to stage 4 seizure at 5 and 15 min post injection. Pretreatment of rats with CPT (10 microM), 5 min before CHA (100 microM), reduced the effect of CHA on seizure parameters. CONCLUSION: These results suggested that activity of adenosine A1 receptors in the piriform cortex has an anticonvulsant effect on kindled seizures resulting from electrical stimulation of the CA1 region of the hippocampus.     

Epileptiform activity induced by 4-aminopyridine in entorhinal cortex hippocampal slices of rats with a genetically determined absence epilepsy (GAERS)

Patients with absence epilepsy frequently develop convulsions later in life. We were therefore interested whether tissue from rats with a genetic absence epilepsy is more prone to seizure generation than normal animals. We compared the epileptiform activities induced by 4-aminopyridine (4-AP) induced in hippocampal–entorhinal cortex slices from genetic absence epilepsy rats of Strasbourg (GAERS, age 6 months) in which absence seizures have been present for about 4 months and from control non epileptic rats (NE). 4-AP induced short recurrent discharges in area CA1 of rat hippocampus, seizure-like events and interictal discharges in the entorhinal cortex. The various epileptiform discharges did not differ between the two strains in amplitude, duration and frequency. However, the latency for induction of different epileptiform activities by 50 μM 4-AP was significantly shorter in GAERS (about 16 min) than in NE rats (about 25 min). We also analysed differences in evoked field potentials (fp) in hippocampal area CA1 before, during and after application of 4-AP. Before application of 4-AP, responses to stimulation of Schaffer collateral were smaller in GAERS than in NE rats. Paired pulse potentiation was significantly larger in GAERS than in NE rats. 4-AP in the bath augmented the size of the evoked field potentials and this increase was larger in GAERS than in NE rats. Our findings show a greater excitability of hippocampal area CA1 in GAERS rats and a greater ability to develop 4-AP-induced epileptiform activity in combined hippocampal–enthorhinal cortex slices in GAERS than in NE rats.     

Effects of TRPV1 on the hippocampal synaptic plasticity in the epileptic rat brain

Temporal lobe epilepsy is often presented by medically intractable recurrent seizures due to dysfunction of temporal lobe structures, mostly the temporomesial structures. The role of transient receptor potential vaniloid 1 (TRPV1) activity on synaptic plasticity of the epileptic brain tissues was investigated. We studied hippocampal TRPV1 protein content and distribution in the hippocampus of epileptic rats. Furthermore, the effects of pharmacologic modulation of TRPV1 receptors on field excitatory postsynaptic potentials have been analyzed after induction of long term potentiation (LTP) in the hippocampal CA1 and CA3 areas after 1 day (acute phase) and 3 months (chronic phase) of pilocarpine‐induced status epilepticus (SE). A higher expression of TRPV1 protein in the hippocampus as well as a higher distribution of this channel in CA1 and CA3 areas in both acute and chronic phases of pilocarpine‐induced SE was observed. Activation of TRPV1 using capsaicin (1 µM) enhanced LTP induction in CA1 region in non‐epileptic rats. Inhibition of TRPV1 by capsazepine (10 µM) did not affect LTP induction in non‐epileptic rats. In acute phase of SE, activation of TRPV1 enhanced LTP in both CA1 and CA3 areas but TRPV1 inhibition did not affect LTP. In chronic phase of SE, application of TRPV1 antagonist enhanced LTP induction in CA1 and CA3 regions but TRPV1 activation had no effect on LTP. These findings indicate that a higher expression of TRPV1 in epileptic conditions is accompanied by a functional impact on the synaptic plasticity in the hippocampus. This suggests TRPV1 as a potential target in treatment of seizure attacks. Synapse 69:375–383, 2015. © 2015 Wiley Periodicals, Inc.     

戊四氮点燃癫(癎)大鼠海马巢蛋白和骨形成蛋白-4的表达研究

目的观察巢蛋白(nestin)和骨形成蛋白4(BMP4)基因在戊四氮(PTZ)点燃癫大鼠海马中的表达,并探讨两者与癫发病机制的关系。方法将81只成年雄性SD大鼠随机分为实验组(n=54)和对照组(n=27)。实验组采用PTZ点燃癫大鼠,按点燃中的不同时相点,又随机分为9组。用免疫组化技术、地高辛标记特异性寡核苷酸探针原位杂交组织化学技术,观察海马nestin和BMP4表达的变化。结果nestin阳性细胞在PTZ注射后3d开始出现在齿状回、CA3区和CA1区,到7d达到高峰,以后逐渐减少。BMP4在PTZ注射后7d开始增多,在点燃后1d达到高峰,以后逐渐减少,主要分布在齿状回、CA3区和CA1区。结论PTZ点燃可引起海马内星形胶质细胞增生、活化和神经发生,这可能是癫海马组织胶质化、神经元可塑性的病理基础;BMP4可能在PTZ癫形成过程中起重要作用。