Crucial roles of NGF in dorsal horn plasticity in partially deafferentated cats

Though exogenous nerve growth factor (NGF) has been implicated in spinal cord plasticity, whether endogenous NGF plays a crucial role has not been established in vivo. This study investigated first the role of endogenous NGF in spinal dorsal horn (DH) plasticity following removal of L1-L5 and L7-S2 dorsal root ganglions (DRGs) in cats. Co-culture of chick embryo DRG with DH condition media, protein band fishing by cells as well as western blot showed that NGF could promote neurite growth in vitro. Immunohistochemistry and in situ hybridization technique revealed an increase in the NGF and NGF mRNA immunoreactive cells in the DH after partial deafferentation. Lastly, after blocking with NGF antibody, choleragen subunit B horseradish peroxidase (CB-HRP) tracing showed a reduction in the neuronal sprouting observed in the DH. Our results demonstrated that in the cat, endogenous NGF plays a crucial role in DH plasticity after partial deafferentation.     

部分去背根传入猫脊髓Ⅱ板层神经生长因子的免疫组化研究

为探讨神经生长因子（NGF）在部分去背根传入猫脊髓Ⅱ板层可塑性中的作用，本文采用成年雄猫15只，建立单侧备用根模型（即切断一侧L1～5、L7～S2节段脊髓背根，保留L6背根），术后分别存活3、5和11d，灌流固定，取L5、L6、L7脊髓冻切．用2.5sNGF抗体进行ABC法免疫组化反应．每例在7张切片上测两侧Ⅱ板层内、中、外三个固定大小对应区域的灰度值。结果：三个时间组手术例L6平面Ⅱ板层NGF的平均灰度均显著地小于非手术例（P＜0．05）；而手术侧L5、L7平面Ⅱ板层NGF的平均灰度3d时均大于非手术侧（P＜0．05），5d时无显著差异（P＞0．05），11d时小于非手术侧（P＜0．05），即手术侧L5、L7Ⅱ板层灰度值随时间推移而显著减小。结果表明，手术侧L6Ⅱ板层NGF含量在三个时间组均有增加；而L5、L7Ⅱ板层NGF含量在3d时下降，5d恢复，11d增多．这种内源性NGF含量的变化可能与部分去背根脊髓Ⅱ板层的可塑性变化有关．     

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.     

Changes in trkA expression in the dorsal root ganglion after peripheral nerve injury

Most of the biological effects of nerve growth factor (NGF) are mediated by TrkA, the high affinity receptor for NGF. Previous studies have shown that NGF levels in the dorsal root ganglia (DRG) fluctuate following a peripheral nerve injury. The present study examined changes of TrkA immunoreactivity and trkA mRNA expression in the DRG after segmental nerve ligation. In the normal L5 DRG of the rat, there were, on average, 4700 TrkA-immunoreactive (TrkA-IR) neurons, representing 42% of the total neuronal population. Following L5 spinal nerve ligation, the number of TrkA-IR neurons in the L5 DRG slowly declined, reducing by 25% at 1 week and 35% at 3 weeks postoperation (PO). In contrast, trkA mRNA in these ganglia showed a significant decrease from 3 days to 3 weeks PO and was followed by a full recovery at 2 months PO. The early decrease of trkA mRNA is likely due to deprivation of target-derived NGF, which is caused by nerve ligation, and the recovery might be because substitute sources of NGF become available. Despite the decline in trkA mRNA in the ganglion, 3000 injured DRG neurons sustain TrkA immunoreactivity, suggesting that exogenous NGF can still influence these TrkA expressing neurons, even though they are isolated from the periphery. Accordingly, the effects of endogenous NGF should be as well manifested by local administration of NGF to the ganglion as to the stump of the damaged nerve. Received: 20 October 1998 / Accepted: 10 February 1999     

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     

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。     

Expression of nerve growth factor in spinal dorsal horn following crushed spinal cord injury

Expression of nerve growth factor in spinal dorsal horn following crushed spinal cord injury     

Temporal Profile of Nerve Growth Factor Expression in the Partial Central Nervous System of the Yangtze Alligator Alligator sinensis (Reptilia,Crocodylia) During Early Postnatal Growth

Expression of nerve growth factor (NGF) in structures of the partial central nervous system of the Yangtze alligator, Alligator sinensis (Reptilia, Crocodylia) was examined during early postnatal growth using immunohistochemistry and Western blot assays. In animals 0–2 years of age NGF‐positive cells in the cerebral cortex increased gradually in number and size, and were predominantly distributed in the molecular layer. NGF‐positive cells in the midbrain showed similar increases but with predominant distribution in the ependymal layer. NGF‐positive cells increased in the cerebellum between 0 and 1 years of age, with increased NGF expression being seen during the first 2 years of life mostly in the ependymal layer. NGF‐positive cells were mainly found in the gray matter of the spinal cord with decreasing cell numbers, NGF expression levels being seen from 0 to 2 years and small processes without synaptic connection from 1 to 2 years. These results suggest that NGF is involved in the early postnatal growth of several structures of Yangtze alligator partial central nervous system, suggesting a possible role of NGF in the Yangtze alligator partial central nervous system. Anat Rec, 296:840–845, 2013. © 2013 Wiley Periodicals, Inc.     

Changes in PDGF expression in spared dorsal root ganglia and associated spinal dorsal horns in cats subjected to partial dorsal root ganglionectomy

Changes in the platelet derived growth factor (PDGF) in the spared dorsal root ganglia (DRG) and associated spinal dorsal horns were evaluated in cats subjected to unilateral removal of L1-L5 and L7-S2 DRG, sparing the L6 DRG. The number of PDGF immunopositive neurons and protein expression decreased significantly in the spared DRG and associated dorsal horns of the L3 and L6 cord segments at 3 days post-operation (dpo). It bottomed to the lowest level at 7 dpo in the DRG, then returned to the control level at 14 dpo; while in the L6 dorsal horn, it rapidly increased at 7 dpo and exceeded the control level at 14 dpo. This showed a significant upregulation in the spared DRG and associated spinal dorsal horns, especially in the L6 cord segment following a transient decrease. Meanwhile, a significant upregulation of PDGF mRNA was also seen in L6 DRG and L3 and L6 dorsal horns at 3 dpo. The upregulation of the endogenous PDGF in the said structures indicated a potential role of this factor in spinal cord plasticity after partial dorsal root ganglia removal in cats.     

Changes in nerve growth factor levels in dorsal root ganglia and spinal nerves in a rat neuropathic pain model

The purpose of this study was to measure the changes in levels of nerve growth factor (NGF) in dorsal root ganglia (DRG) and spinal nerves with the aim of investigating the role of NGF in a rat neuropathic pain model. Nerve injuries were made by tight ligation of the left L5 and L6 spinal nerves using 6-0 silk thread in male Sprague-Dawley rats. Before surgery and 1, 3, 5, 7, and 14 days after surgery, tissue samples collected included the L3-6 DRGs bilaterally, segments of the ipsilateral L5-6 spinal nerves proximal and distal to ligation sites, and corresponding sites of the contralateral L3-6 and the ipsilateral L3-4 spinal nerves. NGF levels in the DRGs of the injured spinal nerves (the left L5 and L6) did not change significantly from control values. The spinal nerve segments distal to ligation sites had higher levels of NGF than the control values. Unlesioned sites did not show any significant changes in NGF levels. The increase of NGF in distal segments of injured spinal nerves may be due to an accumulation of retrogradely transported NGF. The maintenance of NGF levels in the DRGs that had lost peripheral connections may reflect local synthesis after nerve injury.     

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。     

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     

Nerve growth factor treatment enhances release of immunoreactive calcitonin gene-related peptide but not substance P from spinal dorsal horn slices in rats

The effect of systemic nerve growth factor (NGF) on neuropeptide content and capsaicin-evoked release of neuropeptide from in vitro spinal cord dorsal horn slices was examined. Rats were injected subcutaneously every other day with murine NGF (mNGF) 1 mg/kg or saline for 7 days, or mNGF 0.1/kg, mNGF 1 mg/kg or saline for 13 days. Lumbar dorsal horn slices of the rat spinal cord from all groups showed a significant increase in immunoreactive calcitonin gene-related peptide (CGRP) release upon exposure to capsaicin. This release was enhanced in rats pretreated with mNGF 1 mg/kg for 7 days, but not after 13 days. No enhancement was seen after 7 or 13 days in any treatment group for immunoreactive substance P release. Upon examination of neuropeptide content in dorsal horn, no significant differences were noted between treatment groups. The increased iCGRP release from dorsal horn slices suggests a preferential release of CGRP and provides further evidence that NGF indirectly plays a role in the modulation of inflammation through the regulation of neuropeptide release.     

Spinal cord long-term potentiation is attenuated by the NMDA-2B receptor antagonist Ro 25-6981

Aim: The NR2B‐containing N‐methyl‐d ‐aspartate (NMDA) receptors may be involved in a variety of phenomena including synaptic plasticity, memory formation and pain perception. Here we used the NMDA‐2B receptor antagonist Ro 25‐6981 to investigate the role of the NR2B‐containing NMDA receptors in spinal nociception. Methods: Extracellular single unit recordings were performed from dorsal horn wide dynamic range (WDR) neurones in intact urethane‐anaesthetized Sprague–Dawley rats. The responses of the WDR neurones evoked by C‐fibre activation after sciatic nerve stimulation were defined according to latencies. To block the dorsal horn NMDA‐2B receptors, the antagonist Ro 25‐6981 was applied topically onto the spinal cord. High‐frequency stimulation (HFS) of the sciatic nerve was used to induce spinal long‐term potentiation (LTP). Results: Spinal administration of the NMDA‐2B receptor antagonist Ro 25‐6981 had a clear antinociceptive effect at the spinal level (P < 0.05, C‐fibre evoked responses after 4 mm Ro 25‐6981 vs. C‐fibre evoked responses in baseline). Moreover, spinal administration of this antagonist clearly attenuated the magnitude of spinal cord LTP after HFS conditioning (P < 0.05, C‐fibre evoked responses after HFS vs. C‐fibre evoked responses after 8 mm Ro 25‐6981 + HFS). Conclusion: The present study indicates that expression of full LTP in dorsal horn neurones obtained by HFS conditioning may be dependent on the NMDA receptors containing the NR2B subunit. This suggests that activation of dorsal horn NR2B‐containing NMDA receptors may be involved in use‐dependent sensitization at the spinal level.     

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.     

Localization of NADPH-diaphorase containing neurons in the spinal dorsal horn and spinal sensory ganglia of the turtle Chrysemys d’orbigny

 NADPH-diaphorase positive (NDP) neurons and nerve fibers were found in the spinal dorsal horn (DH) and sensory ganglia of the turtle Chrysemys d’orbigny. Three well-defined types of NDP neurons were found in the DH: (a) elongated nerve cells with two radially arranged dendritic branches, (b) neurons with rostro-caudal dendritic branches, (c) bitufted neurons with two, practically symmetric branches that project to the ipsilateral and contralateral dorsal horns. A combination of the techniques that reveal NADPH-diaphorase activity with the horseradish peroxidase transganglionic labeling of the dorsal root collaterals, suggested that NDP neurons of the DH are second-order cells of the spinal sensory pathway. NDP neurons were also found in the spinal sensory ganglia at all metameric levels. Our findings indicate that the DH of turtles, like that of mammals, contains both the enzymatic machinery and the neural connections required to postulate the participation of nitric oxide in ”plastic phenomena” such as hyperalgesia and central sensitization. Two other alternatives or complementary hypotheses are discussed: (a) NDP neurons in the DH and sensory ganglia may represent specific cell populations involved in the processing of sensory visceral information; (b) NADPH-diaphorase reactivity may indicate sustained levels of neuronal activity. Received: 12 February 1996 / Accepted: 2 August 1996     

备用背根猫脊髓Ⅱ板层组织对鸡胚背根节神经突起生长的影响

用１０只存活５ｄ的单侧后肢备用根猫（切除Ｌ１～Ｌ５、Ｌ７～Ｓ２背根节，保留Ｌ６背根），取手术侧（实验组）和非手术侧（对照组）脊髓Ⅱ板层组织块及提取液分别与Ｈａｍｂｕｒｇｅｒ３５期鸡胚背根节进行悬滴培养，并以不加植块的背根节培养作参照。比较各组在同一观测时间的差异．结果：（１）各组背根节从培养２４ｈ到４８ｈ，其神经突起均明显增长；（２）同一观测时间内，对照组与参照组的神经突起少而短，从背根节迁出的细胞较多，而实验组神经突起多且长，迁出的细胞较少；（３）在两个观测时间，植块与提取液培养的实验组背根节神经突起平均长度均显著长于对照组者，而对照组与参照组背根节神经突起长度的差异无显著性．表明部分腰骶背根切除猫，其脊髓Ⅱ板层及其提取液的促神经突起生长活性增强．     

Conditioning lesions enhance growth state only in sensory neurons lacking calcitonin gene-related peptide and isolectin B4-binding

A conditioning lesion improves regeneration of central and peripheral axons of dorsal root ganglion (DRG) neurons after a subsequent injury by enhancing intrinsic growth capacity. This enhanced growth state is also observed in cultured DRG neurons, which support a more sparsely and rapidly elongating mode of growth after a prior conditioning lesion in vivo. Here we examined differences in the capacity or requirements of specific types of sensory neurons for regenerative growth, which has important consequences for development of strategies to improve recovery after injury. We showed that after partial or complete injury of the sciatic nerve in mice, an elongating mode of growth in vitro was activated only in DRG neurons that did not express calcitonin gene-related peptide (CGRP) or bind Bandeiraea simplicifolia I-isolectin B4 (IB4). We also directly examined the response of conditioned sensory neurons to nerve growth factor (NGF), which does not enhance growth in injured peripheral nerves in vivo. We showed that after partial injury, NGF stimulated a highly branched and linearly restricted rather than elongating mode of growth. After complete injury, the function of NGF was impaired, which immunohistochemical studies of DRG indicated was at least partly due to downregulation of the NGF receptor, tropomyosin-related kinase A (TrkA). These results suggest that, regardless of the type of conditioning lesion, each type of DRG neuron has a distinct intrinsic capacity or requirement for the activation of rapidly elongating growth, which does not appear to be influenced by NGF.