切割穹窿海马伞大鼠海马内BLBP的表达变化

为了观察切割大鼠右侧穹窿海马伞后,切割侧与正常侧海马齿状回内脑脂结合蛋白(BLBP)的表达变化,本研究应用Western blot和免疫组织化学方法检测双侧海马内BLBP蛋白表达水平的变化,以及海马齿状回门区和颗粒下层中BLBP免疫阳性细胞数和灰度值。结果显示:正常大鼠双侧海马各区和颗粒下层细胞BLBP仅有微量表达,切割穹窿海马伞后第1 d双侧差异不明显;3 d时切割侧颗粒下层阳性细胞及染色深度较正常侧加深;5 d时切割侧颗粒下层阳性细胞数量明显增多,染色较深,并达到最高水平;7 d后BLBP免疫阳性细胞的数量和染色深度开始降低,14 d时接近正常侧水平。而切割后3、5 d时双侧门区BLBP免疫阳性细胞的数量差异不大,但切割侧染色较深,7 d后也逐渐降低,14 d时降至正常侧水平。上述结果提示,切割穹窿海马伞阻断了隔区与海马齿状回的纤维联系后,可引起海马齿状回门区和颗粒下层BLBP表达增强,可能引发了放射状胶质细胞的增殖和激活,构成的支架有助于神经干细胞的迁移和向神经元的分化。     

Jagged1在大鼠切割穹窿海马伞后海马内的表达变化

目的:探讨切割穹窿海马伞后不同时相点海马内Jagged1的动态表达变化。方法:切割SD大鼠双侧穹窿海马伞,于切割后第3、7、14、21 d分别提取海马组织总RNA和总蛋白,应用RT-PCR和Western Blot的方法分别检测Jagged1基因和蛋白的表达变化;切割SD大鼠右侧穹窿海马伞,7 d后通过免疫组织化学的方法检测海马齿状回颗粒下层和门区中Jagged1阳性细胞的数目和平均光密度值(MOD)。结果:Jagged1基因和蛋白在切割穹窿海马伞后的第3 d表达开始增高,第7 d时达到最高水平,第14 d后表达下降至正常水平;切割穹窿海马伞后的第7 d,切割侧海马齿状回颗粒下层和门区中Jagged1阳性细胞数为167.89±22.11,平均光密度值为0.11±0.02;正常侧阳性细胞数为140.45±22.63,平均光密度值为0.06±0.01;切割侧阳性细胞数和平均光密度值与正常侧均明显增高(P0.05)。结论:切割穹窿海马伞后Jagged1基因和蛋白的表达呈现出先增高后降低的变化趋势,提示Jagged1是切割穹窿海马伞后海马内微环境的重要组成分子。     

切割穹窿海马伞大鼠海马内Lhx8 mRNA表达的变化

目的:探讨切割穹窿海马伞大鼠切割侧与正常侧海马内Lhx8 mRNA表达的差异。方法:切割SD大鼠右侧穹窿海马伞。切割后7d制备海马冰冻切片,用体外转录法制备地高辛标记的Lhx8 RNA探针进行原位杂交,分析切割侧和正常侧海马锥体细胞层和齿状回颗粒层中及齿状回门区和颗粒下层中的Lhx8 mRNA阳性细胞的数量和平均光密度值。结果:切割侧和正常侧海马锥体细胞层和齿状回颗粒层Lhx8 mRNA阳性细胞数量无明显差异,但切割侧平均光密度值较正常侧明显增加;在齿状回的门区和颗粒下层,切割侧Lhx8 mRNA阳性细胞数和平均光密度值均较正常侧升高。结论:切割穹窿海马伞后海马中Lhx8 mRNA表达上调,可能与其中的神经干细胞向胆碱能神经元分化的神经再生机制有关。     

穹窿海马伞切割大鼠海马内Brn-4 mRNA 的表达变化

目的探讨切割穹窿海马伞大鼠海马与正常海马内Brn-4 mRNA表达的差异。方法42只SD大鼠随机分成正常对照组和切割海马伞后1、3、7、14、21及28d组。取各组大鼠的海马组织,提取总RNA,采用半定量RT-PCR法分析穹窿海马伞切割后海马内Brn-4 mRNA表达水平的变化。结果海马内Brn-4 mRNA的表达量在切割后第3d开始升高,14d达到最高水平,随后下降,28d左右恢复至正常水平。结论切割穹窿海马伞后海马内Brn-4 mRNA表达明显上调,可能与促进神经干细胞更多地向神经元和AChE阳性神经元分化有关。     

切割穹窿海马伞大鼠海马内Brn-4 mRNA的表达变化——原位杂交法

目的 探讨大鼠穹隆海马伞切割侧与正常侧海马内Brn-4 mRNA表达的差异.方法 切割大鼠右侧穹窿海马伞,切割后14d制备海马冰冻切片,用体外转录法制备地高辛标记的Brn-4 RNA探针进行原位杂交.每只动物随机计数3张切片切割侧和正常侧Brn-4 mRNA的阳性细胞,测定其吸光度(A)值,进行配对t检验分析.结果 切割侧和正常侧海马锥体细胞层和齿状回颗粒层均见Brn-4 mRNA阳性细胞,两侧细胞数无明显差异,但切割侧阳性细胞平均吸光度值较正常侧明显增加(P＜0.01);而在齿状回门区和颗粒下层,切割侧Brn-4 mRNA阳性细胞数和平均吸光度值均较正常侧升高(均P＜0.01).结论 穹窿海马伞切割侧海马锥体细胞层和齿状回颗粒层细胞中Brn-4 mRNA的表达量明显增强,而在齿状回门区和颗粒下层中,其阳性细胞数和表达量均较正常侧明显升高.结合本课题组以往的工作,提示切割穹窿海马伞后,海马中Brn-4 mRNA表达的增高可能与促进其中的神经干细胞向神经元分化有关.     

NOV蛋白在切割海马伞大鼠海马中表达的变化

通过切割右侧海马伞制备大鼠海马伞损伤模型,应用Westernblotting、免疫组织化学技术,观察切割海马伞后不同时程海马中肾母细胞瘤过度表达基因(NOV)蛋白表达的变化,并对结果进行图像处理和统计学分析。Westernblotting结果显示,NOV蛋白在切割海马伞后3d表达开始上升,14d达高峰后缓慢下降。切割海马伞后14d切割侧和正常侧相比较,海马CA1-CA3区的锥体细胞层及齿状回颗粒层NOV阳性细胞数目无显著性差异(P>0.05),但切割侧NOV阳性细胞明显比正常侧深染,两侧比较平均灰度值有显著性差异(P<0.01)。结合本课题组以往的工作,本研究结果提示,切割海马伞后海马中高表达的NOV蛋白可能参与了诱导神经干细胞迁移和向神经元分化的过程。     

切割穹窿海马伞大鼠海马IGF2及其受体IGF2R蛋白的表达变化

目的:观察穹窿海马伞切割后不同时间点海马胰岛素样生长因子2(insulin-like growth factor,IGF2)及其受体IGF2R蛋白的表达变化及定位。方法:应用ELISA、Western blot和免疫荧光组织化学方法检测海马内IGF2及其受体IGF2R蛋白,以及齿状回门区和颗粒下层中IGF2/Hoechst和IGF2R/Hoechst阳性细胞的数量及形态学变化。结果:(1)ELASA结果显示:IGF2在切割后7 d表达开始上升,14 d达最高水平,随后缓慢下降,28 d时降至正常水平。(2)Western blot结果显示:IGF2R也存在一个相应的表达升高与消退的过程,说明两者具有明显的相关性。(3)免疫荧光染色结果显示:正常组海马齿状回门区和颗粒下层IGF2及其受体IGF2R阳性细胞的数量较少,切割穹窿海马伞后第3 d数量稍有增多,但差异不明显;7 d时阳性细胞明显增多;14 d时阳性细胞继续增多,并达到最高水平;21 d后阳性细胞的数量开始下降,28 d时接近正常组水平。结论:切割穹窿海马伞后IGF2及其受体IGF2R的高表达可能与海马神经再生中抑制神经干细胞和放射状胶质细胞增殖,从而启动其向神经元等分化有关。     

穹窿海马伞切割侧海马提取液对神经干细胞分化为神经元的促进作用

目的 探讨穹窿海马伞切割侧海马提取液对神经干细胞分化为神经元和AchE阳性神经元的影响。方法 切割SD大鼠右侧穹窿海马伞,14d后取两侧海马制成提取液;将神经干细胞接种于3块24孔培养板中,每板分成切割组、正常组和对照组各8孔,前2组分别加入切割侧和正常侧海马提取液。第1、2块分别于第7d和14d时行MAP-2免疫荧光检测;第3块于10d时行AChE组织化学染色。计数MAP-2和AChE阳性神经元数,观察胞体大小和突起长度。结果 切割组第7d时MAP-2阳性神经元较多,胞体大、突起长,14d时细胞进一步迁移、成熟;正常组第7d时仅见少量突起短的MAP-2阳性神经元,14d时细胞稍增多,突起稍增长：对照组第7d时未见MAP-2阳性神经元,14d时仅有少量胞体小、突起短的MAP-2阳性神经元。第10d切割组AChE阳性神经元较多,突起多而长,正常组仅见少量无突起的AChE阳性神经元,对照组未见AChE阳性神经元。结论 穹窿海马伞切割侧海马提取液可明显促进神经干细胞分化为神经元和AChE阳性神经元。     

穹窿海马伞切割侧海马对植入神经干细胞分化为神经元的影响

为观察成鼠神经干细胞移植入切割穹窿海马伞侧海马和正常侧海马后存活和分化为神经元的状况 ,用无血清培养和单细胞克隆技术获取成年 SD大鼠前脑室下带神经干细胞 ,进行 Brd U标记和扩增。切割 SD大鼠右侧穹窿海马伞 ,术后 2周将标记有 Brd U的神经干细胞植入双侧海马齿状回。 2月后 ,行 Nissl染色、Brd U免疫荧光、NF -2 0 0 / Brd U免疫荧光、β-tubulin- / Brd U免疫组织化学染色和 ACh E组织化学染色。结果发现 ,移植的神经干细胞在海马齿状回中存活并沿颗粒下层迁移 ,切割侧海马齿状回中有较多的 Nissl深染的大胞体神经元样细胞 ,而正常侧多为小胞体胶质样细胞。切割侧海马齿状回颗粒下层中见有数个NF-2 0 0 / Brd U、β-tubulin- / Brd U双标神经元和 ACh E阳性神经元 ,而正常侧海马中则未能见到。上述结果提示 ,移植到海马中的神经干细胞能存活、迁移 ,穹窿海马伞切割侧海马中某些物质的表达增强 ,可诱导植入其内的神经干细胞向神经元或 ACh E阳性神经元分化。     

神经生长因子对穹窿海马伞切割后海马自体神经干细胞增殖和向神经元分化的影响

目的 探讨切割穹窿海马伞及脑室给予神经生长因子(NGF)后,对大鼠海马自体神经干细胞增殖和分化为神经元的影响.方法 12只SD大鼠,随机分成给药组和对照组,每组6只,切割右侧穹窿海马伞,两组切割后即时及第2d、4d向侧脑室分别注射NGF和人工脑脊液, 并在术后第3～7d每日腹腔注射2次BrdU.于术后28d灌注取脑、冷冻切片,行BrdU/NF-200免疫荧光双标检测.结果 海马齿状回内BrdU阳性细胞数,给药组和对照组切割侧明显多于正常侧,给药组切割侧和正常侧分别多于对照组切割侧和正常侧;海马齿状回内的BrdU/NF-200双标神经元数,给药组切割侧最多,给药组正常侧和对照组切割侧较少,而对照组正常侧则无.结论 切割穹窿海马伞后,可致大鼠海马齿状回自体神经干细胞增殖加快并向神经元分化,脑室施加 NGF可促进其增殖和分化.     

穹隆海马伞切割后大鼠海马内RUNX1T1 mRNA和蛋白的表达变化

目的 观察穹隆海马伞切割后不同时间点海马内RUNX1T1 mRNA和蛋白的表达变化及定位。方法 行穹隆海马伞切割术,制备模型大鼠。实验分为正常组、切割3d组、切割7d组、切割14d组、切割21d组、切割28d组。1.提取海马组织总mRNA,用Real-time PCR 检测大鼠海马组织中RUNX1T1mRNA 的表达;2.提取海马组织总蛋白,用Western blotting 检测海马内RUNX1T1蛋白的表达;3.行脑冷冻切片,行RUNX1T1免疫荧光检测和Hoechst标记,观察海马齿状回中RUNX1T1/Hoechst双标阳性细胞。 结果 Real-time PCR检测显示,切割3d组海马组织中RUNX1T1 mRNA的表达明显上调,其余各组差异不明显;Western blotting检测结果显示,正常组海马中有微量RUNX1T1蛋白表达,而切割3d组海马组织中RUNX1T1蛋白表达量明显上升,并达到高峰,7d时仍有较多的表达,此后,14d、21d、28d组中RUNX1T1蛋白表达很快又恢复到正常水平;免疫荧光检测结果表明,正常组海马齿状回中有少量的RUNX1T1/Hoechst阳性细胞,切割3d组海马齿状回中RUNX1T1/Hoechst阳性细胞数量最多,切割7d组逐渐减少,切割14d组、21d组和28d组与正常组差别不明显。结论 穹隆海马伞切割后海马内RUNX1T1 mRNA和蛋白表达在早期呈短暂性上调,并定位于海马齿状回颗粒下层,推测可能与海马神经再生过程中神经干细胞向神经元分化有关。     

大鼠海马神经元核受体-糖皮质激素受体在穹窿切断同时摘除肾上腺后表达的变化

目的:通过建立穹窿切断的动物模型来检测糖皮质激素的浓度和穹窿切断同时摘除肾上腺的动物模型检测海马神经元核受体-糖皮质激素受体(GR)的表达变化,以探讨GR参与下丘脑-垂体-肾上腺(HPA)轴调节的机制。方法:建立大鼠穹窿切断模型,采用ELISA方法检测穹窿切断0,4,7,10d血中糖皮质激素的浓度、建立大鼠穹窿切断同时摘除肾上腺7d的模型,采用免疫组织化学、免疫印迹方法检测海马神经元核受体GR表达变化的观察及定量检测。结果:穹窿切断7d后血中糖皮质激素的浓度升高,穹窿切断同时摘除肾上腺7d后,海马神经元GR表达升高。结论:海马核受体GR的表达变化可能与糖皮质激素的浓度呈负相关。     

大鼠海马神经元核受体-盐皮质激素受体于穹窿切断后表达的变化

目的:建立大鼠穹窿切断模型,研究海马神经元核受体-盐皮质激素受体(MR)表达的变化.方法:建立大鼠穹窿切断模型,于穹窿切断术后0、4、7、10 d取材;同时取材假手术组(非穹窿切断术)、正常组作为对照,应用免疫组织化学、免疫印迹方法分别进行各组海马神经元MR表达变化的观察及定量检测.结果:穹窿切断7 d后,免疫组织化学和免疫印迹结果显爪海马神经元MR表达下调,10 d下调更为显著.结论:穹窿切断后,海马MR表达下调,提示海马对下丘脑-垂体-肾上腺轴的抑制作用可能在穹窿切断后减弱.     

去神经支配大鼠海马内MTSS1的表达变化

目的:探讨切割穹窿海马伞去神经支配大鼠海马内转移抑制蛋白1(metastasis suppressor 1,MTSS1)的表达变化。方法:SD大鼠随机分成正常组和切割穹窿海马伞后1、3、5、7、14 d组,采用Real-time PCR和免疫荧光组织化学技术观察穹窿海马伞切割后海马内MTSS1 mRNA的表达变化和MTSS1阳性细胞的分布情况。结果:(1)Real-time PCR结果显示:海马内MTSS1 mRNA的相对表达量在切割后3 d开始升高(P<0.05),5 d达到最高水平(P<0.01),7 d恢复至正常水平;(2)免疫荧光组织化学染色结果显示:MTSS1阳性细胞主要表达于海马齿状回门区及颗粒下层中,切割后5 d MTSS1阳性细胞数开始增多(P<0.01),7 d继续增多并达到高峰(P<0.01),14 d时开始下降至正常水平。结论:结合本课题组以往的工作,上述结果提示切割穹窿海马伞后MTSS1的高表达可能与海马神经再生过程中神经干细胞向神经元的分化有关。     

The organization of the fimbria, dorsal fornix and ventral hippocampal commissure in the rat

Summary The morphological organization of the two principal efferent pathways of the hippocampal formation — the fimbria and the dorsal fornix — and of the interhippocampal fibers that cross in the ventral hippocampal commissure, has been studied autoradiographically following injections of ³H-amino acids into the relevant cytoarchitectonic fields. To a considerable extent the rostrally-directed fibers from each cytoarchitectonic field remain topographically segregated within the fimbria or dorsal fornix, their locations being determined by the relative levels at which they join these bundles. Thus, the fibers from the subicular region tend to be aggregated along the ependymal border of the fimbria while those from the hilar region of the dentate gyrus are medially-placed along its pial margin. Similarly, the efferents from the septal and temporal parts of the various fields are distributed along the proximo-distal dimension of the fimbria, from its base (adjacent to field CA₃) to its tip. The dorsal fornix contains fibers from the septal (dorsal) part of the subiculum and field CA₁; although there appears to be some overlap, the subicular efferents tend to be more medially placed while those from CA₁ are further lateral. The topographic organization of the fibers in the fimbria is reflected in the arrangement of the crossed components in the hippocampal commissure. Those arising at more temporal levels cross most rostrally while those from the septal pole of field CA₃ cross in the caudal part of the commissure; those from the hilar region of the dentate gyrus cross most ventrally, while those from the more lateral parts of field CA₃ (CA_3b and CA_3a) cross at successively more dorsal levels.We estimate that the fimbria contains about 900,000 fibers of which approximately 700,000 are myelinated (with internal diameters ranging between 0.1 and 2.8m; mean 0.78 m) and just over 200,000 are unmyelinated (with diameters between 0.1 and 0.8 m; mean 0.24 m).Supported by Grants EY-01255, NS-10943 and NS-13267 from the National Institutes of Health, and by a grant from the Sloan Foundation. We should like to thank Mrs. LueVern Bell and Mrs. Pamela Seyer for their excellent histological work and Mrs. Mary Murphy for secretarial help     

1H-NMR imaging of fimbria fornix lesions in the rat brain

Summary Mechanical lesions of the fimbria fornix (FF) have been widely used as a model to investigate the recovery of damaged brain tissue.¹H-NMR imaging was employed to non-invasively measure changes in the brain after unilateral FF transection. Rats were subjected to NMR imaging at various times after the lesions were made. The experimental protocol included (multislice) T₂-weighted and diffusion-weighted imaging thereby allowing the construction of two-dimensional maps of the relaxation time T₂ (transverse or spin-spin relaxation time) and the apparent diffusion coefficient (ADC) of water. FF transection induced considerable changes in the status of the brain tissue at a number of different locations which were exclusively present in the affected hemisphere. At 1 day post-lesion the region of the lateral ventricle and hippocampus started to display pronounced changes in that T₂- and diffusion-weighted images showed a hyperintensity and a hypointensity, respectively. These effects were maximal around day 2 to 4 whereafter a slow recovery towards the control situation was observed. Immediately after transection the FF lesion itself could be visualized. These early images pointed to an aspecific disruption of the tissue due to the mechanical intervention. Interestingly, however, from day 2 post-lesion a number of changes became evident in this region which seemed to be localized to specific structures, including the ventricle and hippocampus. After one month the presumably ventricular effect dominated and was predominantly localized to the anterior side of the FF lesion. These findings are indicative of pronounced changes in the status of water (e.g., in its distribution between extra- and intracellular compartments) at a number of locations distant from the site of FF transection. The mechanism by which these changes are brought about and the origin of their time-dependence remain to be elucidated.This research was carried out at the Netherlandsin vivo NMR facility at the Bijvoet Center for Biomolecular Research (Utrecht University) which is supported by the Netherlands Organization for Scientific Research (NWO).Supported by grants from the Netherlands Organization for Scientific Research (NWO).