大鼠海马发育过程中神经元和星形胶质细胞p21Cip1的表达

目的研究大鼠海马发育过程中细胞周期蛋白依赖性激酶抑制因子p21Cip1在神经元和星形胶质细胞中的表达。方法应用免疫双标技术和流式细胞术检测p21Cip1在生后1d(P1),生后11d(P11),成年和老年大鼠的海马神经元和星形胶质细胞中的表达。结果 (1)p21Cip1在各个年龄组大鼠的海马神经元和星形胶质细胞中均有表达;(2)大鼠海马神经元和星形胶质细胞中p21Cip1在不同年龄组的表达趋势类似,均在P1表达水平最高;而从P11到老年组p21Cip1的表达则逐渐增高,且各组间p21Cip1的表达有显著性差异(P0.05)。结论 p21Cip1在大鼠海马神经元和胶质细胞中的表达随着大鼠的发育而变化,这提示p21Cip1可能在大鼠海马的发育过程中起重要作用。     

大鼠海马星形胶质细胞对突触可塑性的影响

目的研究星形胶质细胞在神经系统发育成熟过程中对突触可塑性的调控规律.方法取健康初生、幼年和成年大鼠各10只,每只取脑切片,用免疫组织化学方法观察其海马CA1区的S100、胶质纤维酸性蛋白(GFAP)和P38免疫反应产物强度;HE染色法显示神经元胞体.结果初生大鼠海马CA1区中神经元大量存在,但S100、GFAP和P38表达均少,幼鼠的表达增加,但仍显著少于成鼠的表达(P＜0.01).结论大鼠海马CA1区星形胶质细胞增殖与突触出现时间、突触数目增加及功能成熟有关.     

大鼠脑缺血再灌注后神经细胞的细胞周期特征的比较研究

目的比较观察大鼠局灶性脑缺血后星形胶质细胞和神经元细胞周期的变化特征。方法采用线栓法大鼠大脑中动脉栓塞模型,利用流式细胞技术检测假手术组和缺血再灌注后不同时间点各组大脑皮层和海马中星形胶质细胞和神经元细胞周期的异常激活和动态变化。结果缺血后大脑皮层中神经元24h时即发生明显细胞周期变化,而3d时进入细胞周期的星形胶质细胞才明显增加;海马中星形胶质细胞却先于神经元进入细胞周期,于24h时细胞周期即发生明显变化,与假手术组比较有显著差异(P<0.01)。结论在不同脑区星形胶质细胞和神经元两者对缺血性脑损伤的敏感性互不相同,并且不同脑区的星形胶质细胞对缺血性脑损伤的敏感性也有不同,脑缺血后2种细胞均出现细胞周期的异常激活。     

下调miR-203靶向MEF2C/NF-κB抑制颞叶癫痫大鼠海马胶质细胞的活化和炎症反应

目的 探讨下调miR-203对颞叶癫痫大鼠海马胶质细胞活化和炎症反应的影响。方法 将46只成年SD大鼠随机分为假手术组（n=10）、模型组（n=12）、阴性对照组（n=12）和miR-203低表达组（n=12）。立体定向辅助下,将海人酸注入大鼠左侧海马CA3区建立颞叶癫痫模型,miR-203低表达组和阴性对照组大鼠左侧海马CA3区分别注射携带miR-203 inhibitor的腺相关病毒和空腺病毒载体,7 d后取左侧海马组织,PCR法检测miR-203水平,ELISA法检测炎症因子[白细胞介素-1β（IL-1β）、IL-6和肿瘤坏死因子α（TNF-α）]水平,免疫印迹法检测肌细胞增强因子2c（MEF2C）、核因子-κB（NF-κB）p65蛋白表达,TUNEL法检测神经元凋亡,GFAP/CD11b/c免疫荧光染色分析胶质细胞活化。结果 模型组大鼠海马组织miR-203、NF-κB p65蛋白表达量、炎症因子（IL-1β、IL-6和TNF-α）水平明显增高（P<0.05）,MEF2C蛋白表达量明显降低（P<0.05）,活化的胶质细胞数量、神经元凋亡数量明显增多（P<0.05）。双荧光素酶报告基因实验显示,MEF2C是miR-203的靶基因。miR-203表达低表达组大鼠海马组织miR-203表达量、NF-κB p65蛋白表达量、炎症因子（IL-1β、IL-6和TNF-α）水平明显降低（P<0.05）,MEF2C蛋白表达量明显增高（P<0.05）,活化的胶质细胞数量、神经元凋亡数量明显减少（P<0.05）。结论 下调miR-203,靶向调控MEF2C/NF-κB信号通路,抑制颞叶癫痫大鼠海马胶质细胞的活化、神经元凋亡和炎症反应。     

癫痫大鼠海马神经元和星形胶质细胞的病理演变

目的　探讨癫痫大鼠海马神经元和星形胶质细胞在点燃后各期的病理特点、时序及机制。方法　针对匹罗卡品癫痫大鼠模型,行Nissl、免疫组化和HE染色,观察海马神经元及星形胶质细胞的病理变化。结果　癫痫持续状态后超急性期（4h）,CA3区神经元呈嗜酸性变性、胞浆深染;急性期（24h）,嗜酸性变性最为显著,神经元固缩、核仁消失、突起断裂,星形胶质细胞水肿;缄默期（7d）,CA3、CA1区及门区神经元大量坏死、脱失,胶质增生肥大,海马构筑紊乱;慢性期（6w）,CA3、CA1区出现胶质瘢痕,遗有形态正常的神经元,且颗粒细胞层增厚。结论　癫痫时海马神经元先于星形胶质细胞发生病理改变,二者均参与癫痫发生。     

脑室注射痴呆大鼠海马胶质纤维酸性蛋白表达与学习记忆能力的关系

目的探讨星形胶质细胞在老年性痴呆大鼠海马中的表达与老年性痴呆大鼠学习记忆能力减退的关系。方法雄性SD大鼠20只,随机分为痴呆组与假手术组;用Morris水迷宫检测大鼠的学习、记忆能力;用免疫组化技术定量检测大鼠海马CA1区胶质纤维酸性蛋白(GFAP)的表达;分析海马星形胶质细胞变化与学习、记忆能力的关系。结果假手术组海马CA1区锥体细胞排列紧密有序,细胞核大而圆、染色浅、核仁明显、未见明显胞浆浓染、核固缩等神经元变性受损征象。而痴呆大鼠海马CA1锥体神经细胞排列疏松、数目减少、细胞形态异常,许多细胞出现体积缩小、核浓染、核固缩;痴呆鼠海马CA1区GFAP阳性细胞数目明显增多,胞体肥大,突起增粗、变长现象明显,而假手术组海马CA1区仅见少量GFAP阳性细胞,突起较少、短,染色较淡。计数和测量海马CA1区GFAP阳性细胞数目、总面积、平均光密度,痴呆组与假手术组相比均明显增加,有显著意义(P<0.05);水迷宫测试显示痴呆组大鼠隐藏平台获得时间比假手术组明显延长,空间探索时间明显缩短,具有显著意义(P<0.05);显示痴呆组大鼠学习记忆能力与假手术组比较均明显下降;将痴呆大鼠学习成绩与海马CA1区GFAP表达数目之间进行相关分析,两者间存在负相关关系,认为星形胶质细胞参与了学习记忆过程。结论提示海马星形胶质细胞的过度表达可能影响痴呆大鼠的学习记忆能力。     

丁咯地尔对慢性脑缺血大鼠海马CA1区星形胶质细胞及认知障碍的影响

目的研究丁咯地尔对慢性脑缺血大鼠海马CA1区星形胶质细胞和认知障碍的影响。方法采用双侧颈总动脉永久性结扎制备慢性脑缺血模型，治疗组大鼠给与丁咯地尔灌胃，免疫组化法多克隆抗血清GFAP标记海马CA1区星形细胞，用Y-型迷宫测定大鼠的认知功能变化。实验研究为持久性2VO2个月。结果慢性脑缺血2个月后大鼠海马CA1区星形胶质细胞大量增生肥大，认知能力明显下降，丁咯地尔治疗后，星形胶质细胞的活动明显减少，认知功能明显提高。结论丁咯地尔能抑制慢性脑缺血大鼠海马CA1区星形胶质细胞反应，改善其认知功能障碍。     

7蛋白酶体抑制剂对星形胶质细胞周期素D1和周期素依赖性激酶4表达的影响

目的：研究蛋白酶体抑制对体外培养的星形胶质细胞周期素Dl（cyclinD1）和周期素依赖性激酶4（CDK4）表达的影响。方法：SD乳鼠皮质星形胶质细胞原代培养,并纯化鉴定;予不同浓度（2和4μmol·L^-1）的蛋白酶体抑制剂（lactacystin）对第二代星形胶质细胞进行短期（12h）急性干预处理,应用免疫荧光及Westernblot检测星形胶质细胞cyclinD1和CDK4表达的水平。结果：纯化传代的皮质星形胶质细胞经胶质纤维酸性蛋白（GFAP）免疫荧光鉴定,其阳性率可达99％;lactacystin2和4μmol·L^-1可诱导星形胶质细胞cyclinDl和CDK4表达的下降,与对照组相比差异有显著统计学意义（P〈0．01）。结论：一定程度蛋白酶体活性抑制可诱导培养的星形胶质细胞cyclinD1和CDK4表达的减少,从而影响胶质细胞细胞周期,促进胶质细胞分化。提示蛋白酶体功能障碍后可能通过影响胶质细胞细胞周期来参与阿尔茨海默病的病理改变。     

β淀粉样蛋白、炎性细胞因子与阿尔茨海默病

阿尔茨海默病主要病理特征是大脑皮层与海马区出现老年斑和神经元纤维缠结。β淀粉样蛋白是老年斑 的主要成分,能激活小胶质细胞与星形胶质细胞,使炎症细胞因子过度表达,导致炎症反应,而抗炎治疗具有积极 作用。     

炎性细胞因子在早期母子分离对成年后 大鼠认知功能影响中的作用

目的 研究早期母子分离对成年雄性大鼠认知功能的影响,以及海马区炎性细胞因子在 其中的作用,以探讨生命早期应激对神经发育影响的机制。方法 新生SD大鼠随机分成母子分离组（MS 组）和空白对照组（NMS 组）,MS 组幼鼠在出生后第3～22 天,每天与母鼠分离3 h。NMS 组不做处理。 10 周龄时,对两组成年大鼠进行Morris水迷宫行为学测试,NeuN免疫荧光染色观察两组大鼠海马齿状 回（DG 区）正常及变性神经元,GFAP/Iba-1 免疫荧光染色观察星形胶质细胞和小胶质细胞,Ki67/DCX 免 疫荧光染色观察神经元增殖、分化情况,蛋白电泳法检测两组大鼠大脑海马区IL-1β、IL-6、TNF-α含 量。结果 相对于NMS 组,行为学测试提示MS 组大鼠学习、记忆能力下降,表现为MS 组大鼠有更长的 逃逸潜伏期,更少的目标象限停留时间和穿越平台次数（P＜ 0.05）;海马DG 区正常及变性神经元的数目 无明显变化（P ＞ 0.05）,但星形胶质细胞及小胶质细胞的数目增加（P ＜ 0.01）,且神经元增殖减少、分化 减缓（P＜ 0.01）;海马区IL-1β、TNF-α表达增高（P＜ 0.01）,IL-6 表达无明显变化（P＞ 0.05）。结论 生 命早期重复母子分离能够引起大鼠海马区神经炎性反应,增加星形胶质细胞和小胶质细胞数目,增高 海马区炎性细胞因子的表达,导致成年后大鼠认知功能的改变。     

Maternal choline availability alters the localization of p15Ink4B and p27Kip1 cyclin-dependent kinase inhibitors in the developing fetal rat brain hippocampus

Previously we have shown that changes in maternal dietary choline are associated with permanent behavioral changes in offspring. Importantly, in adult male rats, feeding a choline-deficient diet increases the localization of cyclin-dependent kinase inhibitors (CDKIs) in the liver, whereas young adult CDKI knockout mice (p15Ink4B or p27Kip1) exhibit behavioral abnormalities. Thus, maternal dietary choline-CDKI interactions could underlie the changes we observe in fetal hippocampal development and cognitive function in offspring. Here, timed-pregnant rats on embryonic day E12 were fed the AIN-76 diet with varying levels of dietary choline for 6 days, and, on E18, fetal brain sections were collected, and the localization of CDKI proteins was studied using immunohistochemistry and an unbiased image analysis method. In choline-supplemented animals compared to controls, the number of cells with nuclear immunoreactivity for p15Ink4b CDKI protein was decreased 2- to 3-fold in neuroepithelial ventricular zones and adjacent subventricular zones corresponding to the fimbria, primordial dentate gyrus and Ammon's horn regions in the fetal hippocampus. In contrast, maternal dietary choline deficiency significantly decreased nuclear p15Ink4b immunoreactivity in the neuroepithelial layer of the dentate gyrus. Unlike p15Ink4b, the CDKI protein p27Kip1 was observed almost exclusively in the cytoplasm, though the protein was distributed throughout the proliferating and postmitotic zones in the E18 fetal hippocampus. Maternal dietary choline supplementation decreased the cytoplasmic staining intensity for p27Kip1 throughout the fetal hippocampus compared to control animals. Choline deficiency increased the staining intensity of p27Kip1 throughout the hippocampus in association with increased expression of MAP-1 and vimentin proteins. These results link maternal dietary choline availability to CDKI protein immunoreactivity and commitment to differentiation during fetal hippocampal development.     

Expression of Na(+)-K(+)-Cl(-) cotransporter in rat brain during development and its localization in mature astrocytes

Na(+)-K(+)-Cl(-) cotransporter has been proposed to play an important role in the regulation of intracellular Cl(-) concentration in neurons during development. In this study, the expression pattern of the cotransporter in different regions of rat brain was examined at birth (P0), postnatal days 7 (P7), P14, P21, and adult by Western blotting analysis. In cortex, thalamus, cerebellum and striatum, the cotransporter expression level was low at P0 and significantly increased at P14 (P<0.05). The expression peaked at P21 and was maintained at the same level in adulthood. However, in hippocampus, a peak level of the cotransporter expression was detected in adult brain. The immunocytochemistry study of adult rat brain revealed that an intense staining of the Na(+)-K(+)-Cl(-) cotransporter protein was observed in dendritic processes of CA1-CA3 hippocampal pyramidal neurons. In contrast, abundant immuno-reactive signals of the cotransporter were found in somata of thalamic nucleus. Immunofluorescence double staining demonstrates that the Na(+)-K(+)-Cl(-) cotransporter was expressed in astrocytes within cortex, corpus callosum, hippocampus and cerebellum. In addition, co-localization of the cotransporter and glial fibrillary acidic protein (GFAP), or with aquaporin 4, was found in perivascular astrocytes of cortical cortex and white matter. The results indicate that a time-dependent expression of the Na(+)-K(+)-Cl(-) cotransporter protein occurs not only in cortex but also in hippocampus, striatum, thalamus and cerebellum. In addition, the cotransporter is expressed in astrocytes and perivascular astrocytes of adult rat brain.     

Glial expression of fibroblast growth factor-9 in rat central nervous system.

We examined the expression of fibroblast growth factor (FGF)-9 in the rat central nervous system (CNS) by immunohistochemistry and in situ hybridization studies. FGF-9 immunoreactivity was conspicuous in motor neurons of the spinal cord, Purkinje cells, and neurons in the hippocampus and cerebral cortex. In addition to the neuronal localization of FGF-9 immunoreactivity that we reported previously, the present double-label immunohistochemistry clearly demonstrated that the immunoreactivity was present in glial fibrillary acidic protein (GFAP)-positive astrocytes preferentially present in the white matter of spinal cord and brainstem of adult rats and in CNPase-positive oligodendrocytes that were arranged between the fasciculi of nerve fibers in cerebellar white matter and corpus callosum of both adult and young rats. There was a tendency for FGF-9 immunoreactivity in oligodendrocytes to be more pronounced in young rats than in adult rats. The variation of oligodendrocyte FGF-9 immunoreactivity in adult rats was also more pronounced than that in young rats. With in situ hybridization, FGF-9 mRNA was observed in astrocytes in the white matter of rat spinal cord and oligodendrocytes in the white matter of cerebellum and corpus callosum of adult and young rats. The expression of FGF-9 mRNA in glial cells was lower than in neurons, and not all glial cells expressed FGF-9. In the present study, we demonstrated that FGF-9 was expressed not only in neurons but also in glial cells in the CNS. FGF-9 was considered to have important functions in adult and developing CNS.     

Region-specific expression of cell cycle inhibitors in the adult brain

In the adult brain, neural proliferation is almost absent and neurons are generally not renewed. By contrast, in the olfactory organ, olfactory neurons are produced continuously throughout life. To investigate whether specific cell cycle inhibitors are involved in the control of neural quiescence in adulthood, we compared their expression either in different regions of the adult brain weakly or non neurogenic or, for comparison, in the olfactory mucosa. We show that numerous cell cycle inhibitors are expressed in the adult brain either in an ubiquitous fashion (as p19Ink4d) or in specific brain regions (p15Ink4b in the forebrain, p27Kip1 and p21Cip1 in the cerebellum). By contrast p18Ink4c was expressed detectably only in the highly neurogenic olfactory epithelium. The present data suggest that various CDK inhibitors may be involved in a region-specific fashion in the maintenance of nerve cell quiescence in adults.     

Localization of insulin-like growth factor I (IGF-I)-like immunoreactivity in the developing and adult rat brain

The cellular distribution of insulin-like growth factor I (IGF-I) immunoreactivity was examined in the rat brain from embryonic day 15 to maturity. IGF-I immunoreactivity was found in the perikarya of neurons distributed along the entire extension of the neuronal tube in all the embryonic ages studied (E15, E17, E19 and E21). In E21 animals, the majority of immunoreactive neurons was located in the olfactory bulb, cerebral cortex, hippocampus, striatum, diencephalon, mesencephalic colliculi, trigeminal ganglion and in motoneurons of the brainstem. In 10- and 20-day-old rats, in addition to the above areas, IGF-I immunoreactivity was also observed in capillary walls, ependymal cells, choroid plexus, glial cells and most fiber paths. In postnatal ages, immunoreactivity in neuronal somas mainly restricted to the cell nuclei. However, IGF-I immunoreactivity in the neuron cytoplasm was observed in 20-day-old rats treated with colchicine while fiber paths and neuronal cell nuclei were negative in these animals. In the telencephalon of 20-day-old rats injected with colchicine, the most intense immunoreactive neurons were observed in the olfactory bulb, cerebral cortex, tenia tecta, hippocampus, islands of Calleja, septal nuclei, striatum, endopyriform nucleus and amygdala. Most diencephalic nuclei, the substantia nigra, the mesencephalic colliculi, Purkinje cells in the cerebellar cortex and several nuclei in mesencephalon, pons and medulla oblongata were also immunoreactive. In adult rats injected with colchicine, IGF-I immunoreactivity was located in the same areas as in 20-day-old rats. The number of immunoreactive cells and the intensity of the staining was reduced in adult rats as compared to that found in young postnatal animals. Glial cells were negative in adults. The distribution of IGF-I in the developing and mature rat brain supports the proposed roles of this peptide as a neuromodulator and neurotrophic factor.     

马桑内酯激活的星形胶质细胞条件培养液对大鼠脑内谷氨酸及其受体GluR2表达的影响

目的 揭示马桑内酯(CL)激活的星形胶质细胞(Ast)条件培养液(ACM)对大鼠脑内谷氨酸(Glu)及其受体GluR2表达的影响.方法 取成年健康雄性SD大鼠48只,采用随机数字表法分为对照组(16只)和CL组(32只),对照组侧脑室注射未加任何刺激物的ACM 10μL,CL组侧脑室注射CL激活的ACM 10μL;按注射后取材时间不同又分为2h、4h、8h和12h四个亚组,对照组每亚组4只,CL组每亚组8只.观察两组大鼠的行为表现,用免疫组化、免疫荧光检测脑内Glu和GluR2表达的变化,Western blot检测脑内GluR2含量的变化.结果 CL组大鼠有痫样发作,而对照组无痫样发作;免疫组化和免疫荧光检测结果显示,CL组皮质和海马区Glu表达较对照组显著增强,4h时差异有统计学意义(P<0.05),而CL组皮质和海马区GluR2的表达较对照组弱,4h时差异有统计学意义(P<0.05).Western blot结果显示,CL组4个时间点的GluR2表达均较对照组含量显著降低,差异有统计学意义(P<0.05).结论 CL激活的ACM能显著增强脑内神经元Glu的表达,降低GIuR2的表达,进而诱发癫痫.     

脑缺血对大鼠皮层及海马铜蓝蛋白表达的影响

目的 探讨脑缺血对大鼠皮层及海马中铜蓝蛋白(Ceruloplasmin,Cp)表达的影响.方法 雄性Wistar大鼠60只,随机分为脑缺血1、3、7、28 d组和假手术对照组,每组各12只.实验组结扎双侧颈总动脉造成大鼠脑缺血,假手术对照组仅分离出双侧颈总动脉但不结扎.采用反转录聚合酶链反应(RT-PCR)检测皮层及海马组织中Cp mRNA的表达,免疫组织化学观察皮层及海马组织中Cp的表达.结果 大鼠皮层和海马均表达Cp mRNA.皮层和海马Cp mRNA的表达随缺血时间的延长逐渐降低,缺血1、3、7、28 d组表达均低于假手术组(P＜0.01).脑组织脉络丛细胞、室管膜细胞、皮层和海马的星形胶质细胞、血管内皮细胞均表达Cp;而皮层和海马的锥体细胞和颗粒细胞均不表达Cp.缺血1 d组皮层及海马Cp表达与对照组差异不显著(P＞0.05);缺血3 d组皮层和海马Cp表达低于假手术组(P＜0.05);缺血第7、28 d组Cp表达减少极为显著(P＜0.01).脑缺血大鼠皮层和海马中铁含量与Cp的表达呈负相关,相关系数分别为-0.831(P＜0.01)和-0.809(P＜0.01).结论 脑缺血可诱导大鼠皮层及海马中Cp表达降低.脑缺血后Cp表达减少可能参与了脑缺血引起的铁含量升高及神经元铁沉积的过程.     

Constitutive activity and differential localization of p38alpha and p38beta MAPKs in adult mouse brain

To understand the roles of p38 mitogen-activated protein kinase (p38 MAPK) isoforms in adult mouse brain, in vivo activities and detailed expression patterns of two p38 isoforms, p38alpha and p38beta, were examined by using biochemical and immunohistochemical analyses. The result indicated that the activity of both p38alpha and p38b MAPKs in normal adult mouse brain was remarkably high, and the nuclear pool of the p38 isoforms was primarily responsible for most of the constitutive p38 MAPK activity in brain. Both p38alpha and p38beta were highly expressed in brain areas including cerebral cortex, hippocampus, cerebellum, and few nuclei of the brainstem. At the subcellular level, p38alpha was distributed in dendrites and in cytoplasmic and nuclear regions of cell body of neurons, which is in contrast to p38beta, since p38beta was preferentially expressed in nucleus of neurons. These results suggest that the p38 pathway may play an important role, not only in inflammation and neuronal cell death as previously suggested, but also in normal physiology of adult mouse brain.