小鼠神经胶质细胞培养上清IL-6水平与脑不对称性的关系

目的　 通过体外培养小鼠大脑皮层神经胶质细胞 ,探讨该细胞分泌IL 6与脑不对称性的关系。 方法　分别取新生小鼠左、右两侧大脑皮层胶质细胞培养于 2 4孔板内 ;俟细胞长满孔底后 ,用细菌脂多糖 (LPS)刺激 ,培养 2 4h后收集上清并测定IL 6水平。结果　正常对照组胶质细胞培养上清中可检测出较低水平的IL 6 ,其趋势为右侧高于左侧 ,但统计学上无显著性差别 (P >0 .0 5 ) ;LPS刺激组IL 6水平明显增高 (P <0 .0 5 ) ,且右侧IL 6水平明显高于左侧 (P <0 .0 5 )。结论　 左、右两侧大脑皮层胶质细胞对LPS刺激的反应性存在差异 ,右侧显著高于左侧 ,这种异质性可能部分介导了脑不对称对免疫功能的影响     

海马神经元条件培养液体外诱导大鼠骨髓间质干细胞向神经元样细胞和神经胶质样 细胞的分化：与含b-FGF培养基和无血清培养基的比较*☆

背景：目前关于骨髓间质干细胞能否向神经元方向分化的报道不多,且争论多集中在分化后的神经元是否仅具有神经元形态而不具有神经元功能。 目的：探讨海马神经元条件培养液诱导大鼠骨髓间质干细胞向神经元样细胞和神经胶质样细胞分化的可能性。 方法：将第5代大鼠骨髓间质干细胞分为4组：条件培养基组加入海马神经元和胶质细胞的培养液;b-FGF组加入含b-FGF的DMEM培养基;无血清培养组加入含Neurobasal和B27的无血清培养基;阴性对照组加入含胎牛血清的DMEM。各组诱导12,24 h后,应用免疫细胞化学染色行神经元特异性烯醇化酶、微管相关蛋白2、胶质纤维酸性蛋白的鉴定,Western-blot法检测细胞神经元特异性烯醇化酶、微管相关蛋白2和胶质纤维酸性蛋白的表达。 结果与结论：诱导12,24 h后,条件培养基组、b-FGF组、无血清培养组骨髓间质充干细胞微管相关蛋白2、胶质纤维酸性蛋白、神经元特异性烯醇化酶均呈阳性表达,阴性对照组未见表达。与阴性对照组比较,诱导后24 h,条件培养基组、b-FGF组、无血清培养组微管相关蛋白2表达均明显增强(P < 0.05),且条件培养基组增强幅度显著高于另两组(P < 0.05);条件培养基组、b-FGF组、无血清培养组神经元特异性烯醇化酶及胶质纤维酸性蛋白表达无明显差异。结果证实海马神经元条件培养液可体外诱导大鼠骨髓间质干细胞分化为神经元样细胞和神经胶质样细胞,与含b-FGF的培养基和无血清培养基相比,海马神经元条件培养基诱导的神经元和神经胶质细胞阳性率最高。     

趋化因子CC配基2对α?Synuclein诱导的小胶质细胞增殖及神经元凋亡的影响

目的探讨趋化因子CC配基2(CCL2)对α?突触核蛋白(α?Synuclein)介导的小胶质细胞增殖及神经元凋亡的影响。方法体外分离培养原代小胶质细胞和原代神经元。小胶质细胞分为4组,依次为对照组、CCL2组、α?Synuclein组、CCL2+α?Synuclein组。对照组中加入等量的PBS,CCL2组细胞中加入含有CCL2浓度为0.05ng/μL的培养液。α?Synuclein组细胞中加入含有α?Synuclein浓度为0.2ng/μL的培养液。CCL2+α?Synuclein组中加入含有0.05ng/μL CCL2、0.2ng/μLα?Synuclein的细胞培养液。培养24h后,检测小胶质细胞增殖情况及细胞中α?Synuclein蛋白水平,同时检测细胞培养液中TNF-α、IL-1β、NO含量。用各组小胶质细胞培养液培养原代神经元,观察神经元凋亡情况及神经元中Cleaved Caspase-3、Akt、p-Akt水平。结果 CCL2组、α?Synuclein组、CCL2+α?Synuclein组小胶质细胞增殖活性及分泌TNF-α、IL-1β、NO水平明显高于对照组(P0.05)。CCL2组、CCL2+α?Synuclein组小胶质细胞中α?Synuclein水平明显高于对照组(P0.01)。CCL2组、α?Synuclein组、CCL2+α?Synuclein组小胶质细胞培养液作用后的神经元凋亡率及Cleaved Caspase-3蛋白水平明显高于对照组,pAkt水平低于对照组(P0.01)。结论 CCL2促进α?Synuclein引起的小胶质细胞增殖和分泌TNF-α、IL-1β、NO的能力,对α?Synuclein引起的神经元凋亡也具有促进作用,促凋亡作用机制可能与Akt信号通路有关。     

京尼平对脂多糖诱导小胶质细胞炎症及凋亡的作用及机制研究

目的探讨京尼平(genipin)对脂多糖(LPS)诱导小胶质细胞(BV-2)炎症及凋亡反应的作用及机制。方法采用LPS诱导BV-2小胶质细胞建立中枢神经系统炎症和凋亡反应模型。实验细胞分为4组:空白对照组、京尼平组、LPS组、LPS+京尼平组。通过ELISA、RT-PCR、Western Blot、细胞流式检测细胞的炎症及凋亡反应。结果与空白对照组比较,LPS可以诱导BV-2细胞上清培养基中IL-6、IL-1β和TNF-α释放和细胞内IL-6、IL-1β和TNF-α转录的增加;同时,LPS还可以激活凋亡蛋白Bax并抑制抗凋亡蛋白Bcl-2的表达,导致细胞凋亡率的明显升高。京尼平预处理可以有效抑制小胶质细胞介导的炎症因子(IL-6、IL-1β和TNF-α)激活,并减少LPS诱导的小胶质细胞凋亡。结论 genipin干预可对LPS诱导的小胶质细胞炎症及凋亡反应起到保护作用。     

PINK1介导缺氧损伤时星形胶质细胞对神经元的保护作用

目的探究星形胶质细胞内PTEN诱导假定激酶1(PINK1)缺失对缺血时神经保护作用的影响及其作用机制。方法离体培养原代星形胶质细胞,使用小干扰RNA(si RNA)沉默PINK1表达,氧糖剥夺(OGD)建立细胞缺氧模型,分为4组:PINK1沉默组(si RNA+转染剂)、空质粒组(空质粒+转染剂)、转染剂组(只加转染剂)和对照组(星形胶质细胞),各组均与神经元共培养;另设立神经元单独培养组。免疫荧光染色观察神经元凋亡情况。定量PCR及ELISA检测星形胶质细胞促红细胞生成素(EPO)及血管内皮生长因子(VEGF)表达量;Western blot检测星形胶质细胞内缺血诱导因子(HIF)及核因子κB(NF-κB)通路相关蛋白水平。结果 OGD损伤后神经元凋亡率较高,与星形胶质细胞共培养后神经元凋亡率显著降低(P0.05)。PINK1基因沉默后共培养神经元凋亡增加,星形胶质细胞EPO及VEGF分泌量减少、胞内EPO及VEGF转录水平降低(P0.05);HIF-1、HIF-2与NF-κB通路活化水平均显著降低(P0.05)。结论星形胶质细胞对OGD损伤神经元有保护作用,其作用通过EPO及VEGF实现;PINK1基因沉默后星形胶质细胞对缺血神经元保护作用减弱,可能与NF-κB通路活化水平降低、HIF激活受损进而下调EPO和VEGF表达量有关。     

芹黄素抑制小胶质细胞活化作用研究

目的探讨芹黄素对小胶质细胞活化的抑制作用。方法原代培养SD大鼠小胶质细胞,实验随机分为空白对照组、脂多糖组(LPS组)、LPS+芹黄素(10μM)组、LPS+芹黄素(20μM)组、LPS+芹黄素(50μM)组。MTT法检测芹黄素对小胶质细胞活性的影响;ELISA法检测IL-1、IL-10等炎症相关因子以及BDNF、PDNF等神经营养因子的表达;RTPCR、western blot检测炎症相关基因i NOS转录以及表达水平。结果 MTT检测结果表明,芹黄素对小胶质细胞活性无明显抑制。LPS刺激后,芹黄素预处理组IL-1等炎症因子表达水平明显低于单独LPS组(P0.05);而芹黄素预处理组IL-10的表达则高于单独LPS组(P0.01);与此同时,BDNF、PDNF等神经营养因子的分泌也有相同趋势(P0.05)。芹黄素预处理组i NOS表达和转录水平也低于单独LPS组(P0.05)。结论芹黄素可抑制活化状态小胶质细胞炎症因子以及炎症相关蛋白的表达,并可同时促进与神经元分化成熟相关神经营养因子的分泌。     

β-arrestin 2对多巴胺能神经元的保护作用及机制研究

目的探讨β-arrestin 2对多巴胺能神经元的保护作用及机制。方法分离并培养原代小鼠中脑小胶质细胞,脂多糖(LPS)处理后采用实时定量PCR检测炎性细胞因子IL-6、IL-1β和TNF-α的转录水平,ELISA检测上述因子的表达水平;将原代小鼠中脑小胶质细胞和多巴胺能神经元Transwell共培养(细胞数2∶1),LPS处理后TUNEL法检测多巴胺能神经元凋亡,酪氨酸羟化酶(TH)免疫组织化学法检测多巴胺能神经元。结果原代小胶质细胞经LPS处理后IL-6、IL-1β和TNF-α的转录水平及表达水平均较对照组显著升高,而β-arrestin 2高表达能抑制这种炎症反应;β-arrestin 2高表达能显著抑制LPS处理所致的多巴胺能神经元凋亡,抑制多巴胺能神经元数目的减少。结论β-arrestin 2可能通过抑制小胶质细胞炎性细胞因子的产生,从而对多巴胺能神经元发挥神经保护作用。     

星形胶质细胞与Aβ1-40诱导凋亡的PC12细胞共育条件培养液对神经干细胞分化的影响机制

目的 将星形胶质细胞与β-淀粉样蛋白(Aβ1-40)诱导凋亡的PCI2细胞共育,观察星形胶质细胞条件培养液(ACM)对胚胎大鼠皮层神经干细胞(NSCs)体外定向分化为神经元的比例影响及机制,探讨神经营养素家族蛋白[包括脑源性神经营养因子(BDNF)、神经生长因子(NGF)、神经营养素-3(NT-3)]是否参与此过程. 方法 PC12细胞分别经10 μg/mLAβ1-40诱导不同时间(0、4、6、12、24h)后分为两部分,第一部分应用流式细胞技术检测不同时间点PC12细胞凋亡率;第二部分分别与星形胶质细胞共育2 d,将收集的ACM分为两部分.一部分应用ELISA法检测ACM中BDNF、NGF、NT-3蛋白含量,另一部分以1;3比例同DMEM/F12堵养基混合,对NSCs进行体外诱导分化,应用激光共聚焦显微镜、NSE免疫荧光技术鉴定和计数神经元分化比例. 结果 在Aβ1-40作用6 h时间点,PC12细胞凋亡率达高峰,与其共育的ACM中BDNF蛋白总量明显增高,诱导的NSCs神经元分化比例明显升高,与其他组比较,差异均有统计学意义(P<0.05). 结论 星形胶质细胞与Aβ1-40诱导凋亡的PCI2细胞共育后.ACM提高了NSCs向神经元的分化比例,ACM中BDNF可能参与了这一过程.     

再程序化星形胶质细胞的制备及体外定向分化为神经元的研究

目的探讨再程序化星形胶质细胞制备并在体外诱导其分化为神经元。方法在体外培养大鼠脑皮质来源星形胶质细胞(astrocyte),随后将提纯、鉴定过的第三代星形胶质细胞接种于12孔培养皿中,并分为A、B、C 3组。其中A组为带有绿色荧光蛋白(GFP)的慢病毒载体介导neurogenin2(Ngn2)基因转染的星形胶质细胞,制备再程序化星形胶质细胞;B组为带有GFP基因的空载体病毒转染的星形胶质细胞;C组为未进行慢病毒介导基因转染的星形胶质细胞;转基因1周后加入含细胞生长因子诱导培养基诱导分化15 d,光镜下观察各组细胞形态变化以及定向神经元分化的差异。结果 A组星形胶质细胞转基因后再诱导15 d,很大部分细胞形态呈神经元样改变,胞体呈梭形或椭圆形,有多个突起伸出且突起较长,表达神经元核蛋白(Neu N)、神经丝蛋白(NF)及神经元特异性烯醇化酶(NSE)的比例大大提高,相比B组及C组,差异有统计学意义(均P0.05);而B组与C组神经元分化比例的差异无统计学意义(P0.05)。结论慢病毒介导Ngn2基因体外转染星形胶质细胞可制备出再程序化星形胶质细胞,诱导后具有更强的向神经元定向分化能力。     

骨髓间充质干细胞对酒精诱导海马神经元损伤的作用研究

目的通过体外观察骨髓间充质干细胞(BMMSC)对酒精诱导海马神经元损伤的作用,初步探讨BMMSC治疗酒精性痴呆(AAD)的可行性。方法取胎鼠海马进行原代神经元培养并进行神经元纯度鉴定。酒精组给予100mol/L酒精37℃培养24h,建立酒精诱导海马神经元损伤模型;BMMSC+酒精组在其培养液中同时加入BMMSC条件培养液(终浓度为50%)及100mol/L酒精,37℃培养24h;另设相同浓度的BMMSC条件培养液对照组(BMMSC组)及空白对照组(对照组)。采用四甲基偶氮唑蓝(MTT)比色法检测各组细胞存活率,以碘化丙啶(PI)/Hoechst33342染色观察细胞形态及凋亡情况。结果原代培养的海马神经元高表达神经元特异性烯醇化酶(NSE),低表达神经胶质纤维酸性蛋白(GFAP),提示成功培养了纯度较高的原代海马神经元。酒精组细胞存活率(0.80±0.09)低于对照组(1.00±0.11;t=4.128,P0.01)及BMMSC组(1.04±0.07;t=-6.052,P0.01);BMMSC+酒精组细胞存活率(0.92±0.11)高于酒精组(t=2.555,P0.05)。BMMSC组及BMMSC+酒精组细胞存活率与对照组比较差异无统计学意义(均P0.05)。酒精组可见较多的细胞核呈致密浓染及核碎裂的凋亡细胞,BMMSC+酒精组的凋亡细胞数量明显减少。结论 BMMSC可抑制酒精诱导的海马神经元凋亡发生,提示BMMSC移植治疗AAD可能具有一定可行性。     

Measurement of γ-enolase release, a new method for selective quantification of neurotoxicity independently from glial lysis

We have developed a sensitive enzymatic-immunoassay to quantify the level of gamma-enolase (a specific neuronal enzyme) which is released from cultured cells after exposure to various toxins. We show that this method can estimate selectively neuronal cell death without significantly interfering with glial cell death. Indeed, no gamma-enolase is released when glial cells are killed with free-radical producing agents. Experiments comparing the levels of neuronal cell death induced by NMDA or free-radical producing drugs, performed either by measuring gamma-enolase release or using the classical fluorescein diacetate method, yielded similar results. In addition to selectively follow neuronal death in a mixed population of neurons and glial cells, this method provides a way of determining the cell death kinetics from a single culture dish, since enolase can be measured on small samples taken from the culture medium. Finally, we propose these two methods as being complementary and useful neuronal and other cellular death indexes and also to understand the complex problem of glial influence on neuronal survival or death.     

Ability of retinal Müller glial cells to protect neurons against excitotoxicity in vitro depends upon maturation and neuron‐glial interactions

Glutamate is the most abundant excitatory amino acid in the central nervous system. It has also been described as a potent toxin when present in high concentrations because excessive stimulation of its receptors leads to neuronal death. Glial influence on neuronal survival has already been shown in the central nervous system, but the mechanisms underlying glial neuroprotection are only partly known. When cells isolated from newborn rat retina were maintained in culture as enriched neuronal populations, 80% of the cells were destroyed by application of excitotoxic concentrations of glutamate. Massive neuronal death was also observed in newborn retinal cultures containing large numbers of glia, or when neurons were seeded onto feeder layers of purified cells prepared from immature (postnatal 8 day) rat retina. When newborn retinal neurons were seeded onto feeder layers of purified glial cells prepared from adult retinas, application of excitotoxic amino acids no longer led to neuronal death. Furthermore, neuronal death was not observed in mixed neuron/glial cultures prepared from adult retina. However, in all cases (newborn and adult) application of kainate led to amacrine cell‐specific death. Activity of glutamine synthetase, a key glial enzyme involved in glutamate detoxification, was assayed in these cultures in the presence or absence of exogenous glutamate. Whereas pure glial cultures alone (from young or adult retina) showed low activity that was not stimulated by glutamate addition, mixed or co‐cultured neurons and adult glia exhibited up to threefold higher levels of activity following glutamate treatment. These data indicate that two conditions must be satisfied to observe glial neuroprotection: maturation of glutamine synthetase expression, and neuron‐glial signalling through glutamate‐elicited responses. GLIA 25:229–239, 1999. © 1999 Wiley‐Liss, Inc.     

Oxidative stress promotes proliferation and dedifferentiation of retina glial cells in vitro

Oxidative damage is involved in triggering neuronal death in several retinal neurodegenerative diseases. The recent finding of stem cells in the retina suggests that both preventing neuronal death and replacing lost neurons might be useful strategies for treating these diseases. We have previously shown that oxidative stress induces apoptosis in cultured retinal neurons. We now investigated the response of Müller cells, proposed as retina stem cells, to this damage. Treatment of glial cell cultures prepared from rat retinas with the oxidant paraquat (PQ) did not induce glial cell apoptosis. Instead, PQ promoted their rapid dedifferentiation and proliferation. PQ decreased expression of a marker of differentiated glial cells, simultaneously increasing the expression of smooth muscle actin, shown to increase with glial dedifferentiation, the levels of cell-cycle markers, and the number of glial cells in the cultures. In addition, glial cells protected neurons in coculture from apoptosis induced by PQ and H(2)O(2). In pure neuronal cultures, PQ induced apoptosis of photoreceptors and amacrine neurons, simultaneously decreasing the percentage of neurons preserving mitochondrial membrane potential; coculturing neurons with glial cells completely prevented PQ-induced apoptosis and preserved mitochondrial potential in both neuronal types. These results demonstrate that oxidative damage activated different responses in Müller glial cells; they rapidly dedifferentiated and enhanced their proliferation, concurrently preventing neuronal apoptosis. Glial cells might not only preserve neuronal survival but also activate their cell cycle in order to provide a pool of new progenitor cells that might eventually be manipulated to preserve retinal functionality.     

Oxidized proteins in astrocytes generated in a hyperbaric atmosphere induce neuronal apoptosis

In the present study, we investigated the influence of the oxidative damage to astrocytes on neuronal cell survival using cultures of rat cerebral astrocytes and neurons. The exposure of astrocytes to hyperbaric oxygen induced a time-dependent apoptotic cell death, as observed by DNA ladder assessment. When astrocytes damaged by oxidative stress were cocultured with normal neurons from the cerebrum of a newborn rat, neuronal cell death was markedly induced, although normal astrocytes not subjected to hyperoxia cocultured with normal neurons showed no neuronal cell apoptosis. It was found that either the supernatant from the homogenate of astrocytes cultured in hyperbaric oxygen atmosphere or a protein mixture extracted from the supernatant induced neuronal cell death. The level of protein carbonyls, an index of protein oxidation analysis, in cultured astrocytes increased significantly with oxidative stress, and vitamin E inhibited the increase in the level of such oxidized proteins in astrocytes. Furthermore, a two-dimensional (2D) electrophoresis of a protein mixture extracted from the supernatant showed several changes in proteins. These results imply that reactive oxygen species (ROS) induced by oxidative stress attack astrocytes to induce oxidatively denatured proteins in the cells that act as a neurotoxic factor, and that vitamin E protects neurons by inhibiting astrocyte apoptosis caused by oxidative stress.     

Human immunodeficiency virus type 1 and its coat protein gp 120 induce apoptosis and activate JNK and ERK mitogen-activated protein kinases in human neurons

Detection of apoptotic neurons and microglial cells in the brains of human immunodeficiency virus type 1 (HIV-1)-infected patients has suggested that programmed cell death may be implicated in the physiolpathology of HIV-1 encephalopathy. To analyze in vitro the intracellular signals induced by HIV-1 in human neurons and the associated neuronal death, we tested cultured human central nervous system (CNS) cells for apoptosis induced by HIV-1 and gp120 and for signaling pathways activated by gp120. HIV-1 and gp120 induced apoptosis of neurons and microglial cells but not of astrocytes or transformed microglial cells. Gp120 activated c-Jun N-terminal kinase (JNK) and p42 extracellular regulated kinase (ERK) in primary CNS cells, with an early peak of activation at 2 to 5 minutes that was not present when pure microglial or astrocyte cultures were tested, followed by a late and sustained activation (10 and 60 minutes) in primary and enriched glial cell cultures as well as in transformed microglial cells. This demonstrates that gp120 could be an effector of HIV-1-induced apoptosis in the CNS and act directly on neuronal and glial cells.     

Crayfish mechanoreceptor neuron prevents photoinduced apoptosis of satellite glial cells

Interactions between neurons and glia play a key role in the development, functioning and survival of the nervous system. However, the influence of neurons on glial cells has received less attention than the role of glia in supporting neural functions. We here investigated the role of isolated crayfish stretch receptor neuron in the death of satellite glial cells under photodynamic impact. After staining with aluminum phthalocyanine photosens, the neuronal cell body was locally irradiated with a focused beam of He-Ne (633 nm, 200 W/cm2) or semiconductor laser (650 nm, 50 W/cm2). This rapidly abolished neuronal activity. The whole preparation was then subjected to total laser irradiation with lower intensity (633 nm, 0.3 W/cm2), which induced death of glial cells. Double staining of the preparation with propidium iodide and Hoechst 33342 in the following 6-7h allowed the visualization of necrotic, apoptotic and alive cells. Previous neuron inactivation with the focused laser beam was found to increase photodynamically-induced apoptosis but not necrosis of satellite glial cells enwrapping the axon. Therefore, the intact neuronal cell body protected satellite glial cells against photoinduced apoptosis. Altogether the data indicate that mechanoreceptor neurons release some signaling molecules involved in the prevention of glial apoptosis. This may provide integrity of the stretch receptor organ and its resistance to injurious factors.     

Depolarization or glutamate receptor activation blocks apoptotic cell death of cultured cerebellar granule neurons

Cerebellar granule neurons can be readily maintained in culture if depolarized with high concentrations of K⁺ or subtoxic concentrations of various excitatory amino acids. We now report that these depolarizing stimuli promote cerebellar granule neuron survival by blocking their programmed death via apoptosis. Cerebellar granule neurons maintained in depolarizing conditions and then changed to non-depolarizing conditions, exhibit the morphological and biochemical features of apoptosis, including cytoplasmic blebbing, condensation and aggregation of nuclear chromatin internucleosomal DNA fragmentation. Inhibitors of RNA or protein synthesis greatly attenuate cell death induced by non-depolarizing culture conditions. In contrast, cerebellar granule neurons, when exposed to fresh serum-containing medium or to high concentrations of glutamate, exhibit a delayed-type of neurotoxicity which is non-apoptotic in nature. Given the actions of excitatory amino acid receptor agonists in preventing apoptosis of cultured cerebellar granule neurons, we hypothesize that the functional innervation of postmigratory granule neurons during cerebellar development may prevent further elimination of these neurons by blocking their programmed death.     

Neurons derived from embryonic stem (ES) cells resemble normal neurons in their vulnerability to excitotoxic death

We determined whether embryonic stem (ES) cells could provide a model system for examining neuronal death mediated by glutamate receptors. Although limited evidence indicates that normal neurons can be derived from mouse ES cells, there have been no studies examining pathophysiological responses in mouse ES cell systems. Mouse ES cells, induced down a neural lineage by retinoic acid (RA), were found to have enhanced long-term survival when plated onto a layer of cultured mouse cortical glial cells. In these conditions, the ES cells differentiated into neural cells that appeared normal morphologically and displayed normal features of immunoreactivity when tested for neuron-specific elements. Varying the culture medium generated cultures of mixed neuronal/glial cells or enriched in oligodendrocytes. These cultures were viable for at least four weeks. Real-time PCR analysis of N-methyl-D-aspartate (NMDA) receptor subunits revealed an appropriate age-in-vitro dependent pattern of expression. Neurons derived from ES cells were vulnerable to death induced by a 24-h exposure to the selective glutamate receptor agonists NMDA, kainate, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). This vulnerability to agonist-induced death increased with age in vitro, and related closely to expression of receptor subunits, as it does in cultured primary neurons. Experiments with selective receptor antagonists showed that glutamate receptors mediated the NMDA- and kainate-induced death. Neuronal differentiated ES cells therefore exhibited an excitotoxic response resembling that displayed by central nervous system (CNS) neurons. Thus, ES cells, which are very amenable to genetic manipulation, provide a valid system for studying glutamate receptor-mediated toxicity at the molecular level.     

On the Role of Phosphatidylinositol 3-Kinase,Protein Kinase B/Akt,and Glycogen Synthase Kinase-3β in Photodynamic Injury of Crayfish Neurons and Glial Cells

Photodynamic treatment that causes intense oxidative stress and cell death is currently used in neurooncology. However, along with tumor cells, it may damage healthy neurons and glia. To study the involvement of signaling processes in photodynamic injury or protection of neurons and glia, we used crayfish mechanoreceptor consisting of a single neuron surrounded by glial cells. It was photosensitized with alumophthalocyanine Photosens. Application of specific inhibitors showed that phosphatidylinositol 3-kinase did not participate in photoinduced death of neurons and glia. Akt was involved in photoinduced necrosis but not in apoptosis of neurons and glia. Glycogen synthase kinase-3β participated in photoinduced apoptosis of glial cells and in necrosis of neurons. Therefore, phosphatidylinositol 3-kinase/protein kinase Akt/glycogen synthase kinase-3β pathway was not involved as a whole in photodynamic injury of crayfish neurons and glia but its components, Akt and glycogen synthase kinase-3β, independently and cell specifically regulated death of neurons and glial cells. According to these data, necrosis in this system was a controlled but not a non-regulated cell death mode. The obtained results may be used for the search of pharmacological agents selectively modulating death and survival of normal neurons and glial cells during photodynamic therapy of brain tumors.     

Pathway for interferon-gamma to promote the differentiation of cholinergic neurons in rat embryonic basal forebrain/septal nuclei

BACKGROUND: The supernatant of interferon-gamma (IFNγ) co-cultured with neonatal rat cortical glia can promote the cells in embryonic basal forebrain/septal nuclei to differentiate into cholinergic neurons, but the mechanism is still unclear. OBJECTIVE: To analyze the pathways for IFNγ to promote the differentiation of primarily cultured cholinergic neurons in rat embryonic basal forebrain/septal nuclei through culture in different conditioned medium. DESIGN: A controlled experiment taking cells as the observational target. SETTINGS: Department of Biochemistry and Molecular Biology, Youjiang Medical College for Nationalities; Department of Cell Biology, Beijing University Health Science Center. MATERIALS: Sixty-four pregnant Wistar rats for 16 days (250–350 g) and 84 Wistar rats (either male or female, 5–7 g) of 0–1 day after birth were provided by the experimental animal department of Beijing University Health Science Center. Rat IFNγ were provided by Gibco Company; Glial fibrillary acidic protein by Huamei Company. METHODS: The experiments were carried out in the Department of Cell Biology, Beijing University Health Science Center and Daheng Image Company of Chinese Academy of Science from July 1995 to December 2002. ① Interventions: The nerve cells in the basal forebrain/septal nuclei of the pregnant Wistar rats for 16 days were primarily cultured, and then divided into four groups: Blank control group (not any supernatant and medium was added); Control group (added by mixed glial cell or astrocyte conditioned medium); IFNγ group (added by mixed glial cell or astrocyte conditioned medium+IFNγ). Antibody group (added by mixed glial cell or astrocyte conditioned medium+IFNγ+Ab-IFNγ). Mixed glial cell or astrocyte conditioned medium was prepared using cerebral cortex of Wistar rats of 0–1 day after birth. ② Evaluation: The immunohistochemical method was used to perform the choline acetyltransferase (ChAT) staining of cholinergic neurons. The ChAT positive cells were counted. MAIN OUTCOME MEASURES: Comparison of ChAT positive cells in rat basal forebrain and septal nuclei in different conditioned medium. RESULTS: ① ChAT positive cells in mixed glial cell conditioned medium: The ChAT positive cells in the IFNγ group and antibody group were significantly more than those in the control group (P < 0.01). ② ChAT positive cells in astrocyte conditioned medium: The ChAT positive cells in the IFNγ group were significantly more than those in the control group, but there was no significant difference between the antibody group and control group (P > 0.05). CONCLUSION: IFNγ cannot directly promote the differentiation of cholinergic neurons, but plays a role through activating glial cells (except astrocytes) to produce IFNγ like molecules.