Distribution of Nitrergic Neurons in the Dorsal Root Ganglia of the Bottlenose Dolphin (Tursiops truncatus)

Dorsal root ganglia (DRGs) contain the cell bodies of primary afferent neurons that transmit sensory information from the periphery into the spinal cord. Distinct populations of DRG neurons have been characterized by a variety of different immunohistochemical markers. A subpopulation of ganglionic neurons containing neuronal nitric oxide synthase (nNOS), an enzyme known to generate nitric oxide, has been detected in a number of mammalian species. Despite previous studies, no information is known on the presence and exact distribution of nNOS‐immunoreactive neurons in the DRGs of the bottlenose dolphin. In this investigation, immunoperoxidase for nNOS was used to determine the distribution and the perikaryal size of nitrergic neurons in the DRGs of this species. Double immunofluorescence protocol was used to determine the percentage of nNOS‐immunoreactive (IR) neurons over the total primary afferent neurons. In addition, double immunostaining was used to verify whether there was colocalization of nNOS with substance P (SP). In all DRGs, a subpopulation of small‐ and medium‐sized neurons (about 9%) exhibited nNOS immunoreactivity. Data analysis revealed that the majority of nNOS‐IR neurons (81.3%) expressed SP. The density of nNOS‐immunoreactive and nNOS/SP‐double immunopositive cells was relatively constant throughout the ganglia. However, as observed in others mammals, the number of nitrergic neurons decreased in the caudalmost DRGs. Our results, in conjunction with previous observations, suggest that nNOS‐IR neurons may be involved in the afferent transmission of visceral and nociceptive information as well as in the regulation of the vascular tone. Anat Rec,, 2011. © 2011 Wiley‐Liss, Inc.     

中脑导水管周围灰质和E-W核中SP和CCK样神经元至脊髓的投射——HRP与免疫细胞化学结合法研究

本研究用HRP逆行追踪和免疫细胞化学结合法,研究了大白鼠中脑导水管周围灰质(PAG)以及Edinger-Westphal(E-W)核至脊髓的SP能和CCK能神经投射。结果表明,PAG腹外侧区至脊髓颈、胸、腰段双侧均有少量投射,但以同侧为主。在这些下行投射神经元中,约有48％为SP样免疫反应阳性。E-W核至脊髓的颈、胸、腰段均有广泛投射,其中约有70％为SP样免疫反应阳性,73％为CCK样免疫反应阳性。提示至少有一部分E-W核的脊髓投射神经元含有SP和CCK两种神经递质。     

实验性哮喘豚鼠内脏感觉传入部位SP免疫反应的研究

用免疫组织化学方法结合显微图像分析 ,观察哮喘豚鼠脊神经节、结状神经节、脊髓和孤束核内 P物质的变化。正常豚鼠 C7～ T5 脊神经节及结状神经节内存在 SP阳性反应细胞 ,其细胞质内充满棕色阳性反应颗粒 ,并可见大量的阳性反应纤维。 P物质免疫反应也见于 C7～ T5 脊髓后角板层 、板层 、中间带外侧核 ,呈点状分布 ,偶见阳性纤维。孤束核内可见大量的 P物质阳性反应。哮喘豚鼠结状神经节、C7～ T5 脊神经节和相应节段脊髓后角、孤束核内 P物质阳性反应的密度明显增多而平均灰度值却明显地低于对照组。上述结果表明 ,哮喘豚鼠内脏感觉传入部位 P物质表达增强 ,提示这些部位的 P物质可能参与哮喘的发病过程     

NGF、BDNF及受体trkA、trkB、trkC在正常猴脊髓的表达

采用免疫组织化学方法观察了神经生长因子 (NGF) ,脑源性神经营养因子 (BDNF)以及 NGF家族因子受体 trk A、trk B、trk C的免疫阳性反应在正常猴脊髓的分布。结果表明 :NGF免疫反应阳性的神经元在脊髓灰质各层中均有分布 ,灰、白质内也可见较多的 NGF免疫反应阳性的胶质细胞。 BDNF在脊髓各型神经无有明显的表达 ,特别是前角运动神经元。 trk A、trk B、trk C的免疫阳性反应产物主要分布在灰质的神经元及胶质细胞。本实验结果揭示了在正常猴脊髓中神经营养因子 (NGF、BDNF )及受体 trk A、trk B、trk C的表达状况 ,提示这些神经营养因子及受体在维持猴脊髓神经元的正常生理功能中具有重要作用。     

Caspase-3在成年恒河猴腰髓的分布

目的采用免疫组化SP法研究caspase-3在正常猴腰髓中的表达情况。方法2只正常恒河猴经心脏灌注固定取脊髓,行常规免疫组化(SP法)。结果Caspase-3的阳性反应产物主要分布在脊髓的前角的大型运动神经元和中型神经元的胞浆、胞核和突起;灰质侧角、中间带的神经元和白质内的胶质细胞也有着色。结论Caspase-3广泛存在于正常恒河猴腰髓,参与其凋亡的发生及其它功能的调控。     

Immunohistochemical indications of gastrin releasing peptide--bombesin-like immunoreactivity in the nervous system of the rat. Codistribution with substance P-like immunoreactive nerve terminal systems and coexistence with substance P-like immunoreactivity in dorsal root ganglion cell bodies

Weak to strong gastrin releasing peptide--bombesin (GRP-Bn)-like immunoreactivity was found in fine varicose nerve terminal systems of low to high densities in several parts of the CNS. The highest densities of strongly immunoreactive terminals were found in the marginal layer and in the substantia gelatinosa of the spinal cord, and in parts of the nuc. tractus spinalis nervi trigemini. Morphometrical analysis in the spinal cord demonstrates that GRP-BN-like-immunoreactive and substance P (SP), but not somatostatin (SS)-immunoreactive nerve terminals strikingly codistribute. Coexistence of SP and GRP-BN-like immunoreactivities was demonstrated in trigeminal and spinal ganglion nerve cells. Thus, GRP-BN-like immunoreactivity may coexist with SP in certain SP-immunoreactive nerve terminal systems.     

Early blockade of injured primary sensory afferents reduces glial cell activation in two rat neuropathic pain models

Satellite glial cells in the dorsal root ganglion (DRG), like the better-studied glia cells in the spinal cord, react to peripheral nerve injury or inflammation by activation, proliferation, and release of messengers that contribute importantly to pathological pain. It is not known how information about nerve injury or peripheral inflammation is conveyed to the satellite glial cells. Abnormal spontaneous activity of sensory neurons, observed in the very early phase of many pain models, is one plausible mechanism by which injured sensory neurons could activate neighboring satellite glial cells. We tested effects of locally inhibiting sensory neuron activity with sodium channel blockers on satellite glial cell activation in a rat spinal nerve ligation (SNL) model. SNL caused extensive satellite glial cell activation (as defined by glial fibrillary acidic protein [GFAP] immunoreactivity) which peaked on day 1 and was still observed on day 10. Perfusion of the axotomized DRG with the Na channel blocker tetrodotoxin (TTX) significantly reduced this activation at all time points. Similar findings were made with a more distal injury (spared nerve injury model), using a different sodium channel blocker (bupivacaine depot) at the injury site. Local DRG perfusion with TTX also reduced levels of nerve growth factor (NGF) in the SNL model on day 3 (when activated glia are an important source of NGF), without affecting the initial drop of NGF on day 1 (which has been attributed to loss of transport from target tissues). Local perfusion in the SNL model also significantly reduced microglia activation (OX-42 immunoreactivity) on day 3 and astrocyte activation (GFAP immunoreactivity) on day 10 in the corresponding dorsal spinal cord. The results indicate that early spontaneous activity in injured sensory neurons may play important roles in glia activation and pathological pain.     

Vasoactive intestinal polypeptide increases in areas of the dorsal horn of the spinal cord from which other neuropeptides are depleted following peripheral axotomy

Summary Peripheral nerve section or local capsaicin application produces depletion of substance P and an enzymatic marker, fluoride-resistant acid phosphatase (FRAP), from circumscribed regions of the terminal areas in the spinal cord. We have made use of this phenomenon to map the extent of central termination of subpopulations of primary afferent neurons containing substance P (SP), somatostatin (SOM), cholecystokinin (CCK), vasoactive intestinal polypeptide (VIP) and FRAP in the rat lumbar spinal cord following sciatic nerve section at midthigh level under ether anaesthesia. Between 2 days and 1 year postoperatively, the animals were perfused transcardially and SP, CCK, VIP and SOM were localised in frozen transverse sections of spinal cord segments L1 to S2 and their corresponding ganglia using unlabelled antibody immunohistochemistry. FRAP was localised using a modified Gomori method. SP, SOM, CCK and FRAP were maximally depleted from identical restricted areas of the dorsal horn of the third, fourth and fifth lumbar segments fifteen days after nerve section and remained so for a year. In contrast, VIP staining increased dramatically in the areas from which the other markers were depleted and showed the same time course. Moreover, a large number of neurons in the corresponding ganglia showed positive VIP immunoreactivity after axotomy but were absent from the unoperated side.     

Nogo(N-18)在成年大鼠三叉神经节、背根节及腰段脊髓中分布的免疫组织化学研究

目的：观察Nogo在成年大鼠三叉神经节、背根节及腰段脊髓中的分布，探讨其存在的意义。方法：采用抗Nogo(N-18)多克隆抗体免疫组织化学SP法。结果：成年大鼠三叉神经节、背根节感觉神经元胞浆和胞核以及腰段脊髓灰质神经元胞核内出现Nogo免疫反应。结论：Nogo免疫反应存在于大鼠三叉神经节、背根节感觉神经元胞浆和胞核以及腰段脊髓灰质神经元胞核内，应重新认识Nogo的功能。     

大鼠脊髓后角浅层内六种肽类和5-羟色胺的来源及其功能探讨

分别切断大鼠一侧坐骨神经和半横切颈髓上段,用免疫组织化学技术观察了L_(4-6)后角浅层内SP、CCK、SOM、VIP、L-ENK、NT、5-HT等的量的改变。并半横切L_5节段的吻、尾侧,观察了NT在同节段内的含量改变。 1.切断坐骨神经后,术侧L_(4-6)后角浅层的坐骨神经占位区内SP、CCK、SOM、VIP有不同程度的脱失,SP、CCK最明显。结合文献,作者认为后根节中SP、CCK、SOM、VIP阳性神经元外周突部分分布于外周躯体神经。 2.半横切颈髓后,术侧L_(4-5)后角浅层的5-HT阳性反应物基本消失,而L_6后角浅层内有少量存留。CCK、L-ENK阳性反庆物在L_(4-6)节段背外侧索中明显减少,但后角浅层只有轻微减少。SOM在术侧后角浅层中也似有轻微减少。这些提示脑结构到脊髓后角浅层的下行纤维中除含有5-HT外还含有一些神经肽。 3.L—ENK阳性反应物在切断坐骨神经后轻度增高;SOM无论在切断坐骨神经或半横切劲髓例均变比不大;NT在此两组实验中均无变化,且在L_5节段吻、尾侧半切腰髓后L_5后角浅层内仍无变化。以上提示分布于后角浅层的某些肽类有一部分直接来自脊髓内甚至同节段内的固有神经元。     

Differential galanin upregulation in dorsal root ganglia and spinal cord after graded single ligature nerve constriction of the rat sciatic nerve

Single ligature nerve constriction (SLNC) is a newly developed animal model for the study of neuropathic pain. SLNC of the rat sciatic nerve induces pain-related behaviors, as well as changes in the expression of neuropeptide tyrosine and the Y(1) receptor in lumbar dorsal root ganglia (DRGs) and spinal cord. In the present study, we have analyzed the expression of another neuropeptide, galanin, in lumbar DRGs and spinal cord after different degrees of constriction of the rat sciatic nerve. The nerve was ligated and reduced to 10-30, 40-80 or 90% of its original diameter (light, medium or strong SLNCs). At different times after injury (7, 14, 30, 60 days), lumbar 4 and 5 DRGs and the corresponding levels of the spinal cord were dissected out and processed for galanin-immunohistochemistry. In DRGs, SLNC induced a gradual increase in the number of galanin-immunoreactive (IR) neurons, in direct correlation with the degree of constriction. Thus, after light SLNC, a modest upregulation of galanin was observed, mainly in small-sized neurons. However, following medium or strong SLNCs, there was a more drastic increase in the number of galanin-IR neurons, involving also medium and large-sized cells. The highest numbers of galanin-IR neurons were detected 14 days after injury. In the dorsal horn of the spinal cord, medium and strong SLNCs induced a marked ipsilateral increase in galanin-like immunoreactivity in laminae I-II. These results show that galanin expression in DRGs and spinal cord is differentially regulated by different degrees of nerve constriction and further support its modulatory role on neuropathic pain.     

Immunohistochemical Properties of the Peripheral Neurons Projecting to the Pig Bulbospongiosus Muscle

Retrograde neuronal tracing and single labelling immunofluorescence methods were used to define the neurochemical content of the peripheral autonomic and sensitive neurons projecting to the male pig striated bulbospongiosus muscle (BSM). The retrograde fluorescent neuronal tracer Fast Blue (FB) was injected into the left bulbospongiosus muscle of four intact impuberal pigs. After a 10‐day survival time, the ipsilateral sacral sympathetic trunk ganglia (STGs), the caudal mesenteric ganglion (CMG), and the sacral spinal ganglia (SGs) were collected from each animal. In FB+ neurons of these ganglia, the presence of cathecolamine‐ (tyrosine hydroxylase‐TH), acetylcholine‐ (vesicular acetylcholine transporter‐VChAT), or nitric oxide‐synthesizing (neuronal Nitric Oxide Synthase‐nNOS) enzymes and of some biologically active peptides (calcitonine gene‐related peptide‐CGRP, Leu‐Enkephaline‐LENK, Neuropeptide Y‐NPY, Substance P‐SP and Vasoactive Intestinal Peptide‐VIP) were studied. The ipsilateral STGs FB+ neurons showed immunoreactivity principally for TH and NPY and in decreasing order for VIP, VChAT, SP, CGRP, nNOS, and LENK. The left CMG FB+ neurons were immunoreactive to TH and NPY, and in smaller proportions for VIP, LENK, VChAT, CGRP, nNOS, and SP. The ipsilateral SGs FB+ neurons resulted immunoractive for CGRP, LENK, NPY, nNOS, SP, and VChAT. The heterogeneous neurochemical content of the peripheral neurons projecting to the pig BSM allows us to hypothesize the involvement of autonomic ganglia in the activity of both blood vessels and striated fibers of the muscle and the involvement of sensory ganglia in the afferent transmission from the muscle to spinal cord and in antidromic mechanisms that causes the relaxation of the BSM blood vessels. Anat Rec, 299:1192–1202, 2016. © 2016 Wiley Periodicals, Inc.     

Immunohistochemical studies on the effect of capsaicin on spinal and medullary peptide and monoamine neurons using antisera to substance P, gastrin/CCK, somatostatin, VIP, enkephalin, neurotensin and 5-hydroxytryptamine

Summary After neonatal treatment of rats with capsaicin, the spinal cord, the spinal trigeminal nucleus and spinal and trigeminal ganglia were analysed with immunohistochemistry using antisera to several peptides and 5-hydroxytryptamine. A marked decrease was observed in substance P-, cholecystokinin-, somatostatin- and VIP-like immunoreactivity present in the central branches of primary sensory neurons in the spinal cord and in substance P- and somatostatin-like immunoreactivity in sensory ganglion cells. No definite depleting effect of capsaicin could be established on 5-hydroxytryptamine and peptides, such as enkephalin and neurotensin, present in centrally originating fibres in the dorsal horn of the spinal cord. The results demonstrate that the effects of capsaicin are not confined to substance P immunoreactive primary sensory neurons. The possibility is discussed that capsaicin effects specifically functioning rather than chemically specific primary sensory neurons.     

BDNF及其受体TrkB在成年恒河猴脊髓的表达

目的观察脑源性神经营养因子BDNF及其高亲合力受体TrkB在成年恒河猴胸髓的表达分布,为成年恒河猴脊髓BDNF、TrkB的功能研究提供形态学依据。方法灌注固定成年雄性恒河猴,取其胸段脊髓制作冰冻切片,用BDNF、TrkB特异性抗体行免疫组织化学SP法染色,观察BDNF、TrkB免疫阳性物质在胸段脊髓的分布。结果成年恒河猴胸髓内均可见BDNF、TrkB免疫反应阳性神经元,反应产物呈棕黄色颗粒。BDNF和TrkB免疫反应阳性细胞遍及胸髓灰质各板层,在腹角、侧角以及中央管邻近区域均可见阳性神经元,它们的阳性产物主要定位于神经元胞浆中,胞浆着色深,胞浆与胞核的界线清楚。BDNF免疫反应细胞的胞核未见染色,而不同的TrkB阳性细胞其胞核染色不一。结论成年恒河猴胸髓存在BDNF、TrkB的表达,其作用可能与猴脊髓的正常生理功能和损伤后的修复有关。     

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.     

Coexistence of varying combinations of neuropeptides with 5-hydroxytryptamine in neurons of the raphe pallidus et obscurus projecting to the spinal cord

The coexistence of varying combinations of substance P (SP), somatostatin (SOM), thyrotropin-releasing hormone (TRH) and met-enkephalin-Arg-Gly-Leu (ENK) with 5-hydroxytryptamine (5-HT) as semiquantitatively revealed by immunocytochemistry in neuronal perikarya of the raphe pallidus et obscurus in the guinea-pig was analyzed. SOM coexisted most frequently with 5-HT, followed by SP, ENK and TRH. Many 5-HT neurons were immunoreactive to 2 or more peptides such as SP/SOM, SOM/ENK, SP/ENK, SOM/TRH, SP/TRH or SOM/SP/ENK. Most of these neurons were shown to project to the spinal cord by retrograde HRP labeling combined with immunocytochemistry. After hemisection of the cervical spinal cord at the C5 level, ENK and 5-HT immunoreactive nerve terminals in the ipsilateral intermediolateral nucleus of the thoracic spinal cord were decreased in number. The results indicate that neurons in the raphe pallidus et obscurus projecting to the spinal cord can be classified into subpopulations according to which peptides coexist with 5-HT, and may have different functions.     

Alteration of Glial Fibrillary Acidic Proteins Immunoreactivity in Astrocytes of the Spinal Cord Diabetic Rats

Diabetes affects retinal and nervous glial cells, especially the astrocytes. A key indicator of this response is the alteration in the level of intermediate filament glial fibrillary acidic protein (GFAP) and number of GFAP‐immunoreactive astrocytes. To date, no study has investigated the effect of diabetes on the distribution of GFAP‐immunoreactive astrocytes in the spinal cord. Therefore, the present study investigated the effect of diabetes on the number of GFAP‐immunoreactive astrocytes in the gray matter of the spinal cord of streptozotocin‐induced diabetic Wistar rats. Animals were divided into six groups (n = 7); 6 weeks and 12 weeks diabetic duration groups and their respective age‐matched normal control and sham control groups. Our results demonstrated a significant (P < 0.001) decrease in the number of GFAP‐immunoreactive astrocytes in different areas of the spinal cord sections of the 6 weeks and 12 weeks long diabetic rats when compared with the spinal cord of normal and sham control groups of comparable age. The mean percentage in total number of GFAP‐immunoreactive astrocytes in the whole gray matter areas of the spinal cord of the 6 and 12 weeks diabetic groups were approximately 28% and 41% less than control groups. Furthermore, the 12 weeks diabetic group showed a significant (P < 0.001) reduction in the number of GFAP‐immunoreactive astrocytes when compared with the 6 weeks diabetic animals. These results suggest that the induction of diabetes is associated with a reduction in GFAP‐positive astrocytes in the spinal cord, which may affect the functional support and role of astrocytic cells in the nervous tissue. This in turn may contribute to the pathological changes associated with diabetic state in the central nervous system. Anat Rec, 291:390–399, 2008. © 2008 Wiley‐Liss, Inc.     

A subpopulation of preganglionic parasympathetic neurons in the rat contain adenosine deaminase

Immunohistochemical staining and retrograde fluorescent tracing techniques were used to demonstrate the presence of adenosine deaminase in preganglionic parasympathetic neurons. Both brainstem and sacral spinal cord parasympathetic nuclei were found to contain a subpopulation of neurons immunoreactive for adenosine deaminase. Immunostaining of preganglionic neurons in brainstem was restricted to a group of cells which were shown by retrograde tracing with Fast Blue to project exclusively to the sphenopalatine ganglion. This group was defined as the lacrimo-nasopalatine parasympathetic nucleus. Neurons in all other cranial preganglionic centers were devoid of adenosine deaminase immunoreactivity. In spinal cord adenosine deaminase-immunoreactive neurons were found in the intermediolateral gray matter in the region of the sacral parasympathetic nucleus. Injections of Fast Blue into the pelvic ganglion labeled large numbers of neurons in this nucleus, only some of which contained adenosine deaminase. The majority of neurons immunoreactive for adenosine deaminase were also shown to be immunoreactive for choline acetyltransferase in both brainstem and sacral parasympathetic nuclei. The present results show that a subclass of preganglionic parasympathetic neurons are among the few structures in the central nervous system that express what appear to be high levels of adenosine deaminase. This observation together with evidence suggesting that purines serve as neurotransmitters in some sacral parasympathetic neurons supports the notion that adenosine deaminase may constitute a marker for adenine nucleoside and/or nucleotide neurotransmission.     

孤儿受体酪氨酸激酶在成年大鼠腰段脊髓和背根节中的分布

目的　观察孤儿受体酪氨酸激酶 (Ret)在成年大鼠腰段脊髓和背根节中的分布 ,探讨胶质细胞源性神经营养因子 (GDNF)对大鼠脊髓和背根节神经元的作用。　方法　采用Ret抗体免疫组织化学ABC法。　结果　Ret免疫反应存在于成年大鼠腰段脊髓神经元和背根节神经元中。　结论　Ret在大鼠腰段脊髓和背根节神经元中有一定的分布