Origin of the calcitonin gene-related peptide-immunoreactive nerve fibers in the rat shoulder joint

The rat shoulder joint capsule is innervated by thin sympathetic and sensory nerve fibers, most of which contain calcitonin gene-related peptide (CGRP). In order to establish the origin and distribution of CGRP-immunoreactive (IR) fibers, wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the shoulder joints of rats via a dorsal surgical approach. After WGA-HRP injection, the cervico-thoracic dorsal root ganglia (DRG) were removed and processed using both HRP histochemistry and CGRP immunohistochemistry. In the C4 to C7 DRG, small to medium-sized neurons (20–40 m) were labeled by this combined method. The number and size of the labeled neurons were measured in the cervical 4th–7th DRG. The number of double-labeled neurons was one quarter of the total number of HRP-labeled neurons and 1/20 of the CGRP-IR neurons. Most of the double-labeled cells were located in the C6 ganglion, and the mean number of double-labeled neurons was 13 at this level. The distribution and function of the CGRP-IR fibers in the rat shoulder joint capsule are discussed.     

小鼠脊神经节内P2X2、P2X3受体与降钙素基因相关肽和植物凝集素结合位点的共存

目的:研究小鼠脊神经节内ATP受体P2X2、P2X3与降钙素基因相关肽(CGRP)、植物凝集素IB4(IB4)结合位点的共存。方法:在成年c57/6小鼠脊神经节,用免疫组织化学荧光三标方法结合激光共聚焦显微镜技术观察。结果:脊神经节内可见大量P2X2、P2X3阳性细胞和纤维;P2X2阳性细胞多为大型和小型神经元,P2X3阳性细胞多为小型神经元;有34.2%±3.9%P2X2阳性细胞含有CGRP;但只有4.6%±1.1%P2X3阳性神经元含有CGRP。许多感觉神经元结合IB4;有62.4%±4.3%P2X2和89.5%±4.1%P2X3阳性神经元分别结合IB4。未观察到P2X2/CGRP/IB4或P2X3/CGRP/IB4三标神经元。结论:小鼠脊神经节内P2X2、P2X3的表达与IB4结合位点之间密切相关,部分P2X2受体与CGRP共存。     

大鼠坐骨神经压榨损伤后早期降钙素基因相关肽的变化

目的：研究大鼠坐骨神经压榨损伤后早期降钙素基因相关肽(CGRP)的动态变化及与神经再生的关系。方法：SD大鼠坐骨神经压榨损伤后分别存活1d到21d,免疫组化技术观察CGRP分布和含量的变化。结果：(1)1d组神经CGRP大量堆积,压榨近端明显多于远端,随即下降,21d组基本消失。(2)1d组背根节、脊髓后角和前角CGRP开始增高,并分别在3～5d、5～7d和7d组达峰值,随后渐降,21d组脊髓前角CGRP阳性运动神经元仍明显高于假手术组和对照侧。结论：神经压榨损伤后CGRP表达变化呈明显的时空模式,可能参与了神经元保护并介导了损伤信号的传导。     

二脱氧肌苷引发培养的背根神经节神经元的形态学改变

为探讨二脱氧肌苷(ddI)对培养的背根神经节(DRG)神经元的影响,我们对分散培养的胎鼠DRG神经元培养3d后,再分别以不同浓度的ddI(1μg/ml,5μg/ml,10μg/mll和20μg/ml)孵育3d。终止培养后,行微管相关蛋白2(MAP2)标记,用共聚焦激光扫描显微镜观察神经元胞体和突起的改变。结果表明,DRG神经元用ddI孵育3d,神经元突起的数目减少和长度变短,呈剂量依赖性,而神经元的直径则没有变化。本研究的结果表明,ddI可影响培养的DRG神经元突起的再生和生长。     

Changes in calcitonin gene-related peptide expression following joint immobilization in rats

Long-term immobilization by casting can occasionally cause pathologic pain states in the immobilized side. The underlying neurophysiological mechanisms of immobilization-related pain are not well understood. For this reason, we specifically examined changes of calcitonin gene-related peptide (CGRP) expression in the dorsal root ganglion (DRG), spinal dorsal horn and posterior nuclei (cuneate nuclei) in a long-term immobilization model following casting for 5 weeks. A plastic cast was wrapped around the right limb from the forearm to the forepaw to keep wrist joint at 90°of flexion. In this model, CGRP in immobilized (ipsilateral) side was distributed in larger DRG neurons compared with contralateral side, even though the number of CGRP-immunoreactive (CGRP-IR) neurons did not differ. Spinal laminae III–V, not laminae I–II in ipsilateral side showed significantly high CGRP expression relative to contralateral side. CGRP expression in cuneate nuclei was not significantly different between ipsilateral and contralateral sides. Long-term immobilization by casting may induce phenotypic changes in CGRP expression both in DRG and spinal deep layers, and these changes are partly responsible for pathological pain states in immobilized side.     

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。     

TNF-α在CCD模型大鼠背根神经节中的表达及机制

目的:探讨TNF-α和NF-κB在背根神经节慢性压迫(CCD)模型大鼠背根神经节(DRG)中的表达变化及其对疼痛学行为的影响。方法:建立CCD大鼠模型,采用von Frey纤维丝监测机械痛阈的改变;通过Western blotting检测TNF-α和NF-κB在DRG中的表达变化趋势,分析其与疼痛行为之间的相关性;并采用免疫荧光双染技术研究TNF-α在DRG中的表达位置。结果:CCD组的50%机械缩足阈值在术后1 d即开始明显下降(P0.01),7~14 d达到高峰,其后逐渐上升,直至术后35 d仍明显低于术前及sham组(P0.01)。而DRG上的TNF-α及NF-κB于造模后各时点均显著增多(P0.01),且TNF-α的表达趋势与50%机械缩足阈值显著相关(P0.05)。结论:DRG慢性压迫可促进其上的TNF-α和NF-κB的合成和分泌,进而诱发机械痛觉过敏。因此,TNF-α/NF-κB信号通路可能是CCD模型疼痛形成的重要通路之一。     

体外获得高纯度大鼠背根神经节神经元的原代培养

目的：建立一种切实可行的胚胎大鼠背根神经节神经元培养及纯化方法。方法：用显微解剖获取足够数量的胚胎大鼠背根神经节,通过胰蛋白酶+EDTA消化、交替使用NB培养基与加入了5-氟-2-脱氧尿嘧啶核苷酸/尿苷的抗有丝分裂NB培养基等方法,在体外获得纯化的背根神经节神经元,采用神经丝蛋白免疫细胞化学染色法与DAPI染核的方法鉴定并测定神经元的纯度。结果：获得的背根神经节神经元在体外生长良好,纯度可达到96%以上。结论：本实验方法可以获得大量高度纯化的大鼠背根神经节神经元。     

多节段背根节慢性压迫大鼠机械触刺激诱发痛镜像痛的外周机制

目的:研究多节段背根节慢性压迫(chronic compression of multiple dorsal root ganglia,CCD)大鼠机械触刺激诱发痛镜像痛的外周机制。方法:制作单侧L3-L5 CCD模型大鼠,并应用von Frey filaments检测双侧机械触刺激诱发痛行为,利用免疫组织化学方法检测该模型大鼠同侧及对侧DRG大中型和小型神经元中与痛觉信息传递相关的神经肽-降钙素基因相关肽(calcitonin gene-related peptide,CGRP)的表达情况。结果:CCD大鼠表现出明显双侧机械触刺激诱发痛行为;免疫组织化学结果显示,CCD模型大鼠同侧及对侧DRG大和中等大小神经元内,CGRP的表达与正常组大鼠DRG神经元比较有明显升高(P<0.05),而同侧及对侧DRG小神经元,CGRP的表达与正常组大鼠DRG神经元比较无明显变化(P>0.05)。结论:CCD模型大鼠双侧DRG内传递痛觉信息的神经肽CGRP表达升高,提示外周双侧DRG神经元内CGRP的可塑性变化可能参与多节段CCD模型大鼠机械触刺激诱发痛镜像痛的发生。     

Variations in the lumbosacral ligament and associated changes in the lumbosacral region resulting in compression of the fifth dorsal root ganglion and spinal nerve

Sixty-five lumbosacral regions from adult cadavers were dissected and the position and relations of the lumbosacral ligament noted. The lumbosacral ligament was present in all specimens; in 22 (34%) it extended medially across the ventral ramus of the fifth lumbar nerve, and in six (9%) of these the underlying nerve was compressed and visibly flattened. On two of these specimens the nerve, together with its dorsal root ganglion, was removed, processed, and stained with Masson's trichrome. The compressed nerve showed increased thickness of endoneurial and perineurial connective tissue, and the cells of the dorsal root ganglion were smaller and surrounded by increased connective tissue, particularly at the periphery of the ganglion. Observation of the lumbosacral ligament and surrounding anatomical structures suggests that anatomical variation in this region may be attributed to the health of the lumbosacral articular elements. In those specimens showing compression of the fifth lumbar spinal nerve there was also narrowing of the lumbosacral interspace. In these the disc itself was compressed and showed degenerative changes. The articular processes at the lumbosacral joint were irregular, with thinning and fissuring of the articular cartilage. It is suggested that the processes which lead to the further development of the ligament, by the formation of additional fibrous bands, are mechanical in nature and result from instability at the lumbosacral region itself. Instability subsequently leads to the initiation of a chain of degenerative changes, involving pathology at the lumbosacral disc and zygapophyseal joints. Compression of the dorsal root ganglion occurs either within a narrowed inter vertebral foramen, or in the case described here just external to the foramen, while compression of the nerve occurs in the accessory ligamentous bands formed to resist the instability. A mechanism by means of which compression of the ganglion may give rise to pain is suggested. © 1995 WiIey-Liss, Inc.     

大鼠坐骨神经切断后Robo2在脊髓及背根节的表达变化

目的:研究坐骨神经损伤后Roundabout 2(Robo2)在成年大鼠背根节和脊髓的表达变化。方法:健康成年雌性SD大鼠坐骨神经切断后分别存活3～28d,取其L_(4～6)背根节(DRG)和脊髓;利用RT-PCR和免疫组织化学技术检测Robo2在上述组织中的表达变化。图像分析技术对阳性细胞的灰度值进行测定。结果:正常DRG感觉神经元表达Robo2 mRNA和蛋白质,脊髓前角运动神经元不表达。坐骨神经切断后3 d DRG内Robo2表达增加,7～14 d达高峰,21～28 d恢复到正常水平。结论:坐骨神经切断可导致DRG内Robo2的表达上调,可能与早期的感觉轴突再生有关。     

雪旺细胞源神经营养因子对背根节感觉神经元的保护作用

目的：了解雪旺细胞源神经营养因子对周围神经高位损伤所致脊髓背根节感觉神经元死亡的保护作用。方法：选出生3周SD鼠高位切断L4、L5神经根，神经近侧断端应用雪旺细胞源神经营养因子或生理盐水，4周后观察损伤神经根背根节感觉神经元的存活率和形态学变化。结果：术后4周，营养因子组神经元的存活率是91．8％，生理盐水组是58．6％；生理盐水组存活神经元胞体明显萎缩。结论：雪旺细胞源神经营养因子对受损的背根节感觉神经元有明显的神经营养活性．     

Termination patterns of calcitonin gene-related peptide-immunoreactive nerve fibers in the dorsal horn of the human spinal cord

Summary CGRP-immunoreactive varicose nerve fibers displayed three kinds of termination patterns in the cervical, thoracic and lumbar segments of the human spinal cord. Bundles of immunoreactive fibers formed a loose network in lamina I. A homogenous band of immunoreactive fibers filled lamina II. Multiple bundles of CGRP-positive fibers coursed through the superficial laminae towards deep portions of the grey matter. In the lumbar segments, in contrast to the cervical and thoracic segments, the bundles could be followed deep into the dorsal funiculus. Bundles of varicose immunoreactive fibers were seen to twine around the dendrites of neurons located in lamina I, in the dorsal funiculus of the lumbar segments and deep in the dorsal horn (laminae III–V). The corresponding types of large and medium-sized neurons were found in silver impregnated adjacent spinal cord sections. It is suggested that neurons in the above locations preferentially receive multiple contacts from CGRP-containing nerve fibers along their extensive dendritic arborizations (CGRP-target neurons).     

背根神经节电穿孔转染神经元技术的改进

文题释义：背根神经节：具有一组不同的感觉神经元，向大脑传递不同的感觉刺激，如疼痛、温度、触摸和身体姿势。背根神经节电穿孔是基因转染中通过非病毒的方式以物理方法转染神经元的技术。 周围神经损伤：各种原因导致周围神经支配区域的感觉、运动及营养障碍。文献报道周围神经损伤常伴随损伤近端的神经元胞体的肿胀、尼氏体消失、细胞核偏移；损伤远端轴突发生华勒氏变性、许旺细胞增殖等现象。 背景：背根神经节电穿孔技术是一种研究神经再生的高效基因转染方法，以往背根神经节电穿孔的电压条件使得标记的神经元及轴突数目较少，统计误差较高。 目的：提高神经元及其轴突的标记率，为周围神经再生的研究提供理论依据。 方法：以增强型绿色荧光蛋白为观察指标优化背根神经节电穿孔技术在神经元轴突再生方面的应用。将ICR小鼠随机分为2组，分别行背根神经节电穿孔手术，检测在35 V和60 V电压干预下神经元及其轴突的标记率。 结果与结论：①35 V和60 V电压未造成神经元明显死亡；②与35 V电压相比，60 V电压条件下标记的神经元及其轴突数量显著增加，且通过损伤部位的轴突数量明显增多(P < 0.05)；③60 V电压对实验动物未造成行为功能损害；④结果表明，60 V电压能够使神经元及其轴突的标记率增高，其结果能够为周围神经元轴突再生研究提供依据。 ORCID: 0000-0001-8681-1346(齐士斌) 中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程     

Curcumin protects the dorsal root ganglion and sciatic nerve after crush in rat

The goal of this study was to quantify the histological changes in the dorsal root ganglion (DRG) and the sciatic nerve in rats subjected to sciatic nerve crush (SNC) following curcumin treatment. The rats were divided into four groups, each including five animals, and underwent the following intervention: group I: control animals which received olive oil; group II: sham-operated animals whose skin of the posterior thigh was opened, sutured, and received the vehicle; group III: SNC animals which received the vehicle; and group IV: SNC plus curcumin (100 mg/kg/day) solved in the vehicle. On the 28th day, the fifth lumbar DRG and sciatic nerve were removed. Volume of the ganglion, mean cell volume, total volume of DRG cells (A- and B-cells), and total surface of DRG cells, total number, diameter, and area of the myelinated nerve fibers were estimated using stereological methods. Except for the volume of the ganglion, all other parameters were decreased after nerve crush. In curcumin-treated rats, these parameters decreased, but to a lesser extent, and the values were significantly higher than in the non-treated SNC group (p < 0.04).It can be concluded that in rats after crush, curcumin has a protective effect on the DRG and sciatic nerve.     

大鼠坐骨神经损伤后Src抑制的蛋白激酶C的底物在背根神经节中的表达变化

目的:探讨大鼠坐骨神经损伤后,Src抑制的蛋白激酶C的底物(SSeCKS)在背根神经节(DRG)中的表达变化及其意义。方法:制备成年SD(Sprague-Dawley)大鼠坐骨神经夹伤及切断模型。通过Western印迹法、Real-time PCR及免疫组织化学方法检测坐骨神经损伤后SSeCKS在DRG中表达的时空变化。结果:大鼠坐骨神经夹伤后6h,DRG中可检测到SSeCKS的表达并逐步升高,伤后12h达到高峰,2d后逐渐下降;而大鼠坐骨神经切断后DRG中SSeCKS的表达高峰发生在伤后2周,1d时最低;SSeCKS主要分布于DRG大、中、小神经元胞质;SSeCKS与NeuN、NF200以及GAP43存在共定位。结论:大鼠坐骨神经损伤后,引起DRG中SSeCKS的表达变化,其可能参与疼痛信号转导通路并与周围神经损伤后的再生有关。     

促红细胞生成素/神经生长因子对大鼠压迫性损伤后背根神经节细胞的影响

目的:观察促红细胞生成素(EPO)/神经生长因子(NGF)对慢性压迫中的大鼠背根神经节(DRG)细胞的影响。方法:暴露大鼠多节段DRG,行慢性压迫造模型,并分离DRG,模型组分离的DRG细胞给予外源EPO或NGF,台盼蓝染色检测细胞存活率,TUNEL法检测细胞凋亡,免疫荧光法检测Bcl-2和Bax表达,荧光探针法检测活性氧(ROS)活性,ELISA法检测炎症因子IL-1β、TNF-α、IFN-γ的水平。结果:EPO或NGF可减轻DRG细胞的损伤和凋亡,下调促凋亡因子Bax的表达,并上调抗凋亡因子Bcl-2的表达,降低ROS活性。结论:EPO和NGF有利于受损的DRG细胞抗炎和抗氧化作用。     

成年猕猴背根神经节nNOS免疫阳性神经元的分布

目的:观察正常成年猕猴背根神经节神经元型一氧化氮合酶(nNOS)免疫阳性神经元的分布。方法:ABC免疫细胞化学方法显示nNOS免疫阳性神经元,并用体视方法进行定量分析。结果:猕猴颈、胸、腰各段背根神经节nNOS免疫阳性神经元的分布相似,数量较多,阳性神经元的大小不等,多呈圆形或椭圆形;胞浆着色较深,胞核位于细胞中央,不着色,细胞被神经纤维束分隔成群。nNOS免疫阳性神经元以中型神经元为主,其次为小型神经元,其胞浆呈强阳性染色,细胞直径<50μm,大型神经元较少。颈、胸、腰各段背根神经节nNOS免疫阳性神经元的密度以及阳性细胞与总细胞数的比值均无明显差异。结论:猕猴背根神经节nNOS主要表达在中、小型神经元,提示NO可能主要参与痛觉等浅感觉的传导和调制。     

甲钴胺对大鼠受损背根节A-类神经元自发放电的抑制作用

目的:研究甲钴胺(methylcobalamin,Methyl B12)对损伤背根节(dorsal root ganglion,DRG)A-类神经元的不同模式自发放电的作用。方法:在大鼠L5椎间孔植入不锈钢柱制备大鼠DRG慢性压迫模型(chronic com-pression of DRG,CCD)上,利用在体单纤维细胞外记录方法研究Methyl B12对受损DRG神经元自发放电的影响。结果:(1)CCD模型大鼠出现明显的双侧机械刺激引起缩足反应阈值降低(P<0.001);(2)Methyl B12(300μmol/L)对受损A-类DRG神经元自发放电具有显著的抑制作用(n=13,P<0.05);(3)Methyl B12(300μmol/L)对受损A-类DRG神经元非周期和周期放电模式自发放电均有抑制作用,且两者之间没有明显差异。结论:Methyl B12(300μmol/L)对受损DRG A-类神经元产生的异位自发放电具有抑制作用,且对于非周期和周期放电模式的抑制作用没有差异。     

小鼠背根神经节中痒觉神经元的分布特性

目的观察小鼠背根神经节(DRG)中痒觉特异性Mrgpr A3+神经元的分布特征。方法采用遗传学方法将Mrgpr A3+神经元特异性标记强化绿色荧光蛋白(EGFP)和td Tomato;选取3只纯合Mrgpra3EGFP-Cre;ROSA26td Tomato成年转基因小鼠,分离皮肤和背根神经节组织;采用激光扫描共焦成像技术观察Mrgpr A3+神经元的外周神经纤维在小鼠躯体皮肤的投射分布特征;采用双光子成像技术观察Mrgpr A3+神经元在整体背根神经节中的三维空间分布情况。结果脸颊、背部和脚掌皮肤的Mrgpr A3+神经纤维分布密集,粗且长,分布广泛;颈部和腹部皮肤的Mrgpr A3+神经纤维分布稀疏,短且小,呈散点状分布;Mrgpr A3+神经纤维在皮肤的有毛和无毛区域都有投射分布,且不同部位分布特点不同;几乎所有的Mrgpr A3+神经元都为小直径感觉神经元,且在颈、胸、腰、尾段背根神经节中均有分布;颈段、胸段、腰段和尾段的Z轴成像深度分别为350μm、250μm、400μm和200μm;躯体不同部位背根神经节中的Mrgpr A3+神经元的三维空间分布在不同节段存在明显差异。结论小鼠躯体不同部位皮肤的Mrgpr A3+神经纤维的分布特征和整体背根神经节中Mrgpr A3+神经元的三维空间分布特征都存在较大差异,痒觉神经元和末梢分布的差异可能是不同区域存在痒觉生理反应差异的结构基础。