Calcitonin Gene-related Peptide Nerve Research Articles

Vasculature in the mouse colon and spatial relationships with the enteric nervous system, glia, and immune cells.

The distribution, morphology, and innervation of vasculature in different mouse colonic segments and layers, as well as spatial relationships of the vasculature with the enteric plexuses, glia, and macrophages are far from being complete. The vessels in the adult mouse colon were stained by the cardiovascular perfusion of wheat germ agglutinin (WGA)-Alexa Fluor 448 and by CD31 immunoreactivity. Nerve fibers, enteric glia, and macrophages were immunostained in the WGA-perfused colon. The blood vessels entered from the mesentery to the submucosa and branched into the capillary networks in the mucosa and muscularis externa. The capillary net formed anastomosed rings at the orifices of mucosa crypts, and the capillary rings surrounded the crypts individually in the proximal colon and more than two crypts in the distal colon. Microvessels in the muscularis externa with myenteric plexus were less dense than in the mucosa and formed loops. In the circular smooth muscle layer, microvessels were distributed in the proximal, but not the distal colon. Capillaries did not enter the enteric ganglia. There were no significant differences in microvascular volume per tissue volume between the proximal and distal colon either in the mucosa or muscularis externa containing the myenteric plexus. PGP9.5-, tyrosine hydroxylase-, and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers were distributed along the vessels in the submucosa. In the mucosa, PGP9.5-, CGRP-, and vasoactive intestinal peptide (VIP)-immunoreactive nerves terminated close to the capillary rings, while cells and processes labeled by S100B and glial fibrillary acidic protein were distributed mainly in the lamina propria and lower portion of the mucosa. Dense Iba1 immunoreactive macrophages were closely adjacent to the mucosal capillary rings. There were a few macrophages, but no glia in apposition to microvessels in the submucosa and muscularis externa. In conclusion, in the mouse colon, (1) the differences in vasculature between the proximal and distal colon were associated with the morphology, but not the microvascular amount per tissue volume in the mucosa and muscle layers; (2) the colonic mucosa contained significantly more microvessels than the muscularis externa; and (3) there were more CGRP and VIP nerve fibers found close to microvessels in the mucosa and submucosa than in the muscle layers.

Safety and efficacy of intravesical instillation of resiniferatoxin in healthy cats: A preliminary study.

To evaluate the safety of intravesical application of resiniferatoxin (RTX) in healthy cats and its effects on calcitonin gene-related peptide (CGRP) and substance P (SP) produced by C-fibers. Seven adult female cats received either 25 mL of saline (control; n = 1), or intravesical RTX at 5, 25, or 50 μg in 25 mL of saline to a final concentration of 0.2 μg/mL (318 nM), 1 μg/mL (1,591 nM), and 2 μg/mL (3,181 nM) (n = 2 per group). The treatment was instilled into the urinary bladder for 20 min. Plasma concentrations of RTX were measured at 0, 0.5, 1, and 4 h. Physical exam, complete blood count, and serum biochemical analysis were performed on day 0, 7, and 14. After 14 days, the sacral dorsal root ganglia (DRG) and the urinary bladder were harvested for histological and immunofluorescence analysis. Intravesical RTX was well tolerated and plasma concentrations were below the quantifiable limits except for one cat receiving 1 μg/mL. Mild to moderate histopathological changes, including epithelial changes, edema, and blood vessel proliferation, were observed at lower doses (0.2 and 1 μg/mL), and were more severe at the higher dose (2 μg/mL). C-fiber ablation was observed in the urinary bladder tissue at all doses, as shown by an apparent reduction of both CGRP and SP immunoreactive axons. A dose of 25 μg (1 μg/mL) of RTX instilled in the urinary bladder of healthy cats appeared to decrease the density of SP and CGRP nerve axons innervating bladder and induced moderate changes in the bladder tissue.

Alcohol extract from Vernonia anthelmintica willd (L.) seed counteracts stress-induced murine hair follicle growth inhibition

BackgroundVernonia anthelmintica (L.) willd is a traditional urgur herb in China for a long history. Its alcohol extract (AVE) has been proved to promote hair follicle growth in C57BL/6 mice. We conducted this study to investigate the hair-growth effects of AVE in stressed mice and its possible mechanism of action.MethodsThe hair-follicle growth effects of AVE were examined by in vivo and in vitro study. We exposed C57BL/6 male mice to chronic restraint stress to induce murine hair follicle growth inhibition. The effects of AVE were examined by histological analysis, immunofluorescence for Ki67 and cytokeratin 19 immunoreactivity, western blot assay in tyrosinase and related proteins expressions and immunofluorescence for nerve fibers. In organ culture of mouse vibrissae follicles, we used substance P as a catagen-inducing factor of hair follicle growth, and measured the elongation of hair shafts and expression of neurokinin-1 receptor protein by application of AVE.ResultsOur results showed that AVE counteract murine hair follicle growth inhibition caused by chronic restraint stress via inducing the conversion of telogen to anagen and inhibiting catagen premature, increasing bulb keratinocytes and bulge stem cells proliferation, promoting melanogenesis, and reducing the numbers of substance P and calcitonin gene-related peptide nerve fibers. Furthermore, AVE also counteracted murine hair follicle growth inhibition caused by substance P in organ culture.ConclusionThese results suggest that AVE counteract stress-induced hair follicle growth inhibition in C57BL/6 mice in vivo and in vitro, and may be an effective new candidate for treatment of stress-induced hair loss.

Moxa-stick moxibustion activates mast cells of small intestine tissue in rats

To explore the effect of moxa-stick moxibustion and joss-stick moxibustion at "Guanyuan" (CV4) on the activity of mast cells in the small intestine tissue in rats. Twelve male SD rats were randomly divided into control, joss-stick moxibustion and moxa-stick moxibustion groups (n=4 rats in each group). Joss-stick or moxa-stick moxibustion was applied to CV4 for 10 min. After moxibustion, the skin temperature of the CV4 region was measured immediately with a thermometer. The mast cells and nerve fibers in the small intestine tissue were displayed by immunofluorescence histochemistry. Compared with the control group, the skin temperature of the CV4 region in both the joss-stick and the moxa-stick groups were significantly increased (P<0.05), while the skin temperature of the moxa-stick group was significantly higher than that in the joss-stick group (P<0.05). There were a large number of tryptase-positive mast cells in the small intestine of rats, some of which were co-expressed with lysosomal-associated membrane protein 1, displaying an activated state. The average numbers of mast cells in the control, joss-stick and moxa-stick groups were 9.2±3.6, 10.8±5.3 and 17.1±6.3, respectively, being significantly higher in the moxa-stick group than in the control and joss-stick groups (P<0.05). In addition, calcitonin gene-related peptide(CGRP) and neuropeptide Y(NPY) positive nerve fibers were found around the mast cells in the small intestinal tissues. Moxa-stick moxibustion gives rise a higher temperature at CV4 to activate mast cells surrounded by CGRP and NPY positive nerve fibers in the small intestine tissue in rats, suggesting an involvement of the sensory and sympathetic nervous system in the activation of intestinal mast cells possibly by way of somatic sympathetic reflex.

Evidence for changing nerve growth factor signalling mechanisms during development, maturation and ageing in the rat molar pulp

To examine rat molar pulp innervation and identify complex cellular signalling systems involving nerve growth factor (NGF) and its p75 receptors (NGFR) at different stages of development, maturation and ageing. Decalcified mandibular first molar mesial cusps from Wistar rats of ages 0day; 1, 2, 3, 4, 6, 9, 12 and 24weeks (n=5 per group) were sectioned (10μm) and incubated with antibodies for NGF, NGFR, calcitonin gene-related peptide (CGRP) and neurofilament. Nerve densities in worn and intact regions of 3- to 24-week-old rats were compared by anova, Bonferroni and t-tests. During odontogenesis, differences in NGF and NGFR expression were observed, with no evidence of nerve fibres, suggesting a signalling mechanism controlling cellular differentiation and dentine formation. Tooth wear in 4-week rats was associated with reduced NGF expression and significantly decreased CGRP axons within affected odontoblast regions. The underlying subodontoblasts started expressing NGF which continued until 9weeks. This may promote a significant increase in CGRP nerve density in affected regions. Nerve density in intact odontoblast regions increased gradually and reached significant levels in 12-week rats. Reduction in nerve densities within worn and intact regions of cusps was observed at 24weeks. Age-related changes and responses to tooth wear may be controlled by the NGF signalling mechanism, with roles in odontoblast/subodontoblast communication and control of sensory innervation at different stages of tooth development, maturation and ageing. Greater understanding of cellular and nerve regulation in the injured pulp may promote therapeutic strategies for pulp survival.

Neuropeptide Initiated Mast Cell Activation by Transcutaneous Electrical Acupoint Stimulation of Acupoint LI4 in Rats

Transcutaneous electrical acupoint stimulation (TEAS) has been consistently used clinically for its ease of operation, non-invasiveness and painlessness, in contrast to the characteristics of inserted needles. However, the mechanism remains unknown. The aim of this study was to investigate the local response of TEAS at Hegu acupoint (LI4). Immunohistochemistry was used to measure the expression of tryptase-positive mast cells, neuropeptides of the calcitonin gene-related peptide (CGRP) and substance P (SP) in LI4. Mast cells were also labelled with serotonin (5-HT), neurokinin-1 receptor (NK-1R) and toluidine blue. The results showed that cutaneous CGRP and SP immune-positive (CGRP-IP or SP-IP) nerve fibres in LI4 were more highly expressed. There were high degrees of mast cell aggregation and degranulation with release of 5-HT near the CGRP-IP or SP-IP nerve fibres and blood vessels after TEAS. The degranulation of mast cells (MCs) was accompanied by expression of NK-1R after TEAS. Either mast cell membrane stabilizer (Disodium cromoglycate) or NK-1R antagonist (RP 67580) diminished the accumulation and degranulation of MCs induced by TEAS. Taken together, the findings demonstrated that TEAS induced sensory nerve fibres to express CGRP and SP, which then bound to the NK-1R on MCs, after which MCs degranulated and released 5-HT, resulting in TEAS-initiated acupuncture-like signals.

Characterization of Perivascular Nerve Distribution in Rat Mesenteric Small Arteries.

The distribution pattern of perivascular nerves in some branches of rat mesenteric arteries was studied. Mesenteric arteries isolated from 8-week-old Wistar rats were divided into the 1st-, 2nd-, and 3rd-order branches. The distribution of perivascular nerves in each branch was immunohistochemically evaluated using antibodies against neuropeptide Y (NPY), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), substance P (SP), and neuronal nitric oxide synthase (nNOS). The density of NPY-, TH-, CGRP-, and SP-like immunoreactive (LI) nerves in the 2nd and 3rd branches was significantly greater than that in the 1st branch, and a negative relationship was found between nerve density and arterial diameter, except for TH-LI nerves. The density of NPY- and TH-LI nerves in all branches, which was similar, was greater than that of CGRP- (except for NPY-LI nerves in the 1st branch), SP-, or nNOS-LI nerves. Double immunostaining revealed that TH-LI nerves made contact with nNOS-LI, CGRP-LI, and SP-LI nerves and that CGRP-LI nerves made contact with TH-, NPY-, or nNOS-LI nerves, while TH-LI and CGRP-LI nerves nearly merged with NPY-LI and SP-LI nerves, respectively. These results suggest that the each branch of mesenteric arteries is densely innervated by vasoconstrictor nerves containing NPY, TH, and vasodilator CGRP nerves. They also suggest that the intense density of perivascular nerves in the 2nd and 3rd branches may contribute to maintaining vascular tone.

Maintenance of bladder innervation in diabetes: A stereological study of streptozotocin-treated female rats

Neuropathy and cystopathy are two common conditions in patients with chronic diabetes. Despite obvious bladder sensory and motor nerve dysfunction in diabetes, no studies have selectively explored whether sensory or motor innervation is affected in the bladder. In the present study, we tested the hypothesis that loss of bladder sensory and motor fibers is responsible for bladder sensory and motor dysfunction. Parasympathetic and sensory innervation of the bladder dome and neck were examined using immunohistochemistry (IHC) and stereology in adult female rats 12weeks after induction of diabetes by streptozotocin. Naïve and age matched rats were evaluated as controls. Diabetic rats had mean blood glucose level of >400mg/dl, and bladder weights and thicknesses that were more than doubled compared to naïve rats. In naïve rats, parasympathetic vesicular acetylcholine transporter (VAT) and sensory calcitonin gene-related peptide (CGRP) immunopositive nerve fibers were located in bladder smooth muscle and were more densely distributed in the neck compared to the dome. Within the urothelial region, CGRP nerve fibers were densely distributed while VAT nerve fibers were sparsely distributed in the bladder neck and both were virtually absent in the bladder dome. Streptozotocin induced diabetes did not change the total nerve fiber length of either VAT or CGRP stained fibers in either the neck or dome. These studies indicate that hyperglycemia, induced by streptozotocin treatment, does not result in a loss of parasympathetic VAT or CGRP sensory nerve fibers, per se, but the doubling of bladder weight and mass does indicate a decrease in innervation density.

CGRP Receptor Antagonism and Migraine Therapy

Migraine is the most prevalent of the neurological disorders and can affect the patient throughout the lifetime. Calcitonin gene-related peptide (CGRP) is a neuropeptide that is expressed in the central and peripheral nervous systems. It is now 2 decades since it was proposed to be involved in migraine pathophysiology. The cranial sensory system contains C-fibers storing CGRP and trigeminal nerve activation and acute migraine attacks result in release of CGRP. The CGRP receptor consists of a complex of calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1) and receptor component protein (RCP). At the central synapses in the trigeminal nucleus caudalis, CGRP acts postjunctionally on second-order neurons to transmit pain signals centrally via brainstem and midbrain to thalamus and higher cortical pain regions. CLR and RAMPs are widely expressed throughout the brain, in the trigeminal ganglion and in intracranial arteries. CGRP does not induce neurogenic inflammation or sensitization at peripheral meningeal sites but relays nociceptive information from trigeminal primary afferent neurons to the second-order neurons in the spinal trigeminal nucleus neurons. CGRP receptor antagonists have been developed as novel antimigraine drugs and found to be effective in the treatment of acute migraine attacks. Other ways to stop CGRP activity has been introduced recently through antibodies against CGRP and the CGRP receptor. While the CGRP receptors are expressed both in the CNS and at various places related to the trigeminal system the exact site of action for their therapy effect is still unresolved but the new approaches may resolve this.

Innervation and Immunohistochemical Characteristics of Epididymis in Alpaca Camelid (Vicugna Pacos)

Alpacas (Vicugna pacos) are domesticated camelids indigenous to south America and recently also bred in Europe and Italy for their high quality wool. There is little data available regarding the innervation of the male reproductive tract of this species. In the present study, the distribution of protein gene product 9.5 (PGP 9.5), neuropeptide Y (NPY), tyrosine hydroxilase (TH), calcitonin gene related peptide (CGRP) and substance P (SP) was analyzed in the epididymis by using immunohistochemical methods. Specimens of the caput, corpus and cauda epididymis were fixed in Bouin’s fluid and processed for immunohistochemistry analysis with primary antibodies against PGP 9.5, NPY, TH, CGRP and SP. Immunopositivity to PGP 9.5 and TH and NPY was observed in nerve fibre bundles and in single nerve fibres contained into the peritubular connective tissue. Many TH and NPY immunopositive cells were found to innervate blood vessels. Rare CGRP and SP immunopositive nerves were observed. Several PGP 9,5 and NPY immunopositive epithelial cells were observed in the caput epididymis. The results of the present study suggest a role for the innervations in modulate reproductive functions in the alpaca epididymis.

The Mechanism of Calcitonin Gene-Related Peptide–Containing Nerve Innervation

Calcitonin gene-related peptide (CGRP) is a major neurotransmitter and CGRP-containing primary sensory neurons play an important role in nociception and potent vasodilation. CGRP-containing nerves in mesenteric arteries are decreased in pathological animal models (hypertension, diabetes, and atherosclerosis). In apolipoprotein E–knockout mice, which have atherosclerosis and peripheral sensory nerve defects, nerve growth factor (NGF)-mediated CGRP nerve facilitation was down-regulated, which may have been caused by the impairment of the Akt–NO–cGMP pathway. In addition, NGF-mediated CGRP neurite outgrowth was decreased in fructose-induced insulin-resistant rats. We recently discovered that renin–angiotensin inhibitors improved CGRP innervation in spontaneously hypertensive rats, indicating that rescuing CGRP nerve innervation might improve pathophysiological conditions. To find a novel reagent that facilitates CGRP nerves, a new model, phenol-injured perivascular nerve model rats, was established. Adrenomedullin, hepatocyte growth factor, and angiotensin II type 2 receptor activation induced CGRP nerve distribution in phenol-injured rats. Furthermore, in insulin-resistant model rats, the down-regulation of CGRP nerves was likely due to the depression of phosphoinositide 3-kinase (PI3K)-dependent Akt activation. Administration of candesartan improves CGRPergic function via the PI3K–Akt pathway in insulin-resistant rats. Thus, clarification of the mechanisms of CGRP nerve defects may constitute future therapeutic targets.

Distributions of calcitonin gene-related peptide and substance P in the human maxillary sinus of Japanese cadavers

BackgroundSubstance P (SP) and calcitonin gene-related peptide (CGRP) are released by the nociceptive sensory nerve and are involved in blood flow, pain and inflammation in the nasal mucosa. The purpose of this study was to assess the distribution of the SP and CGRP nerve fibres related to blood supply within human Schneiderian membrane of the maxillary sinus (MS). Material and methodsIn this study, the MS from Japanese cadavers was examined by whole-mount immunohistochemistry. Human male cadavers (ranging in age from 80 to 90years) were used in this study. ResultsSP- and CGRP-positive fibres were found around large vessels of the medialis superior alveolar branches and also within the floor region of the MS. The floor region of the MS was composed of complex branches of these fibres. ConclusionOur results give useful information for surgical sinus floor elevation in this region of the MS. These anatomical features may assist in the execution of a successful surgical procedure.

An immunohistochemical study of the gut neuroendocrine system in juvenile pejerrey Odontesthes bonariensis (Valenciennes)

In this study, several neuropeptides were identified by immunohistochemistry in neuroendocrine cells (NEC) located in the gut epithelium and nerve cell bodies of the enteric nervous system of pejerrey Odontesthes bonariensis, a species that is a promising candidate for intensive aquaculture. The neuropeptides involved in orexigenic or anorexigenic action, i.e. gastrin, cholecystokinin-8, neuropeptide Y and calcitonin gene-related peptide (CGRP), displayed a significantly higher number of immunoreactive NECs in the anterior intestine, suggesting that this region of the gut plays an important role in the peripheral control of food intake. On the other hand, leu-enkephalin and vasoactive intestinal peptide (VIP), both associated with the modulation of the enteric immune system, showed no significant variations in the mean value of immunopositive NECs between the anterior and posterior intestine. This may indicate that their activity is required at a similar level along the entire gut. In addition, CGRP and VIP-immunoreactive neurons and nerve fibres were observed in the myenteric plexus, which might exert synergistic effects with the neuropeptides immunolocalized in NECs.

Histamine H 3 receptor-mediated modulation of perivascular nerve transmission in rat mesenteric arteries

The rat mesenteric artery has been shown to be innervated by adrenergic vasoconstrictor nerves and calcitonin gene-related peptide (CGRP)-containing (CGRPergic) vasodilator nerves. The present study was designed to investigate the involvement of histamine H 3 receptors in the neurotransmission of perivascular adrenergic and CGRPergic nerves. The mesenteric vascular beds without an endothelium isolated from male Wistar rats were perfused with Krebs solution and perfusion pressure was measured. In preparations with resting tension, the selective H 3 receptor agonist ( R)-α-methylhistamine (α-methylhistamine; 10–100 nM) significantly reduced periarterial nerve stimulation (2–8 Hz)-induced vasoconstriction and noradrenaline release in the perfusate without an effect on the vasoconstriction induced by exogenously injected noradrenaline (0.5, 1.0 nmol). In preparations with active tone produced by methoxamine (2 μM) and in the presence of guanethidine (5 μM), the periarterial nerve stimulation (1, 2 Hz)-induced vasodilator response was inhibited by α-methylhistamine (0.1–1 μM) perfusion without affecting vasodilation induced by exogenously injected CGRP (5 pmol). Clobenpropit (histamine H 3 receptor antagonist, 1 μM) canceled the α-methylhistamine-induced decrease in the periarterial nerve stimulation-induced vasoconstriction and noradrenaline release and periarterial nerve stimulation-induced vasodilation. These results suggest that the stimulation of H 3 receptors located in rat perivascular nerves inhibits presynaptically the neurotransmission of not only adrenergic nerves, but also CGRP nerves, by decreasing neurotransmitters.

Colitis induces calcitonin gene-related peptide expression and Akt activation in rat primary afferent pathways

Previous study has shown that colitis-induced increases in calcitonin gene-related peptide (CGRP) immunoreactivity in bladder afferent neurons result in sensory cross-sensitization. To further determine the effects of colitis on CGRP expression in neurons other than bladder afferents, we examined and compared the levels of CGRP mRNA and immunoreactivity in the lumbosacral dorsal root ganglia (DRG) and spinal cord before and during colitis in rats. We also examined the changes in CGRP immunoreactivity in colonic afferent neurons during colitis. Results showed increases in CGRP mRNA levels in L1 (2.5-fold, p < 0.05) and S1 DRG (1.9–2.4-fold, p < 0.05). However, there were no changes in CGRP mRNA levels in L1 and S1 spinal cord during colitis. CGRP protein was significantly increased in L1 (2.5-fold increase, p < 0.05) but decreased in S1 (50% decrease, p < 0.05) colonic afferent neurons, which may reflect CGRP release from these neurons during colitis. In L1 spinal cord, colitis caused increases in the number of CGRP nerve fibers in the deep lamina region extending to the gray commissure where the number of phospho-Akt neurons was also increased. In S1 spinal cord, colitis caused the increases in the intensity of CGRP fibers in the regions of dorso-lateral tract, and caused the increases in the level of phospho-Akt in the superficial dorsal horn of the spinal cord. In spinal cord slice culture, exogenous CGRP increased the phosphorylation level of Akt but not the phosphorylation level of extracellular-signal regulated kinase ERK1/2 even though our previous studies showed that colitis increased the phosphorylation level of ERK1/2 in L1 and S1 spinal cord. These results suggest that CGRP is synthesized in the DRG and may transport to the spinal cord where it initiates signal transduction during colitis.

Re-innervation pattern of the ‘neovagina’ created from the bladder flap in patients with Mayer-Rokitanski-Kistner-Hauser syndrome: An immunochemical study

Background. Cystovaginoplasty (CVP) is a method of vaginal reconstruction in women with Mayer-Rokitansky-Kistner-Hauser Syndrome (MRKHS). The neo-vagina allows normal sexual intercourses, but after CVP, the sexual life of patients with MRKHS does not differ significantly from normal females. Therefore, we decided to elucidate the pattern of sensory re-innervation of the bladder flap used for the surgery.Methods. Biopsies were taken from vaginal vestibule and urinary bladder during the CVP and 1 year later in four patients with MRKHS. The following neurotransmitters were studied calcitonin gene-related peptide (CGRP), galanin (GAL), vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP).Results. CGRP and PACAP nerve fibres were sparse under the urothelium and in submucosal layer of the neovagina, they were more numerous around blood vessels and in the vicinity of smooth muscles. This was similar to the pattern observed in the urinary bladder. VIP- and GAL-positive nerve fibres were most numerous in the submucosa around blood vessels and in smooth muscle bundles of neovagina. They were distinctly less numerous beneath the epithelium. This innervation pattern mimicked that seen in normal vagina and in vaginal vestibule of patients with MRKHS.Conclusions. Our findings demonstrate considerable nervous system plasticity in the bladder flap. Distribution of presumably sensory CGRP and PACAP immunoreactive nerve fibers was similar to the pattern observed within the intact bladder wall, and VIP or GAL immunoreactive nerve fibers (vasomotor functions) were distributed in a manner similar to that observed in the intact vaginal wall.

On the Horizon From the ORS

On the Horizon From the ORS

Depletion of peptidergic innervation in the gastric mucosa of streptozotocin-induced diabetic rats

Autonomic neuropathy affecting the gastrointestinal system is a major presentation of diabetic neuropathy. Changes in the innervation of gastric mucosa or muscle layers can contribute to gastrointestinal symptoms. The present study investigated this issue by quantitatively analyzing the immunohistochemical patterns of the gastric innervation in rats with streptozotocin (STZ)-induced diabetes. In control rats, calcitonin gene-related peptide (CGRP) and substance P (SP) (+) nerve fibers appeared in the gastric mucosa and muscle layers. Double immunohistochemical staining showed that immunoreactivities for SP and CGRP were co-localized with a pan-neuronal marker protein gene product 9.5. Both SP (+) nerve fibers (p<0.001) and CGRP (+) nerve fibers (p<0.005) were decreased in the gastric mucosa within 4 weeks of diabetes; the reduction persisted throughout 24 weeks. Diabetic rats treated with insulin did not show decrease of SP or CGRP (+) fibers in the mucosa 4 weeks after STZ injection (p>0.05). There was no significant change in SP (+) nerve fibers (p>0.05) or CGRP (+) nerve fibers (p>0.05) of the gastric muscle layers. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the expression levels of SP and CGRP mRNA in the thoracic dorsal root ganglia were similar between diabetic and control animals (p>0.05). Qualitative and quantitative ultrastructural examinations on the gastric mucosa documented unmyelinated nerve degeneration. These results suggest the existence of gastric sensory neuropathy in STZ-induced diabetes, and this pathology provides a foundation for understanding diabetic gastropathy.

Pregnancy increases perivascular trigeminal innervation of cerebral arteries without changing reactivity to calcitonin gene‐related peptide (CGRP)

This study investigated the influence of pregnancy and postpartum on trigeminal innervation of posterior cerebral arteries (PCA) and reactivity to CGRP. Third-order PCAs were dissected from nonpregnant (NP, n=35), late-pregnant (LP, day 19–20, n=32), or postpartum (PP, day 3–4, n=32) Sprague Dawley rats and either fixed for immunohistochemical staining of CGRP-reactive nerve fibers or mounted in an arteriograph chamber for concentration-response curves to CGRP (0.1–300nM). Nerve density was determined by Metamorph analysis as intersect points on a grid per area after confocal imaging. Pregnancy had a trophic influence on perivascular CGRP-containing nerve fibers, increasing nerve density from 0.6 ± 0.2 counts/μm2 in NP to 1.3 ± 0.3 counts/μm2 in LP (p<0.01). Nerve density significantly decreased in PP animals to 0.2 ± 0.1 counts/μm2 (p<0.01 vs. LP). The gestation-induced increase in CGRP innervation was not accompanied by a change in reactivity. Percent dilation from spontaneous tone at 300nM CGRP for NP, LP and PP animals was: 70±7%, 69±12% and 74±10% (p>0.05). These results demonstrate that trigeminal innervation of PCAs is increased during gestation that is not accompanied by changes in reactivity. It is possible that increased CGRP nerve density during pregnancy relates more to nociception and a predisposition to the neurologic symptoms of eclampsia than to changes in blood flow. Supported by RO1 NS045940 and AHA 0540081N.

Nitric oxide synthase activity in rat gastric mucosa contributes to mucin synthesis elicited by calcitonin gene-related peptide

The majority of research for the calcitonin gene-related peptide (CGRP) in the stomach has been devoted to the submucosal blood flow, and only slight attention has been paid to its involvement in the gastric epithelial function. In this study, we examined the age-related change in the CGRP-containing nerves and its effects on the mucus metabolism. We compared the immunoreactivity for CGRP in the gastric mucosa of 7-week-old rats (young) to that of 52-week-old animals (middle-aged). The effects of CGRP on the mucin biosynthesis were compared using the stomachs from both young and middle-aged rats. The nitric oxide synthase (NOS) activity was measured in the surface and deep mucosa of the gastric corpus. The density of the CGRP nerve fibers was reduced in both the lamina propria and submucosa of the middle-aged rats compared to the young rats. CGRP stimulated the mucin biosynthesis in the cultured corpus mucosa from the 7-week-old rats, but not from the 52-week-old rats. The total NOS activity of the surface layer in the corpus mucosa was markedly reduced in the middle-aged rats compared to the young rats. These findings demonstrate the age-dependent reduction in the CGRP-induced mucin biosynthesis, as well as in the density of the CGRP fibers in the rat stomach. The decreased NOS activity in the surface layer of the oxyntic mucosa in the aged rats may also be a principal cause for the lack of regulation of the mucin biosynthesis by CGRP.