Electro-acupuncture induced NGF, BDNF and NT-3 expression in spared L6 dorsal root ganglion in cats subjected to removal of adjacent ganglia

This study evaluated the effect of electro-acupuncture (EA) on the NGF, BDNF and NT-3 expression in spared L6 dorsal root ganglion (DRG) in cats subjected to bilateral removal of L1-L5 and L7-S2 DRG, using immunostaining, in situ hybridization and RT-PCR. The positive products of NGF, NT-3 protein and mRNA in the small and large neurons of spared L6 DRG in EA side increased greatly more than that of control side, while the increased BDNF was only noted in small and medium-sized neurons. RT-PCR demonstrated that the mRNA level for three factors was not influenced by EA in intact DRG, when a significant increase was seen in the spared L6 DRG of EA side. As it has been well known that DRG neurons project to the spinal cord wherein morphological plasticity has been present after DRG removal, the present results might have some bearing to the observed phenomenon.     

针刺对备用DRG的NGF及其mRNA阳性大、中、小神经元数量的影响

本研究观察了 NGF及其 m RNA阳性神经元在 DRG大、中、小神经元中的数量变化 ,目的在于探讨 DRG各类神经元NGF变化与针刺促进脊髓可塑性的关系。对 5只成年猫进行双侧备用根手术。术后当日开始针刺一侧 L6 脊神经后肢分布区的两组穴位即足三里和悬中、伏兔和三阴交 ,每天一次 ,每次 3 0 min,连续针刺 7d后处死动物。取针刺侧与非针刺侧 L6 的 DRG制成 2 0μm厚冰冻纵切片。分别用 NGF抗血清和 NGF的 c RNA探针进行免疫组织化学反应及原位杂交染色。计数两侧 DRG相同面积内大 (>5 7μm)、中 (4 2～ 5 7μm)、小 (<42 μm)型 NGF及其 m RNA阳性神经元的数量。结果显示 :针刺侧备用 DRG NGF及其 m RNA阳性大、小型神经元的数量明显增加 (P<0 .0 5 ) ,而对中型神经元数量影响不明显。结论 :针刺促进脊髓可塑性可能与备用 DRG大、小神经元表达 NGF增多有关。     

针刺对脑源性神经营养因子在脊髓Ⅱ板层可塑性中表达的影响

采用免疫组化和原位杂交技术 ,探讨了针刺促进脊髓 板层可塑性过程中脑源性神经营养因子及其 m RNA表达的变化。结果显示 :针刺侧备用背根节的脑源性神经营养因子及其 m RNA阳性中 (4 2～ 5 7μm)、小 (<42 μm)型神经元的百分数有所增加 (P<0 .0 5 ) ;脊髓 板层的脑源性神经营养因子的阳性神经膨体密度明显增多 (P<0 .0 1)而阳性神经元百分数无变化 (P>0 .0 5 ) ;针刺后脊髓 板层未见脑源性神经营养因子 m RNA的杂交信号。提示针刺促进脊髓 板层可塑性可能与背根节中、小神经元表达脑源性神经营养因子的增多有关     

NGF,BDNF,NT-3和GDNF在背根节不同神经元的配布的免疫组织化学证据

为了探讨神经生长因子、脑源性神经生长因子 ,神经营养素 -3,胶原细胞源性神经生长因子在成年猫背根节神经元不同大小细胞的分布。取 L6背根节制成 2 0μm厚冰冻切片 ,用上述四种因子抗血清进行免疫组化反应。计数每一种因子阳性大、中、小型 (小于 42μm者为小型神经元 ;42～ 5 7μm者为中型神经元 ;大于 5 7μm者为大型神经元 )神经元的百分数。结果证明 ,此四种因子在背根节大、中、小神经元的阳性百分数分别为 :1.2± 0 .7,2 .1± 0 .9,3.3± 1.1;1.7± 0 .4,11.2± 1.2 ,30 .0± 1.5 ;2 6 .7± 2 .2 ,6 .2± 1.2 ,13.2± 2 .9;2 7.4± 3.3,8.1± 1.7,2 0 .1± 2 .4。表明 :在成年猫 L6背根节仅存在少数神经生长因子阳性细胞 ,而脑源性神经生长因子则主要配布于中、小神经元 ,神经营养素 -3和胶质细胞源性生长因子的免疫阳性细胞主要是大神经元 ,但也包括一些中小神经元。提示此四种因子在成年猫背根节的生理功能可能与神经元的不同大小有关。     

Roles of BDNF in spinal neuroplasticity in cats subjected to partial dorsal ganglionectomy

This study investigated the role of brain-derived neurotrophic factor (BDNF) in neuroplasticity in cats subjected to the removal of dorsal root ganglia (DRG). Following partial ganglionectomy, the number of BDNF-positive varicosities from spared L6 DRG decreased significantly. This reduction was observed at 3 days post operation (dpo) in spinal lamina II of L3 and L5. Whereas the percentages of positive neurons for BDNF and its mRNA in spared L6 DRG at 10 dpo were significantly increased, and accumulated BDNF was seen on the DRG side of the ligated axons. Importantly, BDNF antibody neutralization in vivo results in a significant reduction in the number of varicosities in spinal lamina II, evidenced by BDNF and calcitonin gene-related peptide immunohistochemical staining. These findings suggested that peripheral-derived BDNF could play a critical role in spinal neuroplasticity in cats subjected to partial ganglionectomy. This may underlie the basis of molecular therapy depending on gene drug-like BDNF release.     

NGF及NGF mRNA在去部分背根猫脊髓和背根节表达的变化

用成年猫单侧备用根模型 (切断一侧 L1～ 5 、L7～ S2 节段脊髓背根 ,保留 L6 背根 ) ,用免疫组织化学和原位杂交技术 ,对脊髓背角 板层、背核和 L6 背根节中神经生长因子的表达进行了动态观察。结果证明 ,部分背根去传入后 :(1)脊髓各节段双侧背角 板层的神经生长因子和神经生长因子 m RNA阳性神经元的数量及体积显著增高、增大 ;(2 )手术侧背核神经生长因子和神经生长因子 m RNA阳性神经元的增高强于非手术侧 ,但术后两时间组的手术侧之间无差异 ;(3 )备用根背根节新出现了神经生长因子 m RNA阳性神经元 ,且神经生长因子阳性神经元数量明显高于正常组 ,且随术后时间增加而递增。以上结果表明 :部分背根去传入猫脊髓背角 板层及背核内神经生长因子水平提高 ,尤其备用根背根节神经元神经生长因子的增加 ,为备用背根传入纤维侧支生芽和突触重建提供了良好的条件 ,有助于解释脊髓初级传入纤维损伤溃变后代偿性机能恢复的机制     

BDNF,NGF和NT-3的mRNA和蛋白在猫左侧腰第六背根节共表达的不同方式(英文)

本实验研究了脑源性神经营养因子(brain derived neurotrophic factor,BDNF),神经生长因子(nerve growth factor,NGF)和神经营养因子-3(neurotrophin-3,NT-3)的mRNA和蛋白在猫左侧腰第六背根节(dorsal root ganglion,DRG)共表达的不同方式,并探讨了共表达的机制,为阐明神经营养因子的表达与脊髓可塑性的关系提供依据。本实验所使用的为未接受任何处理的健康猫。猫的左侧腰第六DRG被取出,行免疫组织化学染色与原位杂交方法结合的双重标记,确定是否有BDNF,NGF和NT-3的蛋白与mRNA共表达。实验结果显示BDNF,NGF和NT-3的蛋白与mRNA在猫DRG均有表达,但这三种神经营养因子mRNA和蛋白共表达的方式是不同的。免疫组化结果显示:BDNF阳性产物主要分布于细胞质和细胞核,细胞核的染色颜色较细胞质浅;NGF阳性产物主要分布于细胞核;NT-3阳性产物主要分布于细胞质。原位杂交结果显示:BDNF和NGF阳性信号主要分布于细胞质;NT-3阳性信号在细胞质和细胞核都有分布。由此可见,BDNF,NGF和NT-3的mRNA和蛋白在猫左侧腰第六DRG有不同的共表达方式,提示它们可能存在与脊髓可塑性有关的自分泌和/或旁分泌机制。     

部分背根切断后备用DRG卫星细胞NGF、BDNF和NT-3的表达变化

为探讨部分去背根猫备用背根节 ( L6 )卫星细胞 NGF、BDNF和 NT-3的表达变化 ,将成年雄猫 2 0只分为正常对照组和术后 3 d、术后 7d及术后 10 d三个实验组。实验组行单侧部分背根切断术 (切除一侧 L1 ～ L5,L7～S2 DRG,保留 L6 背根为备用根 )。取正常组一侧和术后 3 d、7d及 10 d实验组手术侧的 L6 DRG制作 2 0μm厚冰冻切片 ,行免疫组化反应 ;观察 NGF、BDNF和 NT-3在 DRG卫星细胞的分布 ,计数各组 L6 DRG的 NGF、BDNF和 NT-3的阳性卫星细胞数量。结果发现 ,部分去背根术后7d和 10 d组 L6 DRG的 NGF、BDNF和 NT-3的阳性卫星细胞数量较术后 3 d组和正常组者明显增高 ( P<0 .0 1)。表明 ,单侧部分背根切断导致备用 DRG卫星细胞的 NGF、BDNF和 NT-3的表达上调 ,这一变化可能与脊髓可塑性有关。     

Functional roles of intrinsic neurotrophin-3 in spinal neuroplasticity of cats following partial ganglionectomy

This study detected the effects of endogenous neurotrophin-3 (NT-3) on the collateral sprouting derived from the L6 dorsal root ganglion (DRG) after unilateral removal of adjacent DRGs (L1–L5 and L7) in cats. Cholera toxin B tracing revealed significant neurite growth from the spared L6 DRG and axonal sprouting in the dorsal column. There was a significant increase in the number of NT-3 and trkC immunopositive neurons as well as in NT-3 protein level in the spared DRG by immunohistochemistry and enzyme-linked immunoadsorbent assay. NT-3 and its mRNA and trkC were located mainly in large- and medium-sized DRG neurons. NT-3 antibody neutralization in vivo and in vitro results in marked reduction in sprouted fibers. These findings point to an important role of NT-3 in neural plasticity at dorsal column axons.     

Roles of BDNF in spinal neuroplasticity in cats subjected to partial dorsal ganglionectomy

This study investigated the role of brain-derived neurotrophic factor (BDNF) in neuroplasticity in cats subjected to the removal of dorsal root ganglia (DRG). Following partial ganglionectomy, the number of BDNF-positive varicosities from spared L6 DRG decreased significantly. This reduction was observed at 3 days post operation (dpo) in spinal lamina II of L3 and L5. Whereas the percentages of positive neurons for BDNF and its mRNA in spared L6 DRG at 10?dpo were significantly increased, and accumulated BDNF was seen on the DRG side of the ligated axons. Importantly, BDNF antibody neutralization in vivo results in a significant reduction in the number of varicosities in spinal lamina II, evidenced by BDNF and calcitonin gene-related peptide immunohistochemical staining. These findings suggested that peripheral-derived BDNF could play a critical role in spinal neuroplasticity in cats subjected to partial ganglionectomy. This may underlie the basis of molecular therapy depending on gene drug-like BDNF release.     

Functional roles of intrinsic neurotrophin-3 in spinal neuroplasticity of cats following partial ganglionectomy

This study detected the effects of endogenous neurotrophin-3 (NT-3) on the collateral sprouting derived from the L6 dorsal root ganglion (DRG) after unilateral removal of adjacent DRGs (L1–L5 and L7) in cats. Cholera toxin B tracing revealed significant neurite growth from the spared L6 DRG and axonal sprouting in the dorsal column. There was a significant increase in the number of NT-3 and trkC immunopositive neurons as well as in NT-3 protein level in the spared DRG by immunohistochemistry and enzyme-linked immunoadsorbent assay. NT-3 and its mRNA and trkC were located mainly in large- and medium-sized DRG neurons. NT-3 antibody neutralization in vivo and in vitro results in marked reduction in sprouted fibers. These findings point to an important role of NT-3 in neural plasticity at dorsal column axons.     

部分背根切断对备用DRG神经元和胶质细胞NGF表达上调的影响

本研究目的在于探讨部分背根切断后备用背根节(DRG)中各类细胞NGF及其mRNA的量变状况。将成年雄猫10只分为正常组、单侧部分背根切断后7天组等2组,每组5只。实验组切除一侧L1～L5,L7～S2 DRG,保留L6 DRG为备用根。取正常组一侧和术后7天组手术侧的L6 DRG制成20μm厚冰冻纵切片,分别用NGF抗体及其cRNA探针进行免疫组织化学及原位杂交反应。观察NGF及其mRNA在DRG各类细胞的分布,测定NGF及其mRNA的光密度值以反映其相对含量。结果表明,在正常组,L6DRG中仅存在少量的NGF及其mRNA阳性神经元,此外,一些胶质细胞也呈阳性反应。在手术组,各类神经元及胶质细胞内NGF及其mRNA的光密度值较正常者显著升高(P<0.05)。结论:部分背根切断导致备用DRG神经元和非神经元表达NGF增多,提示NGF可能有利于促进备用背根生芽。     

针刺对去部分背根猫脊髓和背根节NGF及NGF mRNA的影响

用免疫组织化学和地高辛标记 c RNA探针原位分子杂交技术 ,观察了针刺对成年备用背根猫 (切断一侧 L1～ 5 、L7～ S2节段脊髓背根 ,保留 L6 背根 )脊髓 板层、背核和背根节内神经生长因子和神经生长因子 m RNA的影响。结果证明 ,针刺促进了备用根背根节的神经生长因子基因的表达 ,使备用背根节内神经生长因子、神经生长因子 m RNA阳性神经元数量明显增多 ,并且针刺时间越长促进作用也越强 ;而对脊髓 板层与背核则无明显影响。本实验结果提示 ,针刺对备用根猫脊髓侧支生芽具有促进作用 ,这种作用主要体现在通过增加背根节神经生长因子基因的表达和神经生长因子的合成来实现的     

部分去背根猫备用背根节和脊髓Ⅱ板层BDNF及其mRNA表达的变化

采用免疫组织化学和原位杂交技术探讨了部分去背根猫术后 3 d和 10 d的备用背根节和脊髓 II板层内 BDNF及其m RNA的表达变化。结果表明 ,正常组 ,BDNF主要分布于背根节的部分中、小神经元及脊髓背角浅层神经元内 ,II板层内还有大量的 BDNF阳性神经膨体。背根节同时也为 BDNF m RNA阳性 ,唯背角阴性。部分去背根后 10 d组 ,备用背根节 BDNF及其 m RNA阳性中、小神经元百分数较正常及 3 d者明显增加 (P<0 .0 5 )。而 L3,L5II板层 BDNF阳性神经膨体密度在术后两组均明显下降 (P<0 .0 5 ) ,唯 BDNF的含量在 3 d组者减少 (P<0 .0 5 ) ,10 d组者恢复 (P>0 .0 5 )。术后 II板层未见 BDNF m RNA的杂交信号。结果提示 :去背根术后 3 d组脊髓 II板层阳性膨体数的下降及 BDNF含量减少 ,可能是因背根切断导致输入脊髓 II板层的 BDNF中断所致 ;而 10 d时 II板层 BDNF合成得以恢复 ,可能来源于备用背根节中、小神经元的 BDNF增多所致 ,BDNF在脊髓可塑性中可能发挥作用     

Effects of electro-acupuncture on NT-4 expression in spinal dorsal root ganglion and associated segments of the spinal dorsal horn in cats subjected to adjacent dorsal root ganglionectomy

It is well known that neuroplasticity occurs in the central nervous system in response to injury. Electro-acupuncture (EA) may also promote neuroplasticity. But little is known about the underlying molecular mechanisms for the beneficial effects of EA. This study investigated the effects of EA on neurotrophin-4 (NT-4) expression in L(6) spinal dorsal root ganglion (DRG) and associated segments of the spinal dorsal horn in cats subjected to unilateral removal of L(1)-L(5) and L(7)-S(2) DRG. NT-4 protein was normally present in the cytoplasm of the L(6) DRG neurons and L(3) and L(6) spinal dorsal horn neurons and glia. Adjacent ganglionectomy leads to a significant decrease in NT-4 expression in the L(6) DRG, but no change in the spinal dorsal horn. Following EA treatment a significant increase occurred in the L(6) DRG at 14 days post-operation (dpo) as well as the L(6) cord segment at 7 and 14 dpo. These findings pointed to a possible association between NT-4 expression and EA promoted spinal cord plasticity in adult cats subjected to partial ganglionectomy.     

Neurotrophin-3 attenuates galanin expression in the chronic constriction injury model of neuropathic pain

We have recently shown that exogenous neurotrophin-3 (NT-3) acts antagonistically to nerve growth factor (NGF) in regulation of nociceptor phenotype in intact neurons and suppresses thermal hyperalgesia and expression of molecules complicit in this behavioral response induced by chronic constriction injury (CCI) of the sciatic nerve. The present study examines whether there is a global influence of NT-3 in mitigating alterations in peptide and NGF receptor expression; molecules believed to also contribute to CCI-associated pain. Thus, the influence of NT-3 on phenotypic changes in dorsal root ganglion (DRG) neurons in rats coincident with CCI was examined using in situ hybridization. Seven days following injury, the incidence of expression of the neuropeptides galanin and pituitary adenylate cyclase-activating polypeptide (PACAP) was increased in L5 sensory neurons ipsilateral to the injury from 12% to 60% and 16% to 37% respectively, in addition to an increased level of expression. In contrast, there was no consistent significant change in tropomyosin-related kinase A (trkA) expression following CCI. Intrathecal infusion of NT-3 globally mitigated both the increased incidence and elevated levels of galanin messenger RNA (mRNA) expression observed following CCI, reducing the former from 60% to 39%. NT-3 infusion resulted in a limited reduction in the incidence and level of neuronal PACAP in medium to large size, but not small size, DRG neurons. NT-3 had no significant net effect on CCI-induced alterations in trkA mRNA expression.     

脊髓半横断损伤后腹角神经元神经生长因子、脑源性神经营养因子、神经营养因子-3和神经营养因子-4表达的变化

目的:探讨大鼠脊髓半横断损伤(htSCI)后脑源性神经营养因子(BDNF)、神经生长因子(NGF)、神经营养因子(NT-3、NT-4)在脊髓腹角神经元表达的早期变化。方法:免疫组织化学ABC法分别染4种神经因子并作阳性细胞计数。结果:NGF主要分布于脊髓腹角神经元的胞核,BDNF、NT-4与NT-3主要分布于胞浆。htSCI前后它们在细胞内的分布范围没有变化。BDNF、NGF与NT-3的3 d在损伤尾侧段脊髓双侧腹角阳性神经元数与对照组相比显著减少。BDNF与NGF的14 d的双侧腹角阳性神经元数量均较正常组明显增多,NT-3与NT-4的14 d~21 d的双侧腹角阳性神经元数量均较正常组明显增多,BDNF7~21 d以及NGF14 d的健侧的阳性神经元数量均分别多于相应的伤侧。结论:内源性BDNF、NGF、NT-3、NT-4增加对脊髓损伤修复具有重要作用,BDNF和NGF在健侧表达的增加说明健侧代偿功能的活跃。     

Fibroblast-like cells from rat plantar skin and neurotrophin-transfected 3T3 fibroblasts influence neurite growth from rat sensory neurons in vitro

Our previous finding that skin-derived and muscle-derived molecules can be used to sort regenerating rat sciatic nerve axons evoked questions concerning neuron-target interactions at the level of single cells, which prompted the present study. The results show that dorsal root ganglion (DRG) neurons co-cultured with fibroblast-like skin-derived cells emit many neurites. These have a proximal linear segment and a distal network of beaded branches in direct relation to skin-derived cells. Electron microscopic examination of such co-cultures showed bundles of neurites at some distance from the target cells and single profiles closely apposed to subjacent cells. RNase protection assay revealed that cultivated skin-derived cells express nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). In co-cultures of DRG neurons and 3T3 fibroblasts overexpressing either of the neurotrophins produced by skin-derived cells the picture varied. NT-3 transfected 3T3 fibroblasts gave a growth pattern similar to that seen with skin-derived cells. Neurons co-cultured with mock-transfected 3T3 fibroblasts were small and showed weak neurite growth. In co-cultures with a membrane insert between skin-derived cells or 3T3 fibroblasts and DRG neurons few neurons survived and neurite growth was very sparse. We conclude that skin-derived cells stimulate neurite growth from sensory neurons in vitro, that these cells produce NGF, BDNF, NT-3 and NT-4 and that 3T3 fibroblasts producing NT-3 mimic the effect of skin-derived cells on sensory neurons in co-culture. Finally the results suggest that cell surface molecules are important for neuritogenesis.     

Early postnatal corticosterone administration regulates neurotrophins and their receptors in septum and hippocampus of the rat

The principal glucocorticoid in rats, corticosterone, interacts with neurons in the limbic system and leads to morphological and behavioral changes. Putative corticosterone-triggered mediators are neurotrophins. In the present study we investigated the effects of early postnatal corticosterone treatment in rats on neurotrophic factors of the nerve growth factor (NGF) family and their receptors. Newborn rats were treated with corticosterone-containing polymers until postnatal day 12. The mRNA and protein levels of the neurotrophins of the NGF family (NGF, BDNF, NT-3 and NT-4/5) and their receptors (trkA, trkB, trkC and p75) were quantified in septum and hippocampus using RT-PCR. In the septal region, we found an unchanged mRNA expression after corticosterone treatment, whereas in the hippocampus there was a general increase in mRNA. Particularly, the gene expression of NGF, NT-3, and the high affinity receptors trkA, trkB and trkC increased significantly. Quantification of the neurotrophin protein levels using an ELISA revealed significant treatment effects for NGF and NT-4/5 in the hippocampus. The present study of corticosterone treatment in young rats demonstrates interactions of steroid hormones with neurotrophic factors and their receptors in the septo-hippocampal system during the first two postnatal weeks.     

Neurotrophin-3 is a target-derived neurotrophic factor for penile erection-inducing neurons

The aim of this study was to determine whether the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3 could act as endogenous target-derived trophic factors for erection-inducing, i.e. penis-projecting major pelvic ganglion (MPG) neurons, and/or penile sensory neurons in adult rat. This was accomplished by studying the expression of NT mRNAs in the penis and their cognate receptors in the MPG and dorsal root ganglia (DRGs), and the retrograde axonal transport of radioiodinated NTs injected into the corpora cavernosa. Northern hybridization showed that NGF, BDNF, and NT-3 mRNAs are expressed in the shaft of the penis. In situ hybridization combined with usage of the retrograde tracer Fluoro-Gold showed that TrkC and p75 receptors are expressed in penis-projecting neurons of the MPG whereas the mRNAs for TrkA and TrkB receptors were undetectable. However, all the NT receptor mRNAs were expressed in penile sensory neurons of sacral level 1 (S1) DRG. (125)I-NT-3 injected into the shaft of the penis was retrogradely transported into the MPG and S1 DRG, whereas radioiodinated NGF and BDNF were transported specifically into the S1 DRG, thus confirming the existence of functional NT receptors in these penile neurons. In conclusion, these data suggest that NT-3 may act as a target-derived neurotrophic factor for both erection-inducing and penile sensory neurons, whereas NGF and BDNF may be more important for the sensory innervation of the penis.