家兔胆囊传入神经的逆行追踪

用12只家兔将39%HRP40μl分5-8点注入胆囊壁内,以下神经节内出现标记细胞:双侧迷走神经下节、腹腔节和双侧背根节(DRG)C_4、T_(4-8);左侧DRG C_(3-4)、T_(2-10);右侧DRG C_4、T_(4-8)、T_(11)。C_4、T_(6-8)为高峰区。标记细胞总数94个(左54,右40)。迷走神经下节出现58个(61.70%),DRG出现36个(38.29%),腹腔节出现100个,因性质未定,未计入总数。     

大鼠卵巢的神经联系(FHRP和CT-HRP法)

本文用FHRP和CT-HRP注射大鼠左或右侧卵巢,在同侧T_(10)—L_2脊神经节见到标记细胞,高峰在T_(13)和L1。在T_(10)-L_2脊髓背角Ⅲ-Ⅳ层见到标记纤维。在双侧结状神经节、双腹腔神经节,双侧迷走神经背核都见到标记细胞。并见到迷走神经背核的树突伸入到同侧弧束核内。在弧束核及弧束连合核内见到标记纤维。在内脏大神经及卵巢上神经内见到标记纤维,在以上两神经的边缘见到标记细胞。     

成年猫L1～L7两侧背根节神经元数量的比较

5只正常成年雄性猫双侧L1～L7背根节(DRG)石蜡包埋,连续切片,显微镜观察计数,比较左、右DRG 神经元数量,为神经系统损伤后对神经元的存活和再生影响的研究提供DRG神经元计数资料.结果1)每只动物各节段左、右DRG神经元数量比较,比率在0.70～1.26 之间,个别节段两侧有显著性差异(P＜0.01),而每只动物左、右DRG神经元总数比较,比率在0.93-1.02(P＞0.5),两侧无显著性差异.2)5只动物L1 ～L7各节段左、右DRG神经元均数比较,两侧无显著性差异(P＞0.5).结论成年猫L1～L7各节段两侧DRG神经元数量呈对称性分布.     

家兔胆总管传入神经的逆行追踪

将HRP(SjgmaVI)粉末涂于15只家兔胆总管壁表面,在以下神经节内出现标记细胞:双侧背根节(DRG)T_(3-11)、迷走神经下节(结状节),腹腔神经节,左侧DRGT_(12)及右侧DRGT_2各一例。颈,腰部DRG内未发现标记细胞。标记细胞在DRG内出现的最高点在T_(5-8)。腹腔节内标记细胞致较多(占标记细胞总数1267个的53.78％),并出现47个双核细胞。在迷走神经背核内出现了3个标记细胞。比较了肝脏、胆囊、胆总管传入神经元胞体在各神经节内的分配。     

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。     

家兔肝脏面传入纤维的逆行追踪—HRP法

将HRP注入15只家兔肝门区后,在以下神经节内出现酶标细胞:全部实验动物的双侧T_(2-12)脊神经节、双侧迷走神经的上节(颈静脉节)和下节(结状节),右侧T_(13)和左侧L_1脊神经节各1例。酶标细胞在脊神经节内出现的高峰在T_(4-8),与传统记载的T_(7-9)不同,并讨论了颈神经节(C_(3-5))和腹腔神经节内未出现酶标细胞等问题。     

外周伤害性感受神经元在体特异性表达GCaMP6f蛋白方法的构建及细胞内钙活动的监测

目的:建立在外周伤害性感受器特异性表达GCa MP6f蛋白的方法,并在疼痛刺激条件下,应用钙成像技术实时监测小鼠伤害性感受神经元的细胞内钙活动。方法:包装并纯化r AAV2/9-CAG-DIO-GCamp6f病毒,并通过立体定位注射该病毒于SNS-Cre小鼠L4/L5背根神经节(dorsal root ganglion,DRG)内。动物存活3~4周,然后制备L4/L5整节DRG标本,检测GCa MP6f蛋白表达情况,并观察疼痛刺激条件下诱致的伤害性DRG神经元的钙反应情况。结果:SNS-Cre小鼠L4/L5 DRG内注射r AAV2/9-CAG-DIO-GCamp6f病毒后,荧光显微镜观察显示GCamp6f病毒特异性地表达在中、小型的DRG神经元。给予DRG神经元高钾刺激(KCl 30 mmol/L)可以诱致神经元胞内的钙离子水平显著上升。进一步给予伤害性DRG神经元特异性标志物TRPV1受体的激动剂Capsaicin(1μmol/L),结果显示其可以诱致GCamp6f蛋白标记的DRG神经元呈现显著的钙反应。结论:本研究建立的SNS-Cre小鼠DRG在体注射r AAV2/9-CAG-DIO-GCamp6f病毒特异性标记伤害性DRG神经元的方法是成功的,该方法的成功建立对于活体特异性监测伤害性DRG神经元的功能活动提供了重要工具。     

大鼠桡神经钳夹伤后背根节和脊髓nNOS免疫阳性神经元的变化

目的研究大鼠桡神经钳夹伤后背根神经节(DRG)和脊髓nNOS免疫阳性神经元的变化,探讨NO是否参与大鼠桡神经钳夹伤后的DRG和脊髓水平的痛觉调制。方法用辣根过氧化物酶追踪大鼠桡神经的由来;大鼠桡神经钳夹伤结合免疫组化法,研究桡神经钳夹伤后DRG鄄和脊髓的nNOS免疫阳性结构变化。结果(1)大鼠桡神经的组成范围在C5～T1;(2)大鼠桡神经钳夹伤后,DRG内nNOS免疫阳性神经元的变化难以分析:脊髓后角nNOS免疫阳性结构数量减少、免疫强度下降。结论大鼠桡神经钳夹伤后,脊髓nNOS免疫阳性结构发生可塑性变化,NO在桡神经钳夹伤后的痛觉调制中有一定的作用。     

坐骨神经分支选择性损伤模型L4～6背根神经节水平不同时间点P2X3mRNA表达变化

目的观察坐骨神经分支选择性损伤(SNI)动物制作型模后不同时间点L4~6背根神经节(DRG)水平P2X3mRNA的表达情况,探讨外周P2X3mRNA在神经病理性痛模型不同阶段中的作用。方法 54只健康雄性SD大鼠完全随机分为空白对照(control)组、假手术(sham surgery)组和手术(surgery)组。通过结扎腓总神经及切断胫神经,保留腓肠神经的方法建立SNI模型。动态观察造模前、造模后1d、3d、7d和14d双侧足跖机械痛阈;Real-time PCR法检测患侧造模后3d、7d和14d L4~6 DRG水平P2X3mRNA表达情况。结果模型制作后各时间点,surgery组大鼠患侧足跖痛阈明显降低(P0.05),sham surgery组大鼠与control组比较差异无显著性(P0.05);各组大鼠健侧足跖痛阈于模型制作后各时间点均无显著变化(P0.05)。模型制作后3d、7d,surgery组大鼠L4、L5、L6 DRG水平P2X3mRNA表达均有不同程度的提高(P0.05);而模型制作后14d,surgery组大鼠L5、L6DRG水平P2X3mRNA表达明显减少(P0.05),L4 DRG水平P2X3mRNA表达仍显著多于sham surgery组(P0.05)。模型制作后各时间点、各DRG水平,sham surgery组和surgery组大鼠P2X3mRNA表达差异均无显著性(P0.05)。结论外周P2X3mRNA参与SNI模型疼痛的产生和维持,且其在不同阶段发挥的作用不同。     

中药筋脉通对糖尿病大鼠背根神经节UCP3和IGF-1表达的影响

目的观察中药筋脉通对链尿佐菌素(STZ)诱导的糖尿病(DM)大鼠背根神经节(DRG)解耦联蛋白3(UCP3)和胰岛素样生长因子(IGF-1)的表达的影响。方法将大鼠随机分为正常对照组、糖尿病模型组(腹腔内注射STZ、筋脉通及牛磺酸治疗组)。成模后每天1次灌胃给药,持续16周。电子Von Frey仪检测机械痛阈值,免疫组化法和real time-PCR法分别检测DRG中UCP3和IGF-1蛋白及mRNA的表达。结果与正常对照组相比,糖尿病大鼠出现机械痛阈值下降,DRG中UCP3和IGF-1蛋白及mRNA表达水平明显下降(P0.01);中药筋脉通组可改善糖尿病大鼠周围神经痛阈下降(P0.01),并且能够使DRG中UCP3和IGF-1蛋白及mRNA表达水平显著回升(P0.01)。结论中药筋脉通可通过增加DRG神经组织中UCP3含量,抑制氧化应激对背根神经的损伤,并通过上调神经营养因子IGF-1的含量,促进糖尿病大鼠周围神经修复能力,而改善其痛觉异常。     

大鼠颈脊神经节中降钙素基因相关肽免疫阳性神经元分支至颈椎关节突关节囊和前肢

目的探讨颈椎关节突关节疼痛综合征并发上臂痛的神经解剖学机制。方法以抗降钙素基因相关肽（CGRP）抗体对SD大鼠C4-T1关节突关节囊作免疫组织化学染色。将荧光标记物快蓝（FB）和核黄（NY）分别注射到SD大鼠右侧桡神经和C1～T1颈椎关节突关节囊。显微镜下观察颈脊神经背根节（DRG）内被逆行荧光标记的神经元，再用抗CGRP抗体行免疫组织化学染色，观察DRG内的CGRP免疫阳性神经元。最后比较荧光照片和免疫组织化学照片上的神经元。结果C4～T1关节突关节囊上分布着丰富的CGRP免疫阳性神经纤维，它们或独立走行或交织成网。在C5～T1各节段的DRG中均可观测到被FB单标（76％）、NY单标（16％）和FB／NY双标（8％）的神经元。约40％的荧光逆行标记神经元在免疫组织化学染色的切片上呈CGRP免疫反应阳性。结论大鼠颈部DRG内一部分含GRP的感觉神经元的周围突分两支，一支支配颈椎关节突关节囊，另一支随桡神经分布到前（上）肢。提示，颈椎关节突关节疼痛综合征并发上臂牵涉痛的神经形态学基础之一可能发生在DRG水平。     

Convergence of sensory processes from the heart and left ulnar nerve onto a single afferent perikaryon: a neuroanatomical study in the rat employing fluorescent tracers

After injection of true blue (TB) into the pericardial sac and nuclear yellow (NY) into the left ulnar nerve of rats, most perikarya in the left eighth cervical and first thoracic dorsal root ganglia are single-labeled with either TB or NY. However, 7.1-14.7% of the sensory perikarya from the cardiac area labeled with TB are simultaneously labeled with NY. This finding indicates that some primary sensory perikarya receive processes from both the left arm and the heart. In addition to other mechanisms that may be operative in the referral of pain of cardiac origin to the medial left arm, these results suggest that sensory neurons with dichotomizing somatic and visceral peripheral processes may also contribute to this phenomenon.     

Axonal ramification of neurons in the nucleus reticularis tegmenti pontis projecting to the paramedian lobule in the rabbit cerebellum

Projections of the nucleus reticularis tegmenti pontis (NRTP) to the cerebellar paramedian lobule were examined in the rabbit by means of the double fluorescent retrograde tract-tracing method. The rabbit NRTP is composed of a medial, large part comprising zones A (dorsomedial), B (central) and C (lateral), and of a lateral, small part (the processus tegmentosus lateralis; PTL). Following unilateral injections of Fast Blue (FB) into the rostral part of the paramedian lobule (rPML) and of Diamidino Yellow (DY) into the caudal part (cPML), known to receive spinal inputs from forelimb and hindlimb, respectively, substantial numbers of single labeled neurons were found in all bilateral NRTP divisions, apart from the zone C. Most projection neurons to the PML were located in the medial and medioventral regions of the zone B. Smaller numbers of projection neurons were located in the PTL, zone A and outside the zone B among fibers of the medial lemniscus. The pattern of FB and DY labeling suggested that neurons projecting to the rPML and cPML originated in common rather than separate regions within the NRTP. In addition, a small percentage (mean 1.3%) of double FB+DY labeled neurons were detected with a clear contralateral preponderance, among single labeled FB or DY cells. In spite of the rarity, all the NRTP neurons giving rise to intralobular collateral projections can be regarded as potential sources of simultaneous modulating influences upon two functional different forelimb (rPML) and hindlimb (cPML) regions. The findings have been discussed in relation to earlier studies in other species and commented on with respect to the possible functional meaning of these projections.     

大鼠脊神经节细胞周围突的分支及其向躯体与内脏的分支投射

本文用大鼠做两组实验。一组用CB-HRP注入其膀胱壁内,TMB法成色,追踪其脊神经节内的标记细胞。发现有的节细胞的周围突在节内出现分支,分二支或多支。另一组实验用荧光素双标记法,分别注射核黄和真蓝于大鼠的坐骨神经干和膀胱壁内,追踪其脊神经节内的标记细胞。结果在L_4—L_6节发现双标细胞,以L_5节最多见,双标细胞出现的比例,均占坐骨神经标记细胞总数的3％左右。结果表明少量脊神经节细胞的周围突有向躯体与内脏的分支投射。     

Axotomized,adult basal forebrain neurons can innervate fetal frontal cortex grafts: A double fluorescent tracer study in the rat

Summary The ability of axonal regeneration of identified adult basal forebrain (BFB) neurons was examined after homotopic grafting of fetal neocortical tissue to a lesion cavity in the frontal neocortex. Using a four step experimental procedure, adult rats first received an injection of the fluorescent dye Fluoro-Gold (FG) into the sensorimotor cortex in order to label those neurons with projections to the area by retrograde axonal transport. After one week the injection area was removed by aspiration, leaving a cavity in the neocortex. One week later a block of fetal (E14) frontal cortical tissue was placed in the cavity. The animals were then allowed to survive for 6 weeks before a second fluorescent tracer, Nuclear Yellow (NY), was injected into the transplant. The animals were sacrificed 24 h later and analyzed by fluorescence microscopy. Both single labeled, FG and NY containing neurons and double labeled neurons containing both tracers were found in the BFB. The results demonstrate that adult BFB neurons can reestablish cortical projections into fetal cortical grafts (double labeled neurons), and they suggest that other BFB neurons, not initially innervating the lesioned cortical area, have sprouted into the transplant (NY labeled neurons).     

与坐骨神经和盆神经对应的后根节中CGRP、SP、NOS样阳性神经元分布特点分析——荧光金逆标和荧光免疫组化结合的研究

为了探索大鼠坐骨神经（躯体性）和盆神经（内脏性）内与传递痛信号有关的初级传入神经元在后根节内的分布特点,本研究采用荧光金逆行追踪与免疫荧光组化技术相结合的方法,对ＣＧＲＰ能、ＳＰ 能和ＮＯＳ样神经元在相应的后根节内（坐骨神经,Ｌ４～Ｌ６ ;盆神经,Ｌ６～Ｓ１）的分布状况进行了分析。结果表明：（１）坐骨神经和盆神经初级传入神经元中有相当数量的ＣＧＲＰ和ＳＰ样阳性细胞,与这二者相比,ＮＯＳ样细胞数量稀少;（２）盆神经初级传入神经元中ＣＧＲＰ／ＦＧ、ＳＰ／ＦＧ、ＮＯＳ／ＦＧ 双标细胞的比率高于坐骨神经,而其前两种双标细胞与各该活性物质单标细胞的比率则低于坐骨神经;（３）三种物质与ＦＧ 的双标神经元以小型为主,少有中型细胞。因为既往的研究证明,分布有大量的ＣＧＲＰ、ＳＰ、ＮＯＳ样终末的骶髓后连合核（ＳＤＣＮ）接受盆腔脏器伤害性信息传入,并且ＣＧＲＰ、ＳＰ都以外周来源为主。故本文结果进一步核实了ＳＤＣＮ 区接受来自外周的ＣＧＲＰ、ＳＰ投射,且确为经盆神经传入的细纤维。     

Diamidino yellow dihydrochloride (DY·2HCl); a new fluorescent retrograde neuronal tracer,which migrates only very slowly out of the cell

Summary Earlier studies showed that Nuclear Yellow (NY), True Blue (TB) and Fast Blue (FB) are transported retrogradely through axons to their parent cell bodies. NY produces a yellow fluorescent labeling of the neuronal nucleus at 360 nm excitation wavelength, while TB and FB produce a blue fluorescence of the cytoplasm at this same wavelength. Therefore, NY may be combined with TB or FB in double-labeling experiments demonstrating the existence of axon collaterals. However, retrograde neuronal labeling with TB or FB requires a relatively long survival time, while NY requires a short survival time since NY migrates rapidly out of the retrogradely labeled neurons. This complicates double-labeling experiments since TB and FB must be injected first and NY later, a short time before the animal is sacrificed. We report a new yellow fluorescent tracer which labels mainly the nucleus and migrates much more slowly out of the retrogradely labeled neurons than NY. This new tracer can be used instead of NY in combination with TB or FB in double-labeling experiments and unlike NY can be injected at the same time as TB or FB. The new tracer is a diamidino compound (no. 28826) which is commercially available. It will be referred to as Diamidino Yellow Dihydrochloride (DY·2HCl). According to the present study DY·2HCl is transported over long distances in rat and cat, and produces a yellow fluorescence of the neuronal nucleus at 360 nm excitation wavelength, resembling that obtained with NY. When combined with TB or FB, DY·2HCl is as effective as NY in double labeling of neurons by way of divergent axon collaterals.Supported in part by Grant 13-46-91 of FUNGO/ZWO Dutch Organization for Fundamental Research in Medicine

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Pontocerebellar projection to the rabbit paramedian lobule by means of axonal collaterals: evidence for intralobular connections

In this study, we utilized a double retrograde axonal tracing technique to investigate the possible existence of collateralized axonal projections from the pontine nuclei (PN) to the rostral (rPML) and caudal (cPML) parts of cerebellar paramedian lobule in the rabbit, known to be the forelimb and hindlimb regions, respectively. Following injections of fluorescent tracers Fast Blue (FB) and Diamidino Yellow (DY) within rPML and cPML, respectively, substantial numbers of FB and DY single labeled neurons were found in the dorsolateral, paramedian, lateral and peduncular pontine nuclei bilaterally with a very clearcut contralateral preponderance. No labeling was observed in the ventral pontine nucleus. Extensive areas of overlap of FB or DY labeled neurons indicated that no somatotopical relationship existed in projection from PN to the two functionally different PML target regions. In addition, a small number of double FB + DY labeled neurons was detected in the common areas of FB and DY single labeling in PN. These neurons give rise to pontocerebellar projections to rPML and cPML simultaneously by way of axonal collaterals and thus they may play a role in the coordination of unilateral forelimb and hindlimb movements.     

Topography and axonal collaterals of trigeminocerebellar projection to the paramedian lobule and uvula in the rabbit cerebellum

To study projections of the trigeminal sensory nuclei (TSN) to the rostral parts of the paramedian lobule (PML) and of the uvula of the rabbit cerebellar cortex, the retrograde double fluorescent labeling method was used. Injections of Fast Blue (FB) into PML and Diamidino Yellow (DY) into the uvula, resulted in prominent labeling neurons with FB bilaterally and with DY ipsilaterally, in the principal trigeminal nucleus, subnucleus oralis, and rostral and caudal subnucleus interpolaris. We observed topographical arrangement of neurons in such a fashion that FB labeled cells were localized in the medial and DY labeled cells in the lateral regions of TSN. Apart from this small number of double FB+DY labeled neurons (n = 138) were found in the narrow common region of single labeling. This implies that PML and the uvula receive independent trigeminal sensory information from neurons in separate regions of TSN. However, some trigeminal neurons may also exert simultaneous influences upon these hemispheral and vermal components by way of axonal branchings.     

大鼠胃的神经支配——荧光双标记法研究

采用荧光双标记法,研究大鼠胃体与幽门的神经支配。延髓迷走神经背核(DmnX)、疑核(nA)和孤束核(nTs)均有标记神经元,部分标本脊髓胸段(T_(3～9))灰质后角V层、脊神经节(T_(0～8))和胸交感链中亦有标记神经元。结合观察结果,对胃的神经支配和外周神经标记的荧光染料选择等问题,进行了讨论。根据DmnX标记细胞的情况分析,表明胃体与幽门分别有迷走神经分支支配,部分神经纤维既有末梢分布于胃体,亦有末梢分布于幽门。支配胃体的神经纤维密度远较幽门部为大。根据胸交感链中存在标记神经元的情况,提出支配胃的节前交感纤维有部分是在交感链中换神经元。