维生素D依赖性钙结合蛋白-D25K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24～27w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-D28K(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24～27w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24～27w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。     

人胎儿和新生儿脊髓和DRG内nNOS阳性神经元的表达和分布

目的：观察24～27w人胎儿和新生儿的脊髓和背根神经节(DRG)内神经元型一氧化氮合酶(nNOS)阳性神经元的表达和分布。方法：ABC免疫细胞化学方法。结果：(1)24～27w人胎儿胸髓和腰1～3节段的中间带外侧核和前角Ⅷ层和Ⅸ层内可观察到nNOS阳性神经元。颈、胸、腰各段DRG内nNOS免疫阳性神经元占DRG细胞总数的84％～88％。(2)新生儿脊髓和DRG内nNOS免疫阳性神经元的分布情况与上述胎儿相似。但DRG内nNOS免疫阳性神经元的体积有所增加，数量明显减少，约占DRG细胞总数的70％～73％。结论：人胎儿脊髓和DRG在发育的24～27w至出生时，nNOS阳性神经元的表达在定位分布上无差异，但随胚胎发育阳性神经元的数量明显减少。     

胰岛素受体在猕猴腰段脊髓及相应背根神经节中的分布

目的:观察胰岛素受体在猕猴脊髓腰段第4~6(L4~L6)节段及相应节段背根神经节(dorsal root ganglion,DRG)中的形态学分布特点。方法:取1只成年猕猴的L4~L6节段脊髓并留取相应节段的DRG置于固定液中。脊髓冷冻水平切片(30μm)平均分为4组,分别行Nissl染色、荧光免疫组织化学染色、免疫组织化学染色和对照组处理,再于光学显微镜下观察;DRG平均分为2组,分别行免疫组织化学染色和对照组处理,再于光学显微镜下观察。结果:胰岛素受体分布于猕猴腰段脊髓L4~L6节段及相应节段的DRG中:(1)在腰段脊髓L4~L6节段,背角可见胰岛素受体阳性神经元胞体及终末,并大量分布于背角浅层(集中在第I、II层),腹角也可观察到散在胰岛素受体阳性神经元;(2)在L4~L6节段对应的DRG中,胰岛素受体阳性产物主要集中分布于中型(20μm直径≤40μm)、小型(直径≤20μm)神经元。结论:本研究为进一步研究灵长类动物脊髓和DRG中胰岛素受体的功能提供形态学依据。     

甘油二酯激酶ζ在发育大鼠脊髓和背根神经节的表达和亚细胞定位

目的:探讨甘油二酯激酶ζ(DGKζ)的表达模式和亚细胞定位及其在大鼠脊髓和背根神经节(DRG)发育中的作用。方法:取不同胎龄和出生时间的Wistar大鼠,用DGKζ-c、DGKζ-n抗体对其脊髓及DRG冰冻切片进行免疫组织化学染色。结果:DGKζ-c和DGKζ-n免疫组化结果显示,在脊髓,E15.5时DGKζ呈较强阳性广泛表达,神经上皮、基板腹侧、基板和翼板交界处的细胞胞浆中等阳性、胞核强阳性; E18.5时表达阴性;出生后P0.5-P3,DGKζ再次广泛表达在灰质和白质,灰质内细胞多为胞核表达强于胞浆; P5-P8,DGKζ的表达再次明显降低,在前角和后角细胞的胞核呈弱阳性; P14时,DGKζ在后角细胞胞核的表达增强。在DRG,E15.5时DGKζ在神经元胞浆和胞核表达,多数细胞胞核表达强于胞浆; E18.5时,仅少量神经元胞浆呈阳性表达;出生后P0.5-P3,DGKζ的表达多样,阴性细胞、胞核弱阳性细胞、胞浆或胞核阳性细胞、胞核和胞浆强阳性细胞共存; P5时,DGKζ主要表达在中型神经元胞浆、或胞浆与胞核; P8-P14,DGKζ主要表达在小型神经元胞浆、或胞浆与胞核。结论:DGKζ在大鼠发育中脊髓和DRG的表达不完全同步,核内和胞浆内定位的DGKζ可能在DRG内神经元分化和脊髓内细胞增殖时发挥作用,核内定位的DGKζ可能参与脊髓神经元分化。     

前列腺酸性磷酸酶在慢性痛大鼠脊髓背角和背根神经节的表达变化

目的:观察前列腺酸性磷酸酶(prostatic acid phosphatase,PAP)在多种慢性痛大鼠脊髓背角(spinaldorsal horn,SDH)和背根神经节(dorsal root ganglion,DRG)内的表达变化。方法:应用免疫组织化学染色法以及免疫荧光多重染色技术在多种慢性痛模型大鼠观察PAP的表达变化。结果:在正常大鼠,PAP阳性反应产物主要位于DRG的中、小型的非肽能神经元,PAP阳性神经元约占DRG神经元总数的64±4.3%;在脊髓背角,PAP阳性纤维和终末主要位于Ⅱ层。在神经病理性痛模型大鼠,术侧脊髓背角Ⅱ层的PAP阳性初级传入终末较对侧减少甚至消失,DRG内PAP阳性神经元较对侧明显减少。在慢性炎性痛模型大鼠,双侧脊髓背角和DRG内PAP的表达未见明显改变。结论:PAP特异地定位于DRG神经元以及脊髓背角Ⅱ层,可能与神经病理性痛信号的传递和加工密切相关。     

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。     

不同损伤模型内源性BDNF在背根节和损伤脊髓中的表达及其作用

目的:探讨不同损伤模犁中脑源性神经营养因子(Brain-derived neurotrophin,BDNF)在背根节(dorsal rootganglion,DRG)和损伤脊髓的表达及作用.方法:建立单纯坐骨神经损伤模型、单纯脊髓背索损伤模型以及合并损伤(坐骨神经损伤1周后冉损伤脊髓背索)模型,用免疫荧光组织化学方法和酶联免疫吸附实验(ELISA)方法观察不同模型中BDNF在DRG和损伤脊髓的表达,从脊髓损伤处嘴侧5mm处注入快蓝(Fast Blue,FB)以逆行标记DRG中感觉神经元,并结合FB逆行示踪观察FB阳性神经元BDNF的表达情况.结果:坐骨神经损伤1周后损伤侧的腰4、5背根节中BDNF蛋白表达上调.与单纯脊髓损伤组相比,合并损伤组在脊髓损伤的尾侧端可见较多的BDNF阳性纤维,这些纤维呈膨体样结构,主要分布于坐骨神经损伤侧脊髓尾侧端的白质里,合并损伤组FB标记的感觉神经元数目也较单纯脊髓损伤组多,且大部分FB标记的感觉神经元是BDNF阳性的神经元.结论:坐骨神经损伤后内源性的BDNF在DRG和损伤脊髓中表达上调,可能参与坐骨神经损伤引起的脊髓损伤后再生过程.     

大鼠脊髓钙调蛋白Parvalbumin和Calbindin-28KD阳性神经元的形态与分布特征

目的 观察证实钙调蛋白Parvalbumin( PV)和Calbindin-28KD (CB)阳性神经元在大鼠脊髓不同节段不同区域内的形态及分布特征.方法 成年雄性SD大鼠常规灌注固定,取第5～8颈髓、3～6胸髓和1～5腰髓节段;振动切片之后进行免疫组化PAP单标记;光镜观察阳性神经元的形态和分布特征、并进行计数和测量;用Excel软件对阳性神经元数量、胞体大小、突起数量进行统计处理.结果 PV阳性神经元主要存在于后角的Ⅱ层和胸核、中间带外侧、中间内侧核、前角的Ⅷ层;CB阳性神经元主要存在于后角Ⅰ～Ⅱ层、中间带,前角的Ⅷ～Ⅸ层之间.后角细胞较中间带及前角细胞数量多,胞体小,突起少.在不同区域,两种阳性神经元的细胞数量,胞体大小,突起数目都存在统计学差异(P＜0.05);而在不同节段同一区域的比较中,只有腰前角PV阳性神经元的胞体比较大(P＜0.05).结论 PV阳性神经元在胸核及中间内侧核的聚集以及阳性纤维在薄楔束和后角Ⅱ层胶状质内的密集分布提示其与机体痛觉传入、本体感觉及内脏感觉的联系;CB阳性神经元在前角Ⅷ～Ⅸ层之间的特征性分布说明其与闰绍细胞功能有关;两种钙调蛋白在脊髓不同节段之间的形态及分布基本无差别提示它们的功能并没有特异地对应于躯体或内脏.     

单侧坐骨神经完全结扎对大鼠腰段背根神经节内VGluT1阳性神经元的影响

本实验将大鼠分为两组(正常对照组和单侧坐骨神经完全结扎组),采用免疫细胞化学和中性红复染的方法分别观察了正常大鼠和单侧坐骨神经完全结扎后存活不同时间组大鼠腰4(L4)节段的背根神经节(DRG)内I型囊泡膜谷氨酸转运体(VGluT1)样阳性神经元的分布及其数量的变化。结果如下:(1)正常大鼠L4节段的DRG内可观察到VGluT1样阳性产物呈点状或斑状分布于胞浆内,有大约71.5%的DRG细胞表达VGluT1样免疫阳性,其中以大型(>40μm)和中等大小(20~40μm)的神经元为主(分别占整个VGluT1样阳性细胞总数的30.7%和65.9%);(2)坐骨神经结扎后第1、2d,在结扎同侧L4节段的DRG内未检测到VGluT1样阳性神经元数量的明显变化;但自术后第4d开始,VGluT1样阳性神经元的数量随术后存活时间的延长逐渐减少。结扎1~4周大鼠DRG内VGluT1样阳性神经元数量在同一个动物的手术侧与对照侧相比有明显减少(P<0.01);而结扎1~4周大鼠的手术侧DRG内VGluT1样阳性神经元的数量也明显低于结扎1~3d大鼠的手术侧(P<0.05或P<0.01)。以上结果表明,DRG内合成VGluT1样阳性物质的神经元主要是大、中型细胞,DRG细胞可通过轴浆流将VGLluT1向周围突运输,故外周神经的损伤很易影响到DRG神经元内VGluT1的合成。     

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。     

Downregulation of TrkA expression in primary sensory neurons after unilateral lumbar spinal nerve transection and some rescuing effects of nerve growth factor infusion

Peripheral nerve injury results in sprouting of sympathetic and sensory nerve terminals around large diameter neurons in the dorsal root ganglia (DRG), but the underlying mechanism is not clear. Current study sought to examine changes of the nerve growth factor (NGF) receptor TrkA in DRG and spinal cord after a spinal nerve transection by an immunohistochemical technique and to investigate effects of NGF on the expression of TrkA protein in the same animal model. In the control rat, TrkA immunoreactivity was localized to about 55 +/ -1% of total neurons in DRG and to laminae I and II of the spinal cord. The percentage of TrkA immunoreactive neurons in DRG and TrkA staining intensity of spinal cord were reduced 1 week after the nerve lesion. The changes became maximal 2 weeks, but recovered partially 4 weeks after the lesion. The size of TrkA immunoreactive neurons dramatically shifted to smaller sizes, becoming more remarkable 4 weeks after the lesion. In the contralateral DRG, the percentage of TrkA immunoreactive neurons also decreased significantly. Exogenous NGF delivered to DRG for 2 weeks partially reversed the reduction of TrkA expression as well as atrophy of TrkA immunoreactive neurons. No TrkA immunoreactive basket was found around neuronal somata. Our data show that unilateral peripheral nerve injury results in dynamic downregulation of TrkA in sensory neurons in bilateral DRG and spinal cord, and that TrkA expression in sensory neurons is partially regulated by target-derived NGF.     

Localization of vasoactive intestinal peptide immunoreactivity in human foetus and newborn infant spinal cord

Using an indirect immunofluorescence method the distribution of vasoactive intestinal peptide (VIP) immunoreactivity was studied in human foetus and newborn infant spinal cord and dorsal root ganglia. Further, for comparison some newborn infant brains were also investigated. Vasoactive intestinal peptide-like immunoreactive fibres were exclusively found in the caudal spinal cord and corresponding dorsal root ganglia. No immunoreactive cell bodies were detected. The first appearance of VIP-like immunoreactive fibres in both spinal cord and dorsal root ganglia was suggested during the fourth month of foetal life. Most immunolabelled fibres, concentrated in the sacral segment, were distributed in the Lissauer tract, along the dorsolateral gray border, in the intermediolateral areas and near the central canal in the dorsolateral commissure. A few VIP-like immunoreactive fibres were also seen in the dorsal funiculus and occasionally in the ventral gray horn and ventral roots. Further, a large population of VIP-like immunoreactive fibres occurs longitudinally in dorsal root, in ganglia and in the spinal nerve exit zone. These findings indicate the early appearance of VIP-like immunoreactive fibres in the human foetus spinal cord and corresponding ganglia. Moreover, they emphasize that in both foetus and newborn infant spinal cord VIP-like immunoreactive fibre distribution is limited to the lumbosacral segment.     

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.     

吗啡依赖大鼠背根节神经元胞体的光镜和电镜定量研究

在吗啡依赖条件下 ,观察了脊髓 板层发生轴突终末侧支生芽和建立新突触的背根节神经元的胞体是否出现相应的形态变化。应用光镜体视学方法 ,结合电镜观察和测量 ,比较吗啡依赖组与对照组背根节体积及其神经元胞体、胞核、核仁、粗面内质网和线粒体的体积变化。结果显示 :吗啡依赖组背根节体积与对照组比较未发现差异 ,背根节内的亮、暗两类神经元的胞体、胞核、核仁体积以及胞质内粗面内质网和线粒体体积也无显著性差异。结果表明 ,吗啡对大鼠背根节神经元胞体的形态结构没有明显的影响     

Expression of peripherin in solid transplants of foetal spinal cord and dorsal root ganglia grafted to the injured cervical spinal cord of adult rats

The expression of the neuronal type III intermediate filament protein peripherin was studied in E14 spinal cord fragments and E15 dorsal root ganglia 1–30 weeks after their transplantation to the injured cervical spinal cord of the adult rat. In the dorsal root ganglion transplants, the surviving neurons generally appeared as a rather healthy looking population of small strongly immunoreactive cells which are very similar to the small dorsal root ganglion neurons of adult control rats. In the spinal cord transplants, there were only a few peripherin-immunoreactive neurons, morphologically close to the motoneurons or to the preganglionic sympathetic neurons of adult rats. In both types of transplants, peripherin expression of the immunoreactive neurons was apparently correlated with the previously established ability of these transplanted neurons for extensive axonal growth into a co-grafted peripheral nerve.     

Distribution of substance P-like immunoreactivity in the spinal cord and dorsal root ganglia of the human foetus and infant

Using the indirect immunofluorescence method, the distribution of substance P-like-immunoreactivity was studied in spinal cord and dorsal root ganglia of 25 human foetuses ranging from 12 to 29 weeks of gestational age. The spinal cord and dorsal root ganglia of three infants (1 day-, 2 and 4 month-old) were also investigated as a post-natal reference. On the whole, the substance P distribution patterns seen in infants were already visible throughout most of foetal life. The highest density of substance P-like-immunoreactive fibres was localized over the superficial layers of the dorsal grey horn. Punctiform immunofluorescence was often found over the white matter especially in the funiculi dorsalis et lateralis. In the ventral horn, substance P immunoreactive fibres were few and far between in the grey matter and were only detected from foetal stage 16 weeks. In addition, longitudino-frontal sections through the dorsal regions revealed repetitive arrangements of substance P-like-immunoreactive fibres along the whole spinal cord. In dorsal root ganglia only a few immunoreactive cells were observed. These findings demonstrate the wide and early occurrence of substance P-like-immunoreactivity in the human foetus spinal cord and dorsal root ganglia. They suggest that the development of the substance P neuronal system begins early in ontogenesis and is regionally differentiated.     

Hyperalgesia and neural excitability following injuries to central and peripheral branches of axons and somata of dorsal root ganglion neurons

We examined thermal hyperalgesia, excitability of dorsal root ganglion (DRG) neurons, and antinociceptive effects of N-methyl-d-aspartate (NMDA) receptor antagonists in rats with injury to different regions of DRG neurons. The central or peripheral branches of axons of DRG neurons were injured by partial dorsal rhizotomy (PDR) and chronic constriction injury of sciatic nerve (CCI), respectively, or the somata injured by chronic compression of DRG (CCD). Thermal hyperalgesia was evidenced by significantly shortened latencies of foot withdrawal to radiant heat stimulation of the plantar surface. Intracellular recordings were obtained in vitro from L(4) and/or L(5) ganglia. There are four principle findings: 1) PDR as well as CCD and CCI induced thermal hyperalgesia; 2) PDR produced significantly less severe and shorter duration hyperalgesia than CCD and CCI; 3) intrathecal administration of NMDA receptor antagonists d-2-amino-5-phosphonovaleric acid (APV) and dizocilpine maleate (MK-801) inhibited thermal hyperalgesia in PDR, CCD, and CCI rats. Pretreatment of APV and MK-801 delayed the emergence of hyperalgesia for 48-72 h, while posttreatment inhibited hyperalgesia for 24-36 h; and 4) CCD and CCI increased excitability of DRG neurons as judged by the significantly lowered threshold currents and action potential voltage thresholds and increased incidence of repetitive discharges. However, PDR did not alter the excitability of DRG neurons. These findings indicate that injury to the dorsal root, compared with injury to the peripheral nerve or DRG somata has different effects on the development of hyperalgesia. These contributions involve different changes in DRG membrane excitability, but each involves pathways (presumably in the spinal cord) that depend on NMDA receptors.     

Immunohistochemical Localization of Substance P And Cholecystokinin in the Dorsal Root Ganglia and Spinal Cord of the Bottlenose Dolphin (Tursiops truncatus)

The presence of substance P (SP) and cholecystokinin (CCK) immunoreactive neurons was examined in the bottlenose dolphin dorsal root ganglia (DRGs) and spinal cord by immunohistochemical techniques. SP‐positive and CCK‐immunoreactive neurons were respectively ～50% and 1% of the total number of ganglion cells examined and especially belonged to small and medium‐sized cell populations. Using double labeling techniques we observed that SP‐ and CCK‐immunoreactivity coexisted in a very low number of primary afferent neurons (2.7%). Few SP‐immunoreactive (IR) neurons (2.7%) were also CCK‐positive. On the contrary, 65% of CCK‐immunoreactive neurons contained SP. Interestingly, we observed CCK‐immunoreactive satellite glial cells located around large cell class somata. Virtually no SP‐IR and CCK‐positive neurons were surrounded by peripheral CCK‐immunoreactive satellite glial cells. The SP‐IR and CCK‐positive nerve fibers were particularly conspicuous in the superficial layers of the spinal cord. The present study indicates that SP and CCK only partially overlap in the thoracic, lumbar, and caudal DRGs of the bottlenose dolphin, suggesting that the majority of SP‐IR ganglion neurons are lacking in CCK‐immunoreactivity. The role of SP‐containing DRG neurons is discussed also in relation to the huge vascular spinal retia mirabilia typical of cetaceans. Anat Rec, 293:477–484, 2010. © 2010 Wiley‐Liss, Inc.