Calcitonin Gene-related Peptide Expression Levels Research Articles

Electroacupuncture at Sensitized Acupoints Relieves Somatic Referred Pain in Colitis Rats by Inhibiting Sympathetic-Sensory Coupling to Interfere with 5-HT Signaling Pathway.

To investigate whether electroacupuncture (EA) at sensitized acupoints could reduce sympathetic-sensory coupling (SSC) and neurogenic inflammatory response by interfering with 5-hydroxytryptamine (5-HT)ergic neural pathways to relieve colitis and somatic referred pain, and explore the underlying mechanisms. Rats were treated with 5% dextran sodium sulfate (DSS) solution for 7 days to establish a colitis model. Twelve rats were randomly divided into the control and model groups according to a random number table (n=6). According to the "Research on Rat Acupoint Atlas", sensitized acupoints and non-sensitized acupoints were determined. Rats were randomly divided into the control, model, Zusanli-EA (ST 36), Dachangshu-EA (BL 25), and Xinshu (BL 15) groups (n=6), as well as the control, model, EA, and EA + GR113808 (a 5-HT inhibitor) groups (n=6). The rats in the control group received no treatment. Acupuncture was administered on 2 days after modeling using the stimulation pavameters: 1 mA, 2 Hz, for 30 min, with sparse and dense waves, for 14 consecutive days. GR113808 was injected into the tail vein at 5 mg/kg before EA for 10 min for 7 consecutive days. Mechanical sensitivity was assessed with von Frey filaments. Body weight and disease activity index (DAI) scores of rats were determined. Hematoxylin and eosin staining was performed to observe colon histopathology. SSC was analyzed by immunofluorescence staining. Immunohistochemical staining was performed to detect 5-HT and substance P (SP) expressions. The calcitonin gene-related peptide (CGRP) in skin tissue and tyrosine hydroxylase (TH) protein levels in DRG were detected by Western blot. The levels of hyaluronic acid (HA), bradykinin (BK), prostaglandin I2 (PGI2) in skin tissue, 5-HT, tryptophan hydroxylase 1 (TPH1), serotonin transporters (SERT), 5-HT 3 receptor (5-HT3R), and 5-HT 4 receptor (5-HT4R) in colon tissue were measured by enzyme-linked immunosorbent assay (ELISA). BL 25 and ST 36 acupoints were determined as sensitized acupoints, and BL 15 acupoint was used as a non-sensitized acupoint. EA at sensitized acupoints improved the DAI score, increased mechanical withdrawal thresholds, and alleviated colonic pathological damage of rats. EA at sensitized acupoints reduced SSC structures and decreased TH and CGRP expression levels (P<0.05). Furthermore, EA at sensitized acupoints reduced BK, PGI2, 5-HT, 5-HT3R and TPH1 levels, and increased HA, 5-HT4R and SERT levels in colitis rats (P<0.05). GR113808 treatment diminished the protective effect of EA at sensitized acupoints in colitis rats (P<0.05). EA at sensitized acupoints alleviated DSS-induced somatic referred pain in colitis rats by interfering with 5-HTergic neural pathway, and reducing SSC inflammatory response.

URB937 Prevents the Development of Mechanical Allodynia in Male Rats with Trigeminal Neuralgia.

Cannabinoids are proposed for alleviating neuropathic pain, but their use is limited by cannabimimetic side effects. The inhibition of the fatty acid amide hydrolase (FAAH), the degrading enzyme of the endocannabinoid anandamide, has received attention as an alternative to cannabinoids in the treatment of neuropathic pain. Here, we investigated the effect of URB937, a blood-brain barrier impermeant FAAH inhibitor, on experimentally induced mechanical allodynia in an animal model of trigeminal neuralgia. Male Sprague-Dawley rats were subjected to chronic constriction injury of the infraorbital nerve (IoN-CCI); operated animals were treated sub-chronically with URB937 (1 mg/kg, i.p.) or vehicle before or after trigeminal mechanical allodynia establishment. We also assayed mRNA expression levels of the pain neuropeptide calcitonin gene-related peptide (CGRP) and cytokines in the medulla, cervical spinal cord, and trigeminal ganglion ipsilateral to IoN-CCI using rt-PCR. URB937 treatment prevented the development of mechanical allodynia and IoN-CCI-induced changes in mRNA expression levels of CGRP and cytokines in the evaluated areas. When administered after allodynia development, URB937 prevented IoN-CCI-induced changes in CGRP and cytokine gene expression; this was not associated with a significant abrogation of the mechanical allodynia. These findings suggest that URB937 may counteract, but not reverse, the development of allodynia in trigeminal neuralgia. Further research is needed to elucidate the underlying mechanisms.

Effect of moxibustion and scraping on bioactive substances changes of acupoints in knee osteoarthritis rats

To compare the effects of moxibustion and scraping of "Yanglingquan" (GB34) and "Xuehai" (SP10) area on changes of bioactive substances in the region of acupoints in rats with knee osteoarthritis (KOA). SD rats were randomly divided into blank, model, moxibustion, scraping, and moxibustion + scraping (combination) groups, with 8 rats in each group. The KOA model was established by injecting 50 μL 0.9% sodium chloride solution into the right knee cavity. Fourteen days after modeling, GB34 and SP10 on the right limb were stimulated by moxibustion (10 min) or scraping (till regional flush) once every other day for 7 times. The mechanical paw withdrawal threshold (PWT) and thermal withdrawal latency (TWL) were tested by Von Frey and hot stabbing instrument, separately. The pathological changes of the right knee joint were observed by HE staining. The serotonin (5-HT) contents of skin tissues in the region of acupoint GB34 and SP10 were detected by ELISA. The expression levels of substance P (SP) and calcitonin gene-related peptide (CGRP) in GB34 and SP10 region skin tissues were detected by Western blot. Compared with the blank group, the PWT and TWL of the rats in the model group were significantly decreased (P<0.001), while the contents of 5-HT and the expression levels of SP and CGRP in GB34 and SP10 region skin tissues were significantly increased (P<0.001, P<0.01). Following intervention and in comparison the with the model group, the TWL and PWT of rats in the three treatment groups were significantly increased (P<0.01), the content of 5-HT and the expression levels of SP and CGRP in GB34 and SP10 region skin tissues were significantly decreased (P<0.01, P<0.001, P<0.05). Except for the expression levels of CGRP, the above indexes of the combination group were significantly superior to those of the moxibustion and scraping groups (P<0.05, P<0.01). Findings of HE staining showed severe damaged cartilage, few chondrocytes on the surface, with subchondral neovascularization in the model group, which was relatively milder in the moxibustion, scraping, and combination groups. Moxibustion and scraping can relieve knee joint pain in KOA rats, which may be associated with its function in down-regulating the expression levels of SP and CGRP, and the content of 5-HT. The therapeutic effect of moxibustion plus scraping is better than that of moxibustion and scraping alone.

Comparison of stimulation-induced local changes of expression of local Meckel cell-related proteins and neuropeptides and number of mast cells among acupuncture and moxibustion alone or in combination in chronic atrophic gastritis rats

To compare the effects of acupuncture and moxibustion alone or in combination on the number of mast cells and expression levels of cytoketatin 18 (CK18) and CK19 (marker of Meckel cells), and calcitonin gene-related peptide (CGRP), neuropeptide-Y (NPY) and bradykinin (BK) in the local acupoint area of rats with chronic atrophic gastritis (CAG). Fifty male SD rats were randomly divided into normal, CAG model, moxibustion, acupuncture and acupuncture+moxi-bustion groups (10 rats in each group). The CAG model was established by gavage of 1-methyl-3-nitro-1-nitrosoguanidine (170 μg/mL,1 mL/100 g, once a week) and 40% ethanol solution (twice a week) for 12 consecutive weeks. After successful establishment of CAG model, moxibustion, manual acupuncture or acupuncture+moxibustion was applied to bilateral "Zusanli" (ST36) and "Zhongwan"(CV12) for 15 min, once daily for 14 consecutive days. At the end of the experiment, the gastric mucosal tissues were collected for observing histopathological changes of gastric mucosa after H.E. staining, and the tissues of the stimulated ST36 region collected for detecting the expression levels of CK18, CK19, CGRP, NPY and BK and the number of mast cells in the local ST36 region by immunohistochemistry. Compared with the normal group, the number of mast cells, the expression levels of CK19, NPY and BK in the ST36 area were significantly increased (P<0.05), and the expression level of CGRP was apparently decreased (P<0.05) in the model group. In comparison with the model group, the number of mast cells and the expression levels of CGRP and NPY in the moxibustion group, the expression of CGRP in the acupuncture group, and the number of mast cells, as well as the expression levels of CK18, CK19 and CGRP in the acupuncture+moxibustion group were significantly up-re-gulated (P<0.05). The effect of acupuncture combined with moxibustion was obviously superior to that of moxibustion or acupuncture in up-regulating the expression of CK18 and CK19 (P<0.05) and superior to that of moxibustion in down-regulating BK expressio level (P<0.05). No significant changes were found in the expression of CK18 after modeling (vs the normal group), in the expression levels of CK18, CK19 and BK after moxibustion and acupuncture (vs the model group), in the number of mast cells and expression of NPY after acupuncture (vs the model group), and in the expression levels of NPY and BK after acupuncture+moxibustion (vs the model group) (P>0.05). H.E. staining showed infiltration of many lymphocytes in the gastric mucosa and submucosal layers, atrophy and necrosis of lots of main cells with vacuole-like changes, and disordered arrangement of the atrophic glands in the model group, which was milder particularly in the acupuncture + moxibustion group. Acupuncture combined with moxibustion of ST36 can up-regulate the levels of local CK18, CK19 and CGRP proteins and number of mast cells, moxibustion may up-regulate the levels of CGRP and NPY and number of mast cells, while acupuncture may up-regulate the expression of CGRP in the local stimulated area in CAG rats.

Characteristic expression of CGRP and osteogenic and vasculogenic markers in the proximal and distal regions of the rib during male mouse development

BackgroundNeuropeptide calcitonin gene-related peptide (CGRP) is a neurotransmitter related to vasculogenesis and osteogenesis during bone formation and organ development. From the foetal period to the postnatal period, the thorax, which is necessary for lung respiration, forms. The thorax exhibits the same cartilage ossification as the bones of the extremities, but a specific system within the thorax exists as costal cartilage after birth. The relationship among CGRP, osteogenesis and vasculogenic markers in the two rib locations during thorax formation is not fully understood. Materials and methodsIn our study, male mice were used to provide ribs under different development conditions on various embryonic days (E12. 5, E14.5, and E17.5) and postnatal days (P1 and P5). The mRNA expression levels of CGRP, vascular endothelial growth factor (VEGF-A), type 1 collagen (Col1a-1), type 2 collagen (Col2a1), neuropeptide Y (NPY), osteocalcin (OCN) and osteopontin (OPN) were analysed by qRT-PCR. We also analysed the mRNA expression of CGRP, VEGF-A and OPN by in situ hybridization. Multivariate modelling with principal component analysis (PCA) was performed to estimate the interactions among the quantitative real-time RT-PCR data. ResultsThe mRNA expression levels of CGRP, VEGF-A, Col2a, Col1a-1, OCN, and NPY in the male mouse rib gradually increased during development. An antisense probe for CGRP mRNA was strongly detected in the central region of the mouse rib at E12.5 and the hypertrophic and ossification zones at E17.5 by in situ hybridization. VEGF-A was also located in the same region as CGRP at E12.5 and E17.5. OPN was strongly detected at the rib formation stage from E14.5 to E17.5. The expression of CGRP also differed between the proximal and distal regions of the rib at E17.5. As demonstrated by in situ hybridization, CGRP continuously participates in cartilage formation in the distal regions of the rib after birth. The PCA revealed that the mRNA expression of CGRP was related to that of Col1a-1 and VEGF-A during rib formation. ConclusionThis study shows that CGRP is involved in vascular and bone formation during rib development and may also be involved in cartilage formation after birth. The findings suggest that CGRP may temporarily participate in bone formation and continuously participate in cartilage formation in the rib, which may also be related to the formation of the anterior thoracic wall after birth

Effect of electroacupuncture of "Zhiyang" (GV9) and "Jizhong" (GV6) on expression of CGRP and NLRP3 in rats with spinal cord injury

To observe the effect of electroacupuncture (EA) at "Zhiyang" (GV9) and "Jizhong" (GV6) of the Governor Vessel on the expression of calcitonin gene related peptide (CGRP)and NOD-like receptor pyrin containing 3 (NLRP3) in the injured anterior horn (AH) of rats with spinal cord injury (SCI), so as to explore its mechanisms underlying improvement of SCI. Thirty-six male SD rats were randomly divided into sham operation (sham) group, model group and EA group which were further randomly allocated to 7 day (d) and 14 d subgroups (n=6 per subgroup). The SCI model was established by clamping the exposed spinal cord with an aneurysm clip. Rats of the EA group received EA of GV6 and GV9 for 30 min, once daily for 7 and 14 days, respectively. The Basso, Beattie and Bresnahan (BBB) was used to assess changes of the locomotor function on the 1st, 3rd, 7th and 14th day after SCI. The protein expression levels of CGRP, NLRP3, apoptosis-associated speck-like protein (ASC) and cysteinyl aspartate specific protease-1 (caspase-1) in the anterior horn region of the spinal cord on the 7th and 14th after SCI were detected by Western blot. The BBB scores on the 1st, 3rd, 7th and 14th d after SCI were significantly lower in the model group than in the sham group (P<0.05), whereas the expression levels of NLRP3, ASC and caspase-1 proteins on day 7 and 14 were markedly higher in the model group than in the sham group (P<0.05, P<0.01). Compared with the model group, the BBB score and CGRP expression on the 7th and 14th d were significantly increased in the EA group (P<0.05,P<0.01), while the expression levels of NLRP3, ASC and caspase-1 were obviously decreased in the EA group (P<0.01). EA of GV6 and GV9 can improve the locomotion of SCI rats, which may be associated with its function in up-regulating the expression of CGRP and down-regulating the expression of NLRP3, ASC and caspase-1 proteins in the spinal anterior horn tissue.

Calcitonin gene-related peptide and brain-derived serotonin are related to bone loss in ovariectomized rats

ObjectivesPostmenopausal osteoporosis (PMO) and osteoporotic fracture seriously impair human health in developed countries. The present study aims to explore whether sensory nerves, calcitonin gene-related peptide (CGRP), and brain-derived serotonin are related to bone loss in ovariectomized (OVX) rats. MethodsFemale rats were grouped into the ovariectomized (OVX) and sham surgery (SHAM) groups. Immunocytochemistry, western blotting, and qPCR were performed to detect CGRP expression in the femurs. The expression levels of serotonin and CGRP in the spinal cord and brainstem were estimated using western blotting, immunofluorescence, and qPCR. ELISA was used to evaluate the serum biomarkers of bone formation and resorption. Bone mineral density was measured using dual-energy X-ray (DXA) analysis. Femur microstructure was imaged by Micro CT. P values less than 0.05 were considered statistically significant. ResultsELISA showed that serum bone alkaline phosphatase (BALP), tartrate-resistant acid phosphatase (TRAP), β-crosslaps, and β-ctx were increased in the OVX group. In the OVX group, in vivo bone mineral density, trabecular bone mineral density, bone volume fraction (BV/TV), and trabecular number (Tb. N) were significantly decreased, while trabecular spacing (Tb. Sp) and trabecular bone pattern factor (Tb. Pf) were markedly increased. In the OVX group, the expression levels of CGRP of the femur were significantly downregulated. In contrast, CGRP and serotonin expression was increased in the spinal cord of the OVX group. Serotonin expression was increased in the brainstem, brainstem nucleus raphe magnus (RMG), and nucleus raphe dorsalis (DRN). ConclusionOur results indicated that the activation of osteoclast triggered the release of CGRP from nociceptive sensory nerve fibers and transmitted this painful stimulus to the dorsal horn of the spinal cord to release increased CGRP. The descending serotonergic inhibitory system was activated by increased CGRP levels of the spinal cord and promoted serotonin release in the brainstem RMG, DRN, and the spinal cord, contributing to the decreased CGRP level in bone tissue, which revealed a novel mechanism of bone loss in PMO.

The expression of substance P and calcitonin gene-related peptide is associated with the severity of tendon degeneration in lateral epicondylitis

BackgroundIn this study, we investigated whether substance P (SP) or calcitonin gene-related peptide (CGRP) expression is associated with tendon degeneration in patients with lateral epicondylitis.MethodsTwenty-nine patients who underwent surgical treatment for lateral epicondylitis were enrolled in the final analyses. Extensor carpi radialis brevis tendon origins were harvested for histological analysis.ResultsSP and CGRP immunostaining were negative in healthy tendons but positive in degenerative tendons; moreover, their immunoreactivity increased with degeneration severity. Univariate analysis indicated that variables such as the preoperative visual analog scale (VAS) score or SP or CGRP expression levels were significantly associated with the Movin score. However, multivariate analysis revealed that only higher SP and/or CGRP signals were associated with higher Movin scores. Elevations in SP or CGRP expression were also linked with significantly severe preoperative VAS scores.ConclusionWe demonstrated that tendon degeneration severity is associated with increased SP and CGRP expression in the biopsy samples of lateral epicondylitis.

Tibetan medicine Ru-yi-Zhen-bao Pills exhibits anti-migraine effect through mediating PAG anti-nociceptive channel.

Migraine is a disabling neurovascular disorder, which increases risk of cardiovascular events and is a social burden worldwide. The present first-line anti-migraine medications can cause overwhelming side-effects, of which one includes the onset of cardiovascular disease. As one of the marketed Tibetan drugs, Ru-yi-Zhen-bao Pills (RYZBP) have been clinically used to treat cardiovascular disorders and as anti-migraine medication. However, there is currently no research exploring the anti-migraine actions of RYZBP. The current research was designed to assess the anti-migraine roles of RYZBP and explore the underlying mechanisms in a nitroglycerin (NTG)-induced migraine rat model trial. 120 rats were randomly divided into the following six groups of 20 rats each: normal control group, model control group, positive control group, and RYZBP high/medium/low-dose groups (Ru-yi-Zhen-bao Pills; TH 1.00 g/kg, TM 0.50 g/kg and TL 0.25 g/kg). All rats were administered intragastrically for 7 consecutive days, which were subcutaneously injected with the NTG (10mg/kg) after the last gavage (except in the normal control group). 3min after NTG treatment, 30 rats (5 rats from each group) were anesthetized and devoted to electroencephalogram(EEG) testing, which was used to evaluate the analgesic effect of RYZBP. One hour after NTG treatment, the rest of the 90 rats (15 rats from each group) were anesthetized and midbrain tissue sample was dissected. The dissection was then washed with physiological saline and collected. The histopathological changes in the periaqueductal gray(PAG) of 5 tissue samples were determined by aematoxylin-eosin (H&E) staining, as well as an estimation of substance P (SP) and neurokinin 1 receptor (NK1R) expression through immunohistochemically staining(IHC). Another 5 midbrain preparations were carried out to evaluate calcitonin gene-related peptide (CGRP), proenkephalin (PENK), SP, and cholecystokinin (CCK) expressions by real-time quantitative polymerase chain reaction (RT-qPCR). The rest of the 5 brainstem tissues were then used to measure CCK, CGRP, and opioid peptide receptor (DORR) levels by western blotting(WB). In the EEG test, RYZBP (TM 0.50 g / kg) treatment transformed the EEG pain-wave of the NTG-induced migraine model rats in different time period. In the mechanism assay, compared with the model control group, RYZBP pretreatment reduced inflammatory cell infiltration, fibrosis and vacuolation of neuronal cells of PAG tissue seen by HE staining. IHC experiments further showed that RYZBPTM up-regulated SP expression levels and enhanced NK1R levels in the NTG-induced migraine rats (P<0.05). Therapeutic administration of RYZBP also increased PENK mRNA expression and DORR protein level. Both RT-qPCR and western blotting trials indicated that RYZBP treatment significantly decreased CCK and CGRP expression levels (P<0.01 or P<0.05) in the NTG-induced migraine rats. RYZBP has the potential to be an effective anti-migraine treatment through suppressing the EEG pain-wave, increasing the levels of SP, PENK, DORR and reducing expression of CCK and CGRP. Mediating the PAG anti-nociceptive channel and inhibiting central sensitization were the two potential mechanisms, which offers further evidence for clinical therapy.

Impaired AMPK‑CGRP signaling in the central nervous system contributes to enhanced neuropathic pain in high‑fat diet‑induced obese rats, with or without nerve injury.

Obesity is associated with increased sensitivity to pain, including neuropathic pain, but the precise mechanisms are not fully understood. Recent evidence has revealed that AMP-activated protein kinase (AMPK) in the central nervous system (CNS) regulates the neuropeptide calcitonin gene-related peptide (CGRP), a principal neurotransmitter of the class C nerve fiber, which serves an important role in initiating and maintaining neuropathic pain. AMPK has been demonstrated to be downregulated in the CNS in obesity. The present study hypothesized that obesity may lead to increased sensitivity to neuropathic pain by downregulating AMPK and upregulating CGRP expression levels in the CNS. Sprague-Dawley rats consuming a high-fat diet (HF) for 12 weeks developed obesity; they exhibited significantly decreased levels of phospho (p)-AMPK and increased CGRP expression levels in the spinal cord (SC) and dorsal root ganglion (DRG), respectively, compared with rats consuming a low-fat (LF) diet. HF-fed rats that underwent spared nerve injury (SNI) also exhibited lower p-AMPK and higher CGRP expression levels in the SC and DRG, compared with the corresponding LF-diet rats. The 50% paw withdrawal threshold (PWT; as measured by Von Frey testing) was significantly lower in HF-fed compared with LF-fed rats, with or without SNI. Through intrathecal treatment, the AMPK activator 5-aminoimidazole-4-carboxamide riboside (AICAR) or the CGRP antagonist CGRP8-37 decreased CGRP expression levels and increased the 50% PWT; however, the AMPK inhibitor dorsomorphin augmented CGRP expression levels and further reduced the 50% PWT in HF-fed rats, but not LF-fed rats, with or without SNI. The results indicated that blocking the AMPK-CGRP pathway may enhance neuropathic pain in HF-induced obesity, with or without nerve injury. Targeting AMPK in the CNS may be a novel strategy for the prevention and treatment of obesity-associated neuropathic pain.

Expressions and significance of calcitonin gene-related peptide and nerve growth factor in rabbit model of traumatic brain injury complicated with tibial fracture: preliminary results.

This paper aims to establish the rabbit model of traumatic brain injury (TBI) complicated with tibial fracture and to investigate the expressions and significance of calcitonin gene-related peptide (CGRP) and nerve growth factor (NGF) in cerebrospinal fluid (CSF) and serum. 60 rabbits were randomly divided into control group, TBI group, fracture group (F group), and TBI complicated with fracture group (TBI+F group), with 15 white rabbits in each group. After modeling, the expression levels of CGRP and NGF in the CSF, and serum were detected. At the 7th week after the operation, X-ray was used to evaluate the healing of fracture rabbits. Serum NGF content was compared among groups at the same time point. TBI+F group had significantly higher serum NGF content than the other three groups at each time point after the operation (p<0.05). From the 3rd day after the operation, TBI group and F group had significantly higher serum NGF content than control group (p<0.05). On the 7th and 14th days after the operation, TBI group had significantly higher serum NGF content than the F group (p<0.05). The CSF NGF content in TBI group and TBI+F group showed an upward trend, and it was higher in TBI+F group and TBI group than that in F group and control group from the 7th day (p<0.05). On the 0th and 3rd days, TBI+F group had significantly higher serum NGF content than the other three groups (p<0.05). TBI+F group had a significantly higher healing number than F group on the 14th day (p<0.05). NGF and CGRP are mainly present in the CSF. When TBI complicated with F occurs, the serum NGF and CGRP increase, which may be involved in the fracture healing.

The influence of receptor activity-modifying protein-1 overexpression on angiogenesis in mouse brain capillary endothelial cells.

Receptor activity-modifying protein-1 (RAMP1) is highly expressed in the heart and vasculature, indicating that it might be related to the vascular system. However, the effects of RAMP1 on angiogenesis and the intrinsic mechanisms underlying this process remain unclear. Here, we verified that RAMP1 is a critical regulator of angiogenesis in a mouse brain capillary endothelial cell line (bEnd.3). We first constructed a RAMP1 overexpression lentiviral vector system and stably transfected bEnd.3 cells. We further showed that RAMP1 overexpression could lead to bEnd.3 migration and capillary tube formation in Matrigel without exogenous calcitonin gene-related peptide (CGRP) treatment. At the same time, RAMP1 overexpression had little effect on proliferation. More importantly, vascular endothelial growth factor (VEGF) and CGRP expression levels were not significantly higher in RAMP1-overexpressing cells than in control cells (P > 0.05), indicating that RAMP1 did not function through upregulating VEGF or CGRP expression in bEnd.3 cells. Strikingly, RAMP1 transfection increased adrenomedullin 2 (AM2) expression levels ( P < 0.05). Taken together, these data contribute to a better understanding of the molecular mechanisms of RAMP1 in angiogenesis.

Increased Calcitonin Gene-Related Peptide and Macrophages Are Involved in Astragalus membranaceus-Mediated Peripheral Nerve Regeneration in Rats.

Astragalus membranaceus (AM) is one of 50 fundamental herbs in traditional Chinese medicine. Previous studies have shown that AM extract can be a potential nerve growth-promoting factor, being beneficial for the growth of peripheral nerve axons. We further investigated the effects of AM extract on regeneration in a rat sciatic nerve transection model. Rats were divided into three groups ([Formula: see text]): normal saline (intraperitoneal) as the control, and 1.5[Formula: see text]g/kg or 3.0[Formula: see text]g/kg of AM extract (every other day for four weeks), respectively. We evaluated neuronal electrophysiology, neuronal connectivity, macrophage infiltration, expression levels and location of calcitonin gene-related peptide (CGRP), and expression levels of both nerve growth factors (NGFs) and immunoregulatory factors. In the high-dose AM group, neuronal electrophysiological function (measured by nerve conductive velocity and its latency) was significantly improved ([Formula: see text]). Expression levels of CGRP and macrophage density were also drastically enhanced ([Formula: see text]). Expression levels of fibroblast growth factor (FGF), NGF, platelet-derived growth factor (PDGF), transforming growth factor-[Formula: see text], interleukin-1 (IL-1), and interferon (IFN)-[Formula: see text] were reduced in the high-dose AM group ([Formula: see text]), while FGF, NGF, PDGF, IL-1, and IFN-[Formula: see text] were increased in the low-dose AM group ([Formula: see text]). These results suggest that AM can modulate local inflammatory conditions, enhance nerve regeneration, and potentially increase recovery of a severe peripheral nerve injury.

Comparison of the expression of neurotransmitter and muscular genesis markers in the postnatal male mouse masseter and trigeminal ganglion during development

Calcitonin gene-related peptide (CGRP) is released by motor neurons and affects skeletal muscle fiber and transient receptor potential cation channel subfamily V member 1 (TRPV1), an important marker of pain modulation. However, the expression of CGRP and TRPV1 in the trigeminal ganglion (TG) during changes and in feeding patterns has not been described. We used real-time reverse transcription polymerase chain reaction and in situ hybridization to investigate the mRNA expression levels of CGRP and TRPV1 in the TG. The expression of myosin heavy-chain (MyHC) isoforms was also investigated in the masseter muscle (MM) during the transition from sucking to mastication, an important functional trigger for muscle. The mRNA and protein levels of CGRP increased in the MM and TG from postnatal day 10 (P10) to P20 in male mice. The protein levels of TRPV1 were almost constant in the TG from P10 to P20, in contrast to increases in the MM. The mRNA abundance of TRPV1 in the TG and MM was increased from P10 to P20. The localization of an antisense probe was used to count CGRP cell numbers and found to differentiate the ophthalmic, maxillary, and mandibular nerve divisions of the TG. In particular, the number of CGRP+ cells per 10,000 μm2 in the maxillary and mandibular divisions of the TG gradually changed from P10 to P20. The expression of CGRP and TRPV1 in the TG and MM and the patterns of expression of different MyHC isoforms were affected by changes in feeding during male mouse development.

Increase and regulation of synovial calcitonin gene-related peptide expression in patients with painful knee osteoarthritis.

BackgroundRecent studies suggest that the vasodilatory neuropeptide calcitonin gene-related peptide (CGRP) is localized in the synovial tissue and may be involved in the pathology of hip and knee osteoarthritis (OA). However, the regulation and relationship between pain and CGRP expression levels in the synovial tissue of human OA patients are not fully understood.MethodsSynovial tissues were harvested from 74 participants with radiographic knee OA (unilateral Kellgren/Lawrence grades 3–4) during total knee arthroplasty. CGRP-expressing cells in the resected tissue were identified by immunohistochemical analyses. To examine CGRP expression levels, CD14-positive (CD14+) (macrophage-rich cell fraction) and CD14-negative (CD14−; fibroblast-rich cell fraction) cells were isolated from the synovial tissue. To investigate the involvement of prostaglandin E2 (PGE2) in the regulation of CGRP expression, cultured CD14− and CD14+ cells were stimulated with PGE2. In addition, CGRP expression levels in the synovial tissue of OA patients with strong/severe (visual analog scale [VAS]≥6) and mild/moderate pain (VAS<6) were compared.ResultsCGRP-positive cells were detected in the intimal lining layer and comprised both CD14− and CD14+ cells. CGRP expression in non-cultured CD14− fractions was significantly higher than that in CD14+ fractions. The expression levels of CGRP were significantly increased in cultured CD14− cell fractions treated with exogenous PGE2, compared to untreated CD14− cell fractions. In contrast, treatment with PGE2 did not increase CGRP regardless of whether or not CD14+ cells expressed CGRP. Furthermore, CGRP expression in the VAS≥6 group was also significantly higher than that in the VAS<6 group.ConclusionThese findings suggest that CGRP expression in the synovial fibroblasts is regulated by the COX-2/PGE2 pathway and that elevation of synovial CGRP levels may contribute to OA pain.

Expression of calcitonin gene-related peptide in the infrapatellar fat pad in knee osteoarthritis patients

BackgroundThe infrapatellar fat pad (IPFP) has been implicated as a possible source of osteoarthritis (OA) development and knee pain due to the production of inflammatory mediators and the existence of nerve fibers within this structure. Calcitonin gene-related peptide (CGRP) is a vasodilatory neuropeptide that is localized to joint tissues and has recently been implicated in the development of knee OA and OA pain. To date, however, the expression levels of CGRP in the IPFP of human knee OA patients have not been examined.MethodsIFFP and synovial (SYN) tissues were harvested from 100 individuals with radiographic knee OA (unilateral Kellgren/Lawrence [K/L] grades 2–4) during total knee arthroplasty and subjected to immunohistochemical analysis for CGRP localization. In addition, the messenger RNA (mRNA) expression levels of CGRP and cyclooxygenase-2 (COX-2) in the collected tissues were evaluated and compared using real-time PCR analysis of total RNA extracts. CGRP and COX-2 mRNA expression were also compared among individuals with K/L grades 2–4.ResultsCGRP-positive cells were detected in the capillaries within the IPFP and lining layer of SYN tissue. The expression levels of CGRP in the IPFP were positively correlated with COX-2 and were significantly higher than those in SYN tissue. CGRP expression in tissue from the KL4 group was twofold higher than that from the KL2 group.ConclusionsThe IPFP of knee OA patients produces relatively high levels of CGRP, which may be regulated by COX-2 at the transcriptional level. Further studies are needed to determine if CGRP levels are directly linked to OA pathology.

Effects of Taxol on Regeneration in a Rat Sciatic Nerve Transection Model

Recent studies describe taxol as a candidate treatment for promoting central nerve regeneration. However, taxol has serious side effects including peripheral neurotoxicity, and little information is known about the effect of taxol on peripheral nerve regeneration. We investigated the effects of taxol on regeneration in a rat sciatic nerve transection model. Rats were divided into four groups (n = 10): normal saline (i.p.) as the control, Cremophor EL vehicle, and 2 or 6 mg/kg of taxol in the Cremophor EL solution (four times in day-2, 4, 6, and 8), respectively. We evaluated neuronal electrophysiology, animal behaviour, neuronal connectivity, macrophage infiltration, location and expression levels of calcitonin gene-related peptide (CGRP), and expression levels of both nerve growth factors and immunoregulatory factors. In the high-dose taxol group (6 mg/kg), neuronal electrophysiological function was significantly impaired. Licking latencies were significantly changed while motor coordination was unaffected. Neuronal connectivity, macrophage density, and expression levels of CGRP was dramatically reduced. Expression levels of nerve growth factors and immunoregulatory factors was also reduced, while it was increased in the low-dose taxol group (2 mg/kg). These results indicate that taxol can modulate local inflammatory conditions, impair nerve regeneration, and impede recovery of a severe peripheral nerve injury.

Activation of calcitonin gene-related peptide signaling through the prostaglandin E2-EP1/EP2/EP4 receptor pathway in synovium of knee osteoarthritis patients.

BackgroundCalcitonin gene-related peptide (CGRP) is a 37-amino-acid vasodilatory neuropeptide that binds to receptor activity-modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor (CLR). Clinical and preclinical evidence suggests that CGRP is associated with hip and knee joint pain; however, the regulation mechanisms of CGRP/CGRP receptor signaling in synovial tissue are not fully understood.MethodsSynovial tissues were harvested from 43 participants with radiographic knee osteoarthritis (OA; unilateral Kellgren/Lawrence (K/L) grades 3–4) during total knee arthroplasty. Correlationships between the mRNA expression levels of CGRP and those of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and cycloxygenase-2 (COX-2) were evaluated using real-time PCR analysis of total RNA extracted from the collected synovial tissues. To investigate the factors controlling the regulation of CGRP and CGRP receptor expression, cultured synovial cells were stimulated with TNF-α, IL-1β, IL-6, and prostaglandin E2 (PGE2) and were also treated with PGE2 receptor (EP) agonist.ResultsCGRP and COX-2 localized in the synovial lining layer. Expression of COX-2 positively correlated with CGRP mRNA expression in the synovial tissue of OA patients. The gene expression of CGRP and RAMP1 increased significantly in synovial cells exogenously treated with PGE2 compared to untreated control cells. In cultured synovial cells, CGRP gene expression increased significantly following EP4 agonist treatment, whereas RAMP1 gene expression increased significantly in the presence of exogenously added EP1 and EP2 agonists.ConclusionsPGE2 appears to regulate CGRP/CGRP receptor signaling through the EP receptor in the synovium of knee OA patients.

The Involvement of Calcitonin Gene-Related Peptide and Toll-Like Receptor 4 in the Development of Diabetic Sensory Neuropathy

Background: This study was designed to investigate the course and mechanism of diabetes-induced sensory abnormalities in a type 1 diabetic mouse model. Dynamic neuropeptide calcitonin gene-related peptide (CGRP) expression and involvement of toll-like receptor 4 (TLR4) were explored.Methods: C57BL/6J mice were divided in two groups, a control group and a diabetes mellitus (DM) group which was induced with streptozotocin (STZ). Thermal and mechanical nociceptive behavioral techniques were utilized to measure the development of sensitivities. Mice lumbar L4-L6 spinal cord and dorsal root ganglia (DRG) were taken to evaluate the molecular mechanisms of diabetic neuropathy (DN) by using immunocytochemistry and reverse transcription polymerase chain reaction (RT-PCR) methods.Results: DM mice developed both thermal and mechanical hypersensitivities. Thermal sensitivity returned to normal levels at 6 weeks as the mice then gradually developed thermal hypoalgesia in the late stages of DM. However, mechanical allodynia remained from 2 to 10 weeks post-DM, and mice later developed mechanical hypoalgesia. CGRP expression levels in DRG and the dorsal horn of the spinal cord showed a transient increase post-DM, followed by a significant decrease. In DM mice, TLR4 expression level increased in the dorsal horn of the spinal cord and co-localized with microglia and astrocytes in the spinal cord. The TLR4 specific inhibitor, TAK242, alleviated the symptoms of DM-induced pain.Conclusions: Our results demonstrated that hyperglycemia-induced diabetic neuropathic behavior includes early-stage hypersensitivity and late-stage hyposensitivity. CGRP and TLR4 pathways may play a role in early-stage of diabetic neuropathic pain. Citation: Yan-Ping Zhang, Jin-Feng Yang, Yue Yuan, Karla Gomez, Shan-Shan Mei, Yiliam Rodriguez, et al. The involvement of calcitonin gene-related peptide and toll-like receptor 4 in the development of diabetic sensory neuropathy. J Anesth Perioper Med 2016; 3: 18-26. doi: 10.24015/JAPM.2016.0003This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Up-regulation of calcitonin gene-related peptide in trigeminal ganglion following chronic exposure to paracetamol in a CSD migraine animal model

Previously, our group has demonstrated that chronic paracetamol (APAP) treatment induces alterations to the trigeminovascular nociceptive system in the cortical spreading depression (CSD) migraine animal model. The calcitonin gene related peptide (CGRP) is a key neuropeptide involved in the activation of the trigeminovascular nociceptive system. Therefore, this study examined the expression levels of CGRP in the trigeminal ganglion (TG) after chronic APAP exposure (0, 15, and 30 days) using a CSD model. Rats were divided into control, CSD only, APAP only and APAP treatment with CSD groups. A single injection (i.p.) of APAP (200 mg/kg body weight) was given to the 0-day APAP-treated groups, while the other APAP-treated groups received daily injections for 15 and 30 days. CSD was induced by the topical application of KCl to the parietal cortex. The protein expression of CGRP in the TG was evaluated by immunohistochemistry, and the CGRP mRNA level was investigated by real-time quantitative reverse transcription polymerase chain reaction. The results revealed that the induction of CSD significantly increased the level of CGRP protein but had no effect on CGRP mRNA level. Pretreatment with APAP 1 hour before CSD activation significantly reduced CGRP expression induced by CSD. In contrast, chronic treatment with APAP (15 and 30 days) significantly enhanced CGRP expression in both protein and mRNA levels when compared with the control groups. In combination with CSD, the expression of CGRP further increased in the animal with 30 day treatment. These findings indicate that chronic treatment with APAP induces an increase of CGRP expression in the TG. This alteration may be associated with the increased trigeminovascular nociception observed in our previous studies.