银杏内酯对大鼠基底前脑神经元生长发育的影响

目的：观察银杏内酯对培养的胚胎大鼠基底前脑胆碱能神经元和NADPH-d阳性神经元生长发育的作用。方法：取E17Wistar大鼠胚胎基底前脑进行体外细胞培养，并给予银杏内酯。进行乙酰胆碱酯酶(acetyl cholinesterase，AChE)和NADPH-d酶组织化学染色，并在光镜下进行阳性细胞计数和显微测量胞体及突起发育状况。结果：给予银杏内酯处理后，培养物中乙酰胆碱酯酶阳性神经元和NADPH-d阳性神经元数量均较对照组显著增加，且神经元发育状况好于对照组，胞体大，突起多且长。结论：银杏内酯可以促进体外培养的基底前脑神经元中乙酰胆碱酯酶和NADPH-d的表达，并能促进表达这两种酶的神经元生长发育。     

施万细胞对植入大鼠脊髓损伤区内的神经干细胞存活和分化的影响

目的 探讨施万细胞对植入损伤脊髓内的神经干细胞的存活及其分化的影响。方法 分离和克隆新生大鼠海马组织的神经干细胞；同时获取坐骨神经和臂丛神经，从中分离和纯化施万细胞。在移植前先用核荧光(Hoechst33342)标记神经干细胞。实验组为神经干细胞和施万细胞联合移植入大鼠脊髓半横断处，对照组为单独神经干细胞移植。应用免疫组织化学和酶组织化学技术，在移植后7d、14d、21d和30d分别观察神经干细胞的存活和分化情况。结果 实验组的神经干细胞比对照组迁移的更远，分化为神经丝蛋白染色阳性神经元样细胞的数量比对照组的多，并且有较长的突起长出。在30d实验组中，移植的神经干细胞有部分呈乙酰胆碱酯酶(AchE)染色阳性。而在对照组中，移植区内仅有少数呈AchE染色弱阳性的神经干细胞。结论 在脊髓损伤处，施万细胞可促进移植的神经干细胞存活、迁移和向神经元样细胞分化；有些神经元样细胞能长出较长的突起，有些呈现乙酰胆碱酯酶活性。     

影响大鼠胚胎多巴胺能神经元原代培养因素的研究

目的　试图通过方法改进获得高纯度原代多巴胺能神经元 ,并研究 Matrigel及多聚左旋赖氨酸(poly- l- lysine,PL L )等不同基质对于大鼠胚胎 13d中脑黑质神经元突起生长的不同作用。　方法　无菌技术取得孕 13d SD大鼠胚胎 ,采用改进的取材方法 ,得到胎鼠腹侧中脑细胞 ,以较高密度 (1× 10 5 / cm2 )分别种入由 Matrigel及 PL L作为基质的塑料培养皿中。体外培养 5 d后 ,对培养物进行 TH免疫细胞化学染色 ,测量 TH免疫反应突起的长度并计算 TH免疫反应神经元占细胞总数的比例。　结果和结论　 1.采用改进的方法 ,能够得到高纯度的多巴胺能神经元 (多巴胺神经元占培养细胞总数的比例为 15 % ) ;2 .与 PL L相比 ,Matrigel能有效地促进多巴胺能神经元突起的生长 ,但不能特异地促进中脑培养细胞的存活     

大鼠胚胎神经上皮细胞侧脑室移植后存活及分化的实验研究

目的：观察大鼠胚胎神经上皮细胞同种脑移植后的存活及生长分化状况。方法：孕12d大鼠剖腹取胚胎，剥离神经管，胰酶消化后获取神经管壁上的神经上皮细胞，然后植入其父体侧脑室中，分别于移植后10d,14d,21d,28d灌注取脑，肜HF染色及神经元特异烯醇化酶（NSE），胶质纤维酸性蛋白（GFAP）免疫组织化学方法检测胚胎神经上皮细胞移植后的存活及分化状况。结果：神经上皮细胞侧脑室移植后多贴附于脑室壁形成细胞团块，有的随脑脊液循环至第三脑室生长，随时间延长移植物增大，HE染色见脑室内有成团的移植细胞，免疫组织化学染色显示移植细胞团内既有NSE－免疫阳性细胞，也有GFAP－免疫阳性细胞，移植物周围多为GFAP－免疫阳性细胞。结论：胚胎神经上皮细胞侧脑室移植后能够贴附脑室壁存活，并能分化为神经元和神经胶质细胞。     

大鼠脑内乙酰胆碱酯酶的出现和变化

本文用组织化学技术对62个从胚胎早期到成体大鼠脑内乙酰胆碱酯酶(AChE)的出现和变化进行了观察,同时用Nissl法制作19个相应时期的标本作为对比检查,主要结果如下: 一、AChE的出现顺序是循脊髓→延脑、后脑和中脑→间脑→大脑。在大脑,由旧皮质向新皮质发展。这反映出AChE在大鼠神经系统发生中具有头向发展的趋向。二、大鼠脑内具有AChE阳性的神经元,包括胆碱能神经元和胆碱敏感细胞,它们都和神经递质乙酰胆碱(ACh)有关。二、以成体大鼠脑中AChE的分布与前人的工作进行对比,我们显示的阳性核团较多,如三叉神经中脑核、丘脑前背核、丘脑前腹核、丘脑外侧核、丘脑网状核、外侧缰核、视上核、室旁核、束旁核以及红核中,均能见到明显的含AChE的细胞。大脑皮质中亦有少数阳性细胞存在。     

GDNF促进大鼠背根神经元的存活和突起生长

探讨胶质细胞源性神经营养因子(GDNF)对正常胚胎大鼠背根神经节(DRGn)的存活及突起生长的作用。本实验采用神经组织原代分离培养的方法建立体外胚胎大鼠背根神经节单细胞培养体系，从细胞形态学及应用MTT。法观察1μg／L、10μg／L、50μg／L和100μg／L GDNF对体外培养的正常感觉神经元生长的影响。结果表明：GDNF能明显促进体外培养的正常大鼠背根神经节感觉神经元的存活及突起生长。提示GDNF对正常大鼠胚胎发育期感觉神经元具有神经营养作用。     

隔区向缰核的传出及乙酰胆碱酯酶阳性神经元的投射

采用ＷＧＡ－ＨＲＰ顺逆行追踪法和ＷＧＡ－ＨＲＰ与乙酰胆碱酯酶组织化学相结合技术，观察了３９只大鼠隔区主要亚细胞群神经元和乙酰胆碱酯酶阳性神经元向缰内侧核和缰外侧核的投射。结果表明：隔区向缰内侧核发出投射纤维的亚细胞群是隔三角核、隔伞核及斜角带核垂直支腹侧部；隔区向缰外侧核发出投射纤维的亚细胞群为斜角带核垂直支背、腹侧部和斜角带核水平支。其中斜角带核垂直支和斜角带核水平支向缰核发出投射纤维的细胞约二     

BDNF、NGF对体外长期培养的胚基底前脑胆碱能神经元的影响

本文探讨了脑源性神经营养因子、神经生长因子对体外长期培养的胚基底前脑胆碱能神经元是否具有延缓退变的作用。实验分为脑源性神经营养因子组、神经生长因子组、脑源性神经营养因子加神经生长因子组及单纯对照组。取孕 17d SD大鼠胚基底前脑原基制成细胞悬液接种于 2 4孔培养板中 ,按分组加入含相应神经营养因子和不含神经营养因子的 DMEM培养液 ,分别于体外培养 12、18、2 4、3 0 d后进行乙酰胆碱酯酶组织化学反应。显微镜下计数各孔中乙酰胆碱酯酶阳性神经元数 ,每孔随机测量和计数 2 5个乙酰胆碱酯酶阳性神经元的平均胞体直径、发出的突起数和最长突起长度。数据用方差分析和 SNK检验进行统计学处理。结果显示 ,在培养的 4个时期 ,含脑源性神经营养因子组、神经生长因子组和脑源性神经营养因子加神经生长因子组的各项数据均明显地优于单纯对照组 ;脑源性神经营养因子加神经生长因子组的各项数据 ,特别是最长突起长度优于单独使用脑源性营养因子或神经生长因子组。提示 :脑源性神经营养因子和神经生长因子不仅对体外培养的胚胆碱能神经元发育生长具有促进作用 ,而且还可延缓体外长期培养的大鼠胚基底前脑胆碱能神经元的退变 ;两者的联合使用还可对延缓其退变具有协同作用     

小鼠胚胎干细胞诱导的神经前体细胞大脑皮层移植对AD大鼠的治疗作用

目的与方法：将小鼠胚胎干细胞无血清诱导为神经前体细胞后移植到叠氮钠所致的阿尔茨海默氏病（AD）大鼠额叶皮层，采用免疫组化观察移植细胞的存活、分化以及细胞移植对AD大鼠Morris水迷宫记忆功能的作用。结果： 胚胎干细胞形成的胚胎样体经N2选择性培养基选择生长5 d后，85％以上的小鼠胚胎干细胞分化为nestin阳性的神经前体细胞。移植到AD 大鼠额叶皮层后4-6周，神经前体细胞存活良好，大部分移植细胞保持为未分化的nestin阳性的神经前体细胞并呈克隆生长，部分细胞发出类似于神经元的长突起。移植后4周，AD大鼠的空间记忆能力明显提高。结论： 胚胎干细胞来源的神经前体细胞移植到AD大鼠额叶皮层后能存活并分化为神经元，能改善AD大鼠的记忆功能障碍。     

嗅鞘细胞和神经干细胞联合移植阿尔茨海默病大鼠脑内的增殖和定向分化

背景：阿尔茨海默病大鼠脑内神经元减少,神经干细胞移植后增殖和向神经元分化能力有限。 目的：观察联合移植嗅鞘细胞和神经干细胞在阿尔茨海默病大鼠脑内,嗅鞘细胞对神经干细胞的增殖和向胆碱能神经元分化的影响。 方法：体外培养嗅鞘细胞和神经干细胞,移植前用5-溴脱氧尿嘧啶核苷标记神经干细胞。将生理盐水,神经干细胞和神经干细胞+嗅鞘细胞分别移植入阿尔茨海默病模型大鼠海马。移植7,14,21,28 d后,进行大鼠脑组织切片免疫组织化学染色检测BrdU和ChAT阳性表达。 结果与结论：联合移植组神经干细胞的增殖和分化情况明显优于其他两组,联合移植组BrdU阳性细胞数和ChAT阳性细胞数明显高于神经干细胞移植组和生理盐水组(P < 0.01)。提示嗅鞘细胞能促进移植的神经干细胞在阿尔茨海默病大鼠脑内增殖和向胆碱能神经元的分化。     

NGF对新生大鼠隔神经元的神经营养作用

本实验观察了神经生长因子（ＮＧＦ，２．５ｓ）对无血清培养新生大鼠隔神经元生长发育的作用。结果证明，ＮＧＦ能促进新生大鼠隔神经元的存活，增加神经元胞体面积和直径，增加隔培养神经元中ＡＣｈＥ阳性神经元的数目。流式细胞分析显示，ＮＧＦ使神经元平均蛋白含量增加。以上结果表明：ＮＧＦ对新生大鼠隔神经元的生长发育具有促进作用。Ｐ＜０．０１３．ＮＧＦ增加隔培养神经元胞体面积、最长径和最短径图像分析结果可见：在前７ｄ，在神经元胞体面积、最长径和最短径等方面两组之间无明显差别，但在培养１４、２１和３０ｄ时，ＮＧＦ组培养神经元的平均胞体面积、最长径和最短径均大于对照组，经统计学处理有显著意义（表２）。表２对隔培养神经元胞体面积、最长径和最短径的影响（ｘ±ｓ）注：Ｎ＝３０与同时期对照组相比＊Ｐ＜０．０５Ｐ＜０．０１４．ＮＧＦ促进新生大鼠隔细胞的总量白合成用ＦＡＣＳ４４０流式细胞仪测定隔细胞的平均蛋白含量，结果表明：在培养３、７、１４和２１ｄ时，ＮＧＦ组培养细胞的平均蛋白含量均显著高于对照组（表３，Ｆｉｇ．１）。表３ＮＧＦ对隔神经元蛋白合成的影响（ｘ±ｓ）往：Ｎ＝５与同时期对照组相比　＊Ｐ＜０．０５５．ＮＧＦ提高隔神经元的ＡＣｈＥ?     

Recovery of hippocampal cholinergic activity by transplantation of septal neurons in AF64A treated rats

Embryonic septal neurons were transplanted into the hippocampus of adult rats which had received lateral-ventricular administration of AF64A, a cholinergic neurotoxin, and the effects on hippocampal cholinergic activity were studied. One week after AF64A administration, we injected dissociated septal cell suspension into the dorsal hippocampus, unilaterally. About 3 months after the transplantation, acetylcholine (ACh)-rich septal grafts formed extensive acetylcholinesterase (AChE)-positive fibers into the host hippocampus, recovering choline acetyltransferase (ChAT) level only in the grafted side. These results indicate that septal implants can produce a partial recovery of the cholinergic activity in the chemically damaged hippocampus.     

Integration of human model neurons (NT2) into embryonic chick nervous system

Postmitotic neurons were generated from the human NT2 teratocarcinoma cell line in a novel cell aggregate differentiation procedure. Approximately a third of the differentiated neurons expressed cell markers related to cholinergic neurotransmission. To examine whether this human cell model system can be directed toward a motoneuronal fate, postmitotic neurons were co‐cultured with mouse myotubes. Outgrowing neuronal processes established close contact with the myotubes and formed neuromuscular junction‐like structures that bound α‐bungarotoxin. To determine how grafted precursor cells and neurons respond to embryonic nerve tissue, NT2 cells at different stages of neural development were injected into chick embryo neural tube and brain. Grafted NT2 neurons populated both parts of the nervous system, sometimes migrating away from the site of injection. The neural tube appeared to be more permissive for neurite extensions than the brain. Moreover, extending neurites of spinal grafts were approaching the ventral roots, thus resembling motoneuronal projections. Developmental Dynamics 239:496–504, 2010. © 2009 Wiley‐Liss, Inc.     

Neurite Outgrowth is Directed by Schwann Cell Alignment in the Absence of Other Guidance Cues

Schwann cells enhance axonal regeneration following nerve injury in vivo and provide a favorable substrate for neurite outgrowth in vitro. However, much remains unknown about the nature of interactions that occur between Schwann cells and growing neurites. In this paper, we describe direct evidence of the ability of Schwann cell alignment alone to direct neurite outgrowth. Previously, we reported that laminin micropatterns can be used to align Schwann cells and thus create oriented Schwann cell monolayers. In the current study, dissociated rat spinal neurons were seeded onto oriented Schwann cell monolayers, whose alignment provided the only directional cue for growing neurites, and neurite alignment with the underlying Schwann cells was analyzed. The orientation of neurite outgrowth mimicked that of the Schwann cells. Associations observed between neurites and Schwann cells suggest that Schwann cells may guide neurite outgrowth through both topographical and molecular mechanisms. This work demonstrates that Schwann cell alignment can direct neurite outgrowth in the absence of other directional cues, and provides a new method for examining neuronal–Schwann cell interactions in vitro.     

Neurite Outgrowth Is Directed by Schwann Cell Alignment in the Absence of Other Guidance Cues

Schwann cells enhance axonal regeneration following nerve injury in vivo and provide a favorable substrate for neurite outgrowth in vitro. However, much remains unknown about the nature of interactions that occur between Schwann cells and growing neurites. In this paper, we describe direct evidence of the ability of Schwann cell alignment alone to direct neurite outgrowth. Previously, we reported that laminin micropatterns can be used to align Schwann cells and thus create oriented Schwann cell monolayers. In the current study, dissociated rat spinal neurons were seeded onto oriented Schwann cell monolayers, whose alignment provided the only directional cue for growing neurites, and neurite alignment with the underlying Schwann cells was analyzed. The orientation of neurite outgrowth mimicked that of the Schwann cells. Associations observed between neurites and Schwann cells suggest that Schwann cells may guide neurite outgrowth through both topographical and molecular mechanisms. This work demonstrates that Schwann cell alignment can direct neurite outgrowth in the absence of other directional cues, and provides a new method for examining neuronal–Schwann cell interactions in vitro.     

Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum. To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7 days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls. The amphetamine-induced rotational behavior of all 6-OHDA-lesioned animals was monitored at various time points from 18 days before transplantation and up to 80 days after transplantation. Tyrosine hydroxylase (TH) immunostaining of the histologically processed brains served to assess the long-term survival of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 ± 98, mean ± SEM) and grafts of fresh, dissociated cell suspension (806 ± 105, mean ± SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after explantation, with an additional 23.1% loss after grafting, leaving 8.7% of the original number of TH-ir cells in the intracerebral grafts. This is to be compared with a survival rate of 9.1% for the TH-ir cells in the cell-suspension grafts. Immunostaining for the calcium-binding proteins calretinin, calbindin, and parvalbumin showed no differences in the neuronal expression of these proteins between the two graft types. In conclusion, we found comparable dopaminergic cell survival and functional effects of tissue-culture grafts and cell-suspension grafts, which currently is the type of graft most commonly used for experimental and clinical grafting. In this sense the result is promising for the development of an effective in vitro storage of fetal nigral tissue, which at the same time would allow neuroprotective and neurotrophic treatment prior to intracerebral transplantation. Received: 11 March 1997 / Accepted: 19 August 1997     

Ultrastructural evidence for hippocampal target cell-mediated trophic effects on septal cholinergic neurons in reaggregating cell cultures

We have previously demonstrated at the light microscopic level that when embryonic day-15 septal neurons are co-cultured for 21 days with their target cells from the hippocampus, increased numbers of septal cholinergic neurons are present as compared with co-cultures employing cells from the non-target cerebellum. In addition, fine varicose axon-like cholinergic fibers are found to be associated with the hippocampal cells but not with cerebellar cells. We now provide ultrastructural evidence for hippocampal target cell-enhanced cholinergic neuronal survival, axonal proliferation, and synapse formation in this culture system. Dissociated cell suspensions from septal, hippocampal, and cerebellar areas were obtained from 15-day mouse embryos; and hippocampal and cerebellar cells were internally labeled with rhodamine-conjugated wheat germ agglutinin. Combinations of septal and hippocampal cells, and septal and cerebellar cells were allowed to reaggregate in rotation mediated culture for either 15 or 21 days. The reaggregates were then fixed, embedded, sectioned, and processed for acetylcholinesterase-positive acetylcholinesterase-positive cells and fibers, and under fluorescence to locate rhodamine-labeled cell populations. Representative reaggregate profiles were then re-embedded for electron microscopic examination. In both types of reaggregates, either labeled hippocampal target or cerebellar non-target cells segregated from the septal cells so that areas containing each of the respective cell populations could be studied. In sections of septal-hippocampal reaggregates from 15-day cultures, 571 out of 665 (85%) cholinergic neurons examined were intact, whereas 15% of the cells showed some ultrastructural features of degeneration. Similarly, at day 21, 297 out of 335 (88%) of the cholinergic neurons were intact. In sections of septal-cerebellar reaggregates from 15-day cultures, 473 out of 572 (83%) cholinergic neurons were intact. By day 21 of culture, however, only 15 out of 110 (14%) cholinergic neurons examined were intact from the septal-cerebellar reaggregates. In areas of septal-hippocampal reaggregates occupied by rhodamine-labeled hippocampal cells, profiles of acetylcholinesterase-labeled axons were identified, and synaptic specializations were observed between cholinergic terminals and dendrites as well as somata of hippocampal target cells. In contrast, areas of septal-cerebellar reaggregates occupied by rhodamine-labeled cerebellar cells were devoid of cholinergic fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neurotrophic effects of hippocampal target cells on developing septal cholinergic neurons in culture

The influence of hippocampal target cells on the development of cholinergic septal neurons was studied in rotation-mediated reaggregating cell cultures. Brain cells from 15-day-old mouse embryos were obtained from: septum, containing cholinergic cells which project to the hippocampus; hippocampus which contains target cells for the septal cholinergic neurons; and cerebellum, containing cells which are not targets for the septal cholinergic cells. The cells were then cultured for 3 weeks in a rotary incubator in the following combinations: septal cells alone; hippocampal cells alone; cerebellar cells alone; septal-hippocampal cells together; and septal-cerebellar cells together. After harvesting, fixation, and embedding, 50 micron sections were cut and processed for visualization of acetylcholinesterase activity. Sections from reaggregates containing either hippocampal or cerebellar cells alone contained only a few acetylcholinesterase-positive cells, but no positive fibers. Sections from septal-hippocampal coaggregates revealed a pattern of well-defined, fine-caliber acetylcholinesterase-positive fibers with extensive arborizations and varicosities suggesting axonal proliferation. In septal-cerebellar coaggregates, acetylcholinesterase-positive fibers appeared to be degenerating and distinct areas were observed which were essentially devoid of acetylcholinesterase fibers. In some experiments, either cerebellar or hippocampal cells were labeled with wheatgerm agglutinin-rhodamine prior to culture in order to identify these cells in the resulting reaggregates. Analysis of sections from these studies showed that acetylcholinesterase fibers were excluded from regions of coaggregates containing cerebellar cells, but were present in regions of coaggregates containing hippocampal cells. Finally, cell counts of acetylcholinesterase-positive cells in the various combinations revealed that these putative cholinergic neurons were significantly more numerous in septal-hippocampal coaggregates (271 +/- 19 per 10(6) septal cells added) than in septal reaggregates (38 +/- 6 per 10(6) septal cells added) or septal-cerebellar coaggregates (85 +/- 29 per 10(6) septal cells added). These results, taken together, suggest that hippocampal target cells influence the development and survival of cholinergic neurons.