蛛网膜下隙注射胶质细胞源性神经营养因子抑制脊神经结扎大鼠脊髓背角胶质原纤维酸性蛋白表达

目的观察蛛网膜下隙注射胶质细胞源性神经营养因子(GDNF)对脊神经结扎(SNL)大鼠脊髓背角胶质原纤维酸性蛋白(GFAP)的影响。方法采用结扎SD大鼠第5~第6腰脊神经(L5~L6)制备SNL模型。术后隔日一次性蛛网膜下隙注射10μl GDNF2 g·L-1组。术后第3,7和14天采用免疫组织化学和Western免疫印迹法测定脊髓背角处GFAP蛋白表达。结果免疫组化结果显示,SNL组在术后第3天、第7天和第14天脊髓GFAP阳性细胞数目明显增加,可持续至第14天,而正常对照组与假手术组的星形胶质细胞未发生此种改变。GDNF组的阳性细胞显著减少。Western印迹结果显示,正常对照组和假手术组脊髓背角均出现GFAP免疫阳性条带,灰度值较低;SNL在术后第3天即可诱导脊髓背角GFAP蛋白的表达,随着时间的延长,GFAP蛋白的灰度值逐渐升高,术后第3,7,14天分别为2.55±0.33,2.88±0.79和3.12±0.75(P<0.01)。与SNL模型组比较,GDNF可显著降低GFAP的表达水平,术后第3,7,14天分别为1.61±0.38,1.65±0.64和1.57±0.41(P<0.01)。结论蛛网膜下隙注射GDNF减轻大鼠神经病理性疼痛机制可能与其抑制脊髓背角GFAP蛋白表达有关。     

脑内胶质细胞源性神经营养因子及其受体复合物研究进展

胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)在脑内广泛分布,通过其受体复合物介导激活细胞内信号转导通路,发挥维持神经元功能和损伤修复等作用。胶质细胞源性神经营养因子家族受体α(GD-NF family receptorα,GFRα)和RET是其受体复合物的主要成员。GDNF和其受体复合物可能参与多种脑部病变的病理生理过程,是潜在的治疗靶点之一。     

胶质细胞源性神经营养因子的表达、功能和在药物依赖中的作用

1993年,Lin等[1]从大鼠胶质细胞株B49中提纯到一种可促进胚胎中脑多巴胺能神经元存活的神经营养因子,并命名为胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF).GDNF和后来发现的neurturin(NRTN)、persephin(PSPN)、artemin(ARTN)在结构和功能上有很大的相似性,共同构成一个家族,称为GDNF家族.     

胶质细胞源性神经营养因子与神经病理性疼痛

胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)是神经营养因子家族成员之一,GD-NF对中枢和周围神经系统多种神经元的生长、发育、分化、维持和损伤修复起重要作用。另外,GDNF还参与中枢和外周水平神经病理性疼痛的形成和发展。该文主要就GDNF和神经病理性疼痛的研究作一简要综述。     

外源性bFGF、VEGF对大鼠脊髓损伤后内源性神经干细胞增殖分化影响的实验研究

目的制备大鼠脊髓损伤模型,研究外源性碱性成纤维细胞生长因子(bFGF)、血管内皮生长因子(VEGF)联合移植对内源性神经干细胞(ENSCs)是否具有促增殖分化作用。方法成年Wistar大鼠56只,随机分成四组:A组为生理盐水组(n=14);B组为bFGF组(n=14);C组为VEGF组(n=14);D组为bFGF、VEGF组(n=14)。用Allen法在T10节段制备大鼠脊髓损伤模型,损伤后立即分别给予生理盐水、bFGF、VEGF、bFGF和VEGF。术后各时间点BBB评分系统监测大鼠运动功能的恢复,并在术后21d取材,处死前24h腹腔注射5-溴脱氧尿嘧啶核苷(Brdu),取损伤部位近远端各5mm脊髓组织,行HE染色,免疫组织化学法检测Brdu、巢蛋白(Nestin)、神经元特异性烯醇化酶(NSE)的表达情况。结果 (1)损伤后21d,bFGF、VEGF合用治疗组BBB高于其它三组,差异有统计学意义(P<0.05)。(2)损伤后21d时,bFGF、VEGF治疗组组织形态学基本接近正常脊髓组织。(3)免疫组织化学结果:损伤后21d时,bFGF、VEGF治疗组,神经干细胞数量和神经元数量明显高于其它组(P<0.05)。结论联合移植外源性bFGF、VEGF对ENSCs增殖和向神经元方向分化具有促进作用,且二者具有协同作用。     

糖尿病大鼠海马和大脑皮层GDNF蛋白表达的变化

目的 研究糖尿病大鼠不同血糖水平下脑内胶质细胞源性神经营养因子(GDNF)表达的变化.方法 将30只Wistar大鼠随机均分为糖尿病血糖未控制(DMI)组、糖尿病血糖控制(DM2)组和对照(NC)组.12周后检测血糖与糖化血红蛋白(HbA1c)水平,免疫组织化学(SP)法检测GDNF蛋白表达,HE染色观察海马及大脑皮层神经元细胞计数和形态学变化.结果 海马CA区和大脑皮层的GDNF蛋白表达均按NC组＞DM2组＞DM1组依次减弱(P＜0.05),且均与HbA1c水平之间呈负相关(r分别为-0.961和-0.963,P＜0.05).与NC组相比,DM1组和DM2组海马CA区神经元无明显减少,而海马及大脑皮层神经元呈明显退形性形态学改变.结论 长期慢性高血糖可下调海马和大脑皮层GDNF蛋白表达,从而可能引起中枢神经病变.     

碱性成纤维细胞生长因子在大鼠脊髓损伤后的表达及意义

目的:探讨脊髓损伤后的自我保护修复机制.方法:采用改良的Allen’s法建立SD大鼠T11节段脊髓损伤模型,观察术后不同时间段脊髓内碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)蛋白表达分布及变化情况.结果:脊髓损伤各时间段bFGF阳性神经元数较正常组显著增加.7 d达高峰,21 d恢复正常.结论:脊髓损伤后bFGF表达,提示其参与了脊髓损伤后神经元的保护作用.     

不同细胞因子组合对神经干细胞分化的影响

目的:探讨不同细胞因子组合对神经干细胞(NSCs)分化的影响。方法:采用不同组合的细胞因子(bFGF EGF; bFGF PDGF;bFGF BDNF;bFGF)分离培养大鼠海马神经干细胞,并用免疲细胞化学技术研究神经干细胞的分化情况。结果:在bFGF、bFGF EGF、bFGF BDNF及bFGF PDGF不同因子或因子组合中,均能诱导神经干细胞分化,分化后的神经元比例分别为(14.5±1.2)%、(14.7±1.8)%、(23.3±2.1)%、(35.5±2.4)%,神经干细胞的分化比例趋势为bFGF PDGF >bFGF BDNF>bFGF EGF>bFGF(P<0.05)。姑论:PDGF对bFGF促进神经干细胞向神经元定向分化有较好的协同作用。     

普拉克索和罗匹尼罗对体外培养的多巴胺神经元的神经营养作用

目的 :探讨新型选择性多巴胺D3受体激动剂普拉克索和罗匹尼罗对多巴胺神经元的神经营养作用及其机制。方法 :在大鼠的腹侧中脑细胞和不同部位星形胶质细胞培养基中加入普拉克索和罗匹尼罗刺激 ,观察药物对多巴胺神经元存活的影响。结果 :药物直接作用或从黑质区星形胶质细胞培养基中提取的条件培养液均可使酪氨酸羟化酶 (TH )阳性神经元数量增加 ,同时培养液中脑源性神经营养因子 (BDNF)和胶质细胞源性神经营养因子(GDNF)含量增加。而其他脑区的星形胶质细胞不能产生类似作用。结论 :普拉克索和罗匹尼罗对多巴胺神经元具有神经营养作用 ,这可能是由于其使特定区域星形胶质细胞产生并分泌了神经营养因子。     

鞘内注射促皮质素抑制甲醛痛敏大鼠脊髓NOS阳性神经元增多

目的:研究鞘内注射促皮质素(Cor)对甲醛痛敏大鼠脊髓背角一氧化氮合酶(NOS)阳性神经元增多的影响。方法:采用痛级均数(PIR)测定、NADPH-d组织化学法、Fos免疫组织化学法染色,观察鞘内注射(ith)Cor对甲醛痛敏大鼠脊髓背角NOS阳性神经元、Fos免疫反应神经元、NOS/Fos双标记神经元及痛敏的影响。结果:ith Cor(0.5-1.5U)均能显著抑制甲醛引起的大鼠脊髓背角NOS、Fos、NOS/Fos阳性神经元的增多和痛敏反应,其作用为ith NOS底物左旋精氨酸(Arg,5-15nmol)部分翻转。结论:Cor通过抑制大鼠脊髓背角NOS阳性神经元的增多抑制痛敏。     

碱性成纤维细胞生长因子对培养大鼠胚胎脊髓神经细胞的作用

目的探讨碱性成纤维细胞生长因子 (bFGF)对大鼠胚胎脊髓神经细胞的作用。方法在培养孕 18d的大鼠胚胎脊髓神经细胞中加入bFGF ,通过培养细胞并计数存活的脊髓神经细胞集落数 ,检测超氧化物歧化酶(SOD)的活力及丙二醛的含量 ,并观察bFGF对脊髓神经细胞生长发育的影响。结果bFGF可促进脊髓神经细胞分化、增殖 ,SOD活力、丙二醛含量明显变化 ,脊髓神经细胞胞体较大、突起较长 ,培养 14d后脊髓神经细胞的数量也明显增高。结论bFGF能促进脊髓神经细胞生长发育 ,增加脊髓神经细胞的胞体和突起长度 ,增强脊髓神经细胞内SOD活力 ,降低丙二醛含量。     

bFGF基因修饰骨髓间充质干细胞移植对急性脊髓损伤大鼠神经元的影响

目的探讨bFGF基因修饰的骨髓间充质干细胞移植对急性脊髓损伤大鼠神经元的作用。方法清洁级成年雄性Sprague-Dawley大鼠96只,随机分为培养液组（DMEM组）、骨髓间充质干细胞（BMSCs组）、空载体组（pcD-NA3.1组）和目的基因组（pcDNA3.1-bFGF组）,每组24只,采用Allen法制作脊髓损伤模型,10 min后,于损伤点分别注射DMEM培养液、BMSCs质粒修饰的细胞。在注射后第1、7、14、21天行脊髓运动功能Basso-Beatti-Bresnahan（BBB）评分,用RT-PCR法检测bFGF表达情况,应用尼氏体染色后,采用计算机图像分析技术进行定量分析。结果目的基因组术后各时间点脊髓组织中bFGF表达、BBB评分及尼氏体染色阳性细胞的光密度值都明显高于其余3组,组间差异均有统计学意义。结论 BMSCs是脊髓损伤基因治疗可用的受体细胞,bFGF基因修饰BMSCs移植对大鼠脊髓损伤功能恢复有一定促进作用。     

Cilostazol improves cognitive function in mice by increasing the production of insulin-like growth factor-I in the hippocampus

Insulin-like growth factor I (IGF-I) exerts beneficial effects on cognitive function by inducing angiogenesis and neurogenesis in the hippocampus. We demonstrated that stimulation of sensory neurons in the gastrointestinal tract increased IGF-I production in the hippocampus, and thereby improved cognitive function in mice. Since cAMP plays a critical role in stimulation of sensory neurons, the type III phosphodiesterase (PDE3) inhibitor cilostazol might increase IGF-I production in the hippocampus by stimulating sensory neurons and thus improve cognitive function in mice. We tested this hypothesis in the present study. Cilostazol increased the release of calcitonin gene-related peptide (CGRP) and levels of cAMP in dorsal root ganglion (DRG) neurons isolated from wild-type (WT) mice. Tissue levels of cAMP in the DRG and hippocampus and those of CGRP, IGF-I, and IGF-I mRNA in the hippocampus were increased after 4-week oral administration of cilostazol to WT mice. Levels of expression of c-fos in the spinal dorsal horns, parabrachial nuclei, the solitary tract nucleus, and the hippocampus were also increased in these animals. Significant enhancement of angiogenesis and neurogenesis was observed in the dentate gyrus of the hippocampus after cilostazol administration in WT mice. Significant improvement of spatial learning was also observed in WT mice administered cilostazol. However, none of these effects in WT mice were observed in CGRP-knockout mice. These observations suggest that cilostazol may improve cognitive function in mice by increasing the hippocampal production of IGF-I through stimulation of sensory neurons.     

Organotypic spinal cord culture using mice

Amyotrophic lateral scleorsis (ALS) is a progressive neurodegenerative disease, which predominantly affects both upper and lower motor neurons. ALS is usually fatal within a few years after clinical onset. An organotypic slice culture of rat spinal cord, in which glutamate toxicity induces slow loss of spinal motoneurons, has been used for preclinical drug screening for ALS. In this report, we modified the conventional slice culture to put mouse spinal cords to use, as an alternative in vitro model of ALS. L-trans pyrrolidine 2, 4-dicarboxylic acid (PDC), an inhibitor of glutamate uptake, induced slow loss of spinal motoneurons in anterior horns, whereas small neurons in posterior horns were relatively preserved. This technique using mouse allows us to use transgenic and knockout mice. In addition to glutamate toxicity, many other mechanisms including oxidative stress and neurofilamentous disorganization are also considered to be involved in development of this devastating disease. Thus, a better in vitro model of ALS is strongly anticipated. At present, we are making efforts to apply this technique to establish a better in vitro model using spinal cord from transgenic ALS model mice, where spontaneous loss of spinal motoneurons will be observed.     

脊髓火器伤后GDNFmRNA表达的实验研究

目的观察脊髓火器伤后GDNFmRNA的表达变化并探讨其意义。方法利用民用射钉枪建立大鼠脊髓火器损伤模型,不同时间采用半定量RT-PCR方法,观察T12～L1段脊髓GDNFmRNA表达的变化。结果脊髓损伤前,GDNFmRNA在T12～L1段脊髓微量表达,脊髓损伤后表达逐渐减少,伤后1、4、7、10d分别减少25%、71%、82%、86%。结论脊髓GDNFmRNA表达减少,是由于失去靶组织转运GDNFmRNA所造成,这为外源性GDNF治疗脊髓火器伤提供了理论依据。     

石杉碱甲对体外培养大鼠脊髓片前角运动神经元损伤的保护作用

目的:利用选择性运动神经元死亡的脊髓薄片培养模型,观察石杉碱甲对运动神经元的保护作用。方法:乳大鼠腰段脊髓组织薄片分为正常对照组、模型组和石杉碱甲1,10,100μmol·L-15组。用倒置显微镜观察脊髓片形态变化,SMI32免疫组化染色对腹角α运动神经元记数,透射电镜观察α运动神经元的超微结构并测定培养液中乳酸脱氢酶(LDH)的含量。结果:石杉碱甲100μmol·L-1组脊髓片在体外生长良好,形态与正常对照组相近;石杉碱甲1,10μmol·L-1组脊髓片生长不良,形态与模型组相近。与正常对照组相比,模型组脊髓片前角SMI32阳性α运动神经元数减少(P<0.05),培养液中LDH含量增加(P<0.05)。与模型组相比,石杉碱甲100μmol·L-1组脊髓片前角α运动神经元数增加(P<0.05),胞浆内空泡明显减少,培养液中LDH含量减低(P<0.05)。结论:高浓度石杉碱甲能改善乳大鼠脊髓片前角α运动神经元生长,减少其空泡形成及死亡,并降低LDH的释放。     

银杏内酯B促进体外培养胚胎大鼠脊髓神经细胞生长发育的研究

目的探讨GB对体外培养的胚鼠脊髓神经元存活和生长发育的作用。方法胚胎大鼠脊髓神经细胞原代培养,相差显微镜下进行细胞计数和显微测量,观察GB对神经元存活和生长发育的作用。对培养7d细胞行NSE免疫组化染色,光镜下观察GB对NSE染色阳性神经元生长发育的影响。结果GB及NGF能够促进体外培养胚胎大鼠脊髓神经细胞及NSE染色阳性神经元的存活,促进其细胞及突起的生长。其促进脊髓神经细胞的存活、胞体及突起发育的作用与NGF一致,而其促进神经突起分化与生长的作用强于NGF。结论NGF和GB能促进培养大鼠发育期脊髓神经元存活、分化和生长,并且表现出各自作用的特异性。     

Interplay of BDNF and GDNF in the Mature Spinal Somatosensory System and Its Potential Therapeutic Relevance

The growth factors BDNF and GDNF are gaining more and more attention as modulators of synaptic transmission in the mature central nervous system (CNS). The two molecules undergo a regulated secretion in neurons and may be anterogradely transported to terminals where they can positively or negatively modulate fast synaptic transmission. There is today a wide consensus on the role of BDNF as a pro-nociceptive modulator, as the neurotrophin has an important part in the initiation and maintenance of inflammatory, chronic, and/or neuropathic pain at the peripheral and central level. At the spinal level, BDNF intervenes in the regulation of chloride equilibrium potential, decreases the excitatory synaptic drive to inhibitory neurons, with complex changes in GABAergic/glycinergic synaptic transmission, and increases excitatory transmission in the superficial dorsal horn. Differently from BDNF, the role of GDNF still remains to be unraveled in full. This review resumes the current literature on the interplay between BDNF and GDNF in the regulation of nociceptive neurotransmission in the superficial dorsal horn of the spinal cord. We will first discuss the circuitries involved in such a regulation, as well as the reciprocal interactions between the two factors in nociceptive pathways. The development of small molecules specifically targeting BDNF, GDNF and/or downstream effectors is opening new perspectives for investigating these neurotrophic factors as modulators of nociceptive transmission and chronic pain. Therefore, we will finally consider the molecules of (potential) pharmacological relevance for tackling normal and pathological pain.     

Glial cell line-derived neurotrophic factormediated enhancement of noradrenergic descending inhibition in the locus coeruleus exerts prolonged analgesia in neuropathic pain

Background and Purpose

The locus coeruleus (LC) is the principal nucleus containing the noradrenergic neurons and is a major endogenous source of pain modulation in the brain. Glial cell line-derived neurotrophic factor (GDNF), a well-established neurotrophic factor for noradrenergic neurons, is a major pain modulator in the spinal cord and primary sensory neurons. However, it is unknown whether GDNF is involved in pain modulation in the LC.

Experimental Approach

Rats with chronic constriction injury (CCI) of the left sciatic nerve were used as a model of neuropathic pain. GDNF was injected into the left LC of rats with CCI for 3 consecutive days and changes in mechanical allodynia and thermal hyperalgesia were assessed. The α₂-adrenoceptor antagonist yohimbine was injected intrathecally to assess the involvement of descending inhibition in GDNF-mediated analgesia. The MEK inhibitor U0126 was used to investigate whether the ERK signalling pathway plays a role in the analgesic effects of GDNF.

Key Results

Both mechanical allodynia and thermal hyperalgesia were attenuated 24 h after the first GDNF injection. GDNF increased the noradrenaline content in the dorsal spinal cord. The analgesic effects continued for at least 3 days after the last injection. Yohimbine abolished these effects of GDNF. The analgesic effects of GDNF were partly, but significantly, inhibited by prior injection of U0126 into the LC.

Conclusions and Implications

GDNF injection into the LC exerts prolonged analgesic effects on neuropathic pain in rats by enhancing descending noradrenergic inhibition.