Expression of TrkA receptor for neurotrophins in trigeminal neurons innervating the rat gingivomucosal tissue

The purpose of this study was to characterize and evaluate the expression of TrkA receptor in trigeminal ganglion (TG) neurons that innervate the rat gingivomucosal tissue. A retrograde nerve tracer Fluorogold (FG) was injected into the gingiva (group 1) or applied into the gingival sulcus (group 2) of the first right maxillary molar to identify the neurons in TG that innervate the gingivomucosa. After 10 days TG were dissected and FG fluorescence in neurons was observed under UV light microscope. To draw a comparison, approximately 1000 neurons per ganglion from the entire TG (group 3) and approximately 350 neurons per ganglion from the maxillary region in TG (group 4), were analyzed. Expression of TrkA receptor in TG neurons was investigated by immunohistochemistry. About 70% of neurons in groups 1 and 2 contained TrkA receptor, which was statistically significantly more than in groups 3 (41%) and 4 (38%). FG-labeled TrkA-immunopositive neurons were predominantly small or medium-sized (less than 1200microm(2)). However, the neurons innervating the rat gingivomucosa were on average larger than the neurons in the entire TG or in the maxillary region. In conclusion, the majority of neurons in TG that innervate the rat gingivomucosa are small or medium-sized, contain TrkA receptor and are most probably nociceptive.     

Morphological and immunohistochemical characterization of the trigeminal ganglion neurons innervating the cornea and upper eyelid of the rat

The cornea is sensitive to nociceptive stimuli and receives dense sensory innervations from the trigeminal ganglion, which also innervates the upper eyelid. We investigated the morphological and immunohistochemical characterization of the trigeminal ganglion neurons innervating the cornea and upper eyelid. We injected the retrograde tracer Fluoro-Gold (FG) into the cornea and the retrograde tracer cholera toxin subunit b (CTb) into the upper eyelid of the same animal. Less than 10% of the FG-labeled neurons were also labeled with CTb. The FG-labeled neurons were small (29.6 ± 0.6 μm), while the CTb-labeled neurons were large (36.1 ± 0.5 μm). We also characterized the neurons in the trigeminal ganglion with the retrograde tracer FG following its injection into the cornea or the upper eyelid, and immunohistochemical double-labeling with nociception-related neuronal markers, such as calcitonin gene-related peptides (CGRP), transient receptor potentiated vanilloid 1 (TRPV1), and substance P (SP). About 27% of the neurons innervating the cornea were double-labeled with CGRP, about 23% with TRPV1, and about 8% with SP. About 4% of the neurons innervating the upper eyelid were double-labeled for CGRP, about 11% for TRPV1, and 3% for SP. Thus, the percentages of double-labeled neurons for the neurons innervating the cornea were higher than those for the neurons innervating the upper eyelid. These results indicate that the cornea and the upper eyelid receive innervations mainly from different neurons of the trigeminal ganglia. The cornea is innervated by many characteristic sensory neurons containing nociception-related neuronal markers.     

Capsaicin receptor immunoreactivity in the human trigeminal ganglion

The cloned capsaicin receptor, also known as vanilloid receptor subtype 1 (VR1) receptor, has been demonstrated to be an integral membrane protein with homology to a family of putative store-operated calcium channels. The VR1 receptor is activated not only by capsaicin but also by noxious heat and protons, and therefore it is suggested as a molecular integrator of chemical and physical stimuli that elicit pain. In the present study, indirect immunofluorescence detected a small number of neurons that are VR1 receptor immunoreactive (ir) (171 versus 1038 or 16% of all neuronal cell bodies) in the human trigeminal ganglion (TG). In addition, RT-PCR confirmed the presence of VR1 mRNA in the human TG. It has been hypothesized that TG neuronal cell bodies are the source of capsaicin-stimulated release of calcitonin gene-related peptide (CGRP), and hence co-localization experiments were performed. Around 10% of the VR1 receptor-ir is expressed on neurons that contain CGRP-ir (ten among 74) in the human TG, indicating that capsaicin may act through the VR1 receptor to modulate the release of CGRP and in turn to modulate pain. We observed that 8% of the VR1 receptor-ir neuronal cell bodies contain substance P-ir and 5% nitric oxide synthase. Capsaicin can release nitric oxide, CGRP and substance P from sensory nerves and contribute to central sensitization.     

Ontogeny of sensory nerves in the developing skeleton

Background: Previous studies have shown that the neuropeptide calcitonin gene-related peptide (CGRP) have an influence on osteoclastic bone resorption and that CGRP and substance P (SP), both wellknown markers for sensory neurons, behave as growth factors. Materials and Methods: The ontogeny of the sensory nerves in the hindlimb skeleton of the rat was studied from gestational day (GD) 15 to neonatal day (ND) 24 by immunohistochemistry. Neurofilaments and nerve terminals were labelled with protein gene-product 9.5 (PGP 9.5) and synaptophycin (SYN) respectively. Results: PGP 9.5 appeared at GD 15 and SYN at GD 19, both in the perichondrial tissue of the long bones. One week later, at ND 4 nerve fibre, immunoreactive to PGP 9.5 and SYN were observed within the bone organ. Sensory nerves, indicated by CGRP and SP, were first discerned at GD 18–19 in the periosteal tissue of the diaphyseal and metaphseal regions and in the bone organ at ND 4. Approximately at ND 6, vascular as well as non-vascular nerves extended into the metaphyses and at ND 8 into the epiphyses, concomitant with the first signs of mineralization. Conclusions: The study shows that a functional sensory nerve supply of the developing bone organ occurs immediatly prior to partus, apparently parallel with an increasing mineralization. The combined findings may indicate a sensory influence on developmental processes in the skeleton. © 1995 Wiley-Liss, Inc.     

A qualitative and quantitative study of substance P immuno-cytochemistry of the trigeminal ganglion in the monkey

The immunoreactivity of substance P(SP) in the monkey trigeminal ganglion was examined and the distribution of immunoreactive cells determined. The monkey trigeminal ganglion is composed of clusters of sensory cells arranged in cords parallel to the long axis of the nerve fibres. The cells have prominent nuclei and are surrounded by satellite cells. Abundant organelles are randomly distributed throughout the cytoplasm. A striking feature of the ganglion was the presence of some axon-like prolifes containing mainly dense-cored vesicles and some agranular vesicles. Between 16 and 32% of the ganglion cells displayed SP-immunoreactivity. Most of the SP-IR cells were unipolar, small to medium-sized ganglion cells and they had no specific pattern of distribution. The staining of the SP-IR cells varied considerably, ranging from weak or moderate to heavy staining, although the majority of them were moderately stained. Immuno-electron microscopy showed that the SP-IR products were distributed throughout the soma of ganglion cells and not associated with any particular organelles or inclusions. The reaction products were also found in both myelinated and unmyelinated fibres between the ganglion cells. Another remarkable feature of the trigeminal ganglion was the occurrence of some SP-IR nerve fibres forming a rich glomerular network of pericellular arborizations around some of the SP-negative cells. Ultrastructural study showed the presence of some SP-IR nerve terminals in close approximation to some SP-negative cells, but there were no synaptic contacts. The relative frequency of the SP-IR pericellular arborizations paralleled the frequency of all the SP-IR cells. The results may imply that the pericellular arborizations function as a medium of communication between SP-positive and SP-negative sensory cells within the ganglion. It was suggested that the fibres forming the pericellular arborizations may originate from the intrinsic ganglion cells that are SP-positive.     

Depletion of calcitonin gene-related peptide from the caudal trigeminal nucleus of the rat after electrical stimulation of the Gasserian ganglion

Electrical stimulation of the Gasserian ganglion resulted in partial depletion of calcitonin gene-related peptide (CGRP) from ipsilateral central terminals of pseudounipolar primary sensory ganglion cells. Affected terminals exhibit decreased CGRP immunoreactivity as shown by cytophotometric densitomery of the caudal trigeminal nucleus. The decrease in CGRP immunoreactivity is statistically significant only in the medial one-third of the caudal trigeminal nucleus. Since earlier studies have shown that electrical stimulation of the Gasserian ganglion induces first accumulation then depletion of CGRP from perivascular sensory terminals in the dura mater, the present experiments suggest that CGRP is depleted also from central terminals of primary sensory trigeminal neurons, which might be of importance in the pathogenesis of migraine headache. Received: 18 December 1996 / Accepted: 26 June 1997     

经颞下窝入路行三叉神经节和眼-上颌神经显微手术及其应用

目的　建立经大鼠颅外进路行三叉神经节和眼 上颌神经显微手术的手术方法。方法　在研究大鼠三叉神经节对松果体的支配中 ,采用经颞下窝入路显露双侧三叉神经节和眼 上颌神经 ,向三叉神经节引入神经追踪剂行追踪研究 ,并行双侧眼 上颌神经切断术行退变研究。结果　向三叉神经节引入神经追踪剂的手术约需 1～ 2h ,双侧眼 上颌神经切断术约需2～ 3h。即使在眼 上颌神经完全切断的动物 ,其进食能力不受明显的影响。结论　该手术较以往采用的从颅顶去除一块颅骨和一部分脑组织的进路 ,对动物的损伤小 ,使得动物的死亡率下降 ,追踪剂对周围结构的污染减轻     

Gene expression for peptides in neurons of the petrosal and nodose ganglia in rat

Summary In situ hybridization was used to determine whether genes for neuropeptides [substance P/neurokinin A (SP/NKA), calcitonin gene-related peptide (CGRP), somatostatin (SOM), neuropeptide tyrosine (NPY) and cholecystokinin (CCK)] are expressed in inferior ganglia of the vagus (nodose) and glossopharyngeal (petrosal) nerves. Synthetic oligodeoxyribonucleotides, complementary to the cognate, mRNAs were labeled with [³²P] or [³⁵S], and hybridized to 10 m thick sections of unperfused tissue which were then processed for film and emulsion autoradiography. We found numerous, clustered neuronal perikarya throughout the nodose and petrosal ganglia that expressed preprotachykinin A (SP/NKA) and CGRP mRNAs to varying degrees. Neurons expressing preproSOM mRNA were less abundant and more scattered throughout both ganglia. Notably, we found mRNA for NPY in cells (usually 5–10 per section) in both ganglia. To our knowledge, this is first evidence for NPY in these sensory ganglia. In contrast to previous immunohistochemical findings, we found no evidence for expression of preproCCK in either the nodose or petrosal ganglia. The present findings demonstrate that cells of the nodose and petrosal ganglia express the genes for a number of neuropeptides that are presumably involved with transmission of visceral sensory afferent information to higher order neurons of the central nervous system.     

Fine structural features of light and dark cells in the trigeminal ganglion of the rat

Summary Prolonged fixation times and handling procedures designed to minimize the introduction of post mortem artifact were used in preparation of rat trigeminal ganglia for fine structural study. Larger, more numerous, light neurons and smaller, less numerous, dark neurons were observed. Major fine structural differences included a more discrete clumping of Nissl substance and more neurofilaments in the light compared with the dark cell. The results suggest that these differences are not artifacts of preparation.     

Excitatory amino acid binding sites in the trigeminal principal sensory and spinal trigeminal nuclei of the rat.

Quantitative autoradiography was used to examine the density and distribution of excitatory amino acid (EAA) binding site subtypes in the principal sensory and spinal trigeminal nuclei of the rat trigeminal complex. The highest densities of N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), kainate and metabotropic receptors were found in the superficial laminae (I and II) of subnucleus caudalis, a region known to be densely innervated by primary afferent nociceptive terminals. Lower densities of EAA binding sites were observed in spinal subnuclei interpolaris and oralis and within the principal sensory nucleus. These results are consistent with the hypothesis that EAAs are involved in primary afferent nociceptive neurotransmission.     

Regional differences in the distribution of nerve fibers showing substance P- and calcitonin gene-related peptide-like immunoreactivity in the rat larynx

Summary The distribution of substance P (SP) — and calcitonin gene-related peptide (CGRP) — containing nerve fibers in the rat larynx was studied using immunohistochemistry. Double-labeling studies revealed a high degree of co-existence of SP- and CGRP-like immunoreactivity (LI) in the nerve fibers in the larynx. There was a considerable regional difference in the number of immunoreactive nerve fibers in the epithelium and lamina propria. Richly supplied sites were the laryngeal side of the epiglottis and the ventral recess, whilst there was no evidence of nerve fibers in the squamous epithelium of the vocal cords. However, where the squamous epithelium of the vocal cords changed into a cuboidal epithelium, a moderate number of nerve fibers was present, and a large number of fibers was seen where the squamous epithelium of the cords was in close contact with cartilage. Nerve fibers showing SP- and CGRP-LI were also observed close to the acini and ducts of the glands, in the blood vessel walls, close to the perichondrium of all the cartilages, and outside the cricothyroid and cricoarytenoid joints. CGRP-LI was detected in epithelial cells facing the lumen of the airway and in cells in the acini and ducts of glands in the subglottic region and trachea. Unilateral sympathectomy did not affect the pattern of SP- and CGRP-innervation in the larynx, whereas after vagotomy, the SP- and CGRP-innervation almost disappeared ipsilaterally in the upper parts of the epiglottis and aryepiglottic folds.     

Coexistence of substance P and calcitonin gene-related peptide in the frog spinal cord

The localization of substance P (SP) and calcitonin gene-related peptide (CGRP) was studied in the untreated spinal cord of the frog using single or double immunohistochemical stainings. SP and CGRP appear to coexist in the primary afferent fibers and in the marginal and submarginal dorsal horn zones, as well as in the dorsolateral zone. In other parts of the spinal cord CGRP immunoreactivity was scanty while diffuse SP systems were seen, suggesting that the coexistence of the two peptides is restricted to primary afferent fibers.     

Distribution of SP- and CGRP-like immunoreactive nerve fibers in the lower respiratory tract of neonatal foals: evidence for loss during development

The lungs of neonatal foals contain many nerves immunoreactive for substance P and calcitonin gene-related peptide. These nerves are closely associated with the epithelium, bronchial and pulmonary vessels and the airway smooth muscle of all intrathoracic airways, including non-cartilaginous bronchioles. Activation of sensory nerves in the respiratory epithelium could thus potentially affect, via local axon reflexes, vascular and respiratory smooth muscle in neonatal equine airways. Nerves immunoreactive for these peptides are much more widely distributed within the lung than in adult horses; they may thus play a trophic role before birth, or contribute to the post-natal adaptation to breathing.     

Immunohistochemical evidence from co-localization and denervation studies for four types of substance P-containing nervous structures in the rat superior cervical ganglion

Four types of substance P-immunoreactive structures have been distinguished in the rat superior cervical ganglion by double-immunofluorescence microscopy: (1) A major population of mainly varicose fibres enmeshed singly-scattered neuronal perikarya, some of which contained vasoactive intestinal polypeptide-immunoreactivity. These substance P-immunoreactive fibres did not contain colocalized calcitonin gene-related peptide (CGRP) and were absent after transection of the cervical sympathetic trunk. (2) A rather small substance P-immunoreactive fibre population with colocalized calcitonin gene-related peptide-immunoreactivity was distributed in a patchy manner and disappeared after cutting the postganglionic branches. (3) Most of the intraganglionic small intensely fluorescent (SIF) cell clusters were intensely substance P-immunoreactive. SIF cells were not visibly changed in number and fluorescence intensity by either surgical procedure. (4) Immunoreactivity was not visible in principal ganglionic neurons of control ganglia, but occurred in cell bodies after pre- as well as after postganglionic nerve transection. Some of the substance P-immunolabelled perikarya in addition revealed immunostaining to antisera against the catecholamine-synthesizin enyzme tyrosine hydroxylase or against the neuropeptides leu-enkephalin and vasoactive intestinal polypeptide, respectively. The results strongly suggest that, in addition to a substance P-containing preganglionic input (1), and a supply by substance P-containing sensory axon collaterals (2), the superior cervical ganglion of the rat gives origin to a paraganglionic (3) and a postganglionic (4) substance P-immunoreactive intrinsic system, the latter becoming visible only after disconnection of the sympathetic pathway.     

The perikaryal projections of rabbit spinal ganglion neurons

Summary Shape, length and width of the perikaryal projections of spinal ganglion neurons from adult rabbits fixed in situ by perfusion have been evaluated by means of serial section electron microscopy. The results thus obtained have been compared with those obtained by enzymatic removal of ganglionic connective tissue and satellite cells followed by direct observation of the true neuronal surface under the scanning electron microscope. The comparison has shown that the perikaryal projections exhibit a similar shape and similar size with both techniques.     

Organization of sensory neurons to the muscles of mastication in the cat

The sensory neurons in the trigeminal ganglion which project to the muscles of mastication of the cat were studied using the retrograde transport of horseradish peroxidase. Horseradish peroxidase either was injected directly into the masticatory muscles or applied to the nerves supplying them. Numerous retrogradely labeled neurons were found in the ipsilateral trigeminal ganglion. All labeled neurons were sequestered within the posterolateral (mandibular) portion of the ganglion, but the ganglion cells innervating individual muscles were not organized topographically. The diameter of the labeled neurons varied greatly and included profiles considered small, medium and large. The organization of the neurons in the trigeminal mesencephalic and trigeminal motor nuclei which project to individual masticatory muscles also was noted. It is suggested that many of the ganglion cells of small and medium size may be important for the perception of muscle pain from the masticatory muscles in the cat.     

Involvement of peripherally released substance P and calcitonin gene-related peptide in mediating mechanical hyperalgesia in a traumatic neuropathy model of the rat

We hypothesized that neuropeptides released from the peripheral terminals of primary afferents play an important role in mechanical hyperalgesia after peripheral nerve injury. Nerve injury was performed on rats with lumbar 5 spinal nerve lesion (L5 SNL), which was preceded by L5 dorsal rhizotomy (L5 DR) to avoid the potential central effects induced by L5 SNL through the L5 dorsal root. L5 DR produced a short-lasting (<6 days) decrease in paw withdrawal threshold (PWT) while the following L5 SNL produced a persistent (>42 days) PWT decrease. When intraplantar injection to the affected hind paw was given immediately before L5 SNL, antagonists for both neurokinin 1 (NK1) and calcitonin gene-related peptide 1 (CGRP1) receptors delayed the onset of the PWT decrease for 2–4 days. However, when the same injection was given after L5 SNL, CGRP1, but not NK1, receptor antagonist reversed the decreased PWT for 105 min. It is suggested that peripherally released neuropeptides contribute to the generation of neuropathic pain, with substance P and CGRP contributing to its induction phase, but only CGRP to its maintenance phase.     

三叉神经根慢性压迫损伤对三叉神经节细胞髓鞘碱性蛋白及P75表达的影响

目的 探讨三叉神经节中卫星胶质细胞与神经元相互作用对三叉神经痛（TN）的影响。 方法 在SD大鼠假手术组（n=24）和三叉神经根慢性压迫诱导的TN大鼠模型组（n=24）中,运用免疫荧光和Western blotting方法,研究髓鞘碱性蛋白（MBP）、胶质纤维酸性蛋白（GFAP）及P75神经营养因子受体（P75NTR）在三叉神经节中的表达情况。 结果 TN模型组P75NTR 表达较假手术组增高,术后第7天和第14 天的表达增高有统计学意义（P<0.01）;MBP在TN模型组的表达较假手术组均降低,特别在第7和14天的表达降低有统计学意义（P<0.05）;卫星胶质细胞标志物GFAP在TN模型组术后第7、14和21天表达增高（P<0.01）。 结论 大鼠三叉神经根区慢性压迫损伤引起三叉神经节神经元MBP和P75NTR 表达改变和卫星胶质细胞的活化,可能影响神经元-卫星胶质细胞的相互作用,参与TN大鼠口面部周围伤害性信息向中枢的传递。     

Colocalization of 5-HT1F receptor and calcitonin gene-related peptide in rat vestibular nuclei

The aim of this study was to determine whether calcitonin gene-related peptide (CGRP) colocalizes with 5-HT_1F receptor in rat vestibular nuclei using a double immunohistochemical staining procedure. The frequent co-occurrence of migraine and balance disorders suggests a pathophysiologic link between the two. However, the mechanism of migrainous vertigo has not been elucidated, though serotonin (5-HT) and its receptors are believed to involve in the pathogenesis of migrainous vertigo. Furthermore, 5-HT_1F receptor agonists and CGRP receptor antagonists have recently attracted attention as potential treatments for migraine, and CGRP release from trigeminal neurons has been associated with migraine. This study demonstrates the colocalization of 5-HT_1F receptor and CGRP in the rat vestibular nuclei, which suggests that 5-HT_1F receptor regulates the release of CGRP from vestibular nuclei. This finding indicates that 5-HT_1F receptor agonists may ameliorate migrainous vertigo by attenuating elevated levels of CGRP release from vestibular nuclei.