Gene-related Peptide Immunoreactive Nerve Fibers Research Articles

Prednisolone Induces Microglial Activation in the Subnucleus Caudalis of the Rat Trigeminal Sensory Complex

Prednisolone is a member of synthetic glucocorticoids which are widely used to treat chronic inflammatory diseases. In this study, neuronal degeneration and cell death, and glial reaction were investigated in the rat trigeminal ganglion (TG) and brainstem after subcutaneous injection of prednisolone for 7 days. Expression of c-Jun activating transcription factor 3 and caspase-3 was absent or infrequent in the TG, and cranial sensory and motor nuclei of saline- and prednisolone-treated animals. In these animals, distribution of calcitonin gene-related peptide-immunoreactive (-IR) neurons and nerve fibers was similar in the brainstem. In addition, the number of Iba1- and glial fibrillary acidic protein (GFAP)-IR cells with some processes in the brainstem was barely affected by prednisolone treatment. However, the treatment increased ramification of Iba1-IR processes in the subnucleus caudalis of the trigeminal sensory complex. Prednisolone scarcely influenced the morphology of GFAP-IR cells in the brainstem. Expression of p38 mitogen-activated protein kinase was very rare in the brainstem of saline- and prednisolone-treated animals. The present study suggests that microglia are activated by prednisolone in the subnucleus caudalis of the trigeminal sensory complex. The glucocorticoid may affect nociceptive transmission in the brainstem.

Emotional stress and orthodontic tooth movement: effects on apical root resorption, tooth movement, and dental tissue expression of interleukin-1 alpha and calcitonin gene-related peptide immunoreactive nerve fibres in rats

The aim of the study was to investigate the effect of emotional stress on apical root resorption (ARR) and tooth displacement during orthodontic tooth movement in rats. A further area of interest was to evaluate if the expression of interleukin-1 alpha (IL-1alpha) as well as the density and distribution of peptidergic nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP) in the periodontal ligament (PDL) are associated with possible stress-induced changes in root resorption and tooth movement. A total of 52 male Wistar rats, aged 6 weeks, were divided in three experimental and one control group (n = 4). Group 1 had orthodontic tooth movement and received foot shocks (OTMS; n = 16), group 2 had orthodontic tooth movement but received no foot shocks (OTMNS; n = 16), and group 3 had no orthodontic tooth movement and received foot shocks (NOTMS; n = 16). Each group was further divided into four subgroups (n = 4), corresponding to the period of the experiment, i.e. 3, 7, 13, and 21 days. At the end of each experimental period, the blood samples were taken, the animals were sacrificed, and the jaws excised, deminerialized, and processed for immunocytochemistry. One-way analysis of variance was used to detect inter-group differences for all investigated variables. CGRP immunopositive nerve fibres were evaluated qualitatively. All the experimental groups demonstrated higher corticosterone levels than the control group, suggesting a stress-induced experience by orthodontic treatment per se. The OTMS group had the least amount of cellular cementum throughout the experimental periods and showed significant reduction in tooth displacement, especially at 3 and 7 days. No obvious changes were observed in the dental tissue expression of IL-1alpha and CGRP immunoreactive nerve fibres between the stressed and non-stressed orthodontically treated groups.

P2X3 Receptor Mediates Heat Hyperalgesia in a Rat Model of Trigeminal Neuropathic Pain

The present study was undertaken to determine the role of P2X3 receptor (P2X3R) on heat hyperalgesia in a newly developed rat model of trigeminal neuropathic pain. The unilateral infraorbital nerve (IoN) was partially ligated by 6-0 silk. To assess heat sensitivity, a vibrissal pad (VP) was placed on a hot plate and the latency until the rats withdrew their head was measured. Mechanical sensitivity of VP was also assessed by the use of von Frey filament. Both heat and mechanical hyperalgesia were observed at the VP ipsilateral to the IoN ligation. The latency to heat stimuli was prolonged after subcutaneous administration of pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, P2X1,2,3,5,7,1/5,2/3R antagonist) and 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP, P2X1,3,2/3,1/5R antagonist). The latency was shortened after administration of alpha,beta-methylene ATP (alpha,beta-meATP, P2X1,3,2/3R agonist), although no changes appeared after administration of beta,gamma-methylene-L-ATP (beta,gamma-me-L-ATP, P2X1R agonist). The protein gene product-9.5 and calcitonin gene-related peptide immunoreactive nerve fibers significantly decreased in the VP skin of ipsilateral to the IoN ligation. In the ipsilateral trigeminal ganglion, the number of P2X3-immunoreactive neurons significantly increased in the small cell group. In this study, we developed an experimental model of trigeminal neuropathic pain by partial ligation of IoN, which produced heat and mechanical hyperalgesia in the VP. Pharmacological and immunohistochemical studies revealed that the P2X3R plays an important role in the heat hyperalgesia observed in this model. The study describes the development of a novel model of trigeminal neuropathic pain. Heat hyperalgesia in this model was inhibited by peripheral injection of P2XR antagonists. The results suggest that P2X3R is a potential target for development of a novel therapy for trigeminal neuropathic pain.

Brachioradial pruritus is associated with a reduction in cutaneous innervation that normalizes during the symptom-free remissions

There has been a controversy regarding the cause of brachioradial pruritus: is it caused by a nerve compression in the cervical spine or is it caused by a prolonged exposure to sunlight? The purpose was to study clinical features of patients with brachioradial pruritus and to compare the cutaneous innervation in punch biopsies from the itchy skin of patients with the age-matched controls. Skin biopsy specimens from itchy skin of 16 patients with brachioradial pruritus were collected during the early autumn and were compared with corresponding skin specimens from 11 age-matched controls in the early spring, four of the patients being their own controls. The cutaneous innervation was visualized by antibodies against protein gene product 9.5 (general neuronal marker), by antibodies against calcitonin gene-related peptide (marker for thin sensory nerve fibers), and by antibodies against VR1-receptor (marker for capsaicin-sensitive nerve fibers). In all but two of the patients, itching of the arms or shoulders was seasonal. Some patients reported neck pain. In the skin of the lower arm, the number of protein gene product 9.5 immunoreactive nerve fibers was reduced 23% as compared with controls ( P = .03), the number of intraepithelial nerve fibers being reduced by 27% ( P = .03). The number of calcitonin gene-related peptide immunoreactive nerve fibers in the dermis was reduced by 34% ( P = .02) and the number of capsaicin-sensitive nerve fibers by 43% ( P = .008). The innervation of the four patients who were their own controls became normalized during the symptom-free period. The temporal course of the brachioradial pruritus and the histological changes in the skin similar to those caused by ultraviolet light, indicate that sunlight is an eliciting factor and that cervical spine disease can be a predisposing factor.

Dual Sensory Innervation of Pulmonary Neuroepithelial Bodies

The characteristics of the different populations of sensory nerve terminals that selectively contact pulmonary neuroepithelial bodies (NEBs) in rat lungs were investigated after chemical denervation with capsaicin and compared with control lungs. Vagal calbindin D28k and P2X(3) purinoceptor immunoreactive (IR) afferent nerve terminals contacting NEBs appeared to have their origin in the nodose ganglion. Thick CB/P2X(3)-IR nerve fibers were seen to be myelinated and to lose their myelin sheaths just before branching and protruding intraepithelially between the NEB cells. This vagal sensory component of the innervation of NEBs was not affected by capsaicin nor expressed capsaicin receptors (vanilloid receptor subtype 1). A second sensory nerve fiber population that selectively innervates pulmonary NEBs in the rat lung consists of thin unmyelinated nonvagal substance P/calcitonin gene-related peptide IR nerve fibers, contacting mainly the basal pole of pulmonary NEBs, and having their origin in dorsal root ganglia. In concordance with vanilloid receptor 1 expression on these nerve terminals, the spinal sensory substance P/calcitionin gene-related peptide-IR component of the innervation of NEBs was depleted by systemic capsaicin treatment. The complex sensory innervation pattern of pulmonary NEBs characterized in the present study strongly suggests that, physiologically, pulmonary NEBs represent a group of intraepithelial receptors that may be able to accommodate various local and central reflex actions, in relation to both chemo- and mechanosensory stimuli.

Changes in the distribution of the substance P and calcitonin gene-related peptide immunoreactive nerve fibers in the laryngeal mucosa of chronically hypoxic rats.

The distribution and abundance of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive nerve fibers in four different regions of the laryngeal mucosa were compared between normoxic and chronically hypoxic rats (10% O2 and 3.0-4.0% CO2 for 3 months). In the chronically hypoxic laryngeal mucosa, the number of SP and CGRP fibers within and just beneath the epithelium, and around the laryngeal gland was increased in comparison with those in the normoxic controls. Especially in the epiglottic and arytenoid regions, the number of intraepithelial SP fibers was increased remarkably. Most intraepithelial SP and CGRP fibers penetrated into the epithelium to extend to the luminal surface. There was no distinct difference in the distribution and abundance of these peptidergic fibers in the mucosa of the normoxic and chronically hypoxic vocal cord regions. These results suggest that the increased density of SP and CGRP fibers within the epithelium of the upper laryngeal mucosa is a predominant feature of hypoxic adaptation, and this may be involved in airway protection, swallowing, and other functions in the chronically hypoxic environment. In addition, the increased SP and CGRP fibers around the laryngeal gland suggest an enhanced mucous secretion, and this may participate in the airway defense mechanism in low O2 conditions.

Distribution of vasoactive intestinal peptide and calcitonin gene-related peptide immunoreactive nerve fibers and binding sites in the hamster seminal vesicle during post-natal development.

The distribution of vasoactive intestinal peptide (VIP)- and calcitonin gene-related peptide (CGRP)-immunoreactive nerves and 125I-labeled VIP- and CGRP-binding sites was studied in the hamster seminal vesicle of 12-, 30- and 60-day-old animals. In addition, the general innervation of the seminal vesicle was examined using the general neuronal marker synaptophysin. Our results show that the densities of the overall (synaptophysin immunoreactive) and CGRP-immunoreactive innervation is constant during the post-natal development of the gland. However, a significant decrease in VIP-containing nerves is observed at the end of puberty. The autoradiographic study revealed that in 12-day-old animals, the epithelium presents VIP binding sites. However, in 30-day-old animals, VIP binding sites are observed in the epithelium of only a few clumps of acini. In 60-day-old animals, the gland is composed of acini with dilated lumina where VIP binding sites are not detected. In all groups studied the epithelium does not exhibit CGRP binding sites. The seminal vesicle muscle layer displays specific binding sites for both VIP and CGRP at all post-natal developmental times, but the density of VIP binding sites is higher in 12- than in 30- and 60-day-old animals. Our results, showing the presence of specific VIP and CGRP binding sites during the development of the hamster seminal vesicle, suggest that these neuropeptides may be involved in the growth and differentiation of the gland.

Substance P- and Calcitonin Gene-Related Peptide-Containing Nerve Fibers in the Nasal Mucosa of Chronically Hypoxic Rats

The distribution of substance P-immunoreactive and calcitonin gene-related peptide–immunoreactive nerve fibers in the nasal mucosa was compared between normoxic and chronically hypoxic rats (10% O 2 and 3.0–4.0% CO 2 for 3 months). In the normoxic nasal mucosa, substance P- and calcitonin gene-related peptide–immunoreactive nerve fibers were found within and under the epithelium and around the glands and blood vessels in the lamina propria. These immunoreactive fibers have many varicosities. In the chronically hypoxic nasal mucosa, the relative density of intra- and subepithelial substance P-immunoreactive and calcitonin gene-related peptide–immunoreactive fibers and those in the lamina propria was higher than in normoxic mucosa. The length of substance P-positive fibers within the chronically hypoxic olfactory and respiratory epithelium was 1.66 and 2.45 times higher than within the normoxic epithelium, respectively. The length of calcitonin gene-related peptide-immunostained fibers within the chronically hypoxic olfactory and respiratory epithelium was 1.56 and 1.84 times higher, respectively. Because substance P and calcitonin gene-related peptide are the predominant signal peptides of primary sensory neurons, the increased number of these fibers may represent enhanced sensory mechanisms in the hypoxic nasal mucosa. In addition, considered together with the findings in chronically hypoxic tracheal mucosa, the increased density of intraepithelial fibers containing substance P and calcitonin gene-related peptide suggests that this is a predominant feature of hypoxic adaptation throughout the upper and lower respiratory tracts.

Quantitative examination of calcitonin gene-related peptide immunoreactive nerve fibres in the cat knee joint capsule.

The knee joint of the cat has been used extensively to study the morphology and function of primary afferents in a deep somatic tissue. A proportion of these neurones synthesizes various neuropeptides, with calcitonin gene-related peptide being the most prominent. In the present study we examined the distribution and density of nerve fibers immunoreactive for calcitonin gene-related peptide within the medial articular capsule. The fibres were predominantly located in the superficial layer of the capsule. They formed a dense innervation pattern, mainly accompanying blood vessels. Electron microscopy showed that most fibres were in close proximity to small arteries. The highest innervation density was found in parts of the capsule that were located over the epicondyle of the femur with 21 +/- 12 fibres per mm2 (mean +/- SD). In the tissue over the joint cleft this density was lower, with 11 +/- 6 fibres per mm2. In conclusion, the high innervation density of the knee joint capsule by nerve fibres containing calcitonin gene-related peptide supports the hypothesis of an important regulatory function of this peptide in normal tissue.

Distribution of substance P and calcitonin gene-related peptide immunoreactive nerve fibers in the trachea of chronically hypoxic rats

The distribution of substance P and calcitonin gene-related peptide immunoreactive nerve fibers in the trachea was compared between normoxic and chronically hypoxic rats (at 380 mm Hg for 10 weeks). In the normoxic trachea, the immunoreactivity to either peptide was seen in the nerve fibers in four principal locations: a) within and b) under the ciliated epithelium, c) within the smooth muscle bundles in the posterior wall, and d) in the connective tissue and around blood vessels in the lamina propria and submucosa. These immunoreactive fibers within the epithelium and smooth muscle bundles, in the connective tissue, and around blood vessels were thin and displayed some varicosities, and those under the epithelium appeared as thick nerve bundles. When the distribution and density of immunoreactive fibers were compared between normoxic and chronically hypoxic tracheas, there was a difference in number of substance P and calcitonin gene-related peptide immunoreactive fibers penetrating into the epithelium, although there was no difference in the other three locations. The mean number of substance P and calcitonin gene-related peptide immunoreactive intraepithelial fibers per section of the chronically hypoxic trachea was significantly increased. Because substance P and calcitonin gene-related peptide are predominant signal peptides of primary sensory neurons, the increase of substance P and calcitonin gene-related peptide immunoreactive fibers suggests that altered airway reflexes may be a feature of hypoxic adaptation.

Postnatal development of neuropeptide Y- and calcitonin gene-related peptide-immunoreactive nerves in the rat urinary bladder

The postnatal development of neuropeptide Y- and calcitonin gene-related peptide-immunoreactive (NPY-IR and CGRP-IR) nerve fibers in the rat urinary bladder was investigated using whole-mount preparations and cryostat sections. In newborn and 3-day-old rats, many NPY-IR nerve fibers were observed in the subserous and muscle layers. Many NPY-IR nerve cell bodies clustered at branching points of the subserous nerve bundles. Within 4 weeks after birth, these cell bodies drastically decreased in number and spread along the bundles, although the number of NPY-IR nerve fibers increased moderately. In contrast, CGRP-IR nerve fibers in newborn and 3-day-old rats were less developed, and no CGRP-IR nerve cell body was observed in any rat. However, CGRP-IR nerve fiber distribution in the urinary tissues conspicuously increased within 4 weeks after birth. Especially, an increase of the infraepithelial fibers showing a meshwork appearance was prominent in the fundus and corpus of the bladder. The infra- and intraepithelial CGRP-IR nerve meshwork of the ventral wall was more dense than that of the trigone. At 4 weeks, NPY-IR and CGRP-IR nerves were similar to those of the adult rat (8-12 weeks old). The present study suggests a correlation between the development of the peripheral nervous system in the urinary bladder and maturation of micturition behavior in the rat.

Ultrastructural Analysis of Tyrosine Hydroxylase-, Substance P-, and Calcitonin Gene-Related Peptide Immunoreactive Nerve Fibers in the Rat Iris

Different patterns of distribution of chemically different nerve fibers in the rat iris were studied by immunoelectron microscopy. The iris was immunostained either singly for tyrosine hydroxylase (TH), or doubly for substance P (SP) and calcitonin gene-related peptide (CGRP), or triply for TH, SP and CGRP. TH-positive fibers were distributed in the stroma near the iris dilator and within the iris sphincter. SP/CGRP-positive fibers were found mainly in the anterior half of the stroma and partly in the stroma close to the sphincter, but were hardly ever seen in the stroma near the dilator or within the sphincter. Fibers neither positive for TH, SP nor CGRP were observed in the stroma near the dilator and within the sphincter.

Calcitonin gene-related peptide immunoreactive nerve fibers in the developing salivary glands of the rat

The appearance and distribution of calcitonin gene-related peptide immunoreactive (CGRP-IR) nerve fibers was studied by indirect immunofluorescence microscopy in the developing salivary glands of the rat at daily age intervals from the 15th day in utero (i.u.) until birth and subsequently on the 2nd, 5th, 7th, 16th, 30th postnatal (p.n.) days. The findings were compared to samples of adult rats. CGRP-IR fibers appeared on day 20 i.u. both in parotid and submandibular glands. In submandibular glands, fibers were first seen surrounding the developing ductal branches, in the mesenchyme and also in association with developing ducts and blood vessels. In the parotid gland CGRP-IR fibers first appeared around ductal branches and blood vessels on day 20 i.u. and on day 21 i.u. CGRP-IR nerve fibers were found in the mesenchyme and around developing ducts. The density of CGRP-IR fibers was highest between the 2nd and 16th postnatal days, after which the amount of CGRP immunoreactivity slowly decreased to the levels of adult glands. The increase in a number of CGRP-IR nerve fibers especially around the ductal and vascular elements in the developing salivary glands indicates that they may play a role in the functional maturation of salivary glands.

Distribution of calcitonin gene-related peptide immunoreactive nerve fibers in the human submandibular gland

The indirect immunofluorescence technique was used to study the distribution of calcitonin gene-related peptide (CGRP) in human submandibular gland. A relatively low number of thin varicose fibers with intense immunofluorescence for CGRP was seen in samples from seven glands. These CGRP-immunoreactive (CGRP-IR) nerve fibers were mainly seen around or in close contact with intra- and interlobular blood vessels. Some CGRP-IR nerve fibers were also found in association with intra- and interlobular salivary ducts and a few around the submandibular acini. By visual estimation there was no difference in the density of CGRP-IR nerve fibers between specimens of recurrent duct obstruction and laryngeal carcinoma. The present results show that the distribution of CGRP-IR nerve fibers in the stroma and in the glandular secretory elements of the human submandibular gland is quite similar to that seen in the rat and the ferret, which have been reported earlier. Furthermore, the regional distribution of CGRP-IR fibers in the human submandibular gland suggests that CGRP has a physiological role in the regulation of salivary gland function in human salivary glands, e.g. blood flow and secretion.

Substance P and calcitonin gene-related peptide immunoreactive nerve fibers in the mouse ventral root

Substance P and calcitonin gene-related peptide immunoreactive nerve fibers in the mouse ventral root

Distribution of calcitonin gene-related peptide immunoreactive nerve fibers in the mudpuppy cardiac septum

An immunohistochemical study was undertaken to determine the distribution of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the cardiac septum of the mudpuppy, Necturus maculosus. Numerous long, CGRP-immunoreactive nerve fibers course across the septum, run in the nerve trunks connecting clusters of postganglionic parasympathetic cells, form complexes over groups of ganglion cells and make pericellular networks around individual ganglion cells. The postganglionic parasympathetic neurons and small intensely fluorescent (SIF)-like cells did not exhibit CGRP immunoreactivity. Most of the CGRP-immunoreactive nerve fibers also are labeled for substance P. In freshly dissected preparations, the staining pattern for CGRP was not similar to that obtained using an antiserum against synaptic vesicle membrane, which appears to preferentially label cholinergic preganglionic terminals on all postganglionic parasympathetic cells in the mudpuppy preparation. Further, in explanted ganglia (maintained 10 days in culture) almost no reactivity was obtained with the antivesicle antiserum whereas numerous nerve fibers still exhibited CGRP-immunoreactivity. These observations demonstrate that the CGRP-immunoreactive nerve fibers are not parasympathetic preganglionic axons. Rather we suggest that the CGRP-immunoreactive nerve fibers are processes of primary sensory fibers.

Distribution and characterization of calcitonin gene-related peptide immunoreactivity in the digestive system of normal and capsaicin-treated rats

The distribution and characterization of calcitonin gene-related peptide immunoreactivity in the digestive system of normal, capsaicin-treated, and littermate control rats were studied by radioimmunoassay, chromatography, and immunohistochemistry. The highest concentrations of calcitonin generelated peptide immunoreactivity were found in the stomach (45 ± 2.8 pmol/g wet wt, nonsecretory region; 38.7 ± 4.4 pmol/g wet wt, secretory region) and rectum (30.9 ± 1.6 pmol/g wet wt). Significant amounts of peptide were also found in the other regions of the gut and in the pancreas. Neonatal treatment with capsaicin, which causes a permanent degeneration of most of the small-diameter sensory neurons, reduced calcitonin gene-related peptide content by > 95% in the esophagus and stomach, by 60% in the pancreas, and by < 50% in the intestine, when compared with littermate controls. Separation of extracts from the gut, pancreas, and brain by chromatography gave major peaks corresponding to the predicted rat calcitonin gene-related peptide1–37 and small unidentified peaks, which presumably arise from metabolism of the peptide. Immunohistochemical studies demonstrated that in the esophagus and stomach, calcitonin gene-related peptide immunoreactivity is restricted to nerve fibers, whereas in the intestine it is localized in both nerve fibers and enteric ganglion cells. In capsaicintreated rats there was a virtually complete elimination of calcitonin gene-related peptide immunoreactive fibers innervating the esophagus and stomach, whereas in the small and large intestine there was a dramatic reduction and often a complete elimination of those associated with blood vessels and a slighter reduction of the nonvascular immunoreactive fibers. The results of this study indicate that calcitonin gene-related peptide immunoreactive nerve fibers innervating the rat digestive system originate from both intrinsic (enteric) and extrinsic (presumably sensory) sources and that both the intrinsic and extrinsic components appear to contain a substance that corresponds to the predicted calcitonin gene-related peptide1–37.