Sex Differences in Neuropathy: The Paradigmatic Case of MetFormin.

As a widely prescribed anti-diabetic drug, metformin has been receiving novel attention for its analgesic potential. In the study of the complex etiology of neuropathic pain (NeP), male and female individuals exhibit quite different responses characterized by higher pain sensitivity and greater NeP incidence in women. This "gender gap" in our knowledge of sex differences in pain processing strongly limits the sex-oriented treatment of patients suffering from NeP. Besides, the current investigation of the analgesic potential of metformin has not addressed the "gender gap" problem. Hence, this study focuses on metformin and sex-dependent analgesia in a murine model of NeP induced by chronic constriction injury of the sciatic nerve. We investigated sexual dimorphism in signaling pathways involved by 7 days of metformin administration, such as changes in AMP-activated protein kinase and the positive regulation of autophagy machinery, discovering that metformin affected in a sexually dimorphic manner the immunological and inflammatory response to nerve lesion. These effects were complemented by morphological and adaptive changes occurring after peripheral nerve injury. Altogether these data can contribute to explaining a number of potential mechanisms responsible for the complete recovery from NeP found in male mice, as opposed to the failure of long-lasting recovery in female animals.

CXCR2 increases in ALS cortical neurons and its inhibition prevents motor neuron degeneration in vitro and improves neuromuscular function in SOD1G93A mice.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease characterized by depletion of motor neurons (MNs), for which effective medical treatments are still required. Previous transcriptomic analysis revealed the up-regulation of C-X-C motif chemokine receptor 2 (CXCR2)-mRNA in a subset of sporadic ALS patients and SOD1G93A mice. Here, we confirmed the increase of CXCR2 in human ALS cortex, and showed that CXCR2 is mainly localized in cell bodies and axons of cortical neurons. We also investigated the effects of reparixin, an allosteric inhibitor of CXCR2, in degenerating human iPSC-derived MNs and SOD1G93A mice. In vitro, reparixin rescued MNs from apoptotic cell death, preserving neuronal morphology, mitochondrial membrane potential and cytoplasmic membrane integrity, whereas in vivo it improved neuromuscular function of SOD1G93A mice. Altogether, these data suggest a role for CXCR2 in ALS pathology and support its pharmacological inhibition as a candidate therapeutic strategy against ALS at least in a specific subgroup of patients.

Sexually Dimorphic Immune and Neuroimmune Changes Following Peripheral Nerve Injury in Mice: Novel Insights for Gender Medicine.

Neuropathic pain (NeP) in humans is often a life-long condition with no effective therapy available. The higher incidence of female gender in NeP onset is worldwide reported, and although the cause is generally attributed to sex hormones, the actual mechanisms and the players involved are still unclear. Glial and immune cells take part in NeP development, and orchestrate the neuroimmune and inflammatory response, releasing pro-inflammatory factors with chemoattractant properties that activate resident immune cells and recruit immune cells from circulation. The neuro-immune crosstalk is a key contributor to pain hypersensitivity following peripheral nervous system injury. Our previous works showed that in spite of the fact that female mice had an earlier analgesic response than males following nerve lesion, the recovery from NeP was never complete, suggesting that this difference could occur in the very early stages after injury. To further investigate gender differences in immune and neuroimmune responses to NeP, we studied the main immune cells and mediators elicited both in plasma and sciatic nerves by peripheral nerve lesion. After injury, we found a different pattern of distribution of immune cell populations showing either a higher infiltration of T cells in nerves from females or a higher infiltration of macrophages in nerves from males. Moreover, in comparison to male mice, the levels of cytokines and chemokines were differently up- and down-regulated in blood and nerve lysates from female mice. Our study provides some novel insights for the understanding of gender-associated differences in the generation and perseveration of NeP as well as for the isolation of specific neurodegenerative mechanisms underlying NeP. The identification of gender-associated inflammatory profiles in neuropathy is of key importance for the development of differential biomarkers and gender-specific personalized medicine.

Botulinum toxin A increases analgesic effects of morphine, counters development of morphine tolerance and modulates glia activation and µ opioid receptor expression in neuropathic mice.

The use of botulinum neurotoxin type A (BoNT/A) against pain, with emphasis for its possible use in alleviating chronic pain, still represents an outstanding challenge for experimental research. In this study, we examined the effects of BoNT/A on morphine-induced tolerance during chronic morphine treatment in neuropathic CD1 mice subjected to sciatic nerve lesion according to the Chronic Constriction Injury (CCI) model of neuropathic pain. We measured the effects of BoNT/A on CCI-induced allodynia and hyperalgesia and on the expression of glial fibrillary acidic protein (GFAP, marker of astrocytes), complement receptor 3/cluster of differentiation 11b (CD11b, marker of microglia), and neuronal nuclei (NeuN) at the spinal cord level. We also analyzed the colocalized expression of GFAP, CD11b and NeuN with phosphorylated p-38 mitogen-activated protein kinase and with µ-opioid receptor (MOR). A single intraplantar injection of BoNT/A (15 pg/paw) into the injured hindpaw, the day before the beginning of chronic morphine treatment (9 days of twice daily injections of 40 mg/kg morphine), was able to counteract allodynia and enhancement of astrocytes expression/activation induced by CCI. In addition, BoNT/A increased the analgesic effect of morphine and countered morphine-induced tolerance during chronic morphine treatment. These effects were accompanied, in neurons, by re-expression of MORs that had been reduced by repeated morphine administration. The combinatory effects of BoNT/A and morphine could have relevant therapeutic implications for sufferers of chronic pain who could benefit of pain relief reducing tolerance due to repeated treatment with opiates.

Xeomin®, a Commercial Formulation of Botulinum Neurotoxin Type A, Promotes Regeneration in a Preclinical Model of Spinal Cord Injury.

Xeomin® is a commercial formulation of botulinum neurotoxin type A (BoNT/A) clinically authorized for treating neurological disorders, such as blepharospasm, cervical dystonia, limb spasticity, and sialorrhea. We have previously demonstrated that spinal injection of laboratory purified 150 kDa BoNT/A in paraplegic mice, after undergoing traumatic spinal cord injury (SCI), was able to reduce excitotoxic phenomena, glial scar, inflammation, and the development of neuropathic pain and facilitate regeneration and motor recovery. In the present study, as proof of concept in view of a possible clinical application, we studied the efficacy of Xeomin® in the same preclinical SCI model in which we highlighted the positive effects of lab-purified BoNT/A. Data comparison shows that Xeomin® induces similar pharmacological and therapeutic effects, albeit with less efficacy, to lab-purified BoNT/A. This difference, which can be improved by adjusting the dose, can be attributable to the different formulation and pharmacodynamics. Although the mechanism by which Xeomin® and laboratory purified BoNT/A induce functional improvement in paraplegic mice is still far from being understood, these results open a possible new scenario in treatment of SCI and are a stimulus for further research.

Sex-specific adipose tissue's dynamic role in metabolic and inflammatory response following peripheral nerve injury.

Epidemiological data and research highlight increased neuropathy and chronic pain prevalence among females, spanning metabolic and normometabolic contexts, including murine models. Prior findings demonstrated diverse immune and neuroimmune responses between genders in neuropathic pain (NeP), alongside distinct protein expression in sciatic nerves. This study unveils adipose tissue's (AT) role in sex-specific NeP responses after peripheral nerve injury. Metabolic assessments, metabolomics, energy expenditure evaluations, AT proteomic analyses, and adipokine mobilization depict distinct AT reactions to nerve damage. Females exhibit altered lipolysis, fatty acid oxidation, heightened energy expenditure, and augmented steroids secretion affecting glucose and insulin metabolism. Conversely, male neuropathy prompts glycolysis, reduced energy expenditure, and lowered unsaturated fatty acid levels. Males' AT promotes regenerative molecules, oxidative stress defense, and stimulates peroxisome proliferator-activated receptors (PPAR-γ) and adiponectin. This study underscores AT's pivotal role in regulating gender-specific inflammatory and metabolic responses to nerve injuries, shedding light on female NeP susceptibility determinants.

Wheel Running Adversely Affects Disease Onset and Neuromuscular Interplay in Amyotrophic Lateral Sclerosis Slow Progression Mouse Model.

BACKGROUND: Physical activity in Amyotrophic Lateral Sclerosis (ALS) plays a controversial role. In some epidemiological studies, both recreational or professional sport exercise has been associated to an increased risk for ALS but the mechanisms underlying the effects of exercise have not been fully elucidated in either patients or animal models. METHODS: To better reproduce the influence of this environmental factor in the pathogenesis of ALS, we exposed SOD1G93A low-copy male mice to multiple exercise sessions at asymptomatic and pre-symptomatic disease stages in an automated home-cage running-wheel system for about 3 months. RESULTS: Repeated voluntary running negatively influenced disease progression by anticipating disease onset, impairing neuromuscular transmission, worsening neuromuscular decline, and exacerbating muscle atrophy. Muscle fibers and neuromuscular junctions (NMJ) as well as key molecular players of the nerve-muscle circuit were similarly affected. CONCLUSION: It thus appears that excessive physical activity can be detrimental in predisposed individuals and these findings could model the increased risk of developing ALS in predisposed and specific professional athletes.

The novel reversible fatty acid amide hydrolase inhibitor ST4070 increases endocannabinoid brain levels and counteracts neuropathic pain in different animal models.

The effect of the enol carbamate 1-biphenyl-4-ylethenyl piperidine-1-carboxylate (ST4070), a novel reversible inhibitor of fatty acid amide hydrolase (FAAH), was investigated for acute pain sensitivity and neuropathic pain in rats and mice. Brain enzymatic activity of FAAH and the endogenous levels of its substrates, anandamide (AEA; N-arachidonoylethanolamine), 2-arachidonoylglycerol (2-AG), and N-palmitoylethanolamine (PEA), were measured in control and ST4070-treated mice. ST4070 (10, 30, and 100 mg/kg) was orally administered to assess mechanical nociceptive thresholds and allodynia by using the Randall-Selitto and von Frey tests, respectively. Neuropathy was induced in rats by either the chemotherapeutic agent vincristine or streptozotocin-induced diabetes, whereas the chronic constriction injury (CCI) model was chosen to evaluate neuropathy in mice. ST4070 produced a significant increase of nociceptive threshold in rats and counteracted the decrease of nociceptive threshold in the three distinct models of neuropathic pain. In diabetic mice, ST4070 inhibited FAAH activity and increased the brain levels of AEA and PEA, without affecting that of 2-AG. The administration of ST4070 generated long-lasting pain relief compared with pregabalin and the FAAH inhibitors 1-oxo-1[5-(2-pyridyl)-2-yl]-7-phenylheptane (OL135) and cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-ylester (URB597) in CCI neuropathic mice. The antiallodynic effects of ST4070 were prevented by pretreatment with cannabinoid type 1 and cannabinoid type 2 receptor antagonists and by the selective peroxisome proliferator-activated receptor α antagonist [(2S)-2-[[(1Z)-1-methyl-3-oxo-3-[4-(trifluoromethyl)phenyl]-1-propenyl]amino]-3-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]phenyl]propyl]-carbamic acid ethyl ester (GW6471). The administration of ST4070 generated long-lasting neuropathic pain relief compared with pregabalin and the FAAH inhibitors OL135 and URB597. Taken together, the reversible FAAH inhibitor ST4070 seems to be a promising novel therapeutic agent for the management of neuropathic pain.

Botulinum neurotoxin A enhances the analgesic effects on inflammatory pain and antagonizes tolerance induced by morphine in mice.

Over the recent years compelling evidence has accumulated indicating that botulinum neurotoxin serotype A (BoNT/A) results in analgesic effects on neuropathic as well as inflammatory pain, both in humans and in animal models. In the present study, the pharmacological interaction of BoNT/A with morphine in fighting inflammatory pain was investigated in mice using the formalin test. Moreover, the effects of BoNT/A on the tolerance-induced by chronic administration of morphine were tested and the behavioral effects were correlated with immunofluorescence staining of glial fibrillary acidic protein, the specific marker of astrocytes, at the spinal cord level. An ineffective dose of BoNT/A (2 pg/paw) combined with an ineffective dose of morphine (1 mg/kg) exerted a significant analgesic action both during the early and the late phases of formalin test. A single intraplantar injection of BoNT/A (15 pg/paw; i.pl.), administered the day before the beginning of chronic morphine treatment (7 days of s.c. injections of 20 mg/kg), was able to counteract the occurrence of tolerance to morphine. Moreover, BoNT/A reduces the enhancement of the expression of astrocytes induced by inflammatory formalin pain. Side effects of opiates, including the development of tolerance during repeated use, may limit their therapeutic use, the possibility of using BoNT/A for lowering the effective dose of morphine and preventing the development of opioid tolerance would have relevant implications in terms of potential therapeutic perspectives.

Early Social Enrichment Modulates Tumor Progression and p53 Expression in Adult Mice.

Epidemiological evidence indicates that stress and aversive psychological conditions can affect cancer progression, while well-being protects against it. Although a large set of studies have addressed the impact of stress on cancer, not much is known about the mechanisms that protect from cancer in healthy psychological conditions. C57BL/6J mouse pups were exposed to an environmental enrichment condition consisting of being raised until weaning by the biological lactating mother plus a non-lactating virgin female (LnL = Lactating and non-Lactating mothers). The Control group consisted of mice raised by a single lactating mother (L = Lactating). Four months after weaning, mice from LnL and L conditions were exposed to intramuscular injection of 3-methylcolantrene (3MCA), a potent tumorigenic drug, and onset and progression of 3MCA-induced fibrosarcomas were monitored over time. Pups from the LnL compared to the L group received more parental care and were more resilient to stressful events during the first week of life. In association, the onset of tumors in LnL adults was significantly delayed. At the molecular level, we observed increased levels of wild-type p53 protein in tumor samples of LnL compared to L adults and higher levels of its target p21 in healthy muscles of LnL mice compared to the L group, supporting the hypothesis of potential involvement of p53 in tumor development. Our study sustains the model that early life care protects against tumor susceptibility.

Corrigendum: Very Early Involvement of Innate Immunity in Peripheral Nerve Degeneration in SOD1-G93A Mice.

[This corrects the article DOI: 10.3389/fimmu.2020.575792.].

Impact of caloric restriction on peripheral nerve injury-induced neuropathic pain during ageing in mice.

The incidence of peripheral neuropathy development and chronic pain is strongly associated with the arrival of senescence. The gradual physiological decline that begins after the mature stage produces myelin dysregulation and pathological changes in peripheral nervous system, attributed to reduction in myelin proteins expression and thinner myelin sheath. Moreover in elder subjects, when nerve damage occurs, the regenerative processes are seriously compromised and neuropathic pain (NeP) is maintained. We previously demonstrated that caloric restriction (CR) in adult (4 months) nerve-lesioned mice was able to facilitate remyelination and axons regeneration, to have anti-inflammatory action and to prevent NeP chronification. Here, we show CR therapeutic potential on nerve injury-induced neuropathy in mice at the beginning of the senescence (12 months). Long lasting decrease in hypersensitvity induced by peripheral nerve lesion and powerful reduction in proinflammatory circulating agents have been observed. Moreover, our results evidence that CR is able to counteract the ageing-related delay in axonal regeneration, enhancing Schwann cells proliferation and accelerating recovery processes. Differently from adults, it does not affect fibres myelination. In light of a continuous growth in elderly population and correlated health problems, including metabolic disorders, the prevalence of neuropathy is enhancing, generating a significant public cost and social concern. In this context energy depletion by dietary restriction can be a therapeutic option in NeP.

Very Early Involvement of Innate Immunity in Peripheral Nerve Degeneration in SOD1-G93A Mice.

Recent preclinical and clinical evidence suggest that immune system has a role in the progression and prognosis of Amyotrophic Lateral Sclerosis (ALS), but the identification of a clear mechanism and immune players remains to be elucidated. Here, we have investigated, in 30 and 60 days (presymptomatic) and 120 days (symptomatic) old SOD1-G93A mice, systemic, peripheral, and central innate and adaptive immune and inflammatory response, correlating it with the progression of the neurodegeneration in neuromuscular junction, sciatic nerves, and spinal cord. Surprisingly, we found a very initial (45-60 days) presence of IgG in sciatic nerves together with a gradual enhancement of A20/TNFAIP3 (protein controlling NF-κB signalling) and a concomitantly significant increase and activation of circulating mast cells (MCs) as well as MCs and macrophages in sciatic nerve and an enhancement of IL-6 and IL-10. This immunological frame coincided with a myelin aggregation. The 30-60 days old SOD1-G93A mice didn't show real elements of neuroinflammation and neurodegeneration in spinal cord. In 120 days old mice macrophages and monocytes are widely diffused in sciatic nerves, peripheral neurodegeneration reaches the tip, high circulating levels of TNFα and IL-2 were found and spinal cord exhibits clear signs of neural damage and infiltrating immune cells. Our results underpin a clear immunological disorder at the origin of ALS axonopathy, in which MCs are involved in the initiation and sustaining of inflammatory events. These data cannot be considered a mere epiphenomenon of motor neuron degeneration and reveal new potential selective immune targets in ALS therapy.

Revealing the Therapeutic Potential of Botulinum Neurotoxin Type A in Counteracting Paralysis and Neuropathic Pain in Spinally Injured Mice.

Botulinum neurotoxin type A (BoNT/A) is a major therapeutic agent that has been proven to be a successful treatment for different neurological disorders, with emerging novel therapeutic indications each year. BoNT/A exerts its action by blocking SNARE complex formation and vesicle release through the specific cleavage of SNAP-25 protein; the toxin is able to block the release of pro-inflammatory molecules for months after its administration. Here we demonstrate the extraordinary capacity of BoNT/A to neutralize the complete paralysis and pain insensitivity induced in a murine model of severe spinal cord injury (SCI). We show that the toxin, spinally administered within one hour from spinal trauma, exerts a long-lasting proteolytic action, up to 60 days after its administration, and induces a complete recovery of muscle and motor function. BoNT/A modulates SCI-induced neuroglia hyperreactivity, facilitating axonal restoration, and preventing secondary cells death and damage. Moreover, we demonstrate that BoNT/A affects SCI-induced neuropathic pain after moderate spinal contusion, confirming its anti-nociceptive action in this kind of pain, as well. Our results provide the intriguing and real possibility to identify in BoNT/A a therapeutic tool in counteracting SCI-induced detrimental effects. Because of the well-documented BoNT/A pharmacology, safety, and toxicity, these findings strongly encourage clinical translation.

Innovative mouse model mimicking human-like features of spinal cord injury: efficacy of Docosahexaenoic acid on acute and chronic phases.

Traumatic spinal cord injury has dramatic consequences and a huge social impact. We propose a new mouse model of spinal trauma that induces a complete paralysis of hindlimbs, still observable 30 days after injury. The contusion, performed without laminectomy and deriving from the pressure exerted directly on the bone, mimics more closely many features of spinal injury in humans. Spinal cord was injured at thoracic level 10 (T10) in adult anesthetized female CD1 mice, mounted on stereotaxic apparatus and connected to a precision impactor device. Following severe injury, we evaluated motor and sensory functions, and histological/morphological features of spinal tissue at different time points. Moreover, we studied the effects of early and subchronic administration of Docosahexaenoic acid, investigating functional responses, structural changes proximal and distal to the lesion in primary and secondary injury phases, proteome modulation in injured spinal cord. Docosahexaenoic acid was able i) to restore behavioural responses and ii) to induce pro-regenerative effects and neuroprotective action against demyelination, apoptosis and neuroinflammation. Considering the urgent health challenge represented by spinal injury, this new and reliable mouse model together with the positive effects of docosahexaenoic acid provide important translational implications for promising therapeutic approaches for spinal cord injuries.

Publisher Correction: Innovative mouse model mimicking human-like features of spinal cord injury: efficacy of Docosahexaenoic acid on acute and chronic phases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Botulinum Toxin B Affects Neuropathic Pain but Not Functional Recovery after Peripheral Nerve Injury in a Mouse Model.

Clinical use of neurotoxins from Clostridium botulinum is well established and is continuously expanding, including in treatment of pain conditions. Background: The serotype A (BoNT/A) has been widely investigated, and current data demonstrate that it induces analgesia and modulates nociceptive processing initiated by inflammation or nerve injury. Given that data concerning the serotype B (BoNT/B) are limited, the aim of the present study was to verify if also BoNT/B is able not only to counteract neuropathic pain, but also to interfere with inflammatory and regenerative processes associated with the nerve injury. Methods: As model of neuropathic pain, chronic constriction injury (CCI) of the sciatic nerve was performed in CD1 male mice. Mice were intraplantarly injected with saline (control) or BoNT/B (5 or 7.5 pg/mouse) into the injured hindpaw. For comparison, another mouse group was injected with BoNT/A (15 pg/mouse). Mechanical allodynia and functional recovery of the injured paw was followed for 101 days. Spinal cords and sciatic nerves were collected at day 7 for immunohistochemistry. Results and Conclusions: The results of this study show that BoNT/B is a powerful biological molecule that, similarly to BoNT/A, can reduce neuropathic pain over a long period of time. However, the analgesic effects are not associated with an improvement in functional recovery, clearly highlighting an important difference between the two serotypes for the treatment of this chronic pain state.

Effects of caloric restriction on neuropathic pain, peripheral nerve degeneration and inflammation in normometabolic and autophagy defective prediabetic Ambra1 mice.

There is a growing interest on the role of autophagy in diabetes pathophysiology, where development of neuropathy is one of the most frequent comorbidities. We have previously demonstrated that neuropathic pain after nerve damage is exacerbated in autophagy-defective heterozygous Ambra1 mice. Here, we show the existence of a prediabetic state in Ambra1 mice, characterized by hyperglycemia, intolerance to glucose and insulin resistance. Thus, we further investigate the hypothesis that prediabetes may account for the exacerbation of allodynia and chronic pain and that counteracting the autophagy deficit may relieve the neuropathic condition. We took advantage from caloric restriction (CR) able to exert a double action: a powerful increase of autophagy and a control on the metabolic status. We found that CR ameliorates neuropathy throughout anti-inflammatory and metabolic mechanisms both in Ambra1 and in WT animals subjected to nerve injury. Moreover, we discovered that nerve lesion represents, per se, a metabolic stressor and CR reinstates glucose homeostasis, insulin resistance, incomplete fatty acid oxidation and energy metabolism. As autophagy inducer, CR promotes and anticipates Schwann cell autophagy via AMP-activated protein kinase (AMPK) that facilitates remyelination in peripheral nerve. In summary, we provide new evidence for the role of autophagy in glucose metabolism and identify in energy depletion by dietary restriction a therapeutic approach in the fight against neuropathic pain.

Exosomal cargo including microRNA regulates sensory neuron to macrophage communication after nerve trauma.

Following peripheral axon injury, dysregulation of non-coding microRNAs (miRs) occurs in dorsal root ganglia (DRG) sensory neurons. Here we show that DRG neuron cell bodies release extracellular vesicles, including exosomes containing miRs, upon activity. We demonstrate that miR-21-5p is released in the exosomal fraction of cultured DRG following capsaicin activation of TRPV1 receptors. Pure sensory neuron-derived exosomes released by capsaicin are readily phagocytosed by macrophages in which an increase in miR-21-5p expression promotes a pro-inflammatory phenotype. After nerve injury in mice, miR-21-5p is upregulated in DRG neurons and both intrathecal delivery of a miR-21-5p antagomir and conditional deletion of miR-21 in sensory neurons reduce neuropathic hypersensitivity as well as the extent of inflammatory macrophage recruitment in the DRG. We suggest that upregulation and release of miR-21 contribute to sensory neuron-macrophage communication after damage to the peripheral nerve.

Role of extracellular calcitonin gene-related peptide in spinal cord mechanisms of cancer-induced bone pain.

Severe pain is a common and debilitating complication of metastatic bone cancer. Current analgesics provide insufficient pain relief and often lead to significant adverse effects. In models of cancer-induced bone pain, pathological sprouting of sensory fibers at the tumor-bone interface occurs concomitantly with reactive astrocytosis in the dorsal horn of the spinal cord. We observed that calcitonin gene-related peptide (CGRP)-fiber sprouting in the bone was associated with an increase in CGRP content in sensory neuron cell bodies in the dorsal root ganglia (DRG) and increased basal and activity-evoked release of CGRP from their central terminals in the dorsal horn. Intrathecal administration of a peptide antagonist (α-CGRP8-37) attenuated referred allodynia in the hind paw ipsilateral to bone cancer. CGRP receptor components (CLR and RAMP1) were up-regulated in dorsal horn neurons and expressed by reactive astrocytes. In primary cultures of astrocytes, CGRP incubation led to a concentration-dependent increase of forskolin-induced cAMP production, which was attenuated by pretreatment with CGRP8-37. Furthermore, CGRP induced ATP release in astrocytes, which was inhibited by CGRP8-37. We suggest that the peripheral increase in CGRP content observed in cancer-induced bone pain is mirrored by a central increase in the extracellular levels of CGRP. This increase in CGRP not only may facilitate glutamate-driven neuronal nociceptive signaling but also act on astrocytic CGRP receptors and lead to release of ATP.