CGRP Fibers Research Articles

Lingual innervation in male and female marmosets

Several gaps in knowledge exists in our understanding of orofacial pain. Some of these include type of peripheral sensory innervation in specific tissues, differences in innervation between sexes and validation of rodent studies in higher order species. The current study addresses these gaps by validating mouse studies for sensory innervation of tongue tissue in non-human primates as well as assesses sex-specific differences. Tongue and trigeminal ganglia were collected from naïve male and female marmosets and tested for nerve fibers using specific markers by immunohistochemistry and number of fibers quantified. We also tested whether specific subgroups of nerve fibers belonged to myelinating or non-myelinating axons. We observed that similar to findings in mice, marmoset tongue was innervated with nerve filaments expressing nociceptor markers like CGRP and TRPV1 as well as non-nociceptor markers like TrkB, parvalbumin (PV) and tyrosine hydroxylase (TH). Furthermore, we found that while portion of TrkB and PV may be sensory fibers, TH-positive fibers were primarily sympathetic nerve fibers. Moreover, number of CGRP, TrkB and TH-positive nerve fibers were similar in both sexes. However, we observed a higher proportion of myelinated TRPV1 positive fibers in females than in males as well as increased number of PV + fibers in females.Taken together, the study for the first time characterizes sensory innervation in non-human primates as well as evaluates sex-differences in innervation of tongue tissue, thereby laying the foundation for future orofacial pain research with new world smaller NHPs like the common marmoset.

(018) Immunohistochemical Characterization of the Innervation of the Vulvar Vestibule in Patients with Suspected Neuroproliferative Vestibulodynia

Abstract Introduction Provoked vestibulodynia (PVD) is a chronic pain condition characterized by allodynia localized to the vestibule, the endodermal tissue surrounding the vaginal introitus. The finding of increased densities of nerve fibers in the vestibular mucosa of patients with PVD has led to the identification of a neuroproliferative subtype. The etiology of PVD, including neuroproliferative vestibulodynia (NPV), is not fully known which has led to challenges in adequately managing this condition. Significantly, the microscopic innervation of the vestibule remains incompletely described despite the preliminary data which support the role of peripheral innervation in vestibular pain pathogenesis. Objective To characterize the types and distribution of nerves present in vestibule samples from patients with suspected NPV using a suite of markers of general (protein gene product 9.5, PGP9.5), sensory (calcitonin gene-related peptide, CGRP) and autonomic innervation (vasoactive intestinal polypeptide, VIP and tyrosine hydroxylase, TH) and compare this to cadaveric vestibule tissues; and to investigate whether there is an association between this innervation and NPV etiology using markers of neuroproliferation (nerve growth factor, NGF) and immune activation (c-kit). Methods Archival formalin-fixed, paraffin embedded vestibulectomy samples were obtained from six patients who had previous immunohistochemical staining to confirm NPV diagnosis. All samples were stained with nerve markers PGP9.5 and CGRP, in addition to NGF, and mast cell marker c-kit. A subset of samples was stained with nerve markers TH and VIP to further elucidate specific types of innervation present. Vestibule samples were obtained from female cadaveric donors and were stained with the same suite of antibodies for comparison. All tissues were serially sectioned at 5 μm. Results Nerve fibers immunoreactive for all nerve markers were identified in both patient and cadaveric vestibule. Patient samples were characterized by the proliferation of PGP9.5-positive nerve fibers and c-kit positive mast cells which were identified throughout the tissue stroma, including in proximity to nerve bundles. NGF expression was localized to a subset of nerves including those that demonstrated co-expression of sensory and autonomic nerve markers. Cells immunoreactive for NGF were also observed, including those that showed co-expression with putative mast cells that stained positively for c-kit. Relative to the cadaveric tissues, the patient samples were characterized by increased densities of CGRP, TH, and VIP-immunoreactive fibers which were observed throughout the vestibular mucosa. Some heterogeneity in patterns of innervation was observed between the patient samples, including increased densities of autonomic fibers in one patient as indicated by increased VIP and TH-immunoreactive nerve fibers. Conclusions Our results support the existence of a neuroproliferative subtype of PVD that is characterized by the hyperinnervation of the vestibular mucosa and neuro-immune interactions. This investigation highlights that the proliferation of other nerve fiber types in addition to nociceptive, CGRP-positive fibers, including adrenergic and cholinergic nerve fibers, may be important in PVD etiology in some patients. Heterogeneity in patterns of innervation across individuals with NPV could explain, in part, variability in clinical response to treatment and should be further elucidated in ongoing research efforts. Disclosure No

Ultrastructural Evidence for Oxytocin and Oxytocin Receptor at the Spinal Dorsal Horn: Mechanism of Nociception Modulation

Oxytocin is a hypothalamic neuropeptide involved in the inhibition of nociception transmission at spinal dorsal horn (SDH) level (the first station where the incoming peripheral signals is modulated). Electrophysiological, behavioral, and pharmacological data strongly support the role of this neuropeptide and its receptor (the oxytocin receptor, OTR) as a key endogenous molecule with analgesic properties. Briefly, current data showed that oxytocin release from the hypothalamus induces OTR activation at the SDH, inducing selective inhibition of the nociceptive Aδ- and C-fibers (probably peptidergic) activity, but not the activity of proprioceptive fibers (i.e. Aβ-fibers). The above inhibition could be a direct presynaptic mechanism, or a mechanism mediated by GABAergic interneurons. However, the exact anatomical localization of oxytocin and OTR remains unclear. In this context, the present study set out to analyze the role of OTRs, GABAergic cells and CGRP fibers in the SDH in rats by using electron microscopy. Ultrastructural analyses of the SDH tissue show that: (i) oxytocin and OTR are found in asymmetrical synapsis; (ii) OTR is found in GABAergic interneurons (near unmyelinated fibers), CGRPergic fibers and glial cells; (iii) whereas oxytocin is present in supraspinal descending projection fibers. These anatomical data strongly support the notion that oxytocin released at the SDH could presynaptically inhibit the nociceptive input from the peripheral primary afferent fibers. This inhibitory action could be direct or use a GABA interneuron. Furthermore, our findings that OTR is exhibited in glial tissue at the SDH requires further exploration in nociception assays.

SPARC: Determining the Projections and Expression Profiles of Neurons of the Stomach‐Brain Axis in Rat

Effectors in the stomach receive motor innervation from intrinsic (enteric) neurons, nuclei in the brain stem, the spinal cord and other gut regions. Sensory signals are conducted to the brain, spinal cord and sympathetic ganglia. We are analyzing the circuits by selective pathway lesions, orthograde and retrograde tracing, RNAScope and immunohistochemistry. Regions of stomach were denervated by myectomy (removing a region of myenteric ganglia), bilateral vagotomy or sympathectomy, or combined surgeries. Retrogradely transported fluorescent microbeads were injected selectively into the gastric mucosa or muscle layers. Neuronal markers were localized by multi‐labeling immunohistochemistry. When a region of longitudinal muscle and myenteric ganglia was removed, and 7–10 days allowed for fiber degeneration, nerve fibers in the circular muscle and muscularis mucosae were no longer detected. Two major nerve fiber types innervated the muscle, fibers immunoreactive for neuronal NOS and VIP that are deduced to be inhibitory motor neurons and fibers that were labelled by tachykinins and cholinergic markers that are deduced to be excitatory motor neurons. Some inhibitory fibers were also immunoreactive for gastrin releasing peptide (GRP). The muscle was also innervated by sympathetic (tyrosine hydroxylase positive) nerve fibers. The mucosa received a mixed motor and sensory supply from myenteric ganglia, submucosal ganglia, the vagus, and sympathetic pathways. Denervation studies showed that VIP fibers arose from both myenteric ganglia and the nodose ganglia; thus VIP is in subpopulations of motor and of sensory fibers. Some VIP fibers ended in close relation to ghrelin‐containing enteroendocrine cells. There were few sympathetic endings in the mucosa and CGRP fibers were also rare. Arteries in the gastric wall were triply innervated by sympathetic noradrenaline/ NPY fibers, tachykinin fibers and VIP fibers. The origins of the perivascular VIP fibers have yet to be determined. Injection of retrograde tracer into the gastric mucosa labeled large populations of nodose ganglion neurons. Some nodose ganglion neurons were labeled both from the gastric mucosa and from the external muscle. The chemical phenotypes of myenteric and submucosal ganglia are being investigated. VIP‐immunoreactive cell bodies were numerous in submucosal ganglia. We conclude that there is a complex interplay between nerve endings originating in myenteric, submucosal, nodose, sympathetic and spinal ganglia in gastric effector tissues.Support or Funding InformationNIH/SPARC

BDNF Overexpression Exhibited Bilateral Effect on Neural Behavior in SCT Mice Associated with AKT Signal Pathway.

Spinal cord injury (SCI), a severe health problem in worldwide, was commonly associated with functional disability and reduced quality of life. As the expression of brain-derived neurotrophic factor (BDNF) was substantial event in injured spinal cord, we hypothesized whether BDNF-overexpression could be in favor of the recovery of both sensory function and hindlimb function after SCI. By using BDNF-overexpression transgene mice [CMV-BDNF 26 (CB26) mice] we assessed the role of BDNF on the recovery of neurological behavior in spinal cord transection (SCT) model. BMS score and tail-flick test was performed to evaluate locomotor function and sensory function, respectively. Immunohistochemistry was employed to detect the location and the expression of BDNF, NeuN, 5-HT, GAP-43, GFAP as well as CGRP, and the level of p-AKT and AKT were examined through western blot analysis. BDNF overexpressing resulted in significant locomotor functional recovery from 21 to 28days after SCT, compared with wild type (WT)+SCT group. Meanwhile, the NeuN, 5-HT and GAP-43 positive cells were markedly increased in ventral horn in BDNF overexpression animals, compared with WT mice with SCT. Moreover, the crucial molecular signal, p-AKT/AKT has been largely up-regulated, which is consistent with the improvement of locomotor function. However, in this study, thermal hyperpathia encountered in sham (CB26) group and WT+SCT mice and further aggravated in CB26 mice after SCT. Also, following SCT, the significant augment of positive-GFAP astrocytes and CGRP fibers were found in WT+SCT mice, and further increase was seen in BDNF over-expression transgene mice. BDNF-overexpression may not only facilitate the recovery of locomotor function via AKT pathway, but also contributed simultaneously to thermal hyperalgesia after SCT.

Mechanisms of PDGF siRNA-mediated inhibition of bone cancer pain in the spinal cord.

Patients with tumors that metastasize to bone frequently suffer from debilitating pain, and effective therapies for treating bone cancer are lacking. This study employed a novel strategy in which herpes simplex virus (HSV) carrying a small interfering RNA (siRNA) targeting platelet-derived growth factor (PDGF) was used to alleviate bone cancer pain. HSV carrying PDGF siRNA was established and intrathecally injected into the cavum subarachnoidale of animals suffering from bone cancer pain and animals in the negative group. Sensory function was assessed by measuring thermal and mechanical hyperalgesia. The mechanism by which PDGF regulates pain was also investigated by comparing the differential expression of pPDGFRα/β and phosphorylated ERK and AKT. Thermal and mechanical hyperalgesia developed in the rats with bone cancer pain, and these effects were accompanied by bone destruction in the tibia. Intrathecal injection of PDGF siRNA and morphine reversed thermal and mechanical hyperalgesia in rats with bone cancer pain. In addition, we observed attenuated astrocyte hypertrophy, down-regulated pPDGFRα/β levels, reduced levels of the neurochemical SP, a reduction in CGRP fibers and changes in pERK/ERK and pAKT/AKT ratios. These results demonstrate that PDGF siRNA can effectively treat pain induced by bone cancer by blocking the AKT-ERK signaling pathway.

Innervation of cardiac ventricles: an immunohistochemical study in bats (LB46)

Neuroanatomy of cardiac ventricles in bats that are adapted for 5‐7 month long hibernation was not identified so far. We used fluorescent immunolabeling for PGP9.5, TH, ChAT, nNOS, SP, and CGRP in order to detect general, sympathetic, vagal and sensory neural structures on ventricles of three species of hibernating bats. We found ventricles in bats to be highly innervated by a dense ganglionated nerve plexus scattered up to heart apex. Ventricular neurons were mostly positive (+) for ChAT, fewer – for TH or nNOS. Small intensely fluorescent cells, strongly TH(+), were identified as well. Epicardial nerves possessed the CGRP(+) and SP(+) nerve fibers. Employing immunohistochemistry for PGP9.5, we revealed numerous sensory nerve endings distributed widely beneath the mesothelium. These nerve endings were solely positive for PGP 9.5 and possessed a complex morphology that was not demonstrated previously. The present findings offer insights into further studies of the hibernating myocardial cells that are fully resistant to long term hypoxia and apparently controlled by autonomic nervous system in perfection.Grant Funding Source: Supported by the Research Council of Lithuania (MIP‐031/2013)

Long‐Term Effects of Neonatal Capsaicin Treatment on Intraepidermal Nerve Fibers and Keratinocyte Proliferation in Rat Glabrous Skin

Innervation is required to preserve several aspects of skin homeostasis. Previous studies in rodents have shown that sciatic nerve transection leads to epidermal thinning and reduced keratinocyte proliferation. As the sciatic nerve is composed of sensory and motor axons, it is not clear whether skin alterations reflect motor or sensory disturbances. In this study, we used neonatal capsaicin treatment to evaluate whether sensory chemical denervation affects keratinocyte proliferation at 1, 3, and 6 months of age. Using design-based stereological methods, we estimated the total length of intraepidermal nerve fibers (IENF) that were of peptidergic type and the number of bromodeoxyuridine-labeled (BrdU(+) ) nuclei in the hind paw glabrous epidermis of control and capsaicin-treated rats. We found that the treatment decreased the total fiber length of IENF immunoreactive for both protein gene product 9.5 (PGP(+) ) and of IENF immunoreactive for calcitonin gene-related peptide (CGRP(+) ). The length of PGP(+) fibers decreased by 83%, 81%, and 77% and that of CGRP(+) fibers decreased by 48%, 58%, and 58% at 1, 3, and 6 months, respectively. Double-immunofluorescence staining for neural beta III tubulin and CGRP revealed that the majority of the remaining fibers in the epidermis after capsaicin treatment were of peptidergic type. The number of BrdU(+) nuclei was similar in both groups. Our findings suggest that IENF present after capsaicin treatment are sufficient to maintain epidermal replacement.

Multiple sclerosis produces significant changes in urinary bladder innervation which are partially reflected in the lower urinary tract functional status-sensory nerve fibers role in detrusor overactivity

Background Detrusor overactivity is often observed in patients with multiple sclerosis (MS), and neurotoxins are emerging as second-line therapies albeit with different degrees of success per patient basis. Objective To investigate lower urinary tract (LUT) functional status and bladder innervation (calcitonin gene related peptide [CGRP] and substance P [SP] positive nerve fibers) in patients with MS. Method Eighteen MS patients with LUT symptoms underwent urodynamic investigations, and six non-MS patients undergoing cystoscopy due to microscopic hematuria served as controls. Cold cut bladder biopsies were taken from the bladder trigone region. Neurotransmitter expression was determined by individual immunohistochemical staining. Results Two distinct groups could be distinguished: group 1 with pronounced neurogenic detrusor overactivity and mild outflow obstruction; group 2 with some degree of neurogenic detrusor overactivity, detrusor hypocontractility during voiding, and high degree of an outflow obstruction. The presence of SP and CGRP immunoreactive + fiber density was observed in greater numbers in group 1. Conclusion Density of CGRP and SP positive nerve fibers within the urinary bladder of patients with MS may be suggestive of functional status of the lower urinary tract, namely denser innervation is observed in patients with mild outflow obstruction and strong detrusor overactivity. This observation could be useful when planning second-line treatment (neurotoxins) in these patients. Patients with denser innervation probably will respond better to such a therapy.

Caudal end of the rat spinal dorsal horn

We have previously demonstrated that the transformation of the caudal spinal cord through the conus medullaris to the filum terminale takes place in three steps. In the conus medullaris the twin layers of CGRP-immunoreactive and IB4-labeled primary afferent fibers as well as the translucent portion of the superficial dorsal horn equivalent to the substantia gelatinosa discontinue before the complete removal of the dorsal horn. Parallel with these changes VGLUT1-immunoreactive myelinated primary afferent fibers arborize not only in the deep layers but also in the entire extension of the remaining dorsal horn, while scattered CGRP fibers still remains at the margin of and deep in the dorsal horn. PKCgamma-immunoreactive dorsal horn neurons discontinue parallel with the disappearance of the IB4-labeled nerve fibers. These observations suggest that in the dorsal horn certain neurons are linked to the substantia gelatinosa, while others are substantia gelatinosa-independent neurons.

Morphological changes in the rat carotid body 1, 2, 4, and 8 weeks after the termination of chronically hypocapnic hypoxia.

Morphological changes in the rat carotid bodies 1, 2, 4, and 8 weeks after the termination of chronically hypocapnic hypoxia (10% O2 for 8 weeks) were examined by means of morphometry and immunohistochemistry. The rat carotid bodies after 8 weeks of hypoxic exposure were enlarged several fold with vascular expansion. The carotid bodies 1 and 2 weeks after the termination of 8 weeks of hypoxic exposure were diminished in size, although their diameter remained larger than the normoxic controls. The expanded vasculature in chronically hypoxic carotid bodies returned to the normoxic control state. In the carotid bodies 1 week after the termination of chronic hypoxia, the density of NPY fibers was remarkably increased and that of VIP fibers was dramatically decreased in comparison with the density in chronically hypoxic carotid bodies. In the carotid bodies 2 and 4 weeks after the termination of hypoxia, the density of SP and CGRP fibers was gradually increased. In the carotid bodies 8 weeks after the termination of hypoxia, the appearance of the carotid body returned to a nearly normoxic state, and the density of SP, CGRP, VIP, and NPY fibers also recovered to that of normoxic controls. These results suggest that the morphological changes in the recovering carotid bodies start at a relatively early period after the termination of chronic hypoxia, and a part of these processes may be under the control of peptidergic innervation.

Peptide immunoreactivity and ultrastructure of rat urinary bladder nerve fibers after topical desensitization by capsaicin or resiniferatoxin

In the present study the decrease of neuropeptide containing nerve fibers and the increase in the volume threshold to reflex micturition occurring in the rat bladder after intravesical application of capsaicin or resiniferatoxin were compared. The ultrastructure of bladder terminal axons was evaluated at the moment of maximal peptide depletion and compared to that of nerve fibers after systemic capsaicin application. Adult Wistar rats were treated intravesically for 30 min with 0.5 ml of 100 nM RTX, 1 mM capsaicin or 30% ethanol in saline, the vehicle solution. Twenty-four hours and 1, 2, 3, 4 and 8 weeks later the bladders were immunostained for CGRP, SP, VIP and NPY. Cystomanometric studies were performed 24 h and 1, 8, and 12 weeks after vanilloid instillation. Twenty-four hours after systemic capsaicin or intravesical capsaicin or RTX, bladders were prepared for electron microscopic (EM) observation. Intravesical capsaicin or RTX decreased, in a similar way, the number of CGRP and SP-IR (immunoreactive) fibers coursing in the muscular layer and the mucosa. IR fibers amounted to less than 20% of controls at 24 h and returned to normal levels in the eighth week. At the EM level, bladders treated with topical vanilloids did not show morphological changes in terminal axons coursing in the mucosa. In contrast, bladders from animals treated systemically with capsaicin contained numerous grossly degenerated nerve fibers. VIP and NPY-IR fibers were not affected by the treatment. Cystometrograms showed an increase of the volume threshold to reflex micturition that started at 24 h and disappeared at 12 weeks. We conclude that intravesical capsaicin or RTX were equally effective in terms of reducing the number of SP and CGRP-IR fibers and increasing the volume threshold for reflex micturition. Both changes were transient and were not associated with ultrastructural changes of the bladder nerve fibers, excluding terminal axon degeneration as the main mechanism of action of intravesical vanilloids.

Distribution of neuropeptide-containing nerve fibers in the salamander taste organs.

Salamander taste organs were recognized as oval cell clusters within the dorsal surface of the tongue. A moderate number of SP, CGRP, VIP, NPY, and GAL immunoreactive nerve fibers terminated in the cell clusters, and some of them penetrated into the basal half of the cell clusters. Around the glands, VIP, NPY, and GAL fibers were numerous, and SP and CGRP fibers were less numerous. Immunoreactivity of SOM and FMRF was not detected either in the nerve fibers associated with the cell clusters or those around the glands. These findings suggest that the chemosensory mechanisms of the salamander gustatory organs are under the control of peptidergic innervation. In addition, the present study indicates that the caudate taste organs are structurally primitive but functionally mature.

Distribution of CGRP, substance P, VIP and somatostatin immunoreactive nerve fibers in the internal gills of larvae of the bullfrog, Rana catesbeiana.

CGRP, substance P (SP), VIP and somatostatin were demonstrated in the internal gills of larval bullfrogs using the immunohistochemical peroxidase-antiperoxidase method. Three groups of immunoreactive fibers were distinguished by their distribution area. The first group includes CGRP, SP and VIP immunoreactive fibers running in the connective tissue of the gill tufts. These fibers were associated with the capillaries. Some CGRP fibers were distributed similarly to SP fibers, and the density of VIP immunoreactive fibers was low. The second group includes CGRP, SP, VIP and somatostatin fibers distributed in the connective tissue surrounding the ceratobranchialia. The third group includes CGRP, SP and somatostatin fibers located in the branchial muscle. Their terminals appeared to be neuromuscular junctions. No immunoreactivity of leu- or met-enkephalins was found in the internal gills. From these findings, the first group of fibers is presumed to be involved in modulating ion transport of the internal gills. The second group of fibers except for the somatostatin fibers is thought to be continuations of the first group of fibers. The third group of fibers may possibly be involved in the modulation of transmissions at the neuromuscular junction. It is unclear whether somatostatin terminals are also involved in this.

Peptidergic and catecholaminergic fibers in the human corneal epithelium

Innervation of the clinically normal human corneal epithelium was investigated utilizing immunohistochemical and electron microscopic techniques. All corneal epithelial sheets examined demonstrated neuron specific enolase (NSE: a non-specific marker for neural elements), calcitonin gene-related peptide (CGRP: a putative marker for sensory fibers), and tyrosine hydroxylase (TH: a marker for catecholaminergic nerves) immunoreactive fibers. NSE, CGRP, and TH fibers formed a dense basal epithelial plexus. The CGrp fibers tended to have beaded profiles, while TH fibers were smooth. Numerous free nerve endings originating from the basal epithelial plexus og NSE and CGRP fibers terminated throughout the thickness of epithelium. The densities of fibers in the basal epithelial nerve plexus were: NSE greater than CGRP greater than TH. Transmission electron microscopy demonstrated two types of epithelial nerve fibers, one containing large dense-core vesicles and another small dense-core vesicles. Both types contained clear vesicles. These large and small dense-core vesicle fibers appeared to correspond to the CGRP and TH immunoreactive fibers, respectively. These results provide morphological baseline data on the normal sensory and sympathetic corneal epithelial innervation.