星形胶质细胞条件培养液对活性氧所致的神经元损伤的防护作用

本文以活性氧叔丁基脂氢过氧化物tbOOH应激PC12神经元细胞,造成PC12神经元损伤、死亡;以分离、纯化的SD大鼠大脑皮层星形胶质细胞制备的无血清条件培养液培养tbOOH损伤的神经元细胞。采用快速灵敏的MTT比色法测定神经细胞活力,用Olym—pus光学显微镜观察神经细胞的形态学变化。结果发现神经元细胞活力由0．179±0．037上升至0．563±0．025,细胞数目明显增多,细胞死亡残留碎片明显减少,表明星形胶质细胞条件培养液可能有防护tbOOH致的神经元损伤作用,具抗氧化能力。     

脑微血管内皮细胞条件培养液对星形胶质细胞活性的影响

目的:观察脑微血管内皮细胞与星形胶质细胞的相互关系,探讨血脑屏障维持脑内环境稳定的生理学基础.方法:原代培养大鼠脑皮质微血管内皮细胞,传至三代,收集在指数生长期细胞生长48 h后的务件培养液;将条件培养液分别按20%、30%、40%、50%、60%、70%、80%、90%、100%不同浓度作用于星形胶质细胞,MTT法检测不同浓度内皮细胞条件培养液作用于星形胶质细胞24 h、48h后的活性变化.结果:48h时间点的各浓度内皮细胞条件液组与相应的正常对照组相比差异均有显著统计学意义(P<0.01),内皮细胞条件液对星形胶质细胞表现出显著的抑制效应,而24 h的70%、80%、90%、100%浓度组与相应正常对照组相比也有显著统计学意叉的差异(P<0.01),且有浓度依赖性.结论:正常脑微血管内皮细胞条件培养液抑制了正常星形胶质细胞的活性.     

TRPV2激活对缺氧再灌注时体外培养星形胶质细胞神经生长因子释放的影响

神经生长因子对脑缺血后神经元的存活有重要意义。该研究观察了TRPV2激活剂2APB对体外缺血再灌注模型中原代培养大鼠大脑皮层星形胶质细胞神经生长因子释放的影响。将原代培养大鼠大脑皮层星形胶质细胞分为2APB组（0．5mmol／L）和对照组（不含2APB）,在糖氧剥夺情况下培养2h,然后恢复正常全培养基复氧培养48h。用Westem blot检测星形胶质细胞神经生长因子的表达水平;用ELISA检测星形胶质细胞条件培养液中神经生长因子的含量。结果表明,0．5mmol／L2APB可以诱导正常情况下及糖氧剥夺再灌注情况下体外培养星形胶质细胞NGF的合成和释放LP〈0．01）。此外,JNK阻滞剂可抑制糖氧剥夺再灌注情况下2APB诱导的星形胶质细胞神经生长因子的释放。综上．TRPV2激活可以影响糖氧剥夺再灌注情况下体外培养星形胶质细胞神经生长因子的合成和释放。TRPV2有可能成为脑缺血再灌注后的潜在治疗靶点。     

大鼠脑缺血再灌注损伤后神经元和星形胶质细胞的动态变化及Cyclin D1表达的差异

目的研究大鼠局灶性脑缺血再灌注损伤后神经元和星形胶质细胞表达量的动态演变及各自cyclin D1的表达差异。方法建立大鼠大脑中动脉阻塞（MCAO）再灌注模型,随机分为再灌注后1d组,3d组,7d组,14d组和假手术组,应用流式细胞术检测各组再灌注后不同时间点神经元和星形胶质细胞数量变化及各自cyclin D1的表达。结果缺血侧梗死边缘区皮质星形胶质细胞的表达增加,而神经元的表达下降,与假手术组比较有明显差异（P〈0．05）;神经元和星形胶质细胞中各自cyclin D1的表达在再灌注7d、14d后表达上调,且星形胶质细胞中的cyclinD1增加更明显,与假手术组比较有统计学差异（P〈0．05）。结论大鼠脑缺血再灌注后,缺血侧梗死边缘区皮质星形胶质细胞和神经元的cyclinD1表达均有不同程度的上调,星形胶质细胞的cyclin D1表达上调比神经元的更为显著。     

星形胶质细胞的兴奋对神经元树突丝运动的调节机制

神经元树突上树突丝(filopodia)的形成及其运动,是神经元探索胞外环境、寻找突触前膜结构的一种方式.为研究星形胶质细胞的兴奋对神经元树突上树突丝运动的调节机制,在与神经元混合培养的星形胶质细胞中转染光敏感通道(channelrhodopsin-2).Channelrhodopsin-2是一种可表达于细胞膜表面的非选择性阳离子通道,可被特定模式的蓝光激活,导致大量钙离子内流并进一步诱发星形胶质细胞产生钙波,从而实现了选择性激活星形胶质细胞的目的.研究结果显示,在混合培养的神经元与星形胶质细胞模型中,激活的星形胶质细胞可以抑制神经元filopodia的运动,与外源性ATP、谷氨酸的作用效果一致.这表明星形胶质细胞激活后可能通过释放ATP和谷氨酸等递质来抑制神经元filopodia的运动.     

大鼠脑皮质星形胶质细胞的限制性细胞培养

介绍一种新的脑组织星形胶质细胞培养方法即限制性细胞培养（constraint cell culture）。常规分离纯化星形胶质细胞,将其低密度种植,维持在添中低量血清的化学成分限定的培养基中培养,并在长时期内不给予更换或补加培养液。利用波形蛋白(vimentin）和胶质纤维酸性蛋白（glial fibrary acidic protein）抗体的免疫荧光染色法鉴定观察不同培养时期的星形胶质细胞及其形态学变化。结果发现星形胶质细胞在最初的5天之内有一定程度的增殖,未出现过度增殖导致的细胞相互融合现象;接下来的3－5天内细胞形态明显分化,星形胶质细胞突起细长、胞体明显缩小、形态多样,最后细胞突起之间相互连接形成星形胶质细胞网络,并在相当长的时间内保持不变。实验结果显示在限制细胞种植密度和限制给予培养液的培养条件下星形质细胞的体外形态发育与在体的情形基本一致。提示该细胞培养方法可能有助于研究中枢神经系统中星形胶质细胞的生理功能。     

脑微血管内皮细胞条件培养液对星形胶质细胞的影响及通络中药的干预特征

目的:揭示脑微血管内皮细胞生理、病理及通络中药处理后不同状态的培养液对正常星形胶质细胞影响的特征,从细胞间相互作用角度探讨脑微血管内皮细胞与星形胶质细胞的生物学关系,为阐释脑微环境稳定的血脑屏障维护机制以及通络中药通过内皮细胞调节脑内微环境理论假说提供新的证据。方法:制备正常、拟缺血和拟缺血合并通络救脑注射液处理的大鼠脑微血管内皮细胞条件培养液,观察其对星形胶质细胞活性和凋亡率的影响。结果:与正常星形胶质细胞相比,正常内皮细胞条件培养液能够降低正常星形胶质细胞的活性,并促进星形胶质细胞的凋亡;而拟缺血处理的内皮细胞条件培养液能够提高正常星形胶质细胞的活性和凋亡率;拟缺血合并通络药物处理的内皮细胞条件培养液对正常星形胶质细胞的活性有提高作用,并显著降低其凋亡率。结论:三种不同处理方式的内皮细胞条件培养液对正常星形胶质细胞活性和凋亡产生不同的影响,提示不同状态的微血管内皮细胞对脑内微环境产生影响,通络救脑注射液可能通过调节微血管内皮细胞的分泌而对星形胶质细胞发挥作用。     

马桑内酯激活的星形胶质细胞条件培养液对正常大鼠脑内及培养的神经元内PLCβ1的影响

目的揭示星形胶质细胞对大鼠脑内及培养的神经元磷脂酶Cβ1(PLCβ1)的影响及其在癫痫发病中的作用。方法将马桑内酯激活的星形胶质细胞条件培养液(astrocyte-conditioned medium,ACM)注射入正常SD大鼠侧脑室,观察大鼠的行为变化;运用免疫组织化学方法,观察大鼠大脑皮质、海马内PLCβ1免疫反应的变化;将培养的神经元随机分为2组:1.对照组(无血清培养基组),2.ACM组。各组细胞分别培养4、8、12h后,免疫细胞化学方法观察培养神经元内PLCβ1表达的变化,Western blot法检测各组培养神经元PLCβ1含量的变化。结果ACM组大鼠在注射ACM后30 min出现癫痫行为,2 h恢复正常;免疫组织化学显示:ACM作用后4h,大鼠大脑皮质、海马PLCβ1免疫反应阳性神经元数和平均光密度值显著增高(P<0.05);培养神经元的免疫细胞化学染色证明ACM组在作用4h时PLCβ1免疫阳性反应产物明显增加,与对照组比较有明显差异(P<0.05);Western blot结果表明PLCβ1含量在ACM作用4h较对照组明显增多(P<0.05)。结论马桑内酯激活的星形胶质细胞条件培养液可上调大鼠脑内及培养的神经元内PLCβ1的表达,并导致动物痫性发作。     

TNF-α激活的星形胶质细胞条件培养液对培养的海马星形胶质细胞的兴奋作用

目的：揭示星形胶质细胞在癫痫发病中的作用。方法：将TNF-α激活的星形胶质细胞条件培养液(Astrocytic Conditioned Medium,ACM)作用于纯化培养的海马星形胶质细胞,运用免疫细胞化学的方法观察核转录因子NF-kBp65的表达情况。结果：ACM作用后30min即可诱导NF-kBp65的核内表达,2h达高峰。结论:TNF-α激活的星形胶质细胞可通过释放可溶性的神经活性物质使培养的海马星形胶质细胞激活,兴奋性升高。     

星形胶质细胞糖代谢对神经元的支持及保护作用

脑组织有着极其复杂的功能,这些功能的完成有赖于神经元细胞与胶质细胞之间的广泛合作。星形胶质细胞作为人脑内数量最多的细胞,其与神经元细胞之间的相互作用就显得十分重要。葡萄糖代谢途径包括糖酵解,有氧氧化及磷酸戊糖三条途径。其为脑组织维持其正常功能的前提。研究表明星形胶质细胞和神经元在糖代谢方面有着各自的特点,神经元在能量底物及抗氧化应激中对星形胶质细胞糖代谢途径存在一定的依赖性,干扰星形胶质细胞与神经元之间的代谢过程会导致疾病的发生。本综述主要从糖酵解及磷酸戊糖两条糖代谢途径阐述了星形胶质细胞与神经元的关系。这或许会对研究脑的代谢,脑疾病中神经元的损伤机制及如何保护神经元提供全新的视角,并可能为一些疾病的治疗开辟了新的途径。     

Stimulation of GABA-Induced Ca2+ Influx Enhances Maturation of Human Induced Pluripotent Stem Cell-Derived Neurons

Optimal use of patient-derived, induced pluripotent stem cells for modeling neuronal diseases is crucially dependent upon the proper physiological maturation of derived neurons. As a strategy to develop defined differentiation protocols that optimize electrophysiological function, we investigated the role of Ca²⁺ channel regulation by astrocyte conditioned medium in neuronal maturation, using whole-cell patch clamp and Ca²⁺ imaging. Standard control medium supported basic differentiation of induced pluripotent stem cell-derived neurons, as assayed by the ability to fire simple, single, induced action potentials. In contrast, treatment with astrocyte conditioned medium elicited complex and spontaneous neuronal activity, often with rhythmic and biphasic characteristics. Such augmented spontaneous activity correlated with astrocyte conditioned medium-evoked hyperpolarization and was dependent upon regulated function of L-, N- and R-type Ca²⁺ channels. The requirement for astrocyte conditioned medium could be substituted by simply supplementing control differentiation medium with high Ca²⁺ or γ-amino butyric acid (GABA). Importantly, even in the absence of GABA signalling, opening Ca²⁺ channels directly using Bay K8644 was able to hyperpolarise neurons and enhance excitability, producing fully functional neurons. These data provide mechanistic insight into how secreted astrocyte factors control differentiation and, importantly, suggest that pharmacological modulation of Ca²⁺ channel function leads to the development of a defined protocol for improved maturation of induced pluripotent stem cell-derived neurons.     

小脑皮层在兔瞬膜条件反射过程中的调制作用

以音调结合气流刺激兔角膜的训练建立瞬膜条件反射,在条件反射率刚达90%,连续出现三组的学习初始阶段,电解损毁小脑半球第六小叶皮层使 D-I 核的学习相关性电活动和瞬膜条件反射消除,但不影响“非条件”反射,而在经一周巩固训练的动物,损毁小脑皮层上述区域不发生影响。D-I 核的细胞自发电活动在学习初期和记忆巩固时期也有所不同。在学习后期,D-I 核的细胞自发电活动频率减低,和在学习初期与损毁小脑皮层后的频率变化相似。实验结果表明:在瞬膜条件反射过程中,以小脑皮层为主导,对瞬膜条件反射的产生和D-I 核的学习相关性电活动具有调制作用。随着记忆巩固过程,D-I 核脱离皮层的控制而发展成为这一学习模式的记忆痕迹基础部位。     

The Low-Density Lipoprotein Receptor-Related Protein, a Multifunctional Apolipoprotein E Receptor, Modulates Hippocampal Neurite Development

Abstract: The ε4 allele of apolipoprotein E (apoE) is an important risk factor for Alzheimer's disease. A major neuronal receptor for apoE within the brain is the low-density lipoprotein receptor-related protein (LRP). Using primary cultured hippocampal neurons, we examined the role of LRP in early neuronal development. LRP, as well as a 39-kDa protein that regulates its activity, is localized abundantly in developing neurons. Both the 39-kDa protein and an anti-LRP antibody inhibited neurite outgrowth of primary hippocampal neurons cultured in either serum-containing medium or on cortical astrocyte monolayers in serum-free medium. It is noteworthy that microtubule-associated protein-2 immunoreactive process outgrowth was decreased significantly in hippocampal neurons cultured on cortical astrocytes derived from apoE-deficient mice and was not diminished further following incubation with LRP inhibitors. Thus, these results suggest that LRP can influence aspects of neuronal process development and that apoE-containing lipoproteins may be one of the major LRP ligands that can contribute to this process.     

Astrocytes protect neurons from nitric oxide toxicity by a glutathione-dependent mechanism

Nitric oxide (NO) contributes to neuronal death in cerebral ischemia and other conditions. Astrocytes are anatomically well positioned to shield neurons from NO because astrocyte processes surround most neurons. In this study, the capacity of astrocytes to limit NO neurotoxicity was examined using a cortical co-culture system. Astrocyte-coated dialysis membranes were placed directly on top of neuronal cultures to provide a removable astrocyte layer between the neurons and the culture medium. The utility of this system was tested by comparing neuronal death produced by glutamate, which is rapidly cleared by astrocytes, and N-methyl-D-aspartate (NMDA), which is not. The presence of an astrocyte layer increased the LD(50) for glutamate by approximately four-fold, but had no effect on NMDA toxicity. Astrocyte effects on neuronal death produced by the NO donors S-nitroso-N-acetyl penicillamine and spermine NONOate were examined by placing these compounds into the medium of co-cultures containing either a control astrocyte layer or an astrocyte layer depleted of glutathione by prior exposure to buthionine sulfoximine. Neurons in culture with the glutathione-depleted astrocytes exhibited a two-fold increase in cell death over a range of NO donor concentrations. These findings suggest that astrocytes protect neurons from NO toxicity by a glutathione-dependent mechanism.     

Synthesis and release of GABA in cerebral cortical neurons co-cultured with astrocytes from cerebral cortex or cerebellum

Cerebral cortical neurons were co-cultured for up to 7 days with astrocytes after plating on top of a confluent layer of astrocytes cultured from either cerebral cortex or cerebellum (sandwich co-cultures). Neurons co-cultured with either cortical or cerebellar astrocytes showed a high stimulus coupled release of gamma-aminobutyric acid (GABA), which is the neurotransmitter of these neurons. When the astrocyte selective GABA uptake inhibitor 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol was added during the release experiments, an increase in the stimulus coupled GABA release was seen, indicating that the astrocytes take up a large fraction of GABA released from the neurons. The activity of the GABA synthesizing enzyme glutamate decarboxylase, which is a specific marker of GABAergic neurons, was markedly increased in sandwich co-cultures of cortical neurons and cerebellar astrocytes compared to neurons cultured in the absence of astrocytes whereas in co-cultures with cortical astrocytes this increase was less pronounced. Pure astrocyte cultures did not show any detectable glutamate decarboxylase activity. The astrocyte specific marker enzyme glutamine synthetase (GS) was present at high activity in a glucocorticoid-inducible form in pure astrocytes as well as in co-cultures regardless of the regional origin of the astrocytes. When neurons were cultured on top of the astrocytes, the specific activity of GS was lower compared to astrocytes cultured alone, a result compatible with the notion that neurons are devoid of this enzyme. The results show that cortical neurons develop and differentiate when seeded on top of both homotypic and heterotypic astrocytes. Moreover, it could be demonstrated that the two cell types in the culture system communicate with each other with regard to GABA homeostasis during transmitter release.     

CNTF-Treated Astrocyte Conditioned Medium Enhances Large-Conductance Calcium-Activated Potassium Channel Activity in Rat Cortical Neurons

Seizure activity is linked to astrocyte activation as well as dysfunctional cortical neuron excitability produced from changes in calcium-activated potassium (KCa) channel function. Ciliary neurotrophic factor-treated astrocyte conditioned medium (CNTF-ACM) can be used to investigate the peripheral effects of activated astrocytes upon cortical neurons. However, CNTF-ACM’s effect upon KCa channel activity in cultured cortical neurons has not yet been investigated. Whole-cell patch clamp recordings were performed in rat cortical neurons to evaluate CNTF-ACM’s effects upon charybdotoxin-sensitive large-conductance KCa (BK) channel currents and apamin-sensitive small-conductance KCa (SK) channel current. Biotinylation and RT-PCR were applied to assess CNTF-ACM’s effects upon the protein and mRNA expression, respectively, of the SK channel subunits SK2 and SK3 and the BK channel subunits BKα1 and BKβ3. An anti-fibroblast growth factor-2 (FGF-2) monoclonal neutralizing antibody was used to assess the effects of the FGF-2 component of CNTF-ACM. CNTF-ACM significantly increased KCa channel current density, which was predominantly attributable to gains in BK channel activity (p < 0.05). CNTF-ACM produced a significant increase in BKα1 and BKβ3 expression (p < 0.05) but had no significant effect upon SK2 or SK3 expression (p > 0.05). Blocking FGF-2 produced significant reductions in KCa channel current density (p > 0.05) as well as BKα1 and BKβ3 expression in CNTF-ACM-treated neurons (p > 0.05). CNTF-ACM significantly enhances BK channel activity in rat cortical neurons and that FGF-2 is partially responsible for these effects. CNTF-induced astrocyte activation results in secretion of neuroactive factors which may affect neuronal excitability and resultant seizure activity in mammalian cortical neurons.     

Unveiling the effects of the secretome of mesenchymal progenitors from the umbilical cord in different neuronal cell populations

It has been previously shown that the secretome of Human Umbilical Cord Perivascular Cells (HUCPVCs), known for their mesenchymal like stem cell character, is able to increase the metabolic viability and hippocampal neuronal cell densities. However, due to the different micro-environments of the distinct brain regions it is important to study if neurons isolated from different areas have similar, or opposite, reactions when in the presence of HUCPVCs secretome (in the form of conditioned media-CM). In this work we: 1) studied how cortical and cerebellar neuronal primary cultures behaved when incubated with HUCPVCs CM and 2) characterized the differences between CM collected at two different conditioning time points. Primary cultures of cerebellar and cortical neurons were incubated with HUCPVCs CM (obtained 24 and 96 h after three days of culturing). HUCPVCs CM had a higher impact on the metabolic viability and proliferation of cortical cultures, than the cerebellar ones. Regarding neuronal cell densities it was observed that with 24 h CM condition there were higher number MAP-2 positive cells, a marker for fully differentiated neurons; this was, once again, more evident in cortical cultures. In an attempt to characterize the differences between the two conditioning time points a proteomics approach was followed, based on 2D Gel analysis followed by the identification of selected spots by tandem mass spectrometry. Results revealed important differences in proteins that have been previously related with phenomena such as neurl cell viability, proliferation and differentiation, namely 14-3-3, UCHL1, hsp70 and peroxiredoxin-6. In summary, we demonstrated differences on how neurons isolated from different brain regions react to HUCPVCs secretome and we have identified different proteins (14-3-3 and hsp70) in HUCPVCs CM that may explain the above-referred results.     

Dependence of neurones on astrocytes in a coculture system renders neurones sensitive to transforming growth factor beta1-induced glutamate toxicity

Transforming growth factor beta1 (TGF-beta1) has been implicated in formation of astrocyte scars, which prevents axonal regeneration. A coculture system of astrocytes and cerebellar cells was used to investigate possible neurotoxic effects of TGF-beta1. Although not directly neurotoxic, TGF-beta1 was toxic to cerebellar cells in the presence of astrocytes. This toxicity is based on an effect of the cytokine on astrocytes, as conditioned medium from astrocyte cultures treated with TGF-beta1 was more toxic by a similar mechanism. This neurotoxicity was mediated by glutamate present in the culture medium as demonstrated by inhibition by MK-801. Astrocytic ability to metabolise glutamate was compromised by TGF-beta1, as this cytokine increased glutamate concentration. The astrocytes in the coculture system responded to the presence of neurones by secreting neuroprotective interleukin-6, which was partly protective against the TGF-beta1-induced toxicity. In the coculture system, neurones responded to the presence of astrocytes by a reduction in resistance to glutamate toxicity. On addition of TGF-beta1, which compromised astrocytic clearance of glutamate, this reduction in resistance to glutamate toxicity led to a reduction in neuronal survival. These results suggest that when neurones are cocultured with astrocytes they become dependent on astrocytes for survival. This dependence makes neurones susceptible to damage when astrocytes are activated by substances such as TGF-beta1.     

Production of GABA by cultured hippocampal glial cells

Medium conditioned by cultured hippocampal glial contains an inhibitory factor that can hyperpolarize and suppress neuronal activity. Using biochemistry, electrophysiology, pharmacology, and mass spectrometry, we have identified the inhibitory factor as GABA (gamma-aminobutyric acid). Like GABA, the inhibitory factor increases chloride and potassium currents in neurons, which can be blocked by bicuculline. Mass spectrometry analysis of conditioned medium reveals peaks that are identical to that for GABA. Up to 500 micromolar GABA is found in conditioned medium from glial cultures. No GABA is found in conditioned medium from neuronal cultures. Hippocampal glia make much more GABA than cortical glia or glia from other brain regions. It is not clear how hippocampal glia synthesize GABA. Although they express GAD mRNA and adding glutamate to the culture medium increases the amount of GABA produced, other data suggest that glia do not use GAD to make GABA. Identifying the mechanism(s) by which GABA is produced by hippocampal glia would help clarify its role in modulating neuronal activity in the brain.     

Lysophosphatidic acid receptor-dependent secondary effects via astrocytes promote neuronal differentiation

Lysophosphatidic acid (LPA) is a simple phospholipid derived from cell membranes that has extracellular signaling properties mediated by at least five G protein-coupled receptors referred to as LPA(1)-LPA(5). In the nervous system, receptor-mediated LPA signaling has been demonstrated to influence a range of cellular processes; however, an unaddressed aspect of LPA signaling is its potential to produce specific secondary effects, whereby LPA receptor-expressing cells exposed to, or "primed," by LPA may then act on other cells via distinct, yet LPA-initiated, mechanisms. In the present study, we examined cerebral cortical astrocytes as possible indirect mediators of the effects of LPA on developing cortical neurons. Cultured astrocytes express at least four LPA receptor subtypes, known as LPA(1)-LPA(4). Cerebral cortical astrocytes primed by LPA exposure were found to increase neuronal differentiation of cortical progenitor cells. Treatment of unprimed astrocyte-progenitor cocultures with conditioned medium derived from LPA-primed astrocytes yielded similar results, suggesting the involvement of an astrocyte-derived soluble factor induced by LPA. At least two LPA receptor subtypes are involved in LPA priming, since the priming effect was lost in astrocytes derived from LPA receptor double-null mice (LPA(1)((-/-))/LPA(2)((-/-))). Moreover, the loss of LPA-dependent differentiation in receptor double-null astrocytes could be rescued by retrovirally transduced expression of a single deleted receptor. These data demonstrate that receptor-mediated LPA signaling in astrocytes can induce LPA-dependent, indirect effects on neuronal differentiation.