Spinal Calcitonin Gene-related Peptide Research Articles

Blockade of spinal dopamine D1/D2 receptor suppresses activation of NMDA receptor through Gαq and Src kinase to attenuate chronic bone cancer pain

IntroductionSpinal N-methyl-D-aspartate receptor (NMDAR) is vital in chronic pain, while NMDAR antagonists have severe side effects. NMDAR has been reported to be controlled by G protein coupled receptors (GPCRs), which might present new therapeutic targets to attenuate chronic pain. Dopamine receptors which belong to GPCRs have been reported could modulate the NMDA-mediated currents, while their exact effects on NMDAR in chronic bone cancer pain have not been elucidated. ObjectivesThis study was aim to explore the effects and mechanisms of dopamine D1 receptor (D1DR) and D2 receptor (D2DR) on NMDAR in chronic bone cancer pain. MethodsA model for bone cancer pain was established using intra-tibia bone cavity tumor cell implantation (TCI) of Walker 256 in rats. The nociception was assessed by Von Frey assay. A range of techniques including the fluorescent imaging plate reader, western blotting, and immunofluorescence were used to detect cell signaling pathways. Primary cultures of spinal neurons were used for in vitro evaluation. ResultsBoth D1DR and D2DR antagonists decreased NMDA-induced upregulation of Ca2+ oscillations in primary culture spinal neurons. Additionally, D1DR/D2DR antagonists inhibited spinal Calcitonin Gene-Related Peptide (CGRP) and c-Fos expression and alleviated bone cancer pain induced by TCI which could both be reversed by NMDA. And D1DR/D2DR antagonists decreased p-NR1, p-NR2B, and Gαq protein, p-Src expression. Both Gαq protein and Src inhibitors attenuated TCI-induced bone cancer pain, which also be reversed by NMDA. The Gαq protein inhibitor decreased p-Src expression. In addition, D1DR/D2DR antagonists, Src, and Gαq inhibitors inhibited spinal mitogen-activated protein kinase (MAPK) expression in TCI rats, which could be reversed by NMDA. ConclusionsSpinal D1DR/D2DR inhibition eliminated NMDAR-mediated spinal neuron activation through Src kinase in a Gαq-protein-dependent manner to attenuate TCI-induced bone cancer pain, which might present a new therapeutic strategy for bone cancer pain.

Whole-body Vibration at Thoracic Resonance Induces Sustained Pain and Widespread Cervical Neuroinflammation in the Rat.

Whole-body vibration (WBV) is associated with back and neck pain in military personnel and civilians. However, the role of vibration frequency and the physiological mechanisms involved in pain symptoms are unknown. This study asked the following questions: (1) What is the resonance frequency of the rat spine for WBV along the spinal axis, and how does frequency of WBV alter the extent of spinal compression/extension? (2) Does a single WBV exposure at resonance induce pain that is sustained? (3) Does WBV at resonance alter the protein kinase C epsilon (PKCε) response in the dorsal root ganglia (DRG)? (4) Does WBV at resonance alter expression of calcitonin gene-related peptide (CGRP) in the spinal dorsal horn? (5) Does WBV at resonance alter the spinal neuroimmune responses that regulate pain? Resonance of the rat (410 ± 34 g, n = 9) was measured by imposing WBV at frequencies from 3 to 15 Hz. Separate groups (317 ± 20 g, n = 10/treatment) underwent WBV at resonance (8 Hz) or at a nonresonant frequency (15 Hz). Behavioral sensitivity was assessed throughout to measure pain, and PKCε in the DRG was quantified as well as spinal CGRP, glial activation, and cytokine levels at Day 14. Accelerometer-based thoracic transmissibility peaks at 8 Hz (1.86 ± 0.19) and 9 Hz (1.95 ± 0.19, mean difference [MD] 0.290 ± 0.266, p < 0.03), whereas the video-based thoracic transmissibility peaks at 8 Hz (1.90 ± 0.27), 9 Hz (2.07 ± 0.20), and 10 Hz (1.80 ± 0.25, MD 0.359 ± 0.284, p < 0.01). WBV at 8 Hz produces more cervical extension (0.745 ± 0.582 mm, MD 0.242 ± 0.214, p < 0.03) and compression (0.870 ± 0.676 mm, MD 0.326 ± 0.261, p < 0.02) than 15 Hz (extension, 0.503 ± 0.279 mm; compression, 0.544 ± 0.400 mm). Pain is longer lasting (through Day 14) and more robust (p < 0.01) after WBV at the resonant frequency (8 Hz) compared with 15 Hz WBV. PKCε in the nociceptors of the DRG increases according to the severity of WBV with greatest increases after 8 Hz WBV (p < 0.03). However, spinal CGRP, cytokines, and glial activation are only evident after painful WBV at resonance. WBV at resonance produces long-lasting pain and widespread activation of a host of nociceptive and neuroimmune responses as compared with WBV at a nonresonance condition. Based on this work, future investigations into the temporal and regional neuroimmune response to resonant WBV in both genders would be useful. Although WBV is a major issue affecting the military population, there is little insight about its mechanisms of injury and pain. The neuroimmune responses produced by WBV are similar to other pain states, suggesting that pain from WBV may be mediated by similar mechanisms as other neuropathic pain conditions. This mechanistic insight suggests WBV-induced injury and pain may be tempered by antiinflammatory intervention.

Facilitated spinal neuropeptide signaling and upregulated inflammatory mediator expression contribute to postfracture nociceptive sensitization.

Tibia fracture induces exaggerated substance P (SP) and calcitonin gene-related peptide (CGRP) signaling and neuropeptide-dependent nociceptive and inflammatory changes in the hind limbs of rats similar to those seen in complex regional pain syndrome. Inflammatory changes in the spinal cord contribute to nociceptive sensitization in a variety of animal pain models. This study tested the hypothesis that fracture-induced exaggerated neuropeptide signaling upregulates spinal inflammatory mediator expression, leading to postfracture hind limb nociceptive sensitization. At 4 weeks after performing tibia fracture and casting in rats, we measured hind limb allodynia, unweighting, warmth, edema, and spinal cord neuropeptide and inflammatory mediator content. The antinociceptive effects of intrathecally injected neuropeptide and inflammatory mediator receptor antagonists were evaluated in fracture rats. Transgenic fracture mice lacking SP or the CGRP RAMP1 receptor were used to determine the effects of neuropeptide signaling on postfracture pain behavior and spinal inflammatory mediator expression. Hind limb allodynia, unweighting, warmth, edema, increased spinal SP and CGRP, and increased spinal inflammatory mediator expression (TNF, IL-1, IL-6, CCL2, and nerve growth factor) were observed at 4 weeks after fracture in rats. Fracture-induced increases in spinal inflammatory mediators were not observed in fracture mice lacking SP or the CGRP receptor, and these mice had attenuated postfracture nociceptive sensitization. Intrathecal injection of selective receptor antagonists for SP, CGRP, TNF, IL-1, IL-6, CCL2, or nerve growth factor each reduced pain behaviors in the fracture rats. Collectively, these data support the hypothesis that facilitated spinal neuropeptide signaling upregulates the expression of spinal inflammatory mediators contributing to nociceptive sensitization in a rodent fracture model of complex regional pain syndrome.

Microglial content-dependent inhibitory effects of calcitonin gene-related peptide (CGRP) on murine retroviral infection of glial cells.

C57BL/6 (B6) mice develop peripheral neuropathy post-LP-BM5 infection, a murine model of HIV-1 infection, along with the up-regulation of select spinal cord cytokines. We investigated if calcitonin gene-related peptide (CGRP) contributed to the development of peripheral neuropathy by stimulating glial responses. An increased expression of lumbar spinal cord CGRP was observed in vivo, post-LP-BM5 infection. Consequently, in vitro CGRP co-treatments led to a microglial content-dependent attenuation of viral loads in spinal cord mixed glia infected with selected doses of LP-BM5. This inhibition was neither caused by the loss of glia nor induced via the direct inhibition of LP-BM5 by CGRP.

NO‐ting a putative mechanism by which anti‐migraine drugs mitigate trigeminal neuropathic pain

<scp>NO</scp>‐ting a putative mechanism by which anti‐migraine drugs mitigate trigeminal neuropathic pain

Antinociceptive effects of dexmedetomidine via spinal substance P and CGRP.

The aim of this study was to examine the role played by substance P and calcitonin gene-related peptide (CGRP) within the dorsal horn of the spinal cord in engagement of antinociception evoked by dexmedetomidine (DEX). Paw withdrawal threshold (PWT) to mechanical stimulation was determined after chronic intrathecal infusion of DEX and enzyme-linked immunosorbent assay (ELISA) was employed to examine the levels of spinal substance P and CGRP. Our results show that PWT was significantly increased by intrathecal administration of DEX in rats (P < 0.05 vs. vehicle control, n = 20 in each group). Also, intrathecal infusion of DEX significantly decreased the concentrations of substance P and CGRP as compared with vehicle control (P < 0.05 DEX vs. vehicle control, n = 20 in each group). Blocking α2-adrenoreceptors (α2-AR) blunted the decreases of substance P and CGRP levels and the enhancement of PWT evoked by DEX. Additionally, a linear relationship was observed between PWT and the levels of spinal substance P (r = 0.87; P < 0.005) and CGRP (r = 0.85; P < 0.005). Moreover, blocking individual substance P and CGRP receptors amplified PWT without altering substance P and CGRP levels. Thus, DEX plays a role in stimulating α2-AR receptors, which thereby decreases substance P and CGRP levels within the dorsal horn. This contributes to DEX-evoked antinociception.

Hydrogen sulfide inhibits opioid withdrawal-induced pain sensitization in rats by down-regulation of spinal calcitonin gene-related peptide expression in the spine.

Hyperalgesia often occurs in opioid-induced withdrawal syndrome. In the present study, we found that three hourly injections of DAMGO (a μ-opioid receptor agonist) followed by naloxone administration at the fourth hour significantly decreased rat paw nociceptive threshold, indicating the induction of withdrawal hyperalgesia. Application of NaHS (a hydrogen sulfide donor) together with each injection of DAMGO attenuated naloxone-precipitated withdrawal hyperalgesia. RT-PCR and Western blot analysis showed that NaHS significantly reversed the gene and protein expression of up-regulated spinal calcitonin gene-related peptide (CGRP) in naloxone-treated animals. NaHS also inhibited naloxone-induced cAMP rebound and cAMP response element-binding protein (CREB) phosphorylation in rat spinal cord. In SH-SY5Y neuronal cells, NaHS inhibited forskolin-stimulated cAMP production and adenylate cyclase (AC) activity. Moreover, NaHS pre-treatment suppressed naloxone-stimulated activation of protein kinase C (PKC) α, Raf-1, and extracellular signal-regulated kinase (ERK) 1/2 in rat spinal cord. Our data suggest that H2S prevents the development of opioid withdrawal-induced hyperalgesia via suppression of synthesis of CGRP in spine through inhibition of AC/cAMP and PKC/Raf-1/ERK pathways.

P63: Hydrogen sulfide inhibits opioid withdrawal-induced pain sensitization in rats by down-regulation of CGRP expression in the spine

Hyperalgesia often occurs in opioid-induced withdrawal syndrome. In the present study, we found that three hourly injections of DAMGO (a μ -opioid receptor agonist) followed by naloxone administration at the fourth hour significantly decreased rat paw nociceptive threshold, indicating the induction of withdrawal hyperalgesia. Application of NaHS (a hydrogen sulfide donor) together with each injection of DAMGO attenuated naloxone-precipitated withdrawal hyperalgesia. RT-PCR analysis showed that NaHS significantly reversed the up-regulated spinal calcitonin gene related peptide (CGRP) expression in naloxone-treated animals. NaHS also inhibited naloxone-induced cAMP rebound and cAMP response element-binding protein (CREB) phosphorylation in rat spinal cord. In SH-SY5Y neuronal cells, NaHS inhibited forskolin-stimulated cAMP production and adenylate cyclase (AC) activity. Moreover, NaHS pretreatment suppressed naloxone-stimulated activation of protein kinase C (PKC) α , Raf-1, and extracellular signal-regulated kinase (ERK) 1/2 in rat spinal cord. In addition, both repeated DAMGO treatment and naloxone-precipitated withdrawal decreased endogenous H2S level in rat spinal cord, suggesting that the endogenous hydrogen sulfide is important to prevent hyperalgesia. Our data suggest that H2S prevents the development of opioid withdrawal-induced hyperalgesia via suppression of synthesis of CGRP in spine through inhibition of AC/cAMP and PKC/Raf-1/ERK pathways.

Intrathecal PKA-selective siRNA treatment blocks sustained morphine-mediated pain sensitization and antinociceptive tolerance in rats

Sustained morphine treatment has been shown to produce paradoxical pain sensitization (opioid-induced hyperalgesia) and also causes increase in spinal pain neurotransmitter, such as calcitonin gene related peptide (CGRP), concentration in experimental animals. Studies have also shown that cyclic adenosine-monophosphate (cAMP)-dependent protein kinase (PKA) plays a major role in the regulation of presynaptic neurotransmitter (such as CGRP and substance P) synthesis and release. We have previously shown that in cultured primary sensory dorsal root ganglion (DRG) neurons sustained in vitro opioid agonist treatment upregulates cAMP levels (adenylyl cyclase (AC) superactivation) and augments basal and capsaicin evoked CGRP release in a PKA dependent manner. In the present study, we investigated the in vivo role of PKA in sustained morphine-mediated pain sensitization. Our data indicate that selective knock-down of spinal PKA activity by intrathecal (i.th.) pretreatment of rats with a PKA-selective small interference RNA (siRNA) mixture significantly attenuates sustained morphine-mediated augmentation of spinal CGRP immunoreactivity, thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance. The present findings indicate that sustained morphine-mediated activation of spinal cAMP/PKA-dependent signaling may play an important role in opioid induced hyperalgesia.

Sustained morphine‐mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf‐1‐selective siRNA

Long-term morphine treatment enhances pain neurotransmitter [such as calcitonin gene-related peptide (CGRP)] levels in the spinal cord. It has been suggested previously that increased spinal CGRP may contribute to sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance. Previous in vitro studies from our group indicated that Raf-1 kinase-mediated adenylyl cyclase superactivation played a crucial role in sustained morphine-mediated augmentation of basal and evoked CGRP release from cultured primary sensory neurons. The present study was aimed to evaluate the physiological significance of this molecular mechanism in vivo, in rats. Rats were intrathecally (i.th) injected with a Raf-1-selective small interfering RNA (siRNA) mixture for 3 days and were subsequently infused with saline or morphine, s.c. for 7 days. Thermal and mechanical sensory thresholds of the animals were assessed by daily behavioural tests. After final behavioural testing (day 6), spinal cords were isolated from each animal group and spinal CGRP and Raf-1 protein levels were measured using elisa and immunohistochemistry. Selective knockdown of spinal Raf-1 protein levels by i.th Raf-1-selective siRNA pretreatment significantly attenuated sustained morphine-mediated up-regulation of CGRP immunoreactivity in the spinal cord of rats and prevented the development of thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance. Raf-1 played a significant role in sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance in vivo. These findings suggest novel pharmacological approaches to improve the long-term utility of opioids in the treatment of chronic pain.

A-995662 [(R)-8-(4-methyl-5-(4-(trifluoromethyl)phenyl)oxazol-2-ylamino)-1,2,3,4-tetrahydronaphthalen-2-ol], a novel, selective TRPV1 receptor antagonist, reduces spinal release of glutamate and CGRP in a rat knee joint pain model

The TRPV1 antagonist A-995662 demonstrates analgesic efficacy in monoiodoacetate-induced osteoarthritic (OA) pain in rat, and repeated dosing results in increased in vivo potency and a prolonged duration of action. To identify possible mechanism(s) underlying these observations, release of neuropeptides and the neurotransmitter glutamate from isolated spinal cord was measured. In OA rats, basal release of glutamate, bradykinin and calcitonin gene-related peptide (CGRP) was significantly elevated compared to naïve levels, whereas substance P (SP) levels were not changed. In vitro studies showed that capsaicin-evoked TRPV1-dependent CGRP release was 54.7 ± 7.7% higher in OA, relative to levels measured for naïve rats, suggesting that TRPV1 activity was higher under OA conditions. The efficacy of A-995662 in OA corresponded with its ability to inhibit glutamate and CGRP release from the spinal cord. A single, fully efficacious dose of A-995662, 100 μmol/kg, reduced spinal glutamate and CGRP release, while a single sub-efficacious dose of A-995662 (25 μmol/kg) was ineffective. Multiple dosing with A-995662 increased the potency and duration of efficacy in OA rats. Changes in efficacy did not correlate with plasma concentrations of A-995662, but were accompanied with reductions in spinal glutamate release. These findings suggest that repeated dosing of TRPV1 antagonists enhances therapeutic potency and duration of action against OA pain, at least in part, by the sustained reduction in release of glutamate and CGRP from the spinal cord.

Spinal mediators that may contribute selectively to antinociceptive tolerance but not other effects of morphine as revealed by deletion of GluR5

Several groups maintain that morphine tolerance and dependence correlate with increased activity of protein kinases ERK1/2 and P38 MAPK and PKC as well as elevated levels of the neuropeptides dynorphin (DYN), substance P (sP), and calcitonin gene-related peptide (CGRP) in spinal cord dorsal horn (SCDH). They demonstrate that tolerance and dependence can be prevented, and sometimes reversed, by constitutive genetic deletion or pharmacological inhibition of these factors. Recently, we showed that mice with a constitutive deletion of the GluR5 subunit of kainate receptors (GluR5 KO) are not different from wild type (WT) littermates with respect to baseline nociceptive thresholds as well as acute morphine antinociception, morphine physical dependence and conditioned place preference. However, unlike WT, GluR5 KO mice do not develop antinociceptive tolerance following systemic morphine administration. In this report, we examined levels of these mediators in SCDH of WT and GluR5 KO mice following subcutaneous implantation of placebo or morphine pellets. Surprisingly, spinal DYN and CGRP, along with phosphorylated ERK2 (pERK2), P38 (pP38) and PKCgamma (pPKCγ) are elevated by deletion of GluR5. Additionally, chronic systemic morphine administration increased spinal pERK2, pP38 and pPKCγ levels in both tolerant WT and non-tolerant GluR5 KO mice. In contrast, while morphine increased spinal DYN and CGRP in WT mice, DYN remained unchanged and CGRP was reduced in GluR5 KO mice. These observations suggest that spinal ERK2, P38 and PKCγ are likely involved in multiple adaptive responses following systemic morphine administration, whereas DYN and CGRP may contribute selectively to the development of antinociceptive tolerance.

A Segmental Effect Involved in the Changes of Skin Blood Flow Induced by Acupuncture in Normal Health Human

Our previous studies found that segmental effect of spinal nerve plays an important role in acupuncture stimulation (AS) on nail-fold microcirculation. Calcitonin-gene related peptide (CGRP) is a potent endogenous vasodilator. Therefore, the aim of the present study was to investigate the effect of AS on skin blood flow (SBF) and skin temperature (ST), and the involvement of spinal nerve segment and CGRP. A total of 17 healthy adult volunteers were studied, and all of them accepted three assessments as follows: (1) left Waiguan (SJ5) assessment (LW), AS applied to Waiguan acupoint of the left hand with obtaining qi; (2) right Waiguan assessment (RW), AS applied to Waiguan acupoint of the right hand with obtaining qi; (3) sham assessment, AS applied to the cutaneous portion of Waiguan acupoint of the left hand only. Each assessment was divided into before acupuncture period (BP), acupuncture period (AP) and post acupuncture period (PP). Both SBF and skin temperature (ST) were recorded simultaneously for 20 min at central dorsal part of the left hand in each period. Besides, the levels of CGRP in the blood were measured before AS, and at 20 min after AS in the LW assessment. Results indicated that the SBF was less in the PP than that in the BP, but SBF did not significantly vary between AP and BP, and between AP and PP in the LW assessment; SBF is similar among BP, AP, and PP in the sham and RW assessments; the difference of SBF between AP and BP was greater in the LW assessment than that in the sham assessment; the difference of SBF between AP and BP, and between PP and BP was greater in the LW assessment than that in the RW assessment. The levels of CGRP in the BP were similar to that at 20 min after AS; ST was similar among BP, AP and PP in the sham, LW and RW assessments. These results suggested that a segmental effect of spinal nerve is involved in the changes of SBF induced by AS since both the cutaneous part of left Waiguan acupoint and central dorsal part of the left hand belong to the 7th segment of cervical nerve.

Cell‐type specific activation of p38 and ERK mediates calcitonin gene‐related peptide involvement in tolerance to morphine‐induced analgesia

Tolerance to morphine-induced analgesia is a well-established phenomenon, often limiting its usefulness in the long-term treatment of pain. The mechanisms underlying tolerance are not well understood. We previously suggested a possible role for spinal calcitonin gene-related peptide (CGRP) in the development of tolerance to morphine-induced analgesia. In the present study, we demonstrate that CGRP is involved in morphine tolerance by differentially regulating the ERK-dependent up-regulation of IL-1beta, TNF-alpha, and microsomal prostaglandin E synthase-1 (mPGES-1) in astrocytes and p38-dependent up-regulation of IL-6 in microglia in the rat spinal cord. A 7-d treatment with morphine induced tolerance to the antinociceptive effect and increased phosphorylated ERK localized in astrocytes and phosphorylated p38 enriched in microglia, both effects being inhibited by blocking CGRP receptors. Interestingly, the inhibition of the ERK pathway suppressed the development of tolerance and morphine-induced up-regulation of IL-1beta, TNF-alpha, and mPGES-1. Blockade of p38 activity also inhibited the development of tolerance and morphine-induced IL-6 up-regulation. Taken together, these data suggest that chronic morphine induces the synthesis of CGRP, which in turn acts on CGRP receptors located on astrocytes and microglia to stimulate ERK and p38, respectively, leading to increased synthesis and release of proinflammatory mediators resulting in tolerance to morphine-induced analgesia.

Involvement of cannabinoid (CB 1)-receptors in the development and maintenance of opioid tolerance

Sustained exposure to opioid agonists such as morphine increases levels of calcitonin gene-related peptide (CGRP) in the spinal dorsal horn, a response implicated in the development of opioid tolerance and physical dependence. Recent evidence suggests that both the opioid-induced increase in CGRP and the development of opioid physical dependence are suppressed by blockade of spinal cannabinoid (CB 1)-receptors. The present study examined whether CB 1-receptor activity also has a role in the development of opioid tolerance. In rats implanted with spinal catheters, repeated acute injections of morphine (15 μg) delivered over 4 h resulted in a rapid decline of thermal and mechanical antinociception and a significant loss of analgesic potency, reflecting development of acute opioid tolerance. In another set of experiments, chronic administration of spinal morphine (15 μg) once daily for 5 days produced a similar loss of analgesic effect and a marked increase in CGRP-immunoreactivity in the superficial laminae of the dorsal horn. Consistent with the in vivo findings, primary cultures of adult dorsal root ganglion (DRG) neurons exposed to morphine for 5 days showed a significant increase in the number of CGRP-immunoreactive neurons. Co-administration of acute or chronic morphine with a CB 1-receptor antagonist/inverse agonist, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (AM-251), inhibited the development of both acute and chronic analgesic tolerance. In animals already exhibiting tolerance to morphine, intervention with AM-251 restored morphine analgesic potency. Co-administration with AM-251 attenuated the morphine-induced increase in CGRP-immunoreactivity in the spinal cord and in DRG cultured neurons. Collectively, the results of this study suggest that activity of endocannabinoids, mediated via CB 1-receptors, contributes to both the development and maintenance of opioid tolerance by influencing the opioid-induced increase in spinal CGRP.

Spinal modulation of calcitonin gene-related peptide by endocannabinoids in the development of opioid physical dependence

Studies implicate endocannabinoids in the acute and chronic actions of opioid drugs, including the genesis of physical dependence. Previous evidence suggests that spinal release of calcitonin gene-related peptide (CGRP) and activation of its receptors contribute to opioid physical dependence. The release of CGRP at the spinal level is modulated by cannabinoid (CB 1)-receptors. Thus, this study examined whether CB 1-receptor activity mediates changes in CGRP underlying development of opioid physical dependence. Systemic morphine administration for 5-days elevated CGRP-immunoreactivity in the rat spinal dorsal horn. In situ hybridization of dorsal root ganglion (DRG) neurons revealed an increase in CGRP mRNA during initial (day 1–3) but not later phase (day 4–5) of morphine treatment. CGRP-immunoreactivity in DRG neurons, however, was increased in the later phase of morphine treatment. Naloxone challenge to morphine-treated animals precipitated an intense withdrawal syndrome that depleted CGRP-immunoreactivity and increased Fos expression in the dorsal horn. The Fos-response primarily occurred in neurons that expressed CGRP receptor component protein (RCP) suggesting CGRP activity contributes to neuronal activation during precipitated withdrawal. Spinal slices obtained from morphine-treated animals showed higher levels of CGRP release than from saline controls. Intrathecal co-administration of CB 1-receptor antagonists, AM-251 or SR141716A, with daily morphine attenuated the behavioral manifestations of withdrawal. Treatment with AM-251 also reduced the depletion of CGRP, suppressed Fos-induction, and prevented the increase in capsaicin-evoked spinal CGRP release. Altogether, this study suggests that endocannabinoid activity, expressed via CB 1-receptors, contributes to the induction of opioid physical dependence through spinal modulation of CGRP.

The majority of bladder sensory afferents to the rat lumbosacral spinal cord are both IB4- and CGRP-positive

The rat urinary bladder is innervated by neurons in dorsal root ganglia (DRG) that express the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP), and a fraction of bladder afferents can bind the non-peptidergic marker isolectin B4 (IB4). We used histochemical binding and axonal tracing to identify the bladder afferents, and immunocytochemistry to determine the degree of colocalization of CGRP with IB4 in their cell bodies in DRG and in their central axons in the spinal cord. In the L6 DRG, about 60% of CGRP-positive neurons were also positive for IB4. In the spinal cord, IB4 and CGRP colocalized in fibers and terminals in the inner part of lamina II, the lateral collateral path, and the sacral parasympathetic nucleus (SPN). In SPN, the majority of IB4-positive fibers and terminals were also CGRP-positive. After injection of IB4 into the bladder wall, immunoreaction for IB4 was detected in SPN, but not in lamina II. These results suggest that most IB4-positive afferents from the bladder are also CGRP-positive, and that the distinction between peptidergic and non-peptidergic bladder afferents based on IB4 binding is of limited validity.

Peripherally Delivered Glutamic Acid Decarboxylase Gene Therapy for Spinal Cord Injury Pain

Neuropathic pain after spinal cord injury (SCI) represents a difficult problem that is commonly refractory to conventional medical management. To determine if spinal release of γ-amino butyric acid (GABA) could reduce below-level central neuropathic pain after SCI, we constructed a replication-incompetent herpes simplex virus (HSV)-based vector encoding one isoform of human glutamic acid decarboxylase (GAD67). Dorsal root ganglion (DRG) neurons transduced in vitro or in vivo by subcutaneous inoculation produced GAD and released GABA constitutively. T13 spinal cord hemisection resulted in central neuropathic pain manifested by mechanical allodynia and thermal hyperalgesia. Subcutaneous inoculation of the vector into both feet reduced both manifestations of below-level SCI pain; the vector-mediated effect was partially reversed by intrathecal bicuculline or phaclofen at doses that did not affect thresholds in normal or injured uninoculated animals. Vector-mediated GABA release attenuated the increase in spinal calcitonin gene-related peptide immunoreactivity caused by cord hemisection. These results suggest that HSV-mediated gene transfer to DRG could be used to treat below-level central neuropathic pain after incomplete SCI.

214. Additive Effects of HSV Vectors Expressing Proenkephalin, Interleukin-4, and Tumor Necrosis Factor Alpha Soluble Receptor To Treat Bone Cancer Pain

Top of pageAbstract In order to determine whether increasing GABA in the spinal cord by glutamic acid decarboxylase 67 (GAD67) gene transfer to the dorsal root ganglion (DRG) would attenuate below-level central neuropathic pain resulting from spinal cord injury (SCI) we engineered a replication defective HSV based vector, deleted for the essential immediate early genes ICP4 and ICP27 and additionally deficient in expression of immediate early genes ICP22 and ICP47, to express GAD 67 driven by the HCMV IE promoter at the UL41 locus (vector QHGAD67). Transduction of DRG neurons in vitro with QHGAD67 resulted in the release of GABA measured by HPLC. Subcutaneous inoculation of the vector in the foot resulted in GAD67 expression and constitutive release of GABA in microdialysate of the dorsal horn. One week after left T13 spinal hemisection in rats, animals were inoculated subcutaneously into the plantar surface of both hindpaws with 30 microliters QHGAD67 (1x109 pfu/ml) or a lacZ-expressing control vector QOZHG. Mechanical allodynia and thermal hyperalgesia in both hindpaws was assessed with a series of calibrated von Frey filaments and Hargreaves heat apparatus, respectively. Animals inoculated with QHGAD67 demonstrated antiallodynic and antihyperalgesic effects that persisted for several weeks and could be effectively reestablished by reinoculation. The vector-mediated analgesic effects were reduced by intrathecal bicuculline or phaclofen administered at doses that produced neither mechanical allodynia nor thermal hyperalgesia. Vector-mediated GABA release attenuated the increase in spinal calcitonin gene related peptide (CGRP) caused by cord hemisection. These results demonstrate that transduction of DRG with a GAD-expressing HSV vector results in the release of GABA from nerve terminals in the spinal cord to provide an analgesic effect through actions at both GABAA and GABAB receptors, and suggests that gene therapy with a GAD67 expressing HSV based vector may prove useful in the treatment of central neuropathic pain after spinal cord injury.

Flurbiprofen inhibits capsaicin induced calcitonin gene related peptide release from rat spinal cord via an endocannabinoid dependent mechanism

Calcitonin gene related peptide (CGRP) is involved in nociceptive transmission and modulation at the spinal level. In the spinal superperfusion model, Δ 9 tetrahydrocannabinol inhibited capsaicin induced CGRP release in a concentration dependent manner. Similarly, flurbiprofen (3 μM) inhibited spinal CGRP release. This inhibition was reversed by the CB 1 antagonist AM-251 (1 μM), but not by co-administration of prostaglandin E 2 (PGE 2; 285 nM). AM-251 had no modulatory effect on flurbiprofen-induced cyclooxygenase (COX) inhibiting capacity as shown by PGE 2 levels. Furthermore, the phospholipase A 2 inhibitor palmityl trifluromethyl ketone (15 μM) reversed flurbiprofen's inhibitory effect. In conclusion the present work provides evidence on the shift of arachidonic acid metabolism towards endocannabinoids formation in response to COX inhibition as a mechanism for flurbiprofen inhibitory effect on spinal CGRP release.