Spinal CGRP Research Articles

Desloratadine ameliorates paclitaxel-induced peripheral neuropathy and hypersensitivity reactions in mice.

Paclitaxel (PTX) serves as a primary chemotherapy agent against diverse solid tumors including breast cancer, lung cancer, head and neck cancer and ovarian cancer, having severe adverse effects including PTX-induced peripheral neuropathy (PIPN) and hypersensitivity reactions (HSR). A recommended anti-allergic agent diphenhydramine (DIP) has been used to alleviate PTX-induced HSR. Desloratadine (DLT) is a third generation of histamine H1 receptor antagonist, but also acted as a selective antagonist of 5HTR2A. In this study we investigated whether DLT ameliorated PIPN-like symptoms in mice and the underlying mechanisms. PIPN was induced in male mice by injection of PTX (4 mg/kg, i.p.) every other day for 4 times. The mice exhibited 50% reduction in mechanical threshold, paw thermal response latency and paw cold response latency compared with control mice. PIPN mice were treated with DLT (10, 20 mg/kg, i.p.) 30 min before each PTX administration in the phase of establishing PIPN mice model and then administered daily for 4 weeks after the model wasestablished. We showed that DLT administration dose-dependently elevated the mechanical, thermal and cold pain thresholds in PIPN mice, whereas administration of DIP (10 mg/kg, i.p.) had no ameliorative effects on PIPN-like symptoms. We found that the expression of 5HTR2A was selectively elevated in the activated spinal astrocytes of PIPN mice. Spinal cord-specific 5HTR2A knockdown by intrathecal injection of AAV9-5Htr2a-shRNA significantly alleviated the mechanical hyperalgesia, thermal and cold hypersensitivity in PIPN mice, while administration of DLT (20 mg/kg) did not further ameliorate PIPN-like symptoms. We demonstrated that DLT administration alleviated dorsal root ganglion neuronal damage and suppressed sciatic nerve destruction, spinal neuron apoptosis and neuroinflammation in the spinal cord of PIPN mice. Furthermore, we revealed that DLT administration suppressed astrocytic neuroinflammation via the 5HTR2A/c-Fos/NLRP3 pathway and blocked astrocyte-neuron crosstalk by targeting 5HTR2A. We conclude that spinal 5HTR2A inhibition holds promise as a therapeutic approach for PIPN and we emphasize the potential of DLT as a dual-functional agent in ameliorating PTX-induced both PIPN and HSR in chemotherapy. In summary, we determined that spinal 5HTR2A was selectively activated in PIPN mice and DLT could ameliorate the PTX-induced both PIPN- and HSR-like pathologies in mice. DLT alleviated the damages of DRG neurons and sciatic nerves, while restrained spinal neuronal apoptosis and CGRP release in PIPN mice. The underlying mechanisms were intensively investigated by assay against the PIPN mice with 5HTR2A-specific knockdown in the spinal cord by injection of adeno-associated virus 9 (AAV9)-5Htr2a-shRNA. DLT inhibited astrocytic NLRP3 inflammasome activation-mediated spinal neuronal damage through 5HTR2A/c-FOS pathway. Our findings have supported that spinal 5HTR2A inhibition shows promise as a therapeutic strategy for PIPN and highlighted the potential advantage of DLT as a dual-functional agent in preventing against PTX-induced both PIPN and HSR effects in anticancer chemotherapy.

Identification and targeting of a unique NaV1.7 domain driving chronic pain

Small molecules directly targeting the voltage-gated sodium channel (VGSC) NaV1.7 have not been clinically successful. We reported that preventing the addition of a small ubiquitin-like modifier onto the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 function and was antinociceptive in rodent models of neuropathic pain. Here, we discovered a CRMP2 regulatory sequence (CRS) unique to NaV1.7 that is essential for this regulatory coupling. CRMP2 preferentially bound to the NaV1.7 CRS over other NaV isoforms. Substitution of the NaV1.7 CRS with the homologous domains from the other eight VGSC isoforms decreased NaV1.7 currents. A cell-penetrant decoy peptide corresponding to the NaV1.7-CRS reduced NaV1.7 currents and trafficking, decreased presynaptic NaV1.7 expression, reduced spinal CGRP release, and reversed nerve injury-induced mechanical allodynia. Importantly, the NaV1.7-CRS peptide did not produce motor impairment, nor did it alter physiological pain sensation, which is essential for survival. As a proof-of-concept for a NaV1.7 -targeted gene therapy, we packaged a plasmid encoding the NaV1.7-CRS in an AAV virus. Treatment with this virus reduced NaV1.7 function in both rodent and rhesus macaque sensory neurons. This gene therapy reversed and prevented mechanical allodynia in a model of nerve injury and reversed mechanical and cold allodynia in a model of chemotherapy-induced peripheral neuropathy. These findings support the conclusion that the CRS domain is a targetable region for the treatment of chronic neuropathic pain.

Convergence of peptidergic and non-peptidergic protein markers in the human dorsal root ganglion and spinal dorsal horn.

Peripheral sensory neurons are characterized by their size, molecular profiles, and physiological responses to specific stimuli. In mouse, the peptidergic and non-peptidergic subsets of nociceptors are distinct and innervate different lamina of the spinal dorsal horn. The unique molecular signature and neuroanatomical organization of these neurons supports a labeled line theory for certain types of nociceptive stimuli. However, long-standing evidence supports the polymodal nature of nociceptors in many species. We have recently shown that the peptidergic marker, CGRP, and the non-peptidergic marker, P2X3R, show largely overlapping expression at the mRNA level in human dorsal root ganglion (DRG). Herein, our aim was to assess the protein distribution of nociceptor markers, including their central projections, in the human DRG and spinal cord. Using DRGs obtained from organ donors, we observed that CGRP and P2X3R were co-expressed by approximately 33% of human DRG neurons and TrpV1 was expressed in ~60% of human DRG neurons. In the dorsal spinal cord, CGRP, P2X3R, TrpV1, and Nav1.7 proteins stained the entirety of lamina 1-2, with only P2XR3 showing a gradient of expression. This was confirmed by measuring the size of the substantia gelatinosa using Hematoxylin and Eosin staining of adjacent sections. Our findings are consistent with the known polymodal nature of most primate nociceptors and indicate that the central projection patterns of nociceptors are different between mice and humans. Elucidating how human nociceptors connect to subsets of dorsal horn neurons will be important for understanding the physiological consequences of these species differences.

Electroacupuncture intervention of visceral hypersensitivity is involved in PAR-2-activation and CGRP-release in the spinal cord

Electroacupuncture (EA) relieves visceral hypersensitivity (VH) with underlying inflammatory bowel diseases. However, the mechanism by which EA treats ileitis-induced VH is not clearly known. To assess the effects of EA on ileitis-induced VH and confirm whether EA attenuates VH through spinal PAR-2 activation and CGRP release, goats received an injection of 2,4,6-trinitro-benzenesulfonic-acid (TNBS) solution into the ileal wall. TNBS-injected goats were allocated into VH, Sham acupuncture (Sham-A) and EA groups, while goats treated with saline instead of TNBS solution were used as the control. Goats in EA group received EA at bilateral Hou-San-Li acupoints for 0.5 h at 7 days and thereafter repeated every 3 days for 6 times. Goats in the Sham-A group were inserted with needles for 0.5 h at the aforementioned acupoints without any hand manipulation and electric stimulation. Visceromotor responses to colorectal distension, an indicator of VH, were recorded by electromyography. The terminal ileum and thoracic spinal cord (T11) were sampled for evaluating ileitis at days 7 and 22, and distribution and expression-levels of PAR-2, CGRP and c-Fos on day 22. TNBS-treated-goats exhibited apparent transmural-ileitis on day 7, microscopically low-grade ileitis on day 22 and VH at days 7–22. Goats of Sham-A, VH or EA group showed higher (P < 0.01) VH at days 7–22 than the Control-goats. EA-treated goats exhibited lower (P < 0.01) VH as compared with Sham-A or VH group. Immunoreactive-cells and expression-levels of spinal PAR-2, CGRP and c-Fos in the EA group were greater (P < 0.01) than those in the Control group, but less (P < 0.01) than those in Sham-A and VH groups on day 22. Downregulation of spinal PAR-2 and CGRP levels by EA attenuates the ileitis and resultant VH.

(287) Facilitated spinal neuropeptide signaling and upregulated inflammatory mediator expression contributes to post-fracture nociceptive sensitization

Tibia fracture induces exaggerated substance P (SP) and CGRP signaling and neuropeptide-dependent nociceptive and inflammatory changes in the hindlimbs of rats similar to those seen in complex regional pain syndrome (CRPS). Inflammatory changes in the spinal cord also 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 post-fracture hindlimb nociceptive sensitization. At 4 weeks after tibia fracture and casting we measured hindlimb 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 post-fracture pain behavior and spinal inflammatory mediator expression. Hindlimb allodynia, unweighting, warmth, edema, increased spinal SP and CGRP, and increased spinal inflammatory mediator expression (TNF-α, IL-1β, IL-6, CCL2, NGF) 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 post-fracture nociceptive sensitization. Intrathecal injection of selective receptor antagonists for SP, CGRP, TNF-α, IL-1β, IL-6, CCL2, or NGF 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 CRPS.

Intrathecal delivery of resiniferatoxin (RTX) reduces detrusor overactivity and spinal expression of TRPV1 in spinal cord injured animals

Recently, it has been demonstrated that intrathecal delivery of resiniferatoxin (RTX) produces strong analgesia, even in models of bone cancer pain. RTX has been investigated to treat bladder dysfunction of spinal origin, applied by intravesical instillation. However, RTX delivered by this route was not completely satisfactory in controlling urinary incontinence and high intravesical pressure. Thus, the present study assessed the effects of intrathecal injections of RTX in bladder dysfunction in rats with spinal cord transection (SCT). Bladder function was evaluated in SCT rats 24 h following intrathecal administration of RTX. Detrusor overactivity and intravesical pressure were reduced in a dose-dependent manner. This was accompanied by a decrease in spinal cord TRPV1 and CGRP, but not in IB4 binding sensory fibres. Also, intrathecal RTX induced a dose-dependent reduction in spinal cord activation of the ERK pathway. Overall, our results show that intrathecal administration of RTX effectively reduces detrusor overactivity and reduces intravesical pressure in models of complete chronic spinal cord transection by suppressing the activity of TRPV1 expressing afferent fibres. Also, intrathecal RTX decreases sensory input, as shown by reduced spinal ERK activation. These findings might be relevant for the management of patients with spinal cord injuries.

Spinal CGRP1 receptors contribute to supraspinally organized pain behavior and pain-related sensitization of amygdala neurons

CGRP receptor activation has been implicated in peripheral and central sensitization. The role of spinal CGRP receptors in supraspinal pain processing and higher integrated pain behavior is not known. Here we studied the effect of spinal inhibition of CGRP1 receptors on supraspinally organized vocalizations and activity of amygdala neurons. Our previous studies showed that pain-related audible and ultrasonic vocalizations are modulated by the central nucleus of the amygdala (CeA). Vocalizations in the audible and ultrasonic range and hindlimb withdrawal thresholds were measured in awake adult rats before and 5–6 h after induction of arthritis by intra-articular injections of kaolin and carrageenan into one knee. Extracellular single-unit recordings were made from neurons in the latero-capsular division of the CeA (CeLC) in anesthetized rats before and after arthritis induction. CGRP1 receptor antagonists were applied to the lumbar spinal cord intrathecally (5 μl/min) 6 h postinduction of arthritis. Spinal administration of peptide (CGRP8-37, 1 μM) and non-peptide (BIBN4096BS, 1 μM) CGRP1 receptor antagonists significantly inhibited the increased responses of CeLC neurons to mechanical stimulation of the arthritic knee but had no effect under normal conditions. In arthritic rats, the antagonists also inhibited the audible and ultrasonic components of vocalizations evoked by noxious stimuli and increased the threshold of hindlimb withdrawal reflexes. The antagonists had no effect on vocalizations and spinal reflexes in normal rats. These data suggest that spinal CGRP1 receptors are not only important for spinal pain mechanisms but also contribute significantly to the transmission of nociceptive information to the amygdala and to higher integrated behavior.

Role of CGRP and GABA in the hypotensive effect of intrathecally administered anandamide to anesthetized rats

In urethane-anesthetized rats the intrathecal (i.t.) injection of 100 nmol anandamide produced a hypotensive effect (− 19.3 ± 1.6 mm Hg; n = 6) that was mimicked by i.t. administration of 0.25 nmol calcitonin gene-related peptide (CGRP; − 26.2 ± 1.8 mm Hg, n = 4). Both effects were antagonized either by the CGRP receptor antagonist CGRP 8–37 (5 nmol; i.t.) or by the γ-aminobutyric acid (GABA) A receptor antagonist bicuculline (8.8 nmol, i.t) or by the GABA B receptor antagonist 2-hydroxy saclofen (110 nmol; i.t.). On the contrary, blockade of spinal CGRP receptors by CGRP 8–37 did not modify the hypotensive response to either the GABA A-receptor agonist muscimol (8.8 nmol; i.t.) or the GABA B-receptor agonist baclofen (100 nmol; i.t). This result suggests a unidirectional effect of CGRP on the GABAergic system. The response to anandamide remained unaltered after acute inhibition of nitric oxide (NO) synthase activity by either i.t. (1 μmol) or i.v. (10 mg/kg) injection of N G-nitro- l-arginine methyl ester (L-NAME), but increased significantly after long-term L-NAME administration (70 mg/kg/day; four weeks; p.o.), thus suggesting compensatory changes in cardiovascular homeostasis. It is proposed that the hypotensive effect of anandamide in urethane-anesthetized rats could involve the release of CGRP followed by the release of GABA in the spinal cord. NO does not appear to have a direct participation in the spinal mechanisms involved in the decrease of the blood pressure caused by anandamide.

Variable sensitivity to noxious heat is mediated by differential expression of the CGRP gene

Heat sensitivity shows considerable functional variability in humans and laboratory animals, and is fundamental to inflammatory and possibly neuropathic pain. In the mouse, at least, much of this variability is genetic because inbred strains differ robustly in their behavioral sensitivity to noxious heat. These strain differences are shown here to reflect differential responsiveness of primary afferent thermal nociceptors to heat stimuli. We further present convergent behavioral and electrophysiological evidence that the variable responses to noxious heat are due to strain-dependence of CGRP expression and sensitivity. Strain differences in behavioral response to noxious heat could be abolished by peripheral injection of CGRP, blockade of cutaneous and spinal CGRP receptors, or long-term inactivation of CGRP with a CGRP-binding Spiegelmer. Linkage mapping supports the contention that the genetic variant determining variable heat pain sensitivity across mouse strains affects the expression of the Calca gene that codes for CGRPalpha.

Chronic morphine exposure increases the phosphorylation of MAP kinases and the transcription factor CREB in dorsal root ganglion neurons: an in vitro and in vivo study.

Tolerance to opiates reduces their effectiveness in the treatment of severe pain. Although the mechanisms are unclear, overactivity of pro-nociceptive systems has been proposed to contribute to this phenomenon. We have reported that the development of morphine tolerance significantly increased calcitonin-gene-related-peptide-like immunoreactivity (CGRP-IR) in primary sensory afferents of the spinal dorsal horn, suggesting that changes in pain-related neuropeptides in the dorsal root ganglion (DRG) neurons may be involved (Menard et al., 1996, J. Neurosci., 16, 2342-2351). Recently, we have shown that repeated morphine treatments induced increases in CGRP- and substance P (SP)-IR in cultured DRG, mimicking the in vivo effects (Ma et al., 2000, Neuroscience, 99, 529-539). In this study, we investigated the intracellular signal transduction pathways possibly involved in morphine-induced increases in CGRP- and SP-IR in DRG neurons. Repeated morphine exposure (10-20 microm) for 6 days increased the number of neurons expressing phosphorylated (p) mitogen-activated protein (MAP) kinases, including the extracellular signal-regulated kinase (pERK), c-jun N-terminal kinase (pJNK) and P38 (pP38 MAPK). The number of neurons expressing phosphorylated cAMP responsive element binding protein (pCREB) was also markedly increased in morphine-exposed cultured DRG neurons. pERK-, pP38-, pJNK- and pCREB-IR were colocalized with CGRP-IR in cultured DRG neurons. Naloxone effectively blocked these actions of morphine, whereas a selective MEK1 inhibitor, PD98059, inhibited the morphine-induced increase in the phosphorylation of ERK and CREB, and the expression of CGRP and SP. Moreover, in morphine-tolerant rats, the number of pCREB-, CGRP- and SP-IR neurons in the lumbar DRG was also significantly increased. These in vitro and in vivo data suggest that the phosphorylation of MAP kinases and CREB plays a role in the morphine-induced increase in spinal CGRP and SP levels in primary sensory afferents, contributing to the development of tolerance to opioid-induced analgesia.

Decrease in spinal CGRP and substance P is not related to neuropathic pain in a rat model.

We tested the hypothesis that the decrease in spinal levels of SP and CGRP after peripheral nerve injury is related to neuropathic pain. We compared two groups of rats, both of which were subjected to unilateral transection of the inferior and superior caudal trunks between the S1 and S2 spinal nerves. One group exhibited well-developed neuropathic signs after the nerve injury, whereas the other group showed poorly developed signs despite the same nerve injury. The decrease in immunoreactivity of CGRP and SP in the S1 dorsal horn (injured segment) was not significantly different between the two groups. These results suggest that the decrease in spinal levels of CGRP and SP after peripheral nerve injury is not related to neuropathic pain.

Blockade and reversal of spinal morphine tolerance by peptide and non-peptide calcitonin gene-related peptide receptor antagonists.

This study examined the effects of the peptide CGRP receptor antagonist CGRP(8-37) and the newly-developed non-peptide CGRP receptor antagonist BIBN4096BS for their potential to both inhibit the development and reverse tolerance to the antinociceptive action of morphine. Repeated administration of intrathecal morphine (15 microg), once daily, produced a progressive decline of antinociceptive effect and an increase in the ED(50) value in the tailflick and paw pressure tests. Co-administration of CGRP(8-37) (4 microg) or BIBN4096BS (0.05, 0.1 microg) with morphine (15 microg) prevented the decline of antinociceptive effect and increase in ED(50) value in the tailflick test. Intrathecal administration of the CGRP receptor antagonists did not alter the baseline responses in either tests. Acute CGRP(8-37) also did not potentiate the acute actions of spinal morphine. In animals rendered tolerant to intrathecal morphine, subsequent administration of CGRP(8-37) (4 microg) with morphine (15 microg) partially restored the antinociceptive effect and ED(50) value of acute morphine, reflecting the reversal of tolerance. Animals tolerant to intrathecal morphine expressed increased CGRP and substance P-like immunostaining in the dorsal horn of the spinal cord. The increase in CGRP, but not substance P-like immunostaining, was blocked by a co-treatment with CGRP(8-37) (4 microg). In animals already tolerant to morphine, the increase in CGRP but not substance P-like immunostaining was partially reversed by CGRP(8-37) (4 microg). These data suggest that activation of spinal CGRP receptors contributes to both the development and expression of spinal opioid tolerance. CGRP receptor antagonists may represent a useful therapeutic approach for preventing as well as reversing opioid tolerance.

Differential involvement of spinal NMDA, CGRP and NK1 receptors in responses to phasic vs. tonic colorectal distension

Differential involvement of spinal NMDA, CGRP and NK1 receptors in responses to phasic vs. tonic colorectal distension

The role of spinal CGRP as a putative neurotransmitter in visceral hypersensitivity

The role of spinal CGRP as a putative neurotransmitter in visceral hypersensitivity

Calcitonin gene-related peptide (8–37) does not antagonize calcitonin gene-related peptide in rat spinal cord

We have examined the effects of intrathecal (i.t.) human calcitonin gene-related peptide (hCGRP) and its C-terminal fragment hCGRP(8–37), a proposed CGRP antagonist, on the flexor reflex in decerebrate, spinalized, unanesthetized rats. I.t. hCGRP at 26 pmol caused a moderate facilitation of the reflex which was not antagonized by hCGRP(8–37) at doses ranging from 26 pmol to 5.2 nmol. Furthermore, hCGRP(8–37) by itself facilitated the reflex, with no signs of inhibition. It is concluded that the spinal CGRP receptor mediating the spinal facilitatory effect of hCGRP is not antagonized by hCGRP(8–37). Thus, it is unlikely that hCGRP(8–37) can be useful as a spinal analgesic.

Marked depletion of dorsal spinal cord tachykinins and CGRP with intact skin flares in patients with the Shy-Drager syndrome

Marked depletion of dorsal spinal cord tachykinins and CGRP with intact skin flares in patients with the Shy-Drager syndrome