Calcitonin Gene-related Peptide In Dorsal Root Ganglion Neurons Research Articles

Activation of the BMP2-SMAD1-CGRP pathway in dorsal root ganglia contributes to bone cancer pain in a rat model

Peripheral nerve remodeling and sensitization are involved in cancer-related bone pain. As a member of the transforming growth factor-β class, bone morphogenetic protein 2 (BMP2) is recognized to have a role in the development of the neurological and skeletal systems. Our previous work showed that BMP2 is critical for bone cancer pain (BCP) sensitization. However, the mechanism remains unknown. In the current study, we demonstrated a substantial increase in BMP2 expression in the dorsal root ganglia (DRG) in a rat model of BCP. Knockdown of BMP2 expression ameliorated BCP in rats. Furthermore, the DRG neurons of rats with BCP expressed higher levels of calcitonin gene-related peptide (CGRP), and BCP was successfully suppressed by intrathecal injection of a CGRP receptor blocker (CGRP8-37). Downregulation of BMP2 expression reduced the expression of CGRP in the DRG of rats with BCP and relieved pain behavior. Moreover, we revealed that upregulation of CGRP expression in the DRG may be induced by activation of the BMPR/Smad1 signaling pathway. These findings suggest that BMP2 contributes to BCP by upregulating CGRP in DRG neurons via activating BMPR/Smad1 signaling pathway and that therapeutic targeting of the BMP2-Smad1-CGRP pathway may ameliorate BCP in the context of advanced cancer.

Effect of nerve growth factor on elderly degenerative knee osteoarthritis pain

To explore effect of nerve growth factor (NGF) antibody on knee osteoarthritis (KOA) pain model was evaluated by in vitro model. Thirty male SPF rats aged 28-week-old were divided into blank group (10 rats with anesthesia only). The other 20 rats were with monoiodoacetate (MIA) on the right knee joint to establish pain model of OA, and were randomly divided into control group (injected intraperitoneal injection of normal saline) and treatment group (injected anti-NGF) intraperitoneal after successful modeling, and 10 rats in each group. All rats were received retrograde injection of fluorogold (FG) into the right knee joint. Gait was assessed using catwalk gait analysis system before treatment, 1 and 2 weeks after treatment. Three weeks after treatment, right dorsal root ganglia (DRG) were excised on L4-L6 level, immunostained for calcitonin gene-related peptide (CGRP), and the number of DRGS was counted. In terms of gait analysis using cat track system, duty cycle, swing speed and print area ratio in control and treatment group were significantly reduced compared with blank group (P<0.05). Compared with control group, duty cycle and swing speed of treatment group were significantly improved (P<0.05), and there was no significant difference in print area ratio between treatment group and blank group (P>0.05). The number of FG-labeled DRG neurons in control group was significantly higher than that in treatment group and blank group (P<0.05). The expression of CGRP in control group was up-regulated, and differences were statistically significant compared with treatment group (P<0.05). Intraperitoneal injection of anti-NGF antibody inhibited gait injury and upregulation of CGRP in DRG neurons. The results suggest that anti-nerve growth factor therapy may be of value in treating knee pain. NGF may be an important target for the treatment of knee OA pain.

Efficacy of nerve growth factor antibody in a knee osteoarthritis pain model in mice

BackgroundNerve growth factor (NGF) is not only an important factor in nerve growth but also a major contributor to the production of inflammation. It has been reported that inhibiting NGF could reduce several types of pain in several animal models. Here, we aimed to clarify the efficacy of NGF antibody in a knee osteoarthritis (OA) pain model in mice.MethodSix-week-old male C57BR/J mice were used (n = 30). Ten mice comprised the control group, which received saline injection into the right knee joints; the other 20 mice comprised the experimental group, which received monoiodoacetate (MIA) injection into the right knee joints. Three weeks after surgery, the 20 experimental mice were randomly placed into treatment groups which received either sterile saline (non-treat group: 10 mg/kg, i.p.) or an anti-NGF antibody (anti-NGF group: 10 mg/kg, i.p.). Simultaneously, all mice received fluorogold (FG) retrograde neurotracer injection into their right joints. In a behavioral study, we evaluated gait using the CatWalk quantitative gait analysis system before surgery, 3 weeks after surgery (before treatment), 4 weeks after surgery (one week after surgery), and 5 weeks after surgery (2 weeks after surgery). In immunohistochemical analysis, the right dorsal root ganglia (DRGs) from the L4–L6 levels were resected 5 weeks after surgery (2 weeks after surgery). They were immunostained for calcitonin gene-related peptide (CGRP), and the number of FG-labeled or CGRP-immunoreactive (IR) DRG neurons was counted.ResultsOn gait analysis using the CatWalk system, duty cycle, swing speed, and print area were decreased in non-treat group compared with those in control group and improved in the anti-NGF group compared with those in non-treat group. CGRP expression in DRGs was up-regulated in non-treat group compared with that in control group and suppressed in the anti-NGF group compared with that in non-treat group (both p < 0.05).ConclusionsMIA injection into the knee joint induced gait impairment and the up-regulation of CGRP in DRG neurons in a knee OA pain model in mice. Intraperitoneal injection of anti-NGF antibody suppressed this impairment of gait and up-regulation of CGRP in DRG neurons. These finding suggest that anti-NGF therapy might be valuable in the treatment of OA pain in the knee.

Muscle injury in rats induces upregulation of inflammatory cytokines in injured muscle and calcitonin gene-related peptide in dorsal root ganglia innervating the injured muscle.

ABSTRACT Introduction: In this study we evaluated the relationships among the behavioral changes after muscle injury, histological changes, changes in inflammatory cytokines in the injured muscle, and changes in the sensory nervous system innervating the muscle in rats. Methods: We established a model of muscle injury in rats using a dropped weight. Behavior was assessed using the CatWalk system. Subsequently, bilateral gastrocnemius muscles and dorsal root ganglia (DRGs) were resected. Muscles were stained with hematoxylin and eosin, and inflammatory cytokines in injured muscles were assayed. DRGs were immunostained for calcitonin gene–related peptide (CGRP). Results: Changes of behavior and upregulation of inflammatory cytokines in injured muscles subsided within 2 days of injury. Repaired tissue was observed 3 weeks after injury. However, upregulation of CGRP in DRG neurons continued for 2 weeks after injury. Conclusion: These findings may explain in part the pathological mechanism of persistent muscle pain. Muscle Nerve 54: 776–782, 2016

Vertebral Compression Exacerbates Osteoporotic Pain in an Ovariectomy-Induced Osteoporosis Rat Model

Basic pain study using osteoporotic rodent models.To examine alterations in distribution of pain-related neuropeptides after compressive force on osteoporotic vertebrae and their chronic pain-related properties.We previously reported significantly increased production of calcitonin gene-related peptide (CGRP), a marker of inflammatory pain, in the dorsal root ganglia (DRG) of vertebrae in osteoporosis-model ovariectomized (OVX) rats. Here, we hypothesized that longitudinal compressive force on vertebrae can affect osteoporotic pain properties, which has not been examined yet.OVX rats were used as the osteoporosis model. Female Sprague-Dawley rats were prepared and Fluoro-Gold (FG) neurotracer was applied to the periosteal surface of the Co5 vertebra. After FG labeling, the animals were divided into 4 groups: Control, Control + compression, OVX, and OVX + compression. The Control groups were not ovariectomized. In the compression groups, K-wires were stabbed transversely through Co4 and Co6 with Co5 compressed longitudinally by rubber bands bridged between the 2. One, 2, 4, and 8 weeks after surgery, bilateral S1 to S3 DRGs were excised for immunofluorescence assays. Expression of CGRP and activating transcription factor 3, a marker of neuronal injury, were compared among the 4 groups.Sustained upregulation of CGRP in DRG neurons was observed after compression of the Co5 vertebra, and Co5 compression caused significant increase in CGRP production in DRG neurons, whereas a greater level of activating transcription factor 3 upregulation was observed in DRGs in OVX rats after dynamic vertebral compression 8 weeks after surgery, implying potential neuropathic pain.There was sustained upregulation of CGRP and activating transcription factor 3 in DRGs in osteoporotic model rats compared with controls, and levels were further enhanced by dynamic vertebral compression. These findings imply that dynamic compression stress on vertebrae can exacerbate osteoporotic pain by inducing both inflammatory and neuropathic pain mediators.N/A.

The inhibition of subchondral bone lesions significantly reversed the weight-bearing deficit and the overexpression of CGRP in DRG neurons, GFAP and Iba-1 in the spinal dorsal horn in the monosodium iodoacetate induced model of osteoarthritis pain.

BackgroundChronic pain is the most prominent and disabling symptom of osteoarthritis (OA). Clinical data suggest that subchondral bone lesions contribute to the occurrence of joint pain. The present study investigated the effect of the inhibition of subchondral bone lesions on joint pain.MethodsOsteoarthritic pain was induced by an injection of monosodium iodoacetate (MIA) into the rat knee joint. Zoledronic acid (ZOL), a third generation of bisphosphonate, was used to inhibit subchondral bone lesions. Joint histomorphology was evaluated using X-ray micro computed tomography scanning and hematoxylin-eosin staining. The activity of osteoclast in subchondral bone was evaluated using tartrate-resistant acid phosphatase staining. Joint pain was evaluated using weight-bearing asymmetry, the expression of calcitonin gene-related peptide (CGRP) in the dorsal root ganglion (DRG), and spinal glial activation status using glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule-1 (Iba-1) immunofluorescence. Afferent neurons in the DRGs that innervated the joints were identified using retrograde fluorogold labeling.ResultsMIA injections induced significant histomorphological alterations and joint pain. The inhibition of subchondral bone lesions by ZOL significantly reduced the MIA-induced weight-bearing deficit and overexpression of CGRP in DRG neurons, GFAP and Iba-1 in the spinal dorsal horn at 3 and 6 weeks after MIA injection; however, joint swelling and synovial reaction were unaffected.ConclusionsThe inhibition of subchondral bone lesions alleviated joint pain. Subchondral bone lesions should be a key target in the management of osteoarthritic joint pain.

Bradykinin and prostaglandin E1 regulate calcitonin gene-related peptide expression in cultured rat sensory neurons

Primary cultures of adult rat dorsal root ganglia (DRG) sensory neurons were used to determine whether bradykinin and prostaglandins E1 (PGE1), E2 (PGE2) or I2 (PGI2) stimulate long-term calcitonin gene-related peptide (CGRP) mRNA accumulation and peptide release. Treatment (24h) of neurons with either bradykinin or PGE1, significantly increased CGRP mRNA content and iCGRP release. However, PGE2 or PGI2 was without effect. Exposure of the cultured neurons to increasing concentrations of bradykinin or PGE1 demonstrated that the stimulation of CGRP expression was concentration-dependent, while time-course studies showed that maximal levels of CGRP mRNA accumulation and peptide release were maintained for at least 48h. Treatment of the neuronal cultures with a bradykinin B2 receptor antagonist significantly inhibited the bradykinin-induced increase in CGRP expression and release. In addition, preincubation of neuronal cultures with the cyclooxygenase inhibitor indomethacin did not alter the PGE1-mediated stimulation of CGRP but blocked completely the bradykinin-induced increase in CGRP production. Therefore, these data indicate that bradykinin and PGE1 can regulate the synthesis and release of CGRP in DRG neurons and that the stimulatory effects of bradykinin on CGRP are mediated by a cyclooxygenase product(s). Thus, these findings suggest a direct relationship between chronic alterations in bradykinin/prostaglandin production that may arise from pathophysiological causes and long-term changes in CGRP expression.

Increases in transient receptor potential vanilloid‐1 mRNA and protein in primary afferent neurons stimulated by protein kinase C and their possible role in neurogenic inflammation

A recent study by our group demonstrates pharmacologically that the transient receptor potential vanilloid-1 (TRPV(1)) is activated by intradermal injection of capsaicin to initiate neurogenic inflammation by the release of neuropeptides in the periphery. In this study, expression of TRPV(1), phosphorylated protein kinase C (p-PKC), and calcitonin gene-related peptide (CGRP) in dorsal root ganglion (DRG) neurons was visualized by using immunofluorescence, real-time PCR, and Western blots to examine whether increases in TRPV(1) mRNA and protein levels evoked by capsaicin injection are subject to modulation by the activation of PKC and to analyze the role of this process in the pathogenesis of neurogenic inflammation. Capsaicin injection into the hindpaw skin of anesthetized rats evoked increases in the expression of TRPV(1), CGRP and p-PKC in mRNA and/or protein levels and in the number of single labeled TRPV(1), p-PKC, and CGRP neurons in ipsilateral L4-5 DRGs. Coexpressions of TRPV(1) with p-PKC and/or CGRP in DRG neurons were also significantly increased after CAP injection. These evoked expressions at both molecular and cellular levels were significantly inhibited after TRPV(1) receptors were blocked by 5'-iodoresiniferatoxin (5 microg) or PKC was inhibited by chelerythrine chloride (5 microg). Taken together, these results provide evidence that up-regulation of TRPV(1) mRNA and protein levels under inflammatory conditions evoked by capsaicin injection is subject to modulation by the PKC cascade in which increased CGRP level in DRG neurons may be related to the initiation of neurogenic inflammation. Thus, up-regulation of TRPV(1) receptors in DRG neurons seems critical for initiating acute neurogenic inflammation.