RVLM-IML pathway may implicate controlling peripheral airways by melanocortinergic-sympathetic signaling: a transneuronal labeling study using pseudorabies virus

Pseudorabies virus (PRV)-614 was injected into the tracheal wall of male MC4R-GFP transgenic mice, resulting in retrograde infections in spinal cord and autonomic premotor areas of the brain including the rostroventrolateral medulla (RVLM). This polysynaptic pathway to the airway may form the substrate underlying the impact of IML and RVLM on airway function. The neurochemical phenotype of MC4R-GFP-positive neurons was identified using fluorescence immunocytochemical labeling. PRV-614/MC4R-GFP dual labeled neurons were detected in spinal IML and the rostral ventrolateral medulla (RVLM). These data demonstrate the RVLM-IML pathway of synaptically connected neurons extending to the airway through melanocortinergic-sympathetic signaling.     

Melanocortinergic circuits from medial vestibular nuclei to the kidney defined by transneuronal transport of pseudorabies virus

This study was designed to assess whether MC4R signaling existed in vestibular nuclei modulated the activity of kidney by a virally mediated transsynaptic tracing study. Pseudorabies virus (PRV)-614 was injected into the kidney in adult male MC4R-green fluorescent protein (GFP) transgenic mice (n = 5). After a survival time of 5 days, the mice were assigned to humanely sacrifice, and the brainstem were removed and sectioned, and processed for PRV-614 visualization. The neurochemical phenotype of MC4R-GFP-positive neurons was identified using fluorescence immunocytochemical labeling. PRV-614/MC4R-GFP dual labeled neurons were detected in medial vestibular nuclei. Our findings support the hypothesis that there exist melanocortinergic circuits from medial vestibular nuclei to the kidney.     

Central circuits regulating the sympathetic outflow to lumbar muscles in spinally transected mice by retrograde transsynaptic transport

Despite considerable interest in the mechanisms that control the hyperalgesia associated with muscle inflammation, the CNS descending pathways that coordinate autonomic circuits regulating lumbar muscles are not adequately understood. Here we used both pseudorabies virus (PRV)-614 retrograde transsynaptic tracing and spinally transected method in 33 C57BL/6J mice to map the polysynaptic pathways between lumbar muscle and CNS. Tissues were processed for dual-label immunocytochemical detection between PRV-614 and tryptophan hydroxylase (TPH) or tyrosine hydroxylase (TH)-expressing neurons in CNS. In intact mice, PRV-614 was transported to the intermediolateral column (IML) and ventral horn (VH) of spinal cord, with subsequent transport to many brain regions, including the medullary raphe nuclei, rostral ventrolateral medulla (RVLM), A5 cell group regions (A5), locus coeruleus (LC), the medullary and pontine reticular formation nucleus (MRN and PRN), paraventricular nucleus of the hypothalamus (PVN), and other central sites. However, PRV-614 in spinally transected mice produced retrograde infection of IML, with subsequent transport to main brain regions that have been shown to contribute to regulating sympathetic circuits, including RVLM, Lateral paragigantocellular reticular nucleus (LPGi), A5, LC, and PVN, whereas PRV-614 labeling in VH and MRN was eliminated in almost every case. In above five brain regions, dual-labeling immunocytochemistry showed coexpression of PRV-614/TPH and PRV-614/TH immunoreactive (IR) neurons involved in these regulatory circuits. Our results reveal a hierarchical organization of central autonomic circuits controlling the lumbar muscles, thus providing neuroanatomical substrates for the central catecholaminergic and serotonergic system to regulate the lumbar muscles.     

Immunolocalization of alpha-synuclein in the rat spinal cord by two novel monoclonal antibodies

This study provides the first immunohistochemical evidence of the presence and distribution patterns in the rat spinal cord of α-synuclein (α-Syn), a soluble acidic protein, widely expressed in the CNS and closely associated to the pathogenesis of neurodegenerative conditions such as Parkinson's and Alzheimer's diseases. We used two novel homemade monoclonal antibodies (2E3 and 3D5) recognizing two different epitopes of α-Syn. Both antibodies localized α-Syn within the nerve terminals, whereas 3D5 alone also localized it within the neuronal nuclei. α-Syn-immunoreactive nervous elements were widely recognized throughout rat spinal cord and in almost all the gray matter laminae. However, they appeared particularly concentrated within laminae I, II, VII and X and more scattered in the others. Double immunofluorescent labeling showed that α-Syn colocalized with synaptophysin in the presynaptic nerve terminals, with neuropeptide Y (NPY) in lamina I, II, IX and X, and had close relationships with tyrosine hydroxylase (TH) immunoreactive neurons in laminae VII and X. Interestingly, the α-Syn-immunoreactive nerve elements, in lamina X, contained little of calbindin-28KD and calretinin-31KD. Our findings could help in understanding the genesis of some early clinical symptoms of Parkinson's disease (PD), such as pain and dysautonomic disorders, and indicate the spinal cord as their probable starting point, according to the ascending theory of PD, proposed by Braak.     

Retrograde tracing of medial vestibular nuclei connections to the kidney in mice

Vestibular nuclei have been identified as a uniform multifunctional structure in order to maintain physiological homeostasis, including the participation of renal sympathetic activity. In this study, the medial vestibular nuclei (MVe) of 10 adult male C57BL/6J strain mice were mapped retrograde using injections of pseudorabies virus (PRV)-614. The virus, injected into the kidney, was specifically transported to the medial vestibular nuclei (MVe). We used a fluorescence immunohistochemistry to characterize the chemical neuroanatomical substrate of MVe innervating the kidney in the mouse. At five days after PRV-614 injection in the kidney, PRV-614 infected neurons were retrogradely labeled in MVeMC and MVePC; PRV-614/tyrosine hydroxylase (TH) double-labeled neurons located predominantly in MVeMC and not in MVePC, whereas PRV-614/tryptophan hydroxylase (TPH) neurons were not localized in MVeMC and MVePC. Our results revealed direct neuroanatomical evidence to identify catecholaminergic projections from the MVeMC to the kidney, suggesting that medial vestibulo-renal pathway may be catecholaminergic.     

Androgen receptors in areas of the spinal cord and brainstem: A study in adult male cats

Sex hormones, including androgens and estrogens, play an important role in autonomic, reproductive and sexual behavior. The areas that are important in these behaviors lie within the spinal cord and brainstem. Relevant dysfunctional behavior in patients with altered androgen availability or androgen receptor sensitivity might be explained by the distribution of androgens and their receptors in the central nervous system. We hypothesize that autonomic dysfunction is correlated with the androgen sensitivity of spinal cord and brainstem areas responsible for autonomic functions. In this study, androgen receptor immunoreactive (AR-IR) nuclei in the spinal cord and brainstem were studied using the androgen receptor antibody PG21 in four uncastrated young adult male cats. A dense distribution of AR-IR nuclei was detected in the superior layers of the dorsal horn, including lamina I. Intensely stained nuclei, but less densely distributed, were found in lamina X and preganglionic sympathetic and parasympathetic cells of the intermediolateral cell column. Areas in the caudal brainstem showing a high density of AR-IR nuclei included the area postrema, the dorsal motor vagus nucleus and the retrotrapezoid nucleus. More cranially, the central linear nucleus in the pons contained a dense distribution of AR-IR nuclei. The mesencephalic periaqueductal gray (PAG) showed a dense distribution of AR-IR nuclei apart from the most central part of the PAG directly adjacent to the ependymal lining. Other areas in the mesencephalon with a dense distribution of AR-IR nuclei were the dorsal raphe nucleus, the retrorubral nucleus, the substantia nigra and the ventral tegmental area of Tsai. It is concluded that AR-IR nuclei are located in specific areas of the central nervous system that are involved in the control of sensory function and autonomic behavior. Furthermore, damage of these AR-IR areas might explain related dysfunction in humans.     

The central canal of the human spinal cord: a computerised 3-D study

Knowledge of the structure and function of the central canal of the human spinal cord is important in understanding the pathogenesis of syringomyelia. Analysis of the morphology of the central canal is difficult using isolated histological sections. A 3-dimensional reconstruction technique using digitised histological sections was therefore developed to visualise the morphology of the central canal. The technique was used to study the canal in the conus medullaris and filum terminale of 1 sheep and 4 human spinal cords. A variety of morphological features were demonstrated including canal duplication, a terminal ventricle and openings from the canal lumen into the subarachnoid space. The findings suggest the possibility of a functionally important fluid communication in the caudal spinal cord which may have a sink function.     

小鼠脊髓微血管周细胞的体外培养及鉴定

目的应用周细胞培养基(PCM)分离筛选小鼠脊髓微血管周细胞(SCMP),对其生物学功能进行评价。方法10只3周龄C57小鼠,无菌条件下取脊髓去除软脊膜,剪碎成大约1 mm×1 mm×1 mm。两次酶消化后用含20%牛血清白蛋白的DMEM离心获得微血管。用内皮细胞培养基(ECM)培养,传代2次后改用PCM培养。倒置显微镜观察细胞增殖状况。免疫细胞化学法检测血小板源性生长因子受体β(PDGFRβ)、神经元-胶质抗原2(NG2)、von Willebrand因子(v WF)和胶质纤维酸性蛋白(GFAP)的表达。流式检测CD140b、CD31、CD11b和GFAP的表达。将PCM换为10%胎牛血清(FBS)的DMEM培养基,检测细胞α-SMA表达的变化。通过周细胞-内皮细胞共培养成管实验检测细胞成管能力。结果接种48 h细胞爬出,7~9 d汇合,周细胞和内皮细胞伴随状增殖。改用PCM培养后内皮细胞减少,周细胞呈优势生长。免疫细胞化学表明PDGFRβ和NG2阳性,v WF和GFAP阴性;流式结果表明细胞PDGFRβ的阳性率为95.52%±2.55%,GFAP为0.63%±0.26%,CD31为0.80%±0.26%,CD11b为1.02%±0.35%。10%胎牛血清的DMEM促进细胞分化,α-SMA表达升高。成管实验周细胞与内皮细胞共同形成管腔样结构。结论通过PCM筛选法能够成功获得纯度较高的SCMP,所获得的细胞具备明显的周细胞的形态和功能特征。     

Labelling of interneurones by retrograde transsynaptic transport of horseradish peroxidase from motoneurones in rats and cats

Transsynaptic transport of conjugates of wheat germ agglutinin with horseradish peroxidase (WGA-HRP) has been studied in rats and cats. WGA-HRP was injected into a muscle nerve from which it was first transported to motoneurones and along sensory fibres, and secondarily to interneurones. More extensive labelling of interneurones occurred in preparations with only relevant ventral roots intact than in preparations with only dorsal roots intact, which indicates that the transsynaptic transport of WGA-HRP is primarily in the retrograde direction, i.e. from motoneurones to interneurones. Such a transport appeared to be considerably enhanced by neuronal activity.     

不同神经示踪剂组合对大鼠脊髓运动神经元的逆行标记效率比较

目的:探讨神经示踪剂荧光金(FG)、真蓝(TB)和荧光红(FR)两两组合对脊髓运动神经元的标记效率差异,为再生神经重支配准确性研究奠定基础.方法;采用大鼠胫神经示踪模型,采取神经内注射与神经横断后近侧断端浸泡(20 min)2种方式,分别对FG、TB和FR的两两组合进行示踪试验.示踪术后5d,取脊髓腰膨大段冷冻纵切,共聚焦显微镜进行显微成像和计数.结果:FG联合TB示踪标记的运动神经元数量最多,其次为FG联合FR,而FR联合TB组标记细胞数最少,双标比例也最小.神经断端浸泡方式使用示踪剂时标记效率仅为神经内注射的2/3左右.结论:FG联合TB以及FG联合FR示踪对脊髓运动神经元的标记效果较好,且神经内注射使用示踪剂效果优于持续20 min的神经断端浸泡.     

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。     

小鼠脊髓灰质发育过程中细胞迁移与血管之间的关系

目的:探讨小鼠神经系统发育过程中脊髓成熟神经元迁移与血管发育之间的关系。方法:不同年龄小鼠共计75只,应用免疫荧光及墨汁灌注的技术,标记小鼠胚胎E17到P30脊髓神经元和血管。结果:大约在胚胎E17左右,小鼠脊髓灰质内开始出现NeuN阳性的神经元,白质中神经元较少,且此时灰质和白质内血管分布均匀,管径一致,分支较少。随着年龄的增长,脊髓周围的神经元不断向内迁移,灰质内NeuN阳性的神经元数先增多后减少且血管体密度先增加后减小,而白质内的神经元持续减少,血管逐渐稀疏。P14以后,脊髓灰质内的血管密度明显高于白质内的血管密度。同时,研究中还发现部分NeuN阳性细胞紧贴着血管分布。结论:在小鼠脊髓的发育过程中,NeuN阳性细胞由脊髓周围逐渐向中心迁移,和脊髓灰质H形态的形成密切相关。同时,血管扮演着重要的角色,它可能通过与神经元的相互作用,引导神经元的迁移,并且为神经元的迁移提供路径和支架。     

Spinal neurons involved in the control of the seminal vesicles: a transsynaptic labeling study using pseudorabies virus in rats

The seminal vesicles are male accessory sex glands that mainly contribute the seminal fluid of the ejaculate. Previous studies have suggested that seminal vesicles are supplied by both sympathetic and parasympathetic nerves. However, this conclusion was mainly based on studies in pelvic major ganglions and direct neuroanatomical evidence of spinal neurons innervating the seminal vesicles is still lacking. In order to map the spinal nerve circuit innervating the seminal vesicles, the present study used the pseudorabies virus (PRV) retrograde tracing technique in combination with immunohistochemistry. Three groups of rats were prepared: (1) nerves intact; (2) right hypogastric nerve and bilateral pelvic nerves sectioned; (3) right pelvic and bilateral hypogastric nerves sectioned. For the intact group, 3 to 5 days after injection of PRV into the left seminal vesicle in male rats, immunohistochemistry for PRV was performed to map the control circuit. Double immunofluorescence experiments against PRV and choline acetyltransferase (ChAT) were performed to discriminate preganglionic neurons and interneurons. Double detection of PRV and galanin (GAL) was also performed to identify lumbar spinothalamic (LSt) cells. Three days after virus injection, both sympathetic and parasympathetic preganglionic neurons were retrograde-labeled. Four days after injection of PRV into the seminal vesicles, PRV-infected neurons were found in the dorsal horn, ventral horn, dorsal gray commissure (DGC), medial gray matter and intermediolateral cell column (IML) from T13 to S1. For the group with an intact hypogastric nerve, 4 days after injection of PRV into the seminal vesicles, PRV-infected neurons were mainly located in DGC and IML of spinal lumbar segments (L) 1-L2. However, in the group with an intact pelvic nerve, PRV-infected neurons were mainly located in DGC of L5-S1 spinal segments. At the L3-L4 level, most of the virus-labeled neurons around the central canal expressed immunoreactivity for GAL, strongly suggesting that they could be LSt cells. These anatomical data support the idea that the sympathetic and parasympathetic nervous system are both involved in the control of the seminal vesicles and we demonstrated a connection between preganglionic neurons innervating the seminal vesicles and LSt cells which play a crucial role in coordinating the spinal control of ejaculation.     

Non-muscarinic effects of atropine on calcium conductances in cultured mouse spinal cord neurons

Cultured neurons derived from mouse spinal cord were studied using intracellular recording techniques. Effects of muscarinic cholinergic antagonists (atropine) on voltage-dependent membrane events, which could not be related to muscarinic receptors are described. Atropine (in nanomolar to micromolar concentrations) blocks calcium conductances in a manner which is not blocked by carbachol (100 microM). A direct effect of atropine on membrane Ca2+ conductances is suggested.     

Glia to neuron ratio in the posterior aspect of the human spinal cord at thoracic segments relevant to spinal cord stimulation

Spinal cord stimulation (SCS) applied between T8 and T11 segments has been shown to be effective for the treatment of chronic pain of the lower back and limbs. However, the mechanism of the analgesic effect at these medullary levels remains unclear. Numerous studies relate glial cells with development and maintenance of chronic neuropathic pain. Glial cells are electrically excitable, which makes them a potential therapeutic target using SCS. The aim of this study is to report glia to neuron ratio in thoracic segments relevant to SCS, as well as to characterize the glia cell population at these levels. Dissections from gray and white matter of posterior spinal cord segments (T8, T9, intersection T9/T10, T10 and T11) were obtained from 11 human cadavers for histological analyses. Neuronal bodies and glial cells (microglia, astrocytes and oligodendrocytes) were immunostained, microphotographed and counted using image analysis software. Statistical analyses were carried out to establish significant differences of neuronal and glial populations among the selected segments, between the glial cells in a segment, and glial cells in white and gray matter. Results show that glia to neuron ratio in the posterior gray matter of the human spinal cord within the T8–T11 vertebral region is in the range 11 : 1 to 13 : 1, although not significantly different among vertebral segments. Glia cells are more abundant in gray matter than in white matter, whereas astrocytes and oligodendrocytes are more abundant than microglia (40 : 40 : 20). Interestingly, the population of oligodendrocytes in the T9/T10 intersection is significantly larger than in any other segment. In conclusion, glial cells are the predominant bodies in the posterior gray and white matter of the T8–T11 segments of the human spinal cord. Given the crucial role of glial cells in the development and maintenance of neuropathic pain, and their electrophysiological characteristics, anatomical determination of the ratio of different cell populations in spinal segments commonly exposed to SCS is fundamental to understand fully the biological effects observed with this therapy.     

DO11.10转基因小鼠脊髓损伤后的运动功能恢复

目的:通过比较两种小鼠-DO11.10和BALB/c小鼠脊髓损伤后不同的运动功能恢复程度和损伤区的形态学变化来分析T淋巴细胞对脊髓损伤修复的影响。方法:建立小鼠脊髓重度夹伤模型,HE染色,GFAP、CD11b和淋巴细胞免疫组织化学实验分析损伤14 d、21 d后损伤区的变化;在损伤后0 d、7 d、14 d和21 d进行Basso-Beattie-Bresnahan(BBB)运动评分来评价小鼠运动功能恢复情况。结果:脊髓损伤后21 d,BALB/c小鼠产生了较厚的胶质瘢痕并伴有明显的固缩现象;而DO11.10小鼠中只观察到稀薄的胶质瘢痕形成并没出现明显的固缩。DO11.10小鼠脊髓损伤后14 d,巨噬/小胶质细胞浸润较BALB/c小鼠明显增加。脊髓损伤后21 d,DO11.10小鼠脊髓损伤区中的T淋巴细胞较BALB/c小鼠明显减少。BBB运动评分显示,在损伤后21 d内DO11.10小鼠较BALB/c小鼠有显著的运动功能恢复。结论:脊髓损伤后21 d内,针对神经组织抗原的自身反应性T淋巴细胞的浸润和存在不利于脊髓损伤后的运动功能恢复。     

大鼠脊髓损伤后植入人发角蛋白的实验研究

目的：研究人发角蛋白（HHK）在脊髓损伤（SCI）修复中诱导和促进神经元和神经纤维的再生以及HHK在脊髓中的降解机理。方法：选用12只成年雌性SD大鼠,采用改制的II型NYU装置,建立SCI组,SCI后植入HHK组,并设正常对照组,分别于术后14d取材,经HE,Mallory's－磷钨酸－苏木素,Loyer'sSterry Thionin等方法染色,观察其光镜结构的变化。结果：与损伤组相比较,植入HHK组损伤节段的萎缩程度明显,然质中部分神经元残存,有髓神经纤维脱髓鞘现象减轻,HHK周边集聚大量巨噬细胞和胶质细胞,植入的HHK毛小皮膨胀松弛,出现与皮质层分离,皮质层内出现皲裂,中央髓质腔增大,结论：植入HHK具有减轻脊髓损伤后的继发性损伤的作用,为进一步研究HHK在SCI修复过程中的作用及机理提供了形态学基础。     

人脊髓内NF样阳性神经元的形态及分布

目的：观察不同胎龄胎儿脊髓神经丝蛋白(NF)阳性神经元的形态、分布和发育变化，为脊髓-脊髓移植选择适宜的胎龄提供形态学依据。方法：胎儿脊髓19例，SP免疫组化染色，图像分析。结果：脊髓侧角内NF阳性神经元由中央管向外迁移；前角神经元由外向内迁移。NF阳性神经元密度在胚胎早期逐渐升高，晚期呈下降趋势。NF阳性神经元在胎龄16周时，胞体呈圆形、卵圆形，突起少沙，胞核大，有偏极现象，至32周时胞体呈锥形、梭形、多角形；胞体逐渐增大，胞浆逐渐增多，胞核多位居中央；突起增多。结论：未发育成熟的神经元内也有NF的存在。脊髓内NF阳性神经元密度随胎龄增加而逐渐增加，形态逐渐成熟，提示人脊髓-脊髓移植时以16周胎龄作供体较为适宜。     

A double-labelling study of reticular collaterals to the spinal cord and cerebellum of the North American opossum

Injections of horseradish peroxidase into either the spinal cord or cerebellum label neurons in the gigantocellular and lateral reticular nuclei of the North American opossum. In order to determine if neurons which project to the spinal cord and cerebellum are intermingled in these two nuclei and if single neurons provide collaterals to both areas, we have employed fluorescent markers in double-labelling experiments. Our results show that reticular neurons innervating either the spinal cord or cerebellum are often close together and that a few provide collaterals to both areas. Neurons providing such collaterals are rare, however, comprising 2% or less of those innervating either target alone.