CGRP-positive Nerve Fibers Research Articles

Blood-nerve barrier enhances chronic postsurgical pain via the HIF-1α/ aquaporin-1 signaling axis

BackgroundBlood nerve barrier (BNB) participates in the development of neuropathic pain. AQP1 is involved in peripheral pain perception and is negatively correlated with HIF-1α phenotype, which regulates endothelial permeability. However, the role of HIF-1α-AQP1-mediated BNB dysfunction in Chronic Postsurgical Pain (CPSP) has not been reported.MethodsMale Sprague-Dawley rats were randomized into 5 groups: (i) Naive group; (ii) Sham group; (iii) SMIR group: skin/muscle incision and retraction for one hour. Behavioral tests were performed for the three groups, BNB vascular permeability and western blotting were conducted to determine HIF-1α and AQP1 protein expression. (iv) The SMIR + HIF-1α inhibitor group; (v) SMIR + DMSO group. Rats in the two groups were administered with HIF-1α inhibitor (2ME2) or DMSO intraperitoneally on the third day post-SMIR surgery followed by performance of behavioral tests, BNB permeability assessment, and determination of HIF-1α, AQP1 and NF200 protein levels.ResultsThe permeability of BNB was significantly increased and the expression of AQP1 was downregulated on the 3rd and 7th days post-operation. AQP1 is mainly located in neurons and NF200, CGRP-positive nerve fibers. HIF-1α was highly expressed on the third day post-operation. HIF-1α inhibitor reversed the decrease in AQP1 expression and increase in NF200 expression, barrier permeability and hyperalgesia induced by SMIR on the 3rd day post-surgery.ConclusionsEarly dysfunction of BNB mediated by HIF-1α/AQP1 activated by SMIR may be an important mechanism to promote acute postoperative painful transformation of CPSP. Preadaptive protection of endothelial cells around nerve substructures may be an important countermeasure to inhibit CPSP transformation. Early impairment of BNB function mediated by HIF-1α/AQP1 activated by SMIR may be an important mechanism for promoting acute postoperative pain transformation of CPSP.

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.

Distribution of serotonin-positive cells in local cutaneous tissues of different acupuncture points at hindlimbs in rats

To investigate the expression of serotonin (5-HT) in local tissue of different acupoint regions at the hind limbs of rats. A total of 6 male SD rats were used in the present study. The local tissues of "Neiting"(ST44), "Zusanli"(ST36) and "Futu"(ST32) at the hindlimbs were dissected after transcardiac perfusion with fixative solution containing 4% paraformaldehyde, phosphate buffer, etc., fixed again in 4% paraformaldehyde and cut into sections (at 20 µm thick). Then, the tissue sections were subjective into immunofluorescence stain for displaying 5-HT positive cells and calcitonin gene-related peptide (CGRP) -positive fibers, vessel-like structure (labelled with phalloidin) and cellular nucleus (labelled with DAPI), separately. After that, the tissue samples were observed and recorded using a laser confocal microscope. The 5-HT labeled cells presented flat or round form in the local tissues of the 3 acupoint regions. These cells were mainly located in the dermis and subcutaneous tissue, and concentrated around the vessel-like structure and CGRP-positive nerve fibers. The number of cutaneous 5-HT positive cells was most in ST44 (156.6±12.1), and fewer in ST36 (83.5±18.2)and ST32(53.3±5.2)in turn. 5-HT-positive cell is an important member in local tissue of acupoints (ST44, ST36 and ST32), and varies in number according to their locations, suggesting an involvement of different densities of 5-HT-positive cells in different levels of regulatory effects of acupuncture stimulation of these acupoints.

Esophageal mucosal innervation in functional heartburn: Closer to healthy asymptomatic subjects than to non-erosive reflux disease patients.

Mucosal innervation in non-erosive reflux disease (NERD; pathological esophageal acid exposure, normal macroscopic mucosa) is clearly distinct from that of healthy volunteers (HV) and from patients with esophagitis or Barrett's esophagus: The nerves in NERD are situated much closer to the luminal surface of the mucosa. Patients with functional heartburn (FH) have a similar symptom profile to patients with NERD and indistinguishable macroscopic appearances. However, they have physiological acid exposure and no reflux-symptom association. The aim of our study was to delineate the position of esophageal mucosal nerve fibers in patients with FH and compare it with that in NERD and HV. Distal esophageal biopsies from patients with FH were immunohistochemically stained for CGRP. CGRP-positive nerve fibers were identified, and their position relative to the lumen was determined. These results were compared to our previously published cohort of HV and NERD. Eleven patients were included in the FH group with a mean age of 46years (range 33-69); 7F:4M. Nine patients had visible nerve fibers. The location of the afferent nerve fibers in the distal esophageal mucosa (median of 22, range 10.4-28) was similar to the HV group (median 25.5) and significantly deeper than the superficial nerves seen in NERD (median 9.5). The mucosal innervation pattern in FH is more alike that of healthy individuals than that of NERD, with afferent nerves lying deep in the mucosa, away from the luminal surface. This supports the theory that heartburn in FH has a distinct nociceptive pathophysiology.

Mapping the entire nerve architecture of the cat cornea.

To provide a complete nerve architecture and neuropeptide distribution in the cat cornea. Two adult domestic cats. The cat corneas were stained with protein gene product (PGP) 9.5 antibody-a pan marker for nerve fibers-and then divided into four quarters and double labeled with calcitonin gene-related peptide (CGRP) or substance P (SP) antibodies. Relative corneal nerve fiber densities and nerve terminals were evaluated in whole mount images by computer-assisted analysis. An average of 21.5±2.1 thick stromal nerves enters the cornea around the limbus where they split into many branches going up to the anterior stroma. Some branches link to each other, but most of them penetrate the basement membrane in the periphery to give origin to subbasal bundles, which run centripetally and merge to form a whirl-like structure (vortex) at the center. These nerve bundles send out many fine terminals that innervate the epithelial cells. Subbasal nerve density and nerve terminals were greater in the center than in the periphery of the cornea. Additionally, CGRP-positive central epithelial nerve fibers and terminals were more abundant than SP-positive nerves and terminals. The architecture of cat corneal nerves shows similarities to human and mouse cornea innervation. This study provides useful data for researchers who use the cat model to assess corneal nerve pathological alterations, as well as in the veterinary field where corneal opacities, ulcerations, and infections damage the nerves and decrease sensitivity.

Anti-inflammatory role of methotrexate in adjuvant arthritis: effect on substance p and calcitonin gene-related Peptide in thymus and spleen.

To investigate the anti-inflammatory effect of methotrexate (MTX) in rats with adjuvant arthritis through its influence on the expression of proinflammatory neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP) in immune organs, thymus and spleen. Phase-I pre-clinical trial. The Aga Khan University, Karachi, from July to December 2003. Adjuvant arthritis was induced in rats by inoculation with heat-killed mycobacteria. One group of arthritic rats (n=6) was treated with MTX (0.2 mg/kg body weight, subcutaneously) on every 4th day for a period of 18 weeks, while another group of arthritic rats (n=6) was treated with physiological saline served as control. At the end of experiment, animals were sacrificed and thymus and spleen were dissected and prepared for immunohistochemical analysis. The neuronal density of SP and CGRP immunoreactivity in thymus and spleen was assessed by semi-quantitative analysis. There was a marked reduction in hind paw swelling and inflammation in the MTX-treated rats after 18 weeks of treatment. Restoration of joint spaces (tibiotalar and subtalar) was seen after 9 weeks of MTX treatment. CGRP-positive nerve fibres were significantly reduced (p=0.0001) in thymus of rats treated with MTX compared to control rats. SP-positive nerve fibers were also found to be decreased in thymus of rats treated with MTX compared to controls, however, the decrease was not statistically significant. The neuronal density of SP and CGRP-immunoreactivity in spleen was not significantly different in MTX-treated and placebo-treated rats. In arthritic rats, MTX significantly reduced CGRP expression in thymus. Suppression of pro-inflammatory neuropeptides, such as CGRP and probably SP could be another mechanism by which MTX produces its anti-inflammatory effect in adjuvant arthritis.

Newly developed serine protease inhibitors decrease visceral hypersensitivity in a post-inflammatory rat model for irritable bowel syndrome.

Serine proteases have been re suggested as important mediators of visceral pain. We investigated their effect by using newly developed serine protease inhibitors with a well-characterized inhibitory profile in a rat model of post-inflammatory irritable bowel syndrome (IBS). Colitis was induced in rats receiving intrarectal trinitrobenzenesulphonic acid; controls received 0.9% NaCl. Colonoscopies were performed on day 3, to confirm colitis, and later until mucosal healing. Visceral hypersensitivity was quantified by visceromotor responses (VMRs) to colorectal distension, 30min after i.p. injection of the serine protease inhibitors nafamostat, UAMC-00050 or UAMC-01162. Serine proteases, protease-activated receptors (PARs) and TRP channels were quantified by qPCR and immunohistochemistry. Proteolytic activity was characterized using fluorogenic substrates. VMR was significantly elevated in post-colitis rats. Nafamostat normalized VMRs at the lowest dose tested. UAMC-00050 and UAMC-01162 significantly decreased VMR dose-dependently. Expression of mRNA for tryptase-αβ-1and PAR4, and tryptase immunoreactivity was significantly increased in the colon of post-colitis animals. Trypsin-like activity was also significantly increased in the colon but not in the faeces. PAR2 and TRPA1 immunoreactivity co-localized with CGRP-positive nerve fibres in control and post-colitis animals. Increased expression of serine proteases and activity together with increased expression of downstream molecules at the colonic and DRG level and in CGRP-positive sensory nerve fibres imply a role for serine proteases in post-inflammatory visceral hypersensitivity. Our results support further investigation of serine protease inhibitors as an interesting treatment strategy for IBS-related visceral pain.

Transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 contribute to the progression of colonic inflammation in dextran sulfate sodium-induced colitis in mice: Links to calcitonin gene-related peptide and substance P

Transient receptor potential (TRP) vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1), which are non-selective cation channels, play important roles in the sensation of pain. This study investigated the roles of TRPV1 and TRPA1 in dextran sulfate sodium (DSS)-induced murine colitis. DSS (2%) administered for 7 days caused severe colitis that was significantly less severe in TRPV1-deficient (TRPV1KO) and TRPA1-deficient (TRPA1KO) mice than that in wild-type (WT) mice. Similar colitis attenuations were observed in TRPV1KO and TRPA1KO mice but not in WT mice that had been transplanted with bone marrow cells from WT, TRPA1KO, or TRPV1KO mice. DSS treatment upregulated calcitonin gene-relative peptide (CGRP)- and substance P (SP)-positive nerve fibers in the colonic mucosa of WT mice. TRPV1KO and TRPA1KO mice showed significant reductions in the DSS-induced upregulation of SP, but the DSS-induced upregulation of CGRP was not reduced. Sensory deafferentation evoked by pretreatment with high doses of capsaicin markedly exacerbated DSS-induced colitis with reductions in DSS-induced upregulation of SP- and CGRP-positive nerve fibers. These findings suggest that neuronal TRPV1 and TRPA1 contribute to the progression of colonic inflammation. While these responses may be mediated by the upregulation of SP-mediated deleterious mechanisms, CGRP may be associated with protective mechanisms.

Morphology and neurochemistry of rabbit iris innervation

The aim of this study was to map the entire nerve architecture and sensory neuropeptide content of the rabbit iris. Irises from New Zealand rabbits were stained with antibodies against neuronal-class βIII-tubulin, calcitonin gene-related peptide (CGRP) and substance P (SP), and whole-mount images were acquired to build a two-dimensional view of the iridal nerve architecture. After taking images in time-lapse mode, we observed thick nerves running in the iris stroma close to the anterior epithelia, forming four to five stromal nerve rings from the iris periphery to the pupillary margin and sub-branches that connected with each other, constituting the stromal nerve plexus. In the anterior side, fine divisions derivated from the stromal nerves, forming a nerve network-like structure to innervate the superficial anterior border layer, with the pupillary margin having the densest innervation. In the posterior side, the nerve bundles ran along with the pupil dilator muscle in a radial pattern. The morphology of the iris nerves on both sides changed with pupil size. To obtain the relative content of the neuropeptides in the iris, the specimens were double stained with βIII-tubulin and CGRP or SP antibodies. Relative nerve fiber densities for each fiber population were assessed quantitatively by computer-assisted analysis. On the anterior side, CGRP-positive nerve fibers constituted about 61%, while SP-positive nerves constitute about 30.5%, of the total nerve content, which was expressed as βIII tubulin-positive fibers. In addition, in the anterior stroma of the collarette region, there were non-neuronal cells that were positive for SP. On the posterior side, CGRP-positive nerve fibers were about 69% of total nerve content, while SP constituted only up to 20%. Similarly, in the trigeminal ganglia (TG), the number of CGRP-positive neurons significantly outnumbered those that were positive for SP. Also, all the SP-positive neurons were labeled with CGRP. This is the first study to provide a two-dimensional whole mount and a cross-sectional view of the entire iris nerve architecture. Considering the anatomical location, the high expression of CGRP and SP suggests that these neuropeptides may play a role in the pathogenesis of anterior uveitis, glaucoma, cataracts and chronic ocular pain.

Changes in peptidergic fiber density in the synovium of mice with collagenase-induced acute arthritis.

The effect of acute osteoarthritis (OA) on peripheral nerve fibers (NFs) in synovial tissue, and their association with histological changes were investigated in collagenase-induced OA mice. Collagenase (10 U in 5 μL saline) was injected into the right knee, and the same volume of saline was injected into the left knee as the control. Mice were sacrificed 1, 2, 3, and 4 weeks after the collagenase injection. Histopathological changes in the knee joints were evaluated. The numbers of protein gene product (PGP) 9.5-, calcitonin-gene-related peptide (CGRP)-, and substance P (SP)-positive NFs in the synovial tissue were determined, and their densities in the tissue were calculated. The densities of PGP 9.5- and CGRP-positive NFs in the synovium were drastically decreased 1 week after the collagenase injection. However, by week 4, the density of PGP 9.5- and CGRP-positive NFs had recovered to 84% and 79% of their normal levels, respectively. Despite the poor correlation between the synovitis score and the density of CGRP- or SP-positive NFs in the synovium, the ossification rate of chondrophytes in chondro/osteophyte lesions correlated strongly with the density of CGRP-positive NFs (R = 0.855). These results suggest that the ossification of chondrophytes occurred in parallel with the increase in CGRP-positive fiber density in the synovium during the acute phase of collagenase-induced OA.

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.

427 Altered Histamine Receptor Expression in the Esophageal Epithelium During Eosinophilic Esophagitis (EoE): Implications on Disease Pathogenesis

Background: Neuro-immune interaction has evolved as an integral part in the pathophysiology of gut and crosstalk between nerves andmast cells is a typical example of such interactions. We have already reported that CGRP-immunoreactive intrinsic nerve fibers are specifically increased along with the development of food allergy in the colonic mucosa of our food allergy model mice. Furthermore, our previous studies have provided vivid evidences of mucosal mast cells (MMCs) juxtaposed with CGRP-positive enteric nerve fibers in the colonic mucosa of food allergy model mice. IgE-antigen stimulation has not only documented to cause degranulation in mast cells but also, recently, reported to cause activation of neurons in superior cervical ganglion. However, no study so far employed isolated enteric neurons and MMCs to elaborate this interaction which closely mimics to the pathophysiological state of the gut in the allergic condition. In the present study, we cultured both isolated enteric neurons and MMCs and examine their interaction. Material & methods: Enteric neurons were isolated from small intestine of BALB/c (4 to 6 weeks old) and examined by anti-β3tubulin antibody (anti-pan-neuronal marker antibody). Presence of high-affinity IgE receptors (FceRIs) on myentric neurons was examined in longitudinal muscle/myenteric plexus (LMMP) preparations by immunohistochemistry (IHC). Mucosal type bone marrow-derived mast cells (mBMMCs) were prepared from the femurs of BALB/c mice using four cytokines (SCF, IL-3, IL-9 and TGF-β1) and degranulation was performed by incubation with IgEantigen (IgE-DNP) to give mast cell juice (MCJ). Isolated enteric neurons were stimulated with either IgE-DNP or MCJ and analyzed by calcium imaging using fluo-8, a fluorescent calcium indicator. Finally, isolated enteric neurons were co-incubated with IgE-pretreated mBMMCs followed by stimulation with DNP. Only isolated enteric neurons were loaded with Fluo-8. Results: IHC studies revealed the presence of FceRI on the cell body of myentric neurons. Stimulation of isolated enteric neurons with IgE-DNP demonstrated an increase in intracellular calcium concentration ([Ca2+]i). Similarly, treatment of isolated enteric neurons with MCJ also led to an increase in [Ca2+]i. Furthermore, stimulation with DNP in an isolated enteric neuron/IgE-pretreated mBMMC co-culture condition produced an elevation of [Ca2+]i in isolated enteric neurons. Conclusion: We demonstrated here for the first time the activation of isolated enteric neurons by IgE-antigen, MCJ, and activated mBMMCs via calcium imaging. Therefore, we infer both direct and indirect involvement of crosstalk between enteric neurons and mucosal mast cells in allergic and inflammatory/ functional diseases of gut like food allergy or irritable bowel syndrome.

426 Allergic Inflammation-Induced Structural and Functional Changes in Esophageal Epithelium in a Guinea Pig Model of Eosinophilic Esophagitis

Background: Neuro-immune interaction has evolved as an integral part in the pathophysiology of gut and crosstalk between nerves andmast cells is a typical example of such interactions. We have already reported that CGRP-immunoreactive intrinsic nerve fibers are specifically increased along with the development of food allergy in the colonic mucosa of our food allergy model mice. Furthermore, our previous studies have provided vivid evidences of mucosal mast cells (MMCs) juxtaposed with CGRP-positive enteric nerve fibers in the colonic mucosa of food allergy model mice. IgE-antigen stimulation has not only documented to cause degranulation in mast cells but also, recently, reported to cause activation of neurons in superior cervical ganglion. However, no study so far employed isolated enteric neurons and MMCs to elaborate this interaction which closely mimics to the pathophysiological state of the gut in the allergic condition. In the present study, we cultured both isolated enteric neurons and MMCs and examine their interaction. Material & methods: Enteric neurons were isolated from small intestine of BALB/c (4 to 6 weeks old) and examined by anti-β3tubulin antibody (anti-pan-neuronal marker antibody). Presence of high-affinity IgE receptors (FceRIs) on myentric neurons was examined in longitudinal muscle/myenteric plexus (LMMP) preparations by immunohistochemistry (IHC). Mucosal type bone marrow-derived mast cells (mBMMCs) were prepared from the femurs of BALB/c mice using four cytokines (SCF, IL-3, IL-9 and TGF-β1) and degranulation was performed by incubation with IgEantigen (IgE-DNP) to give mast cell juice (MCJ). Isolated enteric neurons were stimulated with either IgE-DNP or MCJ and analyzed by calcium imaging using fluo-8, a fluorescent calcium indicator. Finally, isolated enteric neurons were co-incubated with IgE-pretreated mBMMCs followed by stimulation with DNP. Only isolated enteric neurons were loaded with Fluo-8. Results: IHC studies revealed the presence of FceRI on the cell body of myentric neurons. Stimulation of isolated enteric neurons with IgE-DNP demonstrated an increase in intracellular calcium concentration ([Ca2+]i). Similarly, treatment of isolated enteric neurons with MCJ also led to an increase in [Ca2+]i. Furthermore, stimulation with DNP in an isolated enteric neuron/IgE-pretreated mBMMC co-culture condition produced an elevation of [Ca2+]i in isolated enteric neurons. Conclusion: We demonstrated here for the first time the activation of isolated enteric neurons by IgE-antigen, MCJ, and activated mBMMCs via calcium imaging. Therefore, we infer both direct and indirect involvement of crosstalk between enteric neurons and mucosal mast cells in allergic and inflammatory/ functional diseases of gut like food allergy or irritable bowel syndrome.

425 Activation of Murine Isolated Enteric Neurons by IgE-Antigen and Mediators Released From Mucosal Type Bone Marrow-Derived Mast Cells

Background: Neuro-immune interaction has evolved as an integral part in the pathophysiology of gut and crosstalk between nerves andmast cells is a typical example of such interactions. We have already reported that CGRP-immunoreactive intrinsic nerve fibers are specifically increased along with the development of food allergy in the colonic mucosa of our food allergy model mice. Furthermore, our previous studies have provided vivid evidences of mucosal mast cells (MMCs) juxtaposed with CGRP-positive enteric nerve fibers in the colonic mucosa of food allergy model mice. IgE-antigen stimulation has not only documented to cause degranulation in mast cells but also, recently, reported to cause activation of neurons in superior cervical ganglion. However, no study so far employed isolated enteric neurons and MMCs to elaborate this interaction which closely mimics to the pathophysiological state of the gut in the allergic condition. In the present study, we cultured both isolated enteric neurons and MMCs and examine their interaction. Material & methods: Enteric neurons were isolated from small intestine of BALB/c (4 to 6 weeks old) and examined by anti-β3tubulin antibody (anti-pan-neuronal marker antibody). Presence of high-affinity IgE receptors (FceRIs) on myentric neurons was examined in longitudinal muscle/myenteric plexus (LMMP) preparations by immunohistochemistry (IHC). Mucosal type bone marrow-derived mast cells (mBMMCs) were prepared from the femurs of BALB/c mice using four cytokines (SCF, IL-3, IL-9 and TGF-β1) and degranulation was performed by incubation with IgEantigen (IgE-DNP) to give mast cell juice (MCJ). Isolated enteric neurons were stimulated with either IgE-DNP or MCJ and analyzed by calcium imaging using fluo-8, a fluorescent calcium indicator. Finally, isolated enteric neurons were co-incubated with IgE-pretreated mBMMCs followed by stimulation with DNP. Only isolated enteric neurons were loaded with Fluo-8. Results: IHC studies revealed the presence of FceRI on the cell body of myentric neurons. Stimulation of isolated enteric neurons with IgE-DNP demonstrated an increase in intracellular calcium concentration ([Ca2+]i). Similarly, treatment of isolated enteric neurons with MCJ also led to an increase in [Ca2+]i. Furthermore, stimulation with DNP in an isolated enteric neuron/IgE-pretreated mBMMC co-culture condition produced an elevation of [Ca2+]i in isolated enteric neurons. Conclusion: We demonstrated here for the first time the activation of isolated enteric neurons by IgE-antigen, MCJ, and activated mBMMCs via calcium imaging. Therefore, we infer both direct and indirect involvement of crosstalk between enteric neurons and mucosal mast cells in allergic and inflammatory/ functional diseases of gut like food allergy or irritable bowel syndrome.

Epidermal Nerve Fibers Modulate Keratinocyte Growth via Neuropeptide Signaling in an Innervated Skin Model

Atopic eczema is a chronic inflammatory skin disease characterized by cutaneous nerve fiber sprouting and epidermal hyperplasia, pointing to an involvement of the peripheral nervous system in cutaneous homeostasis. However, the interaction of sensory neurons and skin cells is poorly understood. Using an innervated skin model, we investigated the influence of sensory neurons on epidermal morphogenesis. Neurons induced the proliferation of keratinocytes, resulting in an increase in the epidermal thickness. Inhibition of calcitonin gene-related peptide (CGRP), but not substance P (SP) signaling, reversed this effect. Human CGRP enhanced keratinocyte proliferation and epidermal thickness in skin models, demonstrating a key role of CGRP in modulating epidermal morphogenesis, whereas SP had only a moderate effect. Innervated skin models composed of atopic skin cells showed increased neurite outgrowth, accompanied by elevated CGRP release. As atopic keratinocytes were sensitized to CGRP owing to higher expression levels of the CGRP receptor components, receptor activity-modifying protein 1 (RAMP1) and receptor component protein (RCP), atopic innervated skin models displayed a thicker epidermis than did healthy controls. We conclude that neural CGRP controls local keratinocyte growth. Our results show that the crosstalk of the cutaneous peripheral nervous system and skin cells significantly influences epidermal morphogenesis and homeostasis in healthy and atopic skin.

Effects of CO 2 laser irradiation of the gingiva during tooth movement

It was hypothesized that CO2 laser irradiation may reduce the early responses to nociceptive stimuli during tooth movement. The distribution of Fos-immunoreactive (Fos-IR) neurons in the medullary dorsal horn of rats was evaluated. Two hours after tooth movement, Fos-IR neurons in the ipsilateral part of the medullary dorsal horn increased significantly. CO2 laser irradiation to the gingiva just after tooth movement caused a significant decrease of Fos-IR neurons. PGP 9.5- and CGRP-positive nerve fibers were observed in the PDL of all study groups. It was suggested that CO2 laser irradiation reduced the early responses to nociceptive stimuli during tooth movement and might not have adverse effects on periodontal tissue.

Involvement of Calcitonin Gene-Related Peptide Innervation in the Promoting Effect of Low-Intensity Pulsed Ultrasound on Spinal Fusion Without Decortication

A prospective left-right comparison designed experiment using a rabbit posterolateral intertransverse process fusion model. To investigate the involvement of calcitonin gene-related peptide (CGRP) innervation in the promoting effect of low intensity pulsed ultrasound stimulation (LIPUS) on spinal fusion without decortication. Sensory neuropeptide CGRP is involved in bone repair and ectopic ossification. Comparison of CGRP innervations in ectopic bone between sham LIPUS and LIPUS sides can help us to understand the relationship between sensory nerve innervation and LIPUS. A total of 27 New Zealand white rabbits underwent bilateral posterolateral intertransverse process fusion with implantation of porous poly-D,L-lactic acid blocks loaded with 1.25 μg recombinant human bone morphogenetic protein-4 solution. One side was provided LIPUS daily whereas the other side served as control. Animals were killed and the operated lumbar vertebrae were harvested for histomorphologic evaluation at 3 days (n = 3), 1 week (n = 6), 3 weeks (n = 6), 7 weeks (n = 6), and 12 weeks (n = 6) following surgery, respectively. LIPUS accelerated the invasion of CGRP-positive nerve fibers during ectopic ossification spatially and temporally. Spatially, CGRP-positive nerve fibers were also observed in the new formed cartilage and bone tissues on LIPUS side, whereas they were only detected in the fibrous tissue and bone marrow on sham LIPUS side. Temporally, the density of CGRP-positive nerve fibers was significantly higher on the LIPUS side when compared with the sham LIPUS side. LIPUS promoted the invasion of CGRP sensory nerve in ectopic bone, which may in turn contribute to the promoting effect of LIPUS on ectopic ossification.

Somatostatin, substance P and calcitonin gene-related peptide-positive intramural nerve structures of the human large intestine affected by carcinoma.

The aim of this study was to investigate the arrangement and chemical coding of enteric nerve structures in the human large intestine affected by cancer. Tissue samples comprising all layers of the intestinal wall were collected during surgery form both morphologically unchanged and pathologically altered segments of the intestine (n=15), and fixed by immersion in buffered paraformaldehyde solution. The cryostat sections were processed for double-labelling immunofluorescence to study the distribution of the intramural nerve structures (visualized with antibodies against protein gene-product 9.5) and their chemical coding using antibodies against somatostatin (SOM), substance P (SP) and calcitonin gene-related peptide (CGRP). The microscopic observations revealed distinct morphological differences in the enteric nerve system structure between the region adjacent to the cancer invaded area and the intact part of the intestine. In general, infiltration of the cancer tissue resulted in the gradual (depending on the grade of invasion) first decomposition and reduction to final partial or complete destruction and absence of the neuronal elements. A comparative analysis of immunohistochemically labeled sections (from the unchanged and pathologically altered areas) revealed a statistically significant decrease in the number of CGRP-positive neurons and nerve fibres in both submucous and myenteric plexuses in the transitional zone between morphologically unchanged and cancer-invaded areas. In this zone, a decrease was also observed in the density of SP-positive nerve fibres in all intramural plexuses. Conversely, the investigations demonstrated statistically insignificant differences in number of SP- and SOM-positive neurons and a similar density of SOM-positive nerve fibres in the plexuses of the intact and pathologically changed areas. The differentiation between the potential adaptive changes in ENS or destruction of its elements by cancer invasion should be a subject of further investigations.

Effects of CO2 Laser Irradiation of the Gingiva during Tooth Movement

Patients often feel pain or discomfort in response to orthodontic force. It was hypothesized that CO2 laser irradiation may reduce the early responses to nociceptive stimuli during tooth movement. The distribution of Fos-immunoreactive (Fos-IR) neurons in the medullary dorsal horn of rats was evaluated. Two hrs after tooth movement, Fos-IR neurons in the ipsilateral part of the medullary dorsal horn increased significantly. CO2 laser irradiation to the gingiva just after tooth movement caused a significant decrease of Fos-IR neurons. PGP 9.5- and CGRP-positive nerve fibers were observed in the PDL of all study groups. The maximum temperature below the mucosa during CO2 laser irradiation was less than 40°C. It was suggested that CO2 laser irradiation reduced the early responses to nociceptive stimuli during tooth movement and might not have adverse effects on periodontal tissue.

Observing the bony canal structure of the human maxillary sinus in Japanese cadavers using cone beam CT

We observed the location of the posterior superior alveolar artery (PSAA) and nerve at the macroscopic level between the maxillary sinus (MS) and surrounding bone of the anterior region of the maxilla. This study was completed using cone beam computed tomography (CBCT) imaging of 19 human cadavers with 38 sides of Japanese origin (ranging in age from 59-94 years, mean 77.7 +/- 9.8 years) that were prepared for this study. The bony canal structure of the inner surface of the maxilla was clearly apparent in our results, and the bony canals were classified into three types according to the structure along the course of the PSAA: canal-like, ditch-shaped tunnel and fragmented, and the lest sides were undefined. Calcitonin gene-related peptide (CGRP)-positive fibers were identified along the PSAA in the bony canal of the maxilla by immunohistochemistry. The presence of the bony structure and CGRP-positive nerve fibers along the PSAA suggests that there is risk to the PSAA during surgery involving graft implant in the floor of the maxillary sinus.