Calcitonin Gene-related Peptide Staining Research Articles

Intervertebral Disc Degeneration and Low Back Pain Depends on Duration and Magnitude of Axial Compression.

Purpose The pathological role of axial stress in intervertebral disc degeneration (IDD) is controversial, and there was no quantified study until now. Here, we tried to clarify the correlation between IDD or low back pain (LBP) and axial stress at different duration and magnitude in vitro and in vivo. Method In vitro, the gene expression of aggrecan, matrix metalloproteinase-3 (MMP3), calcitonin gene-related peptide (CGRP), and substance P (SP) was measured when nucleus pulposus cells (NPCs) were compressed under gradual severity. In vivo, a measurable Ilizarov-type compression apparatus was established for single coccygeal (Co) intervertebral disc (IVD) compression of Co7-8 in mouse. Gradient stress was placed at 0.4 Mpa (mild), 0.8 Mpa (moderate), and 1.2 Mpa (severe) for three days to investigate the effect of the magnitude of axial stress. Additionally, mild compression with 3, 7, and 14 days was used to determine the effect of the duration of axial stress. Subsequently, we evaluated the severity of IDD and LBP by radiological X-ray film; histological examination with H&E staining; immunohistochemical analysis with collagen II, aggrecan, and CGRP staining; and western blot analysis with collagen II, aggrecan, MMP-3, and interleukin-1β (IL-1β). Results When NPCs suffered gradual increased mechanical stress, the cells exhibited gradual downregulated expression of extracellular matrix (ECM)-related gene of aggrecan, upregulated expression of IDD-related gene of MMP3, and LBP-related gene of CGRP and SP. In the meantime, with different magnitudes of axial stress, the IVD showed progressively severe IDD and LBP, with gradual narrowing intervertebral height, destruction of IVD anatomy, decreased ECM, and increased catabolic factors and proalgesic peptides. Conclusion Axial compression is one of the critical pathological factors to cause IDD and LBP, and there was a strong positive correlation depended on the duration and magnitude of compression.

Calcitonin Gene-Related Peptide in Charcot Foot Neuroarthropathy

Category:Basic Sciences/Biologics; DiabetesIntroduction/Purpose:Charcot neuroarthropathy (CNA) is a destructive joint condition secondary to neuropathic deficiency. For more than a century, the pathogenesis of CNA has been interpreted as unprotected injury and/or uncontrollable inflammation in a neuropathic joint, with little understanding its cellular and molecular pathology. Histologically, a hallmark of CNA is bone and cartilage fragments engulfed by hyperplastic synovium. Synovium is richly innervated. Neuropeptides, such as calcitonin gene- related peptide (CGRP), are secreted by neuronal and non-neuronal cells of the nervous, endocrine, and immune systems, and regulate inflammation. This study investigated the expression of CGRP in the synovial samples collected from CNA and non-CNA joint conditions, and the effects of CGRP on the proliferation and collagenolysis of fibroblast-like synoviocytes (FLS) isolated from CNA synovium in vitro.Methods:For this study, six CNA and 14 non-CNA synovial samples were collected during foot and ankle surgery. The donors included 10 male and 10 female, age from 14 to 79 years (mean 48). The non-CNA conditions consisted of osteoarthritis, ankle instability, Os Trigonum and osteochondritis dissecans.Western blot and immunohistochemistry of CGRP were performed to detect and localize CGRP expression in the CNA and non- CNA synovium.FLS was isolated from CNA synovium and cultured with, or without, human CGRP (10nM) in the culture media, for comparison of cell proliferation. Additionally, FLS were seeded on the collagen-coated 24-well plate and simulated with CGRP (10nM) and recombinant human tumor necrosis factor-alpha (TNF-α). After 7 days, the residual collagen on the bottom of the culture plate were stained and imaged for area measurements.Data were analyzed with t test or one-way Analysis of Variance, followed with post hoc Tukey's test.Results:By western blot, there was significant CGRP expression in the CNA synovium (5/6; Fig1). Except of an intense CGRP band in one of the osteoarthritis samples, only recognizable CGRP bands were shown in the samples of other non-CNA conditions. The average density of CGRP (in greyscale) in the CNA group was about 2-fold of that in the non-CNA group (10126 +- 5346 vs. 5377+-3734; p < 0.05). Immunohistochemistry demonstrated intense CGRP staining in the intimal layer of the CNA synovium (arrows) but not in the non-CNA synovium (Fig1). Treated with CGRP, the number of FLS in tissue culture increased. Cell number doubling time was 1.0 day (+-0.4) for the CGRP treated FLS and 2.2 days (+-0.5) for the control (p < 0.05). When the culture media were supplemented with CGRP and TNF-α, FLS eroded a larger area of collagen coating comparing with TNF-α alone in the media (p < 0.05).Conclusion:A knowledge gap in understanding the molecular and cellular pathology of CNA hampers the development of disease-modifying therapies. This study showed an increased CGRP, with a concentration in the intimal layer, in the CNA synovium as compared with the synovium in the non-CNA foot conditions. The increased CGRP expression in CNA synovium may be part of the unbalanced neuropeptide signaling in the neuropathic pathology. Functionally, CGRP stimulated FLS proliferation and degrading collagen in vitro. These effects suggest that CGRP may play a role in bone and joint destruction in the Charcot foot.

Neonatal complete Freund's adjuvant-induced inflammation does not induce or alter hyperalgesic priming or alter adult distributions of C-fibre dorsal horn innervation.

Introduction:Inflammation during the neonatal period can exacerbate pain severity following reinjury in adulthood. This is driven by alterations in the postnatal development of spinal and supraspinal nociceptive circuitry. However, the contribution of alterations in peripheral nociceptor function remains underexplored.Objectives:We examined whether neonatal complete Freund's adjuvant (CFA)-induced inflammation induced or altered adult development of hyperalgesic priming (inflammation-induced plasticity in nonpeptidergic C fibres) or altered postnatal reorganization of calcitonin gene-related peptide (CGRP)-expressing and isolectin B4 (IB4)-binding C fibres in the spinal dorsal horn (DH).Methods:After intraplantar injection of CFA at postnatal day (P) 1, we assessed mechanical thresholds in adult (P60) rats before and after intraplantar carrageenan. One week later, intraplantar PGE2-induced hypersensitivity persisting for 4 hours was deemed indicative of hyperalgesic priming. CGRP expression and IB4 binding were examined in adult rat DH after CFA.Results:P1 CFA did not alter baseline adult mechanical thresholds, nor did it change the extent or duration of carrageenan-induced hypersensitivity. However, this was slower to resolve in female than in male rats. Rats that previously received carrageenan but not saline were primed, but P1 hind paw CFA did not induce or alter hyperalgesic priming responses to PGE2. In addition, CFA on P1 or P10 did not alter intensity or patterns of CGRP or IB4 staining in the adult DH.Conclusion:Complete Freund's adjuvant-induced inflammation during a critical period of vulnerability to injury during early postnatal development does not induce or exacerbate hyperalgesic priming or alter the broad distribution of CGRP-expressing or IB4-binding afferent terminals in the adult dorsal horn.

Physical exercise enhances vulnerability to migraine headache associated with CGRP up-expression in trigeminal nucleus caudalis of stressed rats

ABSTRACT Objectives There is conflicting evidence on the effect of physical exercise on migraine development. Present study investigated the impact of treadmill exercise on migraine – associated symptoms and changes in calcitonin gene-related peptide (CGRP) expression in rats with and without maternal deprivation stress (MD). Methods Two days after birth, the male Wistar pups were randomly divided into four groups (n = 6) as follows: intact, exercise, MD, and MD plus exercise. The animals in the MD groups were separated from their dams 4 h per day for 2 weeks. At 8 weeks of age, the rats were exercised on a motor-driven treadmill for 4 weeks. Then, nitroglycerin (NTG) (5 mg/kg/IP) was used to induce migraine and pain-related symptoms were recorded for 90 min. NTG-related thermal hyperalgesia was measured by tail flick and hot plate methods. Finally, immunofluorescence staining of CGRP in trigeminal subnucleus caudalis (Vc) was performed. Results NTG – produced a significant headache symptoms and thermal hypersensitivity, which were aggravated following physical exercise in stressed or unstressed groups. Besides, NTG administration increased CGRP expression in the Vc of rats. Such effect was overpowered by treadmill running only in rats exposed to MD stress. Conclusion These findings highlight the worsening effects of treadmill exercise for migraine in rats with and without MD stress. However, inflammatory response can further exacerbate in stressed rats.

Intra-articular Injection of Pure Platelet-Rich Plasma Is the Most Effective Treatment for Joint Pain by Modulating Synovial Inflammation and Calcitonin Gene-Related Peptide Expression in a Rat Arthritis Model

Background: Platelet-rich plasma (PRP) has emerged as a treatment for osteoarthritis (OA). However, the effect that leukocyte concentrations in PRP have on OA remains unclear. Purpose: To clarify the optimal PRP formulation for OA treatment by comparing pure PRP, leukocyte-poor PRP (LP-PRP), and leukocyte-rich PRP (LR-PRP) in a rat arthritis model. Study Design: Controlled laboratory study. Methods: Knee arthritis was induced bilaterally in male Wistar rats with intra-articular injections of monosodium iodoacetate (MIA) on day 0. Rats were randomly assigned to 1 of 3 treatment groups (pure PRP, LP-PRP, and LR-PRP). On day 1, allogenic PRP was injected into the right knee of rats and phosphate-buffered saline was injected into the left knee as a control. Weight distribution on the hindlimbs was measured for 14 days to assess pain behavior. Rats were euthanized at day 5 or 14 for histological assessment of synovial tissue and cartilage. Immunohistochemical staining of calcitonin gene-related peptide (CGRP) and α-smooth muscle actin was performed to determine the mechanism of pain relief induced by the PRP preparations. Results: In all groups, PRP increased the load-sharing ratio on PRP-injected knees, with pure PRP eliciting the largest effect among the 3 kinds of PRP (P < .05). Structural changes in the synovial tissue were significantly inhibited in the pure-PRP group compared with the control group after both 5 and 14 days (P < .001 and P = .025, respectively), whereas no significant difference was found between the control, LP-PRP, and LR-PRP groups. An inhibitory effect on cartilage degeneration was observed only in the pure-PRP group on day 14. Pure PRP also significantly inhibited expression of CGRP-positive nerve fibers in the infrapatellar fat pad compared with the other groups (P < .05). Conclusion: In an MIA-induced arthritis model, pure PRP injection was the most effective treatment for reduction of pain-related behavior and inhibition of synovial inflammation and pain sensitization. Clinical Relevance: PRP formulations should be optimized for each specific disease. This study shows the superiority of pure PRP for treatment of arthritis and joint pain.

Effects of Ultra-early Hyperbaric Oxygen Therapy on Femoral Calcitonin Gene-Related Peptide and Bone Metabolism of Rats With Complete Spinal Transection.

Seventy-five Sprague-Dawley rats were randomly divided into sham, complete spinal cord transection (CSCT) and hyperbaric oxygen (HBO) groups. Among them, rats in HBO group were further divided into 3 hours group (HBO1) and 12 hours group (HBO2). To study the effects of ultra-early HBO therapy on femoral calcitonin gene-related peptide (CGRP) and bone metabolism of rats with CSCT. Complete spinal cord injury (SCI) is still an unresolved problem in clinical practice. Studies on changes in (calcitonin gene-related peptide) CGRP and bone metabolism and osteoporosis prevention after SCI have important clinical significance. Rats in the sham group underwent laminectomy alone, whereas rats in the other three groups underwent laminectomy and CSCT at the level of the 10th thoracic vertebra. Six weeks after operation, rat blood samples and femoral samples from CSCT area were taken and prepared for immunohistochemical staining of CGRP, quantitative polymerase chain reaction (qPCR) of CGRP mRNA, enzyme-linked immunosorbent assay (ELISA) for the levels of serum bone-specific alkaline phosphatase (sBAP), serum osteocalcin (sOC), serum type-I collagen amino-terminal peptide (sNTX), and urinary deoxypyridinoline (uDPD). These data were statistically analyzed using paired LSD or Tamhane. The number of CGRP-positive cells and expression of CGRP mRNA in femur were significantly reduced, and the levels of sBAP, sOC, sNTX, and uDPD were significantly increased in CSCT, HBO1, and HBO2 groups than in the sham group, (P < 0.05-0.01). In addition, the number of CGRP-positive cells, expression of CGRP mRNA in femur, and the levels of sBAP and sOC were significantly enhanced, but the levels of sNTX and uDPD were significantly lowered in HBO1 group than in HBO2 and CSCT groups (P < 0.05). Ultra-early HBO therapy could improve bone turnover, promote bone formation, and prohibit bone resorption by enhancing CGRP synthesis in the sensory neurons in posterior horn of spinal cord. N/A.

Substance P and Calcitonin Gene-Related Peptide in the Glands of External Auditory Canal Skin.

ObjectivesThe earwax (cerumen) that covers external auditory canal (EAC) skin contains a mixture of ceruminous and sebaceous gland substances, such as lipids, peptides, and proteins. The components secreted from the ceruminous gland that is a modified sweat gland form cerumen and contain several antimicrobial factors. Since substance P (SP) and calcitonin gene-related peptide (CGRP), known as a secretagogue, have been found in sweat glands, our purpose was to determine the expression of SP and CGRP in the glands of EAC skin.Methods Sections of normal human EAC skins were immunostained for the presence of SP and CGRP using polyclonal antibodies. Immunoreactivity was detected using an avidin-biotin peroxidase complex method.Results SP staining was found in ceruminous gland acini and myoepithelial cells. But the SP staining was not found in the sebaceous glands and epidermal region. CGRP was strongly stained in the ceruminous gland and weakly in the sebaceous gland cells. Interestingly, most prominent staining of SP and CGRP was noted in the myoepithelial cells of the ceruminous gland.Conclusion The findings in this study suggest that SP and CGRP are expressed in the glands of the EAC skin and secreted in the process of ceruminous gland secretion.

Evaluation of Histological Changes in Back Muscle Injuries in Rats over Time.

Study DesignAnimal model study.PurposeThe purpose of this study was to evaluate the histological variation in the injured muscle and production of calcitonin gene-related peptide in rats over time.Overview of LiteratureVertebral surgery has been reported to cause atrophy of the back muscles, which may result in pain. However, few reports have described the time series histological variation in the injured muscle and changes in the dominant nerve.MethodsWe used 30 male, 8-week-old Sprague-Dawley rats. The right and left sides of the paravertebral muscle were considered as the injured and uninjured sides, respectively. A 115 g weight was dropped from a height of 1 m on the right paravertebral muscle. Hematoxylin and eosin (H&E) staining of the muscle was performed 1–3 weeks after injury for histological evaluation. Fluoro-Gold (FG) was injected into the paravertebral muscle. The L2 dorsal root ganglia on both sides were resected 1, 2, and 3 weeks after injury, and immunohistochemical staining for calcitonin gene-related peptide was performed.ResultsH&E staining of the paravertebral muscle showed infiltration of inflammatory cells and the presence of granulation tissue in the injured part on the ipsilateral side 1 week after injury. Muscle atrophy occurred 3 weeks after injury, but was repaired via spontaneous replacement of muscle cells/fibers. In contrast, compared with the uninjured side, the percentage of cells double-labeled with FG and calcitonin gene-related peptide in FG-positive cells in the dorsal root ganglia of the injured side was significantly increased at each time point throughout the study period.ConclusionsThese results suggest that sensitization of the dominant nerve in the dorsal root ganglia, which may be caused by cicatrix formation, can protract injured muscle pain. This information may be helpful in elucidating the underlying mechanism of persistent pain after back muscle injury.

Distribution of Neuropeptides in the Synovium of Charcot Neuroarthropathy

Category: Basic Sciences/Biologics Introduction/Purpose: The pathology of Charcot neuroarthropathy (CNA) presents inflammation, and bone and cartilage destruction. It is still unclear how the neuropathic signals lead to joint inflammation and destruction. Neuropeptides are secreted by neurons and involve in the regulation of inflammation. Synovium is richly innervated and known to play a critical role in the pathology of inflammatory arthritis. This study is designed to investigate the distribution of several neuropeptides, such as vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP), in CNA synovium, in comparison with non-CNA synovium, with immunohistochemistry. Methods: Synovial samples were collected from CNA patients (n=3) and patients with osteoarthritis and intraarticular fracture (non-CNA; n=3), during foot and ankle surgery (approved by IRB). The tissue samples were fixed with 4% paraformaldehyde, embedded in O.T.C media and sectioned with a cryostat. From each patient, 6-9 sections were randomly selected for immunohistochemistry. After blocking the tissue sections with normal serum, primary antibody of SP, VIP and CGRP was separately applied and incubated at 4ºC overnight. The biotinylated secondary antibody and ABC kit (Victor Laboratories) were applied subsequently. DAB (3,3’-diaminobenzidine) was used for colorimetric detection (brown) of immunohistochemical reactions. The cell nuclei were stained with hematoxylin. The staining was viewed under a microscope and images were taken with a digital camera. Results: In the non-CNA synovium, SP was stained around neuro-vascular bundles and intensely stained on the surface of the synovium, i.e. the intimal layer. In the CNA synovium, SP staining was increased and more diffuse into the subintimal layer of the synovium (Fig 1). In non-CNA synovium, VIP was detected along the surface area of the synovium but not limited in the intimal layer. In CNA synovium, VIP was a similar staining pattern with a reduced staining intensity. The CGRP staining was primarily limited in the intimal layer in the non-CNA synovium. In CNA synovium, CGRP was stained in the intimal layer with an increased intensity. Conclusion: SP, VIP and CGRP are well-studied neuropeptides and have broad biological and pathological functions. All of them were detected in the intimal layer, where fibroblast-like synoviocytes are located. Fibroblast-like synoviocytes are fundamental elements of joint inflammation and are capable of degradation of bone and cartilage directly and indirectly. The changed patterns of SP, VIP and CGRP distributions in CNA synovium revealed by immunohistochemistry indicate a possible pathological pathway in CNA development: the unbalanced neuropathic signals direct fibroblast-like synoviocytes to an inflammatory state, which trigger the cascade of bone and cartilage destruction in CNA.

Loss of α-calcitonin gene-related peptide (αCGRP) reduces the efficacy of the Vestibulo-ocular Reflex (VOR).

The neuroactive peptide calcitonin-gene related peptide (CGRP) is known to act at efferent synapses and their targets in hair cell organs, including the cochlea and lateral line. CGRP is also expressed in vestibular efferent neurons as well as a number of central vestibular neurons. Although CGRP-null (-/-) mice demonstrate a significant reduction in cochlear nerve sound-evoked activity compared with wild-type mice, it is unknown whether and how the loss of CGRP influence vestibular system function. Vestibular function was assessed by quantifying the vestibulo-ocular reflex (VOR) in alert mice. The loss of CGRP in (-/-) mice was associated with a reduction of the VOR gain of ≈50% without a concomitant change in phase. Using immunohistochemistry, we confirmed that, although CGRP staining was absent in the vestibular end-organs of null (-/-) mice, cholinergic staining appeared normal, suggesting that the overall gross development of vestibular efferent innervation was unaltered. We further confirmed that the observed deficit in vestibular function of null (-/-) mice was not the result of nontargeted effects at the level of the extraocular motor neurons and/or their innervation of extraocular muscles. Analysis of the relationship between vestibular quick phase amplitude and peak velocity revealed that extraocular motor function was unchanged, and immunohistochemistry revealed no abnormalities in motor endplates. Together, our findings show that the neurotransmitter CGRP plays a key role in ensuring VOR efficacy.

Characterization of primary afferent spinal innervation of mouse uterus.

The primary afferent innervation of the uterus is incompletely understood. The aim of this study was to identify the location and characteristics of primary afferent neurons that innervate the uterine horn of mice and correlate the different morphological types of putative primary afferent nerve endings, immunoreactive to the sensory marker, calcitonin gene related peptide (CGRP). Using retrograde tracing, injection of 5–10 μL of 1,1′-didodecyl-3,3,3,3′-tetramethylindocarbocyanine perchlorate (DiI) into discrete single sites in each uterine horn revealed a biomodal distribution of sensory neurons in dorsal root ganglia (DRG) with peak labeling occurring between T13-L3 and a second smaller peak between L6-S1. The mean cross sectional area of labeled cells was 463 μm2 ± s.e.m. A significantly greater proportion of labeled neurons consisted of small cell bodies (<300 μm2) in the sacral spinal cord (S2) compared with peak labeling at the lumbar (L2) region. In both sections and whole mount preparations, immunohistochemical staining for CGRP revealed substantial innervation of the uterus by CGRP-positive nerve fibers located primarily at the border between the circular and longitudinal muscle layers (N = 4). The nerve endings were classified into three distinct types: “single,” “branching,” or “complex,” that often aligned preferentially in either the circular or longitudinal axis of the smooth muscles. Complex endings were often associated with mesenteric vessels. We have identified that the cell bodies of primary afferent neurons innervating the mouse uterus lie primarily in DRG at L2 and S1 spinal levels. Also, the greatest density of CGRP immunoreactivity lies within the myometrium, with at least three different morphological types of nerve endings identified. These findings will facilitate further investigations into the mechanisms underlying sensory transduction in mouse uterus.

High Expression of Calcitonin Gene-Related Peptide and Substance P in Esophageal Mucosa of Patients with Non-Erosive Reflux Disease

Visceral hypersensitivity is an important etiology of non-erosive reflux disease (NERD). Calcitonin gene-related peptide (CGRP) and substance P (SP) are involved in the sensitization of afferent neuronal pathways. The objectives of this study were to evaluate visceral hypersensitivity in NERD patients, investigate the association between visceral hypersensitivity and mucosal expression of SP and CGRP, and assess their involvement in the pathogenesis of NERD. Twenty-six NERD patients and 12 healthy volunteers were recruited. Intraesophageal balloon distention was performed, and initial perception threshold (IPT) and threshold of discomfort (ToD) were determined. Immunohistochemical staining was used to measure the optical density (OD) of CGRP and SP-reactive levels in esophageal mucosa, and the numbers of CGRP and SP-reactive neural fibers. IPT and ToD were 9.6±4.8 and 12.3±3.2ml, respectively, in NERD patients, significantly lower than for controls (13.2±7.5 and 21.6±5.7ml, P<0.05 and P<0.01, respectively). Mean OD values for CGRP and SP staining were significantly higher in NERD than for controls (both P<0.05) and, in NERD, were negatively correlated with IPT and ToD (all P<0.01). Numbers of CGRP and SP-reactive neural fibers in esophageal submucosa of NERD patients were significantly increased (both P<0.05). Expression of esophageal epithelial CGRP and SP is increased, and correlates negatively with perception thresholds in NERD. These findings may aid understanding of peripheral visceral hypersensitivity and the development of new therapeutic approaches for management of NERD.

Smoking Induces Oropharyngeal Narrowing and Increases the Severity of Obstructive Sleep Apnea Syndrome

Smoking is a known risk factor for snoring, and is reported to be associated with an increased prevalence of obstructive sleep apnea syndrome (OSAS). The purpose of this was to determine the relationship of smoking to the severity of OSAS and examine what local histological changes in the uvular mucosa of OSAS patients might influence this relationship. Fifty-seven OSAS subjects were included and classified according to smoking history and OSAS severity. Twenty-eight subjects were heavy smokers and 29 were nonsmokers; these 57 patients were divided according to moderate or severe OSAS. Histologic changes in the uvular mucosa were evaluated in all subjects as well as smoking duration and OSAS severity. Among smokers, moderate-to-severe OSAS was more common, and apnea, hypopnea, and oxygen desaturation indices were higher. Moreover, smoking duration and OSAS severity were significantly correlated. Increased thickness and edema of the uvular mucosa lamina propria were observed in moderate and severe OSAS patients, and only smokers had significant changes in uvular mucosa histology. Positive staining for calcitonin gene-related peptide (CGRP), a neuroinflammatory marker for peripheral nerves, was increased in the uvular mucosa of smokers. Our results suggest that smoking may worsen OSAS through exacerbation of upper airway collapse at the level of the uvula, and that histological changes of the uvular mucosa correlated with smoking might be due to increased CGRP-related neurogenic inflammation.

Calcitonin gene-related peptide (CGRP) and its receptor components in human and rat spinal trigeminal nucleus and spinal cord at C1-level

BackgroundCalcitonin gene-related peptide (CGRP) has a key role in migraine pathophysiology and is associated with activation of the trigeminovascular system. The trigeminal ganglion, storing CGRP and its receptor components, projects peripheral to the intracranial vasculature and central to regions in the brainstem with Aδ- and C-fibers; this constitutes an essential part of the pain pathways activated in migraine attacks. Therefore it is of importance to identify the regions within the brainstem that processes nociceptive information from the trigeminovascular system, such as the spinal trigeminal nucleus (STN) and the C1-level of the spinal cord. Immunohistochemistry was used to study the distribution and relation between CGRP and its receptor components - calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) - in human and rat STN and at the C1-level, using a set of newly well characterized antibodies. In addition, double-stainings with CGRP and myelin basic protein (MBP, myelin), synaptophysin (synaptic vesicles) or IB4 (C-fibers in general) were performed.ResultsIn the STN, the highest density of CGRP immunoreactive fibers were found in a network around fiber bundles in the superficial laminae. CLR and RAMP1 expression were predominately found in fibers in the spinal trigeminal tract region, with some fibers spanning into the superficial laminae. Co-localization between CGRP and its receptor components was not noted. In C1, CGRP was expressed in fibers of laminae I and II. The CGRP staining was similar in rat, except for CGRP positive neurons that were found close to the central canal. In C1, the receptor components were detected in laminae I and II, however these fibers were distinct from fibers expressing CGRP as verified by confocal microscopy.ConclusionsThis study demonstrates the detailed expression of CGRP and its receptor components within STN in the brainstem and in the spinal cord at C1-level, and shows the possibility of CGRP acting postjunctionally in these areas putatively involved in primary headaches.

The immunohistochemical demonstration of parafollicular cells and evaluation of calcium-phosphate balance in patients with thyroid hemiagenesis

Thyroid hemiagenesis (TH) is characterized by the congenital absence of one thyroid lobe. The aim of this study was to evaluate the calcium-phosphate balance in TH. Twenty patients with TH and 20 controls with a bilobed thyroid were studied. Serum concentrations of total calcium, parathormon and calcitonin were measured. Additionally, the immunohistochemical expression of calcitonin, chromogranin A (chA), neuron-specific enolase (NSE) and calcitonin gene-related peptide (CGRP) was evaluated in surgical specimens from patients with TH and controls. There were no significant differences in biochemical parameters between TH and controls. Positive staining for calcitonin was demonstrated in 3/8 thyroid sections from three patients with TH, but only in 2/33 sections from four controls (p < 0.005). All sections from patients with TH positive for calcitonin also expressed chA, NSE and CGRP. Two sections from controls positive for calcitonin presented an additionally positive reaction for chA, and one of them also for NSE. None presented positive staining for CGRP. Of three TH sections, in one, hyperplasia of C cells of medium grade, and in another hyperplasia of C cells of high grade, could be detected. In the controls, hyperplasia of C cells of low and medium grade was observed. TH was associated with slightly enhanced C cells hyperplasia compared to controls, which might indicate compensatory proliferation. However, the calcium-phosphate balance does not seem to be significantly affected.

Cerebellar distribution of calcitonin gene-related peptide (CGRP) and its receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) in rat

Clinical and experimental results have revealed a fundamental role of calcitonin gene-related peptide (CGRP) in primary headaches. CGRP is widely expressed in neurons both in the central nervous system (CNS) and in peripheral sensory nerves. In the CNS there is a wide distribution of CGRP-containing neurons with the highest levels seen in striatum, amygdale and cerebellum. Moreover, in acute attacks of migraine there is evidence of cerebellar activation. To understand the role of CGRP, antibodies towards the CGRP receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein type 1 (RAMP1) have been developed. In the present study we therefore examined immunohistochemically the distribution of CGRP and its receptor components in the cerebellum.CGRP immunoreactivity was only found intracellularly in the cerebellar Purkinje cell bodies, whereas CLR and RAMP1 were detected on the surface of the Purkinje cell bodies and in their processes. The elaborate dendritic tree of Purkinje cell fibers was distinctly visualized with the RAMP1 antibody. In addition, profoundly stained fibers spanning from the molecular layer into the medulla was observed with the RAMP1 antibody. Judged from the high density of immunoreactive cells expressing CGRP, RAMP1 or CLR, and from the double staining of CGRP and RAMP1 it is likely that most, if not all, Purkinje cells express both the peptide and the receptor components. Double staining with RAMP1 and the glial cell markers glial fibrillary acidic protein (GFAP) and S-100 revealed an almost identical staining pattern of the antibodies in the area of the cell body surfaces. However, as judged by confocal microscopy, no double staining was present. Instead, it was discovered that the glial cells tightly surrounded the Purkinje cells which easily could be interpreted as co-localization in the epifluorescence microscope.Our observations demonstrate that there is a rich expression of CGRP and CGRP receptor elements in the cerebellum which points towards a functional role of CGRP in cerebellar Purkinje cells. Recent advances in the biology of the cerebellum indicate that there may be a role in nociception; hence a target of the recently discovered CGRP receptor antagonists that have demonstrated improvement in migraine pain and associated symptoms could be cerebellar CGRP receptors.

Expression of Sensory Neuropeptides in Tendon is Associated with Failed Healing and Activity-Related Tendon Pain in Collagenase-Induced Tendon Injury

Background Increase in expression of substance P (SP) and calcitonin gene-related peptide (CGRP) has been reported in clinical samples of tendinopathy. Purpose To examine the spatial-temporal expression of these neuropeptides as well as their association with activity-related tendon pain, matrix degeneration, failed healing, and pathologic calcification in an established collagenase-induced tendon injury rat model. Study Design Controlled laboratory study. Methods Collagenase or saline was injected into the patellar tendon of rats. At weeks 2, 4, 8, 12, and 16, just before the rats were sacrificed, the double-stance duration of rats was examined by gait analysis method. After sacrifice, the patellar tendons were harvested for histologic analysis and immunohistochemical staining of SP and CGRP. Results There was an increase of SP and CGRP immunopositivity in tendon fibroblasts at week 2. The immunopositive signals decreased at weeks 4 and 8 and were observed in chondrocyte-like cells. At weeks 12 and 16, the immunopositive staining increased again and was observed in cells embedded in calcific deposits in addition to tendon fibroblasts and chondrocyte-like cells. The expression pattern was consistent with matrix degeneration, calcification, and failed healing in the animal model. There were significant positive correlations of immunopositivity of SP (rho = .502, P = .002) and CGRP (rho = .483, P = .003) with double-stance duration after collagenase injection. Conclusion There was increased expression of SP and CGRP after collagenase-induced tendon injury, and their expression was positively associated with double-stance duration. Clinical Relevance Substance P and CGRP might be involved in the pathogenesis and origin of pain of tendinopathy and could be the targets for future intervention.

The Potential Role of Calcitonin Gene-Related Peptide (CGRP) in Breast Carcinogenesis and Its Correlation With 99mTc-(V)DMSA Scintimammography

Experimental data suggest a role for calcitonin gene-related peptide (CGRP) in normal breast development and angiogenesis. This pilot study correlated CGRP with neoangiogenesis and the uptake of the tumor-seeking, proliferation-imaging radiotracer pentavalent technetium-99m dimercaptosuccinate (99mTc-(V)DMSA) in invasive and preinvesive breast lesions. Among women evaluated preoperatively by 99mTc-(V)DMSA scintimammography, 29 invasive ductal carcinomas (IDCs) were retrospectively studied: 15 isolated (Group I); 14 mixed with preinvasive pathologies (ductal carcinoma in situ [DCIS] and/or epithelial hyperplasia [EH]; Group M). CGRP staining and neoangiogenesis were compared between invasive and DCIS/EH regions and were correlated. 99mTc-(V)DMSA displayed a diffusely increased uptake pattern corresponding to DCIS/EH; its lesion-to-background (L/B) ratio was compared between images acquired at 10 and 60 minutes and its retention ratio (RR) was correlated with CGRP. Seven of 15 group I and 10 of 14 group M patients (58.6% of the population) were CGRP-positive. CGRP was prevalent in the DCIS/EH component of mixed-lesions (even in the surrounding normal epithelium of nearly half), with declining intensity as advancing from DCIS/EH to high-grade IDC. Similarly, neoangiogenesis was considerably higher in DCIS/EH than in group I pure IDCs. A significant CGRP-neoangiogenesis correlation was verified only in group I. The diffuse 99mTc-(V)DMSA uptake exhibited significant, time-related L/B increase and a RR positively correlating with CGRP. CGRP expression and neoangiogenesis are intensified in mixed invasive-preinvasive breast lesions; an underlying relation may exist, requiring further investigation. CGRP also appears associated with 99mTc-(V)DMSA's propensity to depict preinvasive pathologies. This relationship could denote an additional proliferative role for CGRP.

Relative contribution of peripheral versus central opioid receptors to antinociception

Opioid effects are mediated by central and peripheral opioid receptors. Here we examine the relative contribution of each receptor population to antinociception elicited by systemically administered centrally penetrating opioids, and by loperamide (a peripherally restricted opioid). Nociception (abdominal writhes) was induced by intraperitoneally (i.p.) injected 0.6% acetic acid in mice. We analyzed opioid receptor expression in peritoneum by immunohistochemistry, antinociception after i.p. injected agonists at mu (morphine, loperamide)-, delta (SNC80)- and kappa (U50488)-receptors, and its reversibility by subcutaneously (s.c.) administered centrally penetrating antagonists β-funaltrexamine (mu), naltrindole (delta) and nor-binaltorphimine (kappa), and by the peripherally restricted antagonist naloxone methiodide (NLXM). NLXM was also injected intracerebroventricularly (i.c.v.) before i.p. loperamide. Mu-, kappa- and, to a lesser degree, delta-receptors were expressed on peripheral nerve terminals in the peritoneum. The anatomical distribution of the opioid receptor staining was very similar to the staining for calcitonin gene-related peptide, a marker of sensory neurons. Morphine, U50488 and, to a lesser degree, SNC80 blocked acetic and acid induced writhes. These effects were reversed by β-funaltrexamine, nor-binaltorphimine and naltrindole, respectively. NLXM (s.c.) reversed antinociceptive effects of morphine, SNC80 and U50488 by 57%, 80% and 47%, respectively. Loperamide (0.05 mg/kg)-induced antinociception was reversed by s.c. β-funaltrexamine and NLXM. Loperamide (0.1 mg/kg)-induced antinociception was completely blocked by s.c. β-funaltrexamine but was only attenuated (by 50%) by s.c. or i.c.v. NLXM. In conclusion, systemically administered centrally penetrating mu-, delta- and kappa-agonists produced a substantial part of antinociception through peripheral opioid receptors. Higher dose loperamide-induced antinociception involved also central opioid receptors.

Neurochemical profile of dorsal root ganglion neurons innervating the canine ventricle

Dorsal root ganglion (DRG) sensory neurons innervating the canine ventricle were identified by retrograde tracing with horseradish peroxidase (HRP and cholera toxin β subunit-conjugated HRP) injected into the anterior left ventricle and right ventricular outflow tract. DRG’s (T1-T4) were harvested 7 days post injection, sectioned and immunostained for HRP and combinations of calcitonin gene-related peptide (CGRP), substance P (SP), or neuronal nitric oxide synthase (nNOS). At the T3 level, 19+6% (mean ±SD) of the DRG neuronal population was immunolabeled for CGRP, 11%±3 for SP, and 6±3% were double labeled for CGRP and SP. Of this population, 2% were immunolabeled for HRP. Of these HRP-labeled DRG neurons, 23±17% exhibited positive staining for CGRP, 3±4% for SP (p<0.02 versus % SP positive in total population), and 13±9% were tripled labeled (HRP, SP and CGRP). Thirty-one percent of HRP-labeled DRG neurons exhibited nNOS immunoreactivity. These data support the emerging concept of afferent heterogeneity in sensory processing of the ventricular milieu. They also suggest that the neurochemical profile for DRG sensory afferents is differentially expressed, depending upon the target organ. (HL71830).