Distribution of five peptides,three general neuroendocrine markers,and two synaptic-vesicle-associated proteins in the spinal cord and dorsal root ganglia of the adult and newborn dog: An immunocytochemical study

This study describes the immunocytochemical distribution of five neuropeptides (calcitonin gene-related peptide [CGRP], enkephalin, galanin, somatostatin, and substance P), three neuronal markers (neurofilament triplet proteins, neuron-specific enolase [NSE], and protein gene product 9.5), and two synaptic-vesicle-associated proteins (synapsin I and synaptophysin) in the spinal cord and dorsal root ganglia of adult and newborn dogs. CGRP and substance P were the only peptides detectable at birth in the spinal cord; they were present within a small number of immunoreactive fibers concentrated in laminae I–II. CGRP immunoreactivity was also observed in motoneurons and in dorsal root ganglion cells. In adult animals, all peptides under study were localized to varicose fibers forming rich plexuses within laminae I–III and, to a lesser extent, lamina X and the intermediolateral cell columns. Some dorsal root ganglion neurons were CGRP- and/or substance P-immunoreactive. The other antigens were present in the spinal cord and dorsal root ganglia of both adult and newborn animals, with the exception of NSE, which, at birth, was not detectable in spinal cord neurons. Moreover, synapsin I/synaptophysin immunoreactivity, at birth, was restricted to laminae I–II, while in adult dogs, immunostaining was observed in terminal-like elements throughout the spinal neuropil. These results suggest that in the dog spinal cord and dorsal root ganglia, peptide-containing pathways complete their development during postnatal life, together with the full expression of NSE and synapsin I/synaptophysin immunoreactivities. In adulthood, peptide distribution is similar to that described in other mammals, although a relative absence of immunoreactive cell bodies was observed in the spinal cord.     

Retinoic acid receptor beta2 promotes functional regeneration of sensory axons in the spinal cord

The embryonic CNS readily undergoes regeneration, unlike the adult CNS, which has limited axonal repair after injury. Here we tested the hypothesis that retinoic acid receptor beta2 (RARbeta2), critical in development for neuronal growth, may enable adult neurons to grow in an inhibitory environment. Overexpression of RARbeta2 in adult rat dorsal root ganglion cultures increased intracellular levels of cyclic AMP and stimulated neurite outgrowth. Stable RARbeta2 expression in DRG neurons in vitro and in vivo enabled their axons to regenerate across the inhibitory dorsal root entry zone and project into the gray matter of the spinal cord. The regenerated neurons enhanced second-order neuronal activity in the spinal cord, and RARbeta2-treated rats showed highly significant improvement in sensorimotor tasks. These findings show that RARbeta2 induces axonal regeneration programs within injured neurons and may thus offer new therapeutic opportunities for CNS regeneration.     

维生素D依赖性钙结合蛋白-D28K样阳性神经元在人胎儿和新生儿脊髓和背根神经节内的表达和分布

目的 :观察 2 4～ 2 7w人胎儿和新生儿的脊髓和背根神经节 (DRG)内维生素D依赖性钙结合蛋白 D2 8K(CB)样阳性神经元的表达和分布。方法 :采用免疫细胞化学ABC法对 2 4～ 2 7w人胎儿和新生儿进行观察。结果 :( 1 )CB样阳性产物的表达在胎儿DRG和脊髓灰质的神经元胞体及树突内可观察到。在脊髓各段有不同分布 ;( 2 )新生儿脊髓和DRG内CB样阳性胞体的数量、大小及染色强度均有所增加 ,但在空间分布上无大的变化。结论 :人胎儿的脊髓和DRG在发育的 2 4～ 2 7w至出生时 ,CB样阳性神经元的表达在定位分布上无差异 ,但随年龄增长 ,CB样阳性神经元的数量、大小和染色强度均有所增加。     

神经元Nogo-A的表达与其髓鞘化关系的研究

目的探讨神经元表达Nogo鄄A蛋白的情况,对Nogo鄄A的功能作初步了解。方法采用免疫组织化学方法,观察神经元表达Nogo鄄A的情况,并设置对照组。结果成年大鼠脊髓内神经元和背根神经节的大﹑中﹑小细胞中都出现Nogo鄄A阳性免疫反应,在胞浆和胞核中表达。结论大鼠神经组织中神经元都可能有Nogo鄄A的表达,表明神经元表达Nogo鄄A与该神经元轴突的髓鞘化没有联系,提示神经元中的Nogo鄄A的功能可能不参与髓鞘形成。     

Studies on the changes of c-fos protein in spinal cord and neurotransmitter in dorsal root ganglion of the rat with an experimental peripheral neuropathy

Animal models for human chronic pain syndromes have been developed and widely used for pain research. One of these neuropathic pain models by Kim and Chung (1992) has many advantages for operation and pain elicitation. In this neuropathic model we have examined the c-fos protein, substance P, CGRP immunoreactivity in dorsal root ganglia and dorsal horn. 50 Sprague-Dawley rats were used for this study. L5 and L6 spinal nerves were ligated tightly to produce the neuropathic pain model. After 2, 4, 8, 16, and 24 hours and 1 week of surgery, rats were anesthetized and sacrificed by perfusion. After confirmation of the roots transected by the surgery, the L5 and L6 dorsal root ganglions and spinal cord were removed and processed for immunohistochemistry. All tissue sections were immunohistochemically stained for substance P, CGRP and c-fos using the peroxidase-antiperoxidase (PAP) method. The number of immunostained substance P and CGRP dorsal root ganglion cells and c-fos immunoreactive dorsal horn cells were counted and analyzed statistically with Mann-Whitney U test. The results are as follows. The number of c-fos protein immunoreactive neurons in the superficial layer of dorsal horn were increased markedly 2 hours after operation, and gradually decreased to normal level 1 week after operation. The number of c-fos protein immunoreactive neurons in the deep layer of the dorsal horn gradually increased to a peak 24 hours after operation, then decreased to the normal level 1 week after operation. The number of substance P and CGRP immunoreactive L5 and L6 dorsal root ganglion neurons were decreased markedly 1 week after the pain model operation. In conclusion, after neuropathic pain model operation, c-fos proteins were immediately expressed in the superficial layer of spinal dorsal horn, thereafter c-fos proteins in the deep layer of spinal dorsal horn were expressed. CGRP and substance P immunoreactive neurons in DRG were decreased markedly 1 week after neuropathic pain model operation. These decrements do not coincide with the other chronic pain models, which show great increases in these pain transmitting substances. Therefore, the relationship between pain and c-fos, SP and CGRP should be investigated further.     

Expression patterns of erythropoietin and its receptor in the developing spinal cord and dorsal root ganglia

Recombinant human erythropoietin (EPO) is neuroprotective in animal models of adult spinal cord injury, and reduces apoptosis in adult dorsal root ganglia after spinal nerve crush. The present work demonstrates that spinal cord and dorsal root ganglia share dynamic expression patterns of EPO and its receptor (EPOR) during development. C57Bl mice from embryonic days (E) 8 (E8) to E19 were studied. In spinal cord and dorsal root ganglia, EPOR expression in all precursor cells preceded the expression of EPO in subsets of neurons. On E11, EPO-immunoreactive spinal motoneurons and ganglionic sensory neurons resided adjacent to EPOR-expressing radial glial cells and satellite cells, respectively. From E12 onwards, EPOR-immunoreactivity decreased in radial glial cells and, transiently, in satellite cells. Simultaneously, large-scale apoptosis of motoneurons and sensory neurons started, and subsets of neurons were labelled by antibodies against EPOR. Viable neurons expressed EPO and EPOR. Up to E12.5, apoptotic cells were EPOR-immunopositive, but variably EPO-immunonegative or EPO-immunopositive. Thereafter, EPO-immunonegative and EPOR-immunopositive apoptotic cells predominated. Our findings suggest that EPO-mediated neuron-glial and, later, neuron–neuronal interactions promote the differentiation and/or the survival of subsets of neurons and glial cells in central as well as in peripheral parts of the embryonic nervous system. Correspondingly, expression of phospho-Akt-1/protein-kinase B extensively overlapped expression sites of EPO and EPOR, but was absent from apoptotic cells. Identified other sites of EPO and/or EPOR expression include radial glial cells that transform to astrocytes, cells of the floor plate and notochord as well as neural crest-derived boundary cap cells at motor exit points and cells of the primary sympathetic chain.     

大鼠脊髓半横断损伤对脊神经节P物质表达的影响

目的探讨脊髓半横断损伤对脊神经节P物质(SP)表达的影响。方法选取成年SD大鼠30只,随机分为5组,每组6只;实验组为4组,正常对照组为1组。实验组:脊髓右侧行半横断损伤,术后3、7、14和28d于各时间点分别取出损伤区手术侧脊神经节,石蜡切片后行免疫组织化学染色,结合图像分析技术观察SP在脊神经节内含量的变化。对照组不做任何处理,7d后检测同上。结果术后3d脊神经节内SP的阳性神经元数、阳性细胞百分率较对照组明显增多,7d组较3d组呈下降趋势,14d进一步下降,28d组SP的阳性神经元数、阳性细胞百分率逐渐回升,但仍低于正常对照组。结论脊髓半横断损伤可导致脊神经节SP表达改变,提示SP作为一种感觉神经的传导物质,可能在脊髓半横断损伤过程中发挥作用。     

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     

Distribution of neurons expressing calcitonin gene-related peptide mRNAs in the brain stem, spinal cord and dorsal root ganglia of rat and guinea-pig

In situ hybridization histochemistry was used to localize calcitonin gene-related peptide mRNAs in spinal cord, brain stem and dorsal root ganglion neurons of the rat and guinea-pig. A 32P-labeled 23-base-long (23mer) oligodeoxyribonucleotide (oligomer) complementary to calcitonin gene-related peptide mRNA sequences encoding residues 23-30 of calcitonin gene-related peptide was used primarily as a probe (CGRP I probe). A 32mer complementary to mRNA sequences for residues 10-20 of calcitonin gene-related peptide (CGRP II probe) was also used as a positive control for specificity of the 23mer for calcitonin gene-related peptide mRNA. In both the guinea-pig and rat calcitonin gene-related peptide mRNA was localized specifically to neurons of the dorsal root ganglion, to spinal motoneurons and to motoneurons of the hypoglossal, facial and accessory facial motor nuclei. Differences in the distribution of calcitonin gene-related peptide mRNA between the rat and guinea-pig included a higher proportion of rat dorsal root ganglion neurons containing calcitonin gene-related peptide mRNA and the localization of calcitonin gene-related peptide mRNA to motoneurons of the ambiguus motor nucleus, parabrachial and peripeduncular nucleus of the rat but not the guinea-pig. In the guinea-pig, in contrast, calcitonin gene-related peptide mRNA was localized also to motoneurons of the abducens, trigeminal, trochlear and oculomotor nerves. The neuronal groups in the intact rat found here to contain calcitonin gene-related mRNA have also been shown previously to contain calcitonin gene-related peptide immunoreactivity in colchicine-treated rats. Colchicine-treated rats, however, have been found to contain additional groups of calcitonin gene-related peptide immunoreactive neurons which, in the intact rats used in the present study, showed no detectable hybridization with the calcitonin gene-related peptide probe.     

大鼠脊髓及脑干内膀胱支配中枢的跨突触示踪研究

目的　跨突触示踪正常大鼠脊髓和脑干内膀胱支配相关中枢,为进一步阐明膀胱功能重建术后中枢重塑奠定研究基础。 方法　成年雌性SD大鼠15只,膀胱壁内分三个点共注射GFP-PRV 4.5 μl（1×108 PFU /ml）。注射后不同时间（72、84、96、108、120 h）分别取大脑、脊髓及背根神经节,荧光显微镜下观察标记结果。 结果　病毒注射后72~120 h,阳性神经元主要分布于L6~S1、L1~L2脊神经背根神经节; L6~S1、L1~L2脊髓节段骶副交感核、中间外侧核及后连合核;脑干的巴氏核、中缝巨细胞核、巨细胞网状核、锥旁网状结构、去甲肾上腺素能细胞群A5及A7、蓝斑、中脑导水管周围灰质和红核腹侧区域。 结论　本实验中标记的相关核团与膀胱存在解剖上的神经联系,可能直接或间接参与膀胱的支配。     

Maturation of opioid sensitivity of fetal mouse dorsal root ganglion neuron perikarya in organotypic cultures: regulation by spinal cord

Opioid agonists selectively decrease the duration of the Ca2+ component of the action potential recorded from embryonic dorsal root ganglion neurons in dissociated cell cultures. In contrast, no significant alterations in the action potentials generated by adult dorsal root ganglion neurons in vivo were detected during opioid exposure. In the present study, the perikaryal opioid sensitivity of fetal mouse dorsal root ganglion neurons was analyzed during maturation in organotypic explant cultures. To determine whether spinal cord might influence this sensitivity, neuron perikarya were tested in ganglia grown: (a) in isolation; (b) attached to spinal cord explants; and (c) attached to spinal cord, but decentralized by a dorsal root transection in mature explants 1-2 weeks before the tests. After 2-8 weeks in culture, the duration of the Ba2+-enhanced Ca2+ component of intracellularly recorded action potentials was measured prior to and during bath exposure to the opioid, [D-Ala2, D-Leu5]enkephalin. Sensitive neurons were characterized by a marked, reversible reduction (averaging about 50%) in the duration of the Ca2+ component (which was antagonized by naloxone). The fraction of opioid-sensitive neuron perikarya in dorsal root ganglia grown attached to cord explants was significantly lower (48%) than in ganglia grown isolated (78%) or decentralized in vitro (79%). The mean duration of the Ca2+ component was significantly shorter in ganglion cells which had been grown attached to cord, or subsequently decentralized, compared to cells grown in isolated ganglia (by 24 and 38%, respectively). This difference was even larger in the opioid-insensitive groups. Although opioid-sensitive perikarya in ganglia grown attached to cord had a significantly longer Ba2+-enhanced Ca2+ component than that of insensitive neurons, some of the insensitive perikarya in all 3 types of explant paradigms displayed Ca2+ components which were as prolonged as those of sensitive cells. The results obtained in this study support the hypothesis that the observed decrease in the fraction of opioid-sensitive perikarya during development of fetal mouse dorsal root ganglia is due to regulation by interactions with their central target tissue, the spinal cord. The developmental decrease in the duration of the Ca2+ component of the action potential of these ganglion cells is also enhanced by the presence of the spinal cord. However, regulation of functional opiate receptors and Ca2+ component duration of the ganglion cell perikarya appear to be independent processes.     

Expression of leukemia inhibitory factor receptor mRNA in sensory dorsal root ganglion and spinal motor neurons of the neonatal rat

Previous studies have shown that the application of leukemia inhibitory factor to the proximal nerve stump prevents the degeneration of axotomized sensory neurons in the dorsal root ganglion and motor neurons in the spinal cord of newborn rats. This study investigated the expression of leukemia inhibitory factor receptor mRNA in these neurons using in situ hybridization. Leukemia inhibitory factor receptor mRNA was detected both in sensory neurons within the dorsal root ganglion and motor neurons of the cervical spinal cord. Twenty-four hours after axotomy these neurons continue to express leukemia inhibitory factor receptor mRNA. This pattern of leukemia inhibitory factor receptor expression provides a mechanism by which endogenous and exogenous leukemia inhibitory factor could act on injured sensory and motor neurons.     

3 Beta-hydroxysteroid dehydrogenase expression in rat spinal cord

In adult male rats, 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3beta-HSD) expressing cells were identified in the spinal cord from the cervical to the sacral segments. An in situ hybridization study, using an oligonucleotide common to the four known isoforms of rat 3beta-HSD, revealed its mRNA in gray matter. Measurements of optical densities in autoradiograms showed the following regional distribution: dorsal horn (layers I-III) > central canal (layer X) > or = ventral horn (layers VIII-IX) > ventral funiculus = lateral funiculus. At the cellular level, the number of grains was higher on the large motoneurons than on small neurons of the dorsal horn, but the grain density per cell was similar. Further evidence for the expression of 3beta-HSD in the spinal cord was obtained by western blot analysis, which revealed an immunoreactive protein of approximately 45 kDa in the dorsal and ventral parts of the spinal cord. Castration and adrenalectomy did not influence the expression of 3beta-HSD mRNA and protein. Gas chromatography/mass spectrometry measurements showed higher levels of pregnenolone and progesterone in the spinal cord than in the plasma. After castration and adrenalectomy, their levels remained elevated in the spinal cord, suggesting that these neurosteroids may be synthesized locally. The wide distribution of 3beta-HSD, and the high levels of pregnenolone and progesterone in the spinal cord even after castration and adrenalectomy, strongly suggest a potential endogenous production of progesterone and an important signalling function of this steroid in the spinal cord.     

神经营养因子-3对大鼠脊髓半横断后背根神经节c-Jun表达的影响

目的：研究神经营养因子-3(NT-3)对脊髓半横断后背根神经节c-Jun表达的影响，探索NT-3促进脊髓修复的作用机制。方法：将实验动物分为：对照组，损伤组和NT-3注射组，应用荧光免疫组化法结合激光扫描共聚焦显微镜，观察各组背根神经节c-Jun的表达，并计数细胞核完整的神经元数目。结果：脊髓损伤后，背根神经节的细胞内c-Jun的表达上调；NT-3注射组脊髓损伤侧背根神经节神经元的c-Jun表达明显上调，背根神经节内细胞核呈完整状态的神经元数量明显增多。结论：(1)c-Jun在轴突损伤后表达上调。(2)NT-3对轴突损伤后的神经元有保护作用。(3)NT-3可能通过使c-Jun表达上调而发挥其促神经再生作用。     

外源性bFGF对坐骨神经钳夹损伤后脊髓DRG感觉神经元CGRP变化的影响

目的:观察外源性碱性成纤维细胞生长因子(bFGF)对坐骨神经损伤后大鼠背根神经节(DRG)和脊髓后角内降钙素基因相关肽(CGRP)变化的影响。方法:成年雄性Wistar大鼠随机分成正常组、阳性对照组和bFGF组。阳性对照组动物右侧坐骨神经钳夹损伤,bFGF组动物右侧坐骨神经损伤后给予bFGF,在不同时间点运用免疫荧光技术结合图像分析检测相应背根节和脊髓后角CGRP的变化。结果:bFGF处理组术侧DRG内中小型神经元和脊髓后角内的CGRP表达明显高于阳性对照组,积分光密度值相比(P<0.05);但DRG内大型神经元内CGRP表达没有明显变化。结论:结果提示外源性bFGF能明显促进损伤后同侧DRG中小型神经元和脊髓后角CGRP的合成,对DRG内大型神经元中CGRP的表达没有明显影响。     

Influence of peripheral and central targets on subpopulations of sensory neurons expressing calbindin immunoreactivity in the dorsal root ganglion of the chick embryo

The influence of central and peripheral target tissues on the expression of calbindin D-28k by sensory neurons of the chick dorsal root ganglia was tested under various experimental conditions. Firstly, dorsal root ganglia of chick embryos were transplanted at two stages of development onto the chorioallantoic membrane of a host embryo for a period of 4 or 8 days. In dorsal root ganglia grafted at E12, 20% of the ganglion cell bodies were immunoreactive to calbindin 4 and 8 days later; the percentage of calbindin-immunostained neurons in grafted dorsal root ganglia was similar to that observed in control dorsal root ganglia of the same embryonic age (E16 or 20). In contrast, when grafted dorsal root ganglia were taken from a donor embryo at E8, no calbindin-immunoreactive neuron was found 4 or 8 days later. However, when dorsal root ganglia at E8 were cotransplanted with musculature cells, 14% of the grafted ganglion cell bodies were again immunoreactive to calbindin 4 or 8 days later. Secondly, peripheral targets of sensory neurons were suppressed by excision of one hindlimb. After excision at E6, virtually all the ipsilateral dorsal root ganglia cells were free of calbindin immunoreaction after 6 days of reincubation. In contrast, when the excision was performed at E11, calbindin was expressed in about 9% of the nerve cell bodies. Thirdly, central connections were destroyed by cauterization of the lumbosacral spinal cord at E6 or E11. Six days after deprivation of central connections, the percentage of calbindin-immunoreactive ganglion cells was the same as in control dorsal root ganglia of the same age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maternal prenatal stress in rats influences c-fos expression in the spinal cord of the offspring

Previous studies in humans have reported a link between maternal stress and disturbed infant physiological behavior. The objective of our study was to examine in experimental rats how maternal prenatal stress induced by a forced swim test affects offspring afferent spinal responses mediated by stimulation of vaginocervical receptors. The activation of spinal cord neurons showing c-fos expression was examined following vaginocervical mechanical stimulation in adult rats, which were the offspring of dams exposed to gestational stress from E10 until delivery. Vaginocervical stimulation of both prenatal-stressed and non-prenatal-stressed rats induced an increase in immunoreactive protein in the spinal cord ranging from T12 to S1 segmental levels. However, a significantly higher (40%) increase in the expression of Fos-immunoreactive neurons was observed in vaginocervical stimulated prenatally stressed rats than in non-stimulated prenatally stressed ones. This increase was higher in L5-S1 levels than in T12-L4. When the regional distribution was examined, results showed that up to 80% of activated neurons were located in the dorsal horn in both non-stimulated prenatally stressed and stimulated prenatally stressed groups, with a significantly higher density in the latter. Our results demonstrate that maternal prenatal stress can have consequences on vaginocervical responses conveyed to the spinal cord. The increase in Fos labeled neurons in T12-S1 in prenatally stressed rats induced by vaginocervical stimulation suggests the hypersensitivity of the genital tract associated with activation of spinal circuits spanning multiple segments.     

Human dorsal root ganglion neurons from embryonic donors extend axons into the host rat spinal cord along laminin-rich peripheral surroundings of the dorsal root transitional zone

Following dorsal root crush, the lesioned axons regenerate in the peripheral compartment of the dorsal root, but stop at the boundary between the peripheral and the central nervous system, the dorsal root transitional zone. We have previously shown that fibres from human fetal dorsal root ganglia grafted to adult rat hosts are able to grow into the spinal cord, but were not able to specify the route taken by the ingrowing fibres. In this study we have challenged the dorsal root transitional zone astrocyte boundary with human dorsal root ganglion transplants from 5–8-week-old embryos. By tracing immunolabelled human fibres in serial sections, we found that fibres consistently grow around the dorsal root transitional zone astrocytes in laminin-rich peripheral surroundings, and extend into the host rat spinal cord along blood vessels, either into deep or superficial laminae of the dorsal horn, or into the dorsal funiculus. Human fibres that did not have access to blood vessels grew on the spinal cord surface. These findings indicate, that in spite of a substantial growth capacity by axons from human embryonic dorsal root ganglion cells as well as their tolerance to non-permissive factors in the mature mammalian CNS, these axons are still sensitive to the repellent effects of astrocytes of the mature dorsal root transitional zone. Furthermore, this axonal ingrowth is consistently associated with laminin-expressing structures until the axons reach the host spinal cord.     

Somatostatin 28(1-14) immunoreactivity in primary afferent neurons of the rat spinal cord

Somatostatin 28(1-14) immunoreactivity is present in dorsal root ganglion cells and in the superficial laminae of the spinal cord in the rat. The distribution of somatostatin 28(1-14) immunoreactive varicosities in the dorsal horn corresponds well to the distribution of somatostatin 14 immunoreactive elements. Some dorsal root ganglion cells exhibit both somatostatin 14 and somatostatin 28(1-14) immunoreactivities.