CNTF对NMDA引起大鼠海马神经元NOS活性改变的影响

为探讨CNTF对NMDA引起大鼠海马神经元一氧化氮合酶(nNOS)表达的作用,以不同浓度的NMDA处理海马神经元,倒置显微镜下观察其形态,并以nNOS免疫细胞化学结合图象分析方法测定nNOS神经元胞体的灰度,揭示NMDA对NOS活性的影响以及在此过程中CNTF发挥的作用。发现(1)NMDA可引起海马神经元的毒性反应,且呈剂量依赖性和时间依赖性;(2)100μmmol/L NMDA 10min组nNOS神经元胞体的灰度值大于对照组(P＜0．01);(3)在NMDA处理前给予CNTF,nNOS神经元的胞体及阳性突起的表现与对照组相似。提示CNTF可能通过抑制nNOS的活性及NO的渗出而减弱NMDA对神经元的毒性作用。     

灯盏花素含药血清对谷氨酸致原代海马神经元损伤的影响

目的:从细胞水平研究注射用灯盏花素对谷氨酸致大鼠原代海马神经元损伤的保护作用及其作用机制。方法:采用中药血清药理学方法,制备含药血清;原代培养大鼠乳鼠大脑海马神经元并经鉴定后,以谷氨酸复制损伤模型,以5%含药血清干预,在透射电镜下及经碘化丙啶和Hoechst33342双染后荧光显微镜下观察海马凋亡神经元的形态学变化,并进行检测:MTT法测定细胞存活率,生化法检测LDH漏出率、丙二醛(MDA)含量、细胞释放的NO量、细胞内tNOS活性和iNOS活性。结果:灯盏花素高、低剂量组均能明显增加海马神经元存活率,而LDH漏出率、丙二醛(MDA)含量、一氧化氮释放、总一氧化氮合酶活性和诱导型一氧化氮合酶活性(p<0.05,p<0.01)明显降低。结论:灯盏花素对谷氨酸致原代培养大鼠海马神经元损伤具有保护作用,其作用机制可能与其能改善能量代谢、稳定细胞膜、抗脂质过氧化、降低一氧化氮合酶的活性、减少一氧化氮释放有关。     

CNTF对NMDA引起大鼠海马神经元蛋白激酶C核转位的影响

目的：探讨睫状神经营养因子(CNTF)对N-甲基-D-天冬氨酸(NMDA)引起大鼠海马神经元蛋白激酶C(PKC)发生核转位的影响。方法：以NMDA及CNTF处理原代培养的大鼠海马神经元,PKCγ、免疫细胞化学并结合图象分析方法测定PKC阳性神经元胞核的灰度。结果：①给予不同浓度NMDA处理不同时间后,神经元核内有不同程度的PKCγ及PKCε表达,其中以100μmol／L NMDA 30min组表现尤为显著;②CNTF 500μmol／L NMDA组神经元胞核PKC灰度与对照组相似。结论：NMDA可引起海马神经元PKCγ、PKCε的核转位,而CNTF则抑制其转位的发生,提示CNTF对海马神经元的保护作用与抑制PKC的核转位右关。     

STAT3信号转导系统介导了CNTF对大鼠尾壳核出血后的神经保护作用

目的研究睫状神经生长因子(CNTF)在中枢神经元抗损伤中的作用及机制。方法对尾壳核出血大鼠进行CNTF治疗,观察海马信号转导和转录激活蛋白-3(STAT3)含量及阳性神经元分布的变化。结果CNTF能显增加尾壳核出血大鼠海马STAT3阳性神经元数量、海马STAT3含量。结论CNTF能通过促进STAT3的表达增强中枢神经元的抗损伤能力。     

脑益康药物血清对谷氨酸诱导海马神经元损伤的保护作用

目的:探讨脑益康药物血清对谷氨酸(Glu)诱导的海马神经元损伤的保护作用。方法:大鼠海马神经元培养后,采用形态学观察、MTT法及DAPI染色法检测脑益康药物血清对Glu损伤细胞活力的影响,采用RT-PCR和免疫组化方法检测脑益康药物血清对Glu损伤细胞PTEN表达的影响。结果:脑益康药物血清可明显提高Glu损伤的海马神经元的细胞活力,减少PTEN的表达。结论:脑益康药物血清对Glu诱导的海马神经元损伤有保护作用,其机制可能与减少PTEN表达,抑制神经元凋亡有关。     

阿糖胞苷对大鼠海马神经元原代培养的影响

目的：探讨在大鼠海马神经元原代培养过程中,阿糖胞苷对培养神经元的影响。方法：将新生24 h大鼠,分离出海马组织,进行原代海马神经元培养,再将细胞分为阿糖胞苷组和对照组,阿糖胞苷组加入1μmol/L阿糖胞苷,通过检测神经元特异性标志物微管相关蛋白-2（Map-2）计算培养神经元的数量,通过台盼蓝染色法观察细胞的存活率。结果：培养第7天,阿糖胞苷组神经元数量为（11±3）个,对照组为（10±4）个,两组无明显差异;阿糖胞苷组神经元细胞在培养第14天时存活率为74%,培养第21天时存活率为49%,而对照组神经元14天时存活率为96%,21天存活率为88%,两组神经元存活率差异明显。结论：原代培养海马神经元时,阿糖胞苷对神经元产量及形态影响不明显,但是由于阿糖胞苷的毒性作用,明显缩短神经元的存活时间,影响长期培养神经元的存活率。     

锌对体外应激海马神经元MTs亚型表达的影响

目的:观察不同锌水平对体外应激海马神经元金属硫蛋白(MTs)亚型表达的影响。方法:取新生1dWis-tar大鼠海马组织进行体外神经元培养,无血清培养24h后,分别向培养液中加入皮质酮、Zn2+特异鳌合剂TPEN,使二者的最终浓度均为1×10-5mol/L,然后加入不同浓度的ZnSO4溶液,使Zn2+的最终浓度分别为1×10-5mol/L、1×10-4mol/L和2×10-4mol/L,作用24h后检测培养液中IL-6和NO含量,以蛋白印迹法检测细胞MTs含量,以RT-PCR检测细胞MT-1mRNA和MT-3mRNA的表达水平。结果:在海马神经元培养液中加入TPEN后,MTs的表达出现明显降低,皮质酮刺激也未见其表达升高。在补锌组,MTs的含量均明显增加,其中以10-4mol/LZn2+组的表达量最高。海马神经元MT-1mRNA和MT-3mRNA的表达水平在皮质酮应激组和补锌组均出现明显升高。另外,锌缺乏和皮质酮刺激可使海马神经元培养上清中的IL-6和NO水平均出现明显升高。结论:不同锌水平对应激海马神经元金属硫蛋白及其亚型mRNA的表达具有调控作用,缺锌可降低金属硫蛋白的表达,而补锌可增加金属硫蛋白的表达。     

大鼠海马神经元体外缺糖缺氧模型的建立

目的：建立体外培养大鼠海马神经元缺糖缺氧模型。方法：取培养12d的海马神经元,在缺糖缺氧条件下分别培养0．5～4h后取出,换原神经元培养液,在常氧下继续培养24h后测定培养液中乳酸脱氢酶活性。测定神经元形态变化,并计算神经元存活百分率。同时用原位末端标记(TUNEL)法检测神经元凋亡。结果：缺糖缺氧后海马神经元胞体逐渐肿胀,培养液中LDH释放量逐渐增多,细胞存活率逐渐减少。恢复糖和氧供应后24h,凋亡神经元百分率明显增多。结论：用改进的无血清、无糖人工脑脊液成功建立了大鼠海马神经元离体缺糖缺氧模型。     

乳酸对培养大鼠大脑皮层神经元缺氧/复氧损伤的影响

目的:观察不同浓度乳酸对原代培养大鼠大脑皮层神经元缺氧/复氧(H/R)损伤的影响及其机制。方法:原代培养大鼠大脑皮层神经元缺氧8、12、24h后在培养液中添加不同浓度乳酸(终浓度分别为0、0.5、1.0、2.0、5.0mmol/L),复氧24h,以细胞存活率和乳酸脱氢酶(LDH)释放量为指标,观察不同浓度乳酸对神经元H/R损伤的影响,并以盐酸模拟乳酸解离的H 的作用,探讨乳酸对大脑皮层神经元H/R损伤影响的机制。结果:5.0mmol/L乳酸和盐酸引起常氧神经元损伤并加重神经元H/R损伤;1.0mmol/L乳酸对缺氧12h和24h神经元H/R损伤具有保护作用,相同浓度盐酸对神经元H/R损伤无影响。结论:较低浓度乳酸对神经元H/R损伤具有保护作用,其机制与H 的作用无关;较高浓度乳酸加重神经元H/R损伤,其机制可能包括H 的作用。     

镁离子对谷氨酸诱发的海马神经元损伤的保护作用

目的 :探讨镁离子 (Mg2 )在 0 .1mmol/L谷氨酸诱发的海马神经元损伤中的作用。方法 :将神经元从新生SD大鼠海马中分离后培养 6～ 9d ,即随机分成三组 :A .单纯培养基 ;B .培养基 谷氨酸 ;C .培养基 Mg2 ,30min后再加入谷氨酸。结果 :①B组海马神经元存活率与A组相比显著地呈剂量依赖性降低。②与B组对照 ,C组加用低浓度的Mg2 可提高海马神经元的存活率。结论 :低浓度的Mg2 能保护谷氨酸诱发损伤的海马神经元     

睫状神经营养因子对NO引起海马神经元毒性反应的影响

本研究采用原代培养大鼠海马神经元,观察睫状神经营养因子（ｃｉｌｉａｒｙ ｎｅｕｒｏｔｒｏｐｈｉｃ ｆａｃｔｏｒ,ＣＮＴＦ）对ＮＯ引起细胞毒性反应的影响。ＮＯ供体硝普钠与Ｓ－亚硝基－乙酰青霉胺,ＮＯＳ底物Ｌ－Ａｒｇ及钙载体ｉｏｎｏｍｙｃｉｎ,均可引起海马神经元存活率下降,ＬＤＨ漏出增加;提前２４ｈ给予不同浓度ＣＮＴＦ,均能提高神经元的存活率,减少ＬＤＨ漏出,其作用呈剂量依赖性。     

CNTF对严重烫伤海马神经元损伤的保护作用

目的：观察大鼠体表严重伤后海马神经元的病理变化,探讨ＣＮＴＦ对其保护作用。方法：ＳＤ大鼠侧脑室埋管,制成３０％体表面积Ⅲ度烫伤模型,伤后３天,测定脑组织含水量,海马乳酸脱氢酶（ＬＤＨ）和一氧化氮（ＮＯ）的含量,做病理切片,尼氏染色。结果：大鼠严重烫伤后,脑组织含水量增加,海马出现明显的病理变化,尼氏小体减少或消失,胞体肿胀,ＬＤＨ和Ｎ煌含量增加,给予ＣＮＴＦ后,脑水肿、海马的病理变化有明显改善,并     

睫状神经营养因子对应激引起大鼠海马神经元损伤的保护作用及其机制的研究

本实验用Ｎｉｓｓｌ染色法、Ｂｉｅｌｓｃｈｏｗｓｋｙ－Ｇｒｏｓ－Ｌａｗｒｅｎｔｊｅｗ染色法、常规透射电镜、行为活动测定、双侧海马微量给药、海马神经元原代培养、活细胞连续照相、全细胞膜片钳记录、细胞内游离Ｃａ＾２＋浓度测定及Ｐ５３蛋白免疫组化测定等方法,观察了睫状神经营养因子（ＣＮＴＦ）对应激引起动物行为变化和海马神经元形态学变化的影响,探讨了ＣＮＴＦ的部分作用机制。结果表明,急性应激不引起大鼠海马神     

Ciliary neurotrophic factor induces cholinergic differentiation of rat sympathetic neurons in culture

Ciliary neurotrophic factor (CNTF) influences the levels of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) in cultures of dissociated sympathetic neurons from newborn rats. In the presence of CNTF both the total and specific activity of ChAT was increased 7 d after culture by 15- and 18-fold, respectively, as compared to cultures kept in the absence of CNTF. Between 3 and 21 d in culture in the presence of CNTF the total ChAT activity increased by a factor of greater than 100. Immunotitration demonstrated that the elevated ChAT levels were due to an increased number of enzyme molecules. In contrast to the increase in ChAT levels, the total and specific activity levels of TH were decreased by 42 and 36%, respectively, after 7 d in culture. Half-maximal effects for both ChAT increase and TH decrease were obtained at CNTF concentrations of approximately 0.6 ng and maximal levels were reached at 1 ng of CNTF per milliliter of medium. The effect of CNTF on TH and ChAT levels were seen in serum-containing medium as well as in serum-free medium. CNTF was shown to have only a small effect on the long-term survival of rat sympathetic neurons. We therefore concluded that the effects of CNTF on ChAT and TH are not due to selective survival of cells that acquire cholinergic traits in vitro, but are rather due to the induction of cholinergic differentiation of noradrenergic sympathetic neurons.     

The cholinergic neuronal differentiation factor from heart cell conditioned medium is different from the cholinergic factors in sciatic nerve and spinal cord

Environmental cues play an important role in determining the transmitter phenotype of developing sympathetic neurons. Several factors have been described which can induce cholinergic function in cultured sympathetic neurons. We have compared certain biological and immunological properties of three of them, cholinergic differentiation factor (CDF), membrane-associated neurotransmitter-stimulating factor (MANS), and ciliary neurotrophic factor (CNTF), to determine whether they are different. As previously reported, all three increased acetylcholine synthesis in cultured sympathetic neurons. In addition, MANS as well as CNTF and CDF decreased catecholamine synthesis. CNTF and MANS, but not CDF, promoted the survival of embryonic chick ciliary neurons. Affinity-purified antibodies raised against a synthetic peptide corresponding to the N-terminal sequence of CDF immunoprecipitated CDF, but not MANS or CNTF. These results indicate that although CDF, MANS, and CNTF have similar effects on transmitter synthesis by cultured sympathetic neurons, CDF lacks the ciliary neurotrophic activity of MANS and CNTF. Further, CDF possesses an N-terminal epitope which is absent from both MANS and CNTF. Thus, CDF is distinct from MANS and CNTF, and at least two factors exist which can alter the transmitter phenotype of sympathetic neurons in vitro.     

The Cytokine Ciliary Neurotrophic Factor (CNTF) Activates Hypothalamic Urocortin-Expressing Neurons Both In Vitro and In Vivo

Ciliary neurotrophic factor (CNTF) induces neurogenesis, reduces feeding, and induces weight loss. However, the central mechanisms by which CNTF acts are vague. We employed the mHypoE-20/2 line that endogenously expresses the CNTF receptor to examine the direct effects of CNTF on mRNA levels of urocortin-1, urocortin-2, agouti-related peptide, brain-derived neurotrophic factor, and neurotensin. We found that treatment of 10 ng/ml CNTF significantly increased only urocortin-1 mRNA by 1.84-fold at 48 h. We then performed intracerebroventricular injections of 0.5 mg/mL CNTF into mice, and examined its effects on urocortin-1 neurons post-exposure. Through double-label immunohistochemistry using specific antibodies against c-Fos and urocortin-1, we showed that central CNTF administration significantly activated urocortin-1 neurons in specific areas of the hypothalamus. Taken together, our studies point to a potential role for CNTF in regulating hypothalamic urocortin-1-expressing neurons to mediate its recognized effects on energy homeostasis, neuronal proliferaton/survival, and/or neurogenesis.     

All‐trans retinoic acid upregulates the expression of ciliary neurotrophic factor in retinal pigment epithelial cells

Retinopathy of prematurity, a leading cause of visual impairment in low birth‐weight infants, remains a crucial therapeutic challenge. Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that promotes rod and cone photoreceptor survival and cone outer segment regeneration in the degenerating retina. Ciliary neurotrophic factor expression is regulated by many factors such as all‐trans retinoic acid (ATRA). In this study, we found that ATRA increased CNTF expression in mouse retinal pigment epithelial (RPE) cells in a dose‐ and time‐dependent manner, and PKA signaling pathway is necessary for ATRA‐induced CNTF upregulation. Furthermore, we showed that ATRA promoted CNTF expression through CREB binding to its promoter region. In addition, CNTF levels were decreased in serum of retinopathy of prematurity children and in retinal tissue of oxygen‐induced retinopathy mice. In mouse RPE cells cultured with high oxygen, CNTF expression and secretion were decreased, but could be recovered after treatment with ATRA. In conclusion, our data suggest that ATRA administration upregulates CNTF expression in RPE cells.     

Growth factor dependent cholinergic function and survival in primary mouse spinal cord cultures

In primary embryonic spinal cord cultures, synaptic transmission can be conveniently studied by monitoring radiolabeled neurotransmitter release or by recording of electrophysiological responses. However, while the mature spinal cord contains an appreciable number of cholinergic motoneurons, cultures of embryonic spinal cord have a paucity of these neurons and release little or no acetylcholine upon stimulation. To determine whether the proportion of cholinergic neurons in primary mouse spinal cord cultures can be augmented, the effects of several classes of growth factors were examined on depolarization- and Ca(2+)-evoked release of choline/acetylcholine (Ch/ACh). In the absence of growth factors, little or no evoked release of radiolabeled Ch/ACh could be demonstrated. Media supplemented with brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF) were examined for their ability to preserve the population of neurons in culture. CNTF was found to increase the number of surviving neurons and to enhance the release of radiolabeled Ch/ACh; the other factors were without effect. The action of CNTF was transient, and the neuronal population decreased to levels observed in cultures lacking growth factor after 20 days in vitro. The correlation between enhanced neuron survival and increased Ch/ACh release suggests that CNTF protected cholinergic neurons, albeit transiently, from cell death.     

Involvement of ras p21 in neurotrophin-induced response of sensory, but not sympathetic neurons

Little is known about the signal transduction mechanisms involved in the response to neurotrophins and other neurotrophic factors in neurons, beyond the activation of the tyrosine kinase activity of the neurotrophin receptors belonging to the trk family. We have previously shown that the introduction of the oncogene product ras p21 into the cytoplasm of chick embryonic neurons can reproduce the survival and neurite-outgrowth promoting effects of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and of ciliary neurotrophic factor (CNTF). To assess the potential signal- transducing role of endogenous ras p21, we introduced function-blocking anti-ras antibodies or their Fab fragments into cultured chick embryonic neurons. The BDNF-induced neurite outgrowth in E12 nodose ganglion neurons was reduced to below control levels, and the NGF- induced survival of E9 dorsal root ganglion (DRG) neurons was inhibited in a specific and dose-dependent fashion. Both effects could be reversed by saturating the epitope-binding sites with biologically inactive ras p21 before microinjection. Surprisingly, ras p21 did not promote the survival of NGF-dependent E12 chick sympathetic neurons, and the NGF-induced survival in these cells was not inhibited by the Fab-fragments. The survival effect of CNTF on ras-responsive ciliary neurons could not be blocked by anti-ras Fab fragments. These results indicate an involvement of ras p21 in the signal transduction of neurotrophic factors in sensory, but not sympathetic or ciliary neurons, pointing to the existence of different signaling pathways not only in CNTF-responsive, but also in neurotrophin-responsive neuronal populations.     

Evidence for Multiple, Local Functions of Ciliary Neurotrophic Factor (CNTF) in Retinal Development: Expression of CNTF and Its Receptor and In Vitro Effects on Target Cells

Abstract: There is increasing, although largely indirect, evidence that neurotrophic factors not only function as target-derived survival factors for projection neurons, but also act locally to regulate developmental processes. We studied the expression of ciliary neurotrophic factor (CNTF) and the CNTF-specific ligand-binding α-subunit of the CNTF receptor complex (CNTFRα) in the rat retina, a well-defined CNS model system, and CNTF effects on cultured retinal neurons. Both CNTF and CNTFRα (mRNA and protein) are expressed during phases of retinal neurogenesis and differentiation. Retina-specific Müller glia are immunocytochemically identified as the site of CNTF production and CNTFRα-expressing, distinct neuronal cell types as potential CNTF targets. Biological effects on corresponding neurons in culture further support the conclusion that locally supplied CNTF plays a regulatory role in the development of various retinal cell types including ganglion cells and interneurons.