Role of pattern recognition receptors in chemotherapy-induced neuropathic pain.

Progress in the development of effective chemotherapy is producing a growing population of patients with acute and chronic painful chemotherapy-induced peripheral neuropathy (CIPN), a serious treatment-limiting side effect for which there is currently no US Food and Drug Administration-approved treatment. CIPNs induced by diverse classes of chemotherapy drugs have remarkably similar clinical presentations, leading to the suggestion they share underlying mechanisms. Sensory neurons share with immune cells the ability to detect damage associated molecular patterns (DAMPs), molecules produced by diverse cell types in response to cellular stress and injury, including by chemotherapy drugs. DAMPs, in turn, are ligands for pattern recognition receptors (PRRs), several of which are found on sensory neurons, as well as satellite cells, and cells of the immune system. In the present experiments, we evaluated the role of two PRRs, TLR4 and RAGE, present in dorsal root ganglion (DRG), in CIPN. Antisense (AS)-oligodeoxynucleotides (ODN) against TLR4 and RAGE mRNA were administered intrathecally before ('prevention protocol') or 3 days after ('reversal protocol') the last administration of each of three chemotherapy drugs that treat cancer by different mechanisms (oxaliplatin, paclitaxel and bortezomib). TLR4 and RAGE AS-ODN prevented the development of CIPN induced by all three chemotherapy drugs. In the reversal protocol, however, while TLR4 AS-ODN completely reversed oxaliplatin- and paclitaxel-induced CIPN, in rats with bortezomib-induced CIPN it only produced a temporary attenuation. RAGE AS-ODN, in contrast, reversed CIPN induced by all three chemotherapy drugs. When a TLR4 antagonist was administered intradermally to the peripheral nociceptor terminal, it did not affect CIPN induced by any of the chemotherapy drugs. However, when administered intrathecally, to the central terminal, it attenuated hyperalgesia induced by all three chemotherapy drugs, compatible with a role of TLR4 in neurotransmission at the central terminal but not sensory transduction at the peripheral terminal. Finally, since it has been established that cultured DRG neurons can be used to study direct effects of chemotherapy on nociceptors, we also evaluated the role of TLR4 in CIPN at the cellular level, using patch-clamp electrophysiology in DRG neurons cultured from control and chemotherapy-treated rats. We found that increased excitability of small-diameter DRG neurons induced by in vivo and in vitro exposure to oxaliplatin is TLR4-dependent. Our findings suggest that in addition to the established contribution of PRR-dependent neuroimmune mechanisms, PRRs in DRG cells also have an important role in CIPN.

A novel anti-pruritic: Topical co-administration of high molecular weight hyaluronan (HMWH) with protamine, a transdermal transport enhancer.

Pruritis, the sensation of itch, is produced by multiple substances, exogenous and endogenous, that sensitizes specialized sensory neurons (pruriceptors and pruri-nociceptors). Unfortunately, many patients with acute and chronic pruritis obtain only partial relief when treated with currently available treatment modalities. We recently demonstrated that the topical application of high molecular weight hyaluronan (HMWH), when combined with vehicles containing transdermal transport enhancers, produce potent long-lasting reversal of nociceptor sensitization associated with inflammatory and neuropathic pain. In the present experiments we tested the hypothesis that the topical formulation of HMWH with protamine, a transdermal transport enhancer, can also attenuate pruritis. We report that this topical formulation of HMWH markedly attenuates scratching behavior at the nape of the neck induced by serotonin (5-hydroxytryptamine, 5-HT), in male and female rats. Our results support the hypothesis that topical HMWH in a transdermal transport enhancer vehicle is a strong anti-pruritic.

Sensitization of human and rat nociceptors by low dose morphine is toll-like receptor 4-dependent.

While opioids remain amongst the most effective treatments for moderate-to-severe pain, their substantial side effect profile remains a major limitation to broader clinical use. One such side effect is opioid-induced hyperalgesia (OIH), which includes a transition from opioid-induced analgesia to pain enhancement. Evidence in rodents supports the suggestion that OIH may be produced by the action of opioids at Toll-like Receptor 4 (TLR4) either on immune cells that, in turn, produce pronociceptive mediators to act on nociceptors, or by a direct action at nociceptor TLR4. And, sub-analgesic doses of several opioids have been shown to induce hyperalgesia in rodents by their action as TLR4 agonists. In the present in vitro patch-clamp electrophysiology experiments, we demonstrate that low dose morphine directly sensitizes human as well as rodent dorsal root ganglion (DRG) neurons, an effect of this opioid analgesic that is antagonized by LPS-RS Ultrapure, a selective TLR4 antagonist. We found that low concentration (100 nM) of morphine reduced rheobase in human (by 36%) and rat (by 26%) putative C-type nociceptors, an effect of morphine that was markedly attenuated by preincubation with LPS-RS Ultrapure. Our findings support the suggestion that in humans, as in rodents, OIH is mediated by the direct action of opioids at TLR4 on nociceptors.

Contribution of G-Protein α-Subunits to Analgesia, Hyperalgesia, and Hyperalgesic Priming Induced by Subanalgesic and Analgesic Doses of Fentanyl and Morphine.

While opioids produce both analgesia and side effects by action at µ-opioid receptors (MORs), at spinal and supraspinal sites, the potency of different opioids to produce these effects varies. While it has been suggested that these differences might be because of bias for signaling via ß-arrestin versus G-protein α-subunits (Gα), recent studies suggest that G-protein-biased MOR agonists still produce clinically important side effects. Since bias also exists in the role of Gα subunits, we evaluated the role of Gαi/o subunits in analgesia, hyperalgesia, and hyperalgesic priming produced by fentanyl and morphine, in male rats. We found that intrathecal treatment with oligodeoxynucleotides antisense (AS-ODN) for Gαi2, Gαi3, and Gαo markedly attenuated hyperalgesia induced by subanalgesic dose (sub-AD) fentanyl, while AS-ODN for Gαi1, as well as Gαi2 and Gαi3, but not Gαo, prevented hyperalgesia induced by sub-AD morphine. AS-ODN for Gαi1 and Gαi2 unexpectedly enhanced analgesia induced by analgesic dose (AD) fentanyl, while Gαi1 AS-ODN markedly reduced AD morphine analgesia. Hyperalgesic priming, assessed by prolongation of prostaglandin E2-induced hyperalgesia, was not produced by systemic sub-AD and AD fentanyl in Gαi3 and Gαo AS-ODN-treated rats, respectively. In contrast, none of the Gαi/o AS-ODNs tested affected priming induced by systemic sub-AD and AD morphine. We conclude that signaling by different Gαi/o subunits is necessary for the analgesia and side effects of two of the most clinically used opioid analgesics. The design of opioid analgesics that demonstrate selectivity for individual Gαi/o may produce a more limited range of side effects and enhanced analgesia.SIGNIFICANCE STATEMENT Biased µ-opioid receptor (MOR) agonists that preferentially signal through G-protein α-subunits over ß-arrestins have been developed as an approach to mitigate opioid side effects. However, we recently demonstrated that biased MOR agonists also produce hyperalgesia and priming. We show that oligodeoxynucleotide antisense to different Gαi/o subunits play a role in hyperalgesia and analgesia induced by subanalgesic and analgesic dose (respectively), of fentanyl and morphine, as well as in priming. Our findings have the potential to advance our understanding of the mechanisms involved in adverse effects of opioid analgesics that could assist in the development of novel analgesics, preferentially targeting specific G-protein α-subunits.

Neuroendocrine Stress Axis-Dependence of Duloxetine Analgesia (Anti-Hyperalgesia) in Chemotherapy-Induced Peripheral Neuropathy.

Duloxetine, a serotonin and norepinephrine reuptake inhibitor, is the best-established treatment for painful chemotherapy-induced peripheral neuropathy (CIPN). While it is only effective in little more than half of patients, our ability to predict patient response remains incompletely understood. Given that stress exacerbates CIPN, and that the therapeutic effect of duloxetine is thought to be mediated, at least in part, via its effects on adrenergic mechanisms, we evaluated the contribution of neuroendocrine stress axes, sympathoadrenal and hypothalamic-pituitary-adrenal, to the effect of duloxetine in preclinical models of oxaliplatin- and paclitaxel-induced CIPN. Systemic administration of duloxetine, which alone had no effect on nociceptive threshold, both prevented and reversed mechanical hyperalgesia associated with oxaliplatin- and paclitaxel-CIPN. It more robustly attenuated oxaliplatin CIPN in male rats, while it was more effective for paclitaxel CIPN in females. Gonadectomy attenuated these sex differences in the effect of duloxetine. To assess the role of neuroendocrine stress axes in the effect of duloxetine on CIPN, rats of both sexes were submitted to adrenalectomy combined with fixed level replacement of corticosterone and epinephrine. While CIPN, in these rats, was of similar magnitude to that observed in adrenal-intact animals, rats of neither sex responded to duloxetine. Furthermore, duloxetine blunted an increase in corticosterone induced by oxaliplatin, and prevented the exacerbation of CIPN by sound stress. Our results demonstrate a role of neuroendocrine stress axes in duloxetine analgesia (anti-hyperalgesia) for the treatment of CIPN.SIGNIFICANCE STATEMENT Painful chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating dose-dependent and therapy-limiting side effect of many of the cytostatic drugs used to treat cancer (Argyriou et al., 2010; Marmiroli et al., 2017). Duloxetine is the only treatment for CIPN currently recommended by the American Society of Clinical Oncology (Hershman et al., 2014). In the present study, focused on elucidating mechanisms mediating the response of oxaliplatin- and paclitaxel-induced painful peripheral neuropathy to duloxetine, we demonstrate a major contribution to its effect of neuroendocrine stress axis function. These findings, which parallel the clinical observation that stress may impact response of CIPN to duloxetine (Taylor et al., 2007), open new approaches to the treatment of CIPN and other stress-associated pain syndromes.

PI3Kγ/AKT Signaling in High Molecular Weight Hyaluronan (HMWH)-Induced Anti-Hyperalgesia and Reversal of Nociceptor Sensitization.

High molecular weight hyaluronan (HMWH), a well-established treatment for osteoarthritis pain, is anti-hyperalgesic in preclinical models of inflammatory and neuropathic pain. HMWH-induced anti-hyperalgesia is mediated by its action at cluster of differentiation 44 (CD44), the cognate hyaluronan receptor, which can signal via phosphoinositide 3-kinase (PI3K), a large family of kinases involved in diverse cell functions. We demonstrate that intrathecal administration of an oligodeoxynucleotide (ODN) antisense to mRNA for PI3Kγ (a Class I PI3K isoform) expressed in dorsal root ganglia (DRGs), and intradermal administration of a PI3Kγ-selective inhibitor (AS605240), markedly attenuates HMWH-induced anti-prostaglandin E2 (PGE2) hyperalgesia, in male and female rats. Intradermal administration of inhibitors of mammalian target of rapamycin (mTOR; rapamycin) and protein kinase B (AKT; AKT Inhibitor IV), signaling molecules downstream of PI3Kγ, also attenuates HMWH-induced anti-hyperalgesia. In vitro patch-clamp electrophysiology experiments on cultured nociceptors from male rats demonstrate that some HMWH-induced changes in generation of action potentials (APs) in nociceptors sensitized by PGE2 are PI3Kγ dependent (reduction in AP firing rate, increase in latency to first AP and increase in slope of current ramp required to induce AP) and some are PI3Kγ independent [reduction in recovery rate of AP afterhyperpolarization (AHP)]. Our demonstration of a role of PI3Kγ in HMWH-induced anti-hyperalgesia and reversal of nociceptor sensitization opens a novel line of research into molecular targets for the treatment of diverse pain syndromes.SIGNIFICANCE STATEMENT We have previously demonstrated that high molecular weight hyaluronan (HMWH) attenuates inflammatory hyperalgesia, an effect mediated by its action at cluster of differentiation 44 (CD44), the cognate hyaluronan receptor, and activation of its downstream signaling pathway, in nociceptors. In the present study, we demonstrate that phosphoinositide 3-kinase (PI3K)γ and downstream signaling pathway, protein kinase B (AKT) and mammalian target of rapamycin (mTOR), are crucial for HMWH to induce anti-hyperalgesia.

Probiotics attenuate alcohol-induced muscle mechanical hyperalgesia: Preliminary observations.

Alcohol use disorder (AUD) is a major health problem that causes millions of deaths annually world-wide. AUD is considered to be a chronic pain disorder, that is exacerbated by alcohol withdrawal, contributing to a high (∼80%) relapse rate. Chronic alcohol consumption has a marked impact on the gut microbiome, recognized to have a significant effect on chronic pain. We tested the hypothesis that modulating gut microbiota through feeding rats with probiotics can attenuate alcohol-induced muscle mechanical hyperalgesia. To test this hypothesis, rats were fed alcohol (6.5%, 4 days on 3 days off) for 3 weeks, which induced skeletal muscle mechanical hyperalgesia. Following alcohol feeding, at which time nociceptive thresholds were ∼37% below pre-alcohol levels, rats received probiotics in their drinking water, either Lactobacillus Rhamnosus GG (Culturelle) or De Simone Formulation (a mixture of 8 bacterial species) for 8 days; control rats received plain water to drink. When muscle mechanical nociceptive threshold was evaluated 1 day after beginning probiotic feeding, nociceptive thresholds were significantly higher than rats not receiving probiotics. Mechanical nociceptive thresholds continued to increase during probiotic feeding, with thresholds approaching pre-alcohol levels 5 days after starting probiotics; nociceptive threshold in rats not receiving probiotics remained low. After probiotics were removed from the drinking water, nociceptive thresholds gradually decreased in these two groups, although they remained higher than the group not treated with probiotic (21 days after ending alcohol feeding). These observations suggest that modification of gut microbiota through probiotic feeding has a marked effect on chronic alcohol-induced muscle mechanical hyperalgesia. Our results suggest that administration of probiotics to individuals with AUD may reduce pain associated with alcohol consumption and withdrawal, and may be a novel therapeutic intervention to reduce the high rate of relapse seen in individuals with AUD attempting to abstain from alcohol.

Mechanisms Mediating High-Molecular-Weight Hyaluronan-Induced Antihyperalgesia.

We evaluated the mechanism by which high-molecular-weight hyaluronan (HMWH) attenuates nociceptor sensitization, in the setting of inflammation. HMWH attenuated mechanical hyperalgesia induced by the inflammatory mediator prostaglandin E2 (PGE2) in male and female rats. Intrathecal administration of an oligodeoxynucleotide antisense (AS-ODN) to mRNA for cluster of differentiation 44 (CD44), the cognate hyaluronan receptor, and intradermal administration of A5G27, a CD44 receptor antagonist, both attenuated antihyperalgesia induced by HMWH. In male rats, HMWH also signals via Toll-like receptor 4 (TLR4), and AS-ODN for TLR4 mRNA administered intrathecally, attenuated HMWH-induced antihyperalgesia. Since HMWH signaling is dependent on CD44 clustering in lipid rafts, we pretreated animals with methyl-ß-cyclodextrin (MßCD), which disrupts lipid rafts. MßCD markedly attenuated HMWH-induced antihyperalgesia. Inhibitors for components of intracellular signaling pathways activated by CD44, including phospholipase C and phosphoinositide 3-kinase (PI3K), also attenuated HMWH-induced antihyperalgesia. Furthermore, in vitro application of HMWH attenuated PGE2-induced sensitization of tetrodotoxin-resistant sodium current, in small-diameter dorsal root ganglion neurons, an effect that was attenuated by a PI3K inhibitor. Our results indicate a central role of CD44 signaling in HMWH-induced antihyperalgesia and suggest novel therapeutic targets, downstream of CD44, for the treatment of pain generated by nociceptor sensitization.SIGNIFICANCE STATEMENT High-molecular-weight-hyaluronan (HMWH) is used to treat osteoarthritis and other pain syndromes. In this study we demonstrate that attenuation of inflammatory hyperalgesia by HMWH is mediated by its action at cluster of differentiation 44 (CD44) and activation of its downstream signaling pathways, including RhoGTPases (RhoA and Rac1), phospholipases (phospholipases Cε and Cγ1), and phosphoinositide 3-kinase, in nociceptors. These findings contribute to our understanding of the antihyperalgesic effect of HMWH and support the hypothesis that CD44 and its downstream signaling pathways represent novel therapeutic targets for the treatment of inflammatory pain.

Sexual dimorphic role of the glucocorticoid receptor in chronic muscle pain produced by early-life stress.

Fibromyalgia and other chronic musculoskeletal pain syndromes are associated with stressful early life events, which can produce a persistent dysregulation in the hypothalamic-pituitary adrenal (HPA) stress axis function, associated with elevated plasm levels of corticosterone in adults. To determine the contribution of the HPA axis to persistent muscle hyperalgesia in adult rats that had experienced neonatal limited bedding (NLB), a form of early-life stress, we evaluated the role of glucocorticoid receptors on muscle nociceptors in adult NLB rats. In adult male and female NLB rats, mechanical nociceptive threshold in skeletal muscle was significantly lower than in adult control (neonatal standard bedding) rats. Furthermore, adult males and females that received exogenous corticosterone (via dams' milk) during postnatal days 2-9, displayed a similar lowered mechanical nociceptive threshold. To test the hypothesis that persistent glucocorticoid receptor signaling in the adult contributes to muscle hyperalgesia in NLB rats, nociceptor expression of glucocorticoid receptor (GR) was attenuated by spinal intrathecal administration of an oligodeoxynucleotide (ODN) antisense to GR mRNA. In adult NLB rats, GR antisense markedly attenuated muscle hyperalgesia in males, but not in females. These findings indicate that increased corticosterone levels during a critical developmental period (postnatal days 2-9) produced by NLB stress induces chronic mechanical hyperalgesia in male and female rats that persists in adulthood, and that this chronic muscle hyperalgesia is mediated, at least in part, by persistent stimulation of glucocorticoid receptors on sensory neurons, in the adult male, but not female rat.

A role for gut microbiota in early-life stress-induced widespread muscle pain in the adult rat.

Adult rats that experienced neonatal limited bedding (NLB), a form of early-life stress, experience persistent muscle mechanical hyperalgesia. Since there is a growing recognition that the gut microbiome regulates pain and nociception, and that early-life stress produces a long-lasting impact on the gut microbiome, we tested the hypothesis that persistent muscle hyperalgesia seen in adult NLB rats could be ameliorated by interventions that modify the gut microbiome. Adult NLB rats received probiotics, either Lactobacillus rhamnosus GG (10 billion CFU/150 ml) or De Simone Formulation (DSF) (112.5 billion CFU/150 ml mixture of 8 bacterial species), in their drinking water, or non-absorbable antibiotics, rifaximin or neomycin, admixed with cookie dough, to provide 50 mg/kg. Mechanical nociceptive threshold in the gastrocnemius muscle was evaluated before and at several time points after administration of probiotics or antibiotics. Adult NLB rats fed probiotics L. Rhamnosus or DSF, antibiotics, as well as rats fed non-absorbable antibiotics rifaximin or neomycin, had markedly attenuated muscle mechanical hyperalgesia. We hypothesize that persistent skeletal muscle hyperalgesia produced by NLB stress may be, at least in part, due to a contribution of the gut microbiome, and that modulation of gut microbiome using probiotics or non-absorbable antibiotics, may be novel therapeutic approaches for the treatment of chronic musculoskeletal pain.

Role of Nociceptor Toll-like Receptor 4 (TLR4) in Opioid-Induced Hyperalgesia and Hyperalgesic Priming.

In addition to analgesia, opioids produce opioid-induced hyperalgesia (OIH) and neuroplasticity characterized by prolongation of inflammatory-mediator-induced hyperalgesia (hyperalgesic priming). We evaluated the hypothesis that hyperalgesia and priming induced by opioids are mediated by similar nociceptor mechanisms. In male rats, we first evaluated the role of nociceptor Toll-like receptor 4 (TLR4) in OIH and priming induced by systemic low-dose morphine (LDM, 0.03 mg/kg). Intrathecal oligodeoxynucleotide antisense to TLR4 mRNA (TLR4 AS-ODN) prevented OIH and prolongation of prostaglandin E2 hyperalgesia (priming) induced by LDM. In contrast, high-dose morphine (HDM, 3 mg/kg) increased nociceptive threshold (analgesia) and induced priming, neither of which was attenuated by TLR4 AS-ODN. Protein kinase C ε (PKCε) AS-ODN also prevented LDM-induced hyperalgesia and priming, whereas analgesia and priming induced by HDM were unaffected. Treatment with isolectin B4 (IB4)-saporin or SSP-saporin (which deplete IB4+ and peptidergic nociceptors, respectively), or their combination, prevented systemic LDM-induced hyperalgesia, but not priming. HDM-induced priming, but not analgesia, was markedly attenuated in both saporin-treated groups. In conclusion, whereas OIH and priming induced by LDM share receptor and second messenger mechanisms in common, action at TLR4 and signaling via PKCε, HDM-induced analgesia, and priming are neither TLR4 nor PKCε dependent. OIH produced by LDM is mediated by both IB4+ and peptidergic nociceptors, whereas priming is not dependent on the same population. In contrast, priming induced by HDM is mediated by both IB4+ and peptidergic nociceptors. Implications for the use of low-dose opioids combined with nonopioid analgesics and in the treatment of opioid use disorder are discussed.SIGNIFICANCE STATEMENT Opioid-induced hyperalgesia (OIH) and priming are common side effects of opioid agonists such as morphine, which acts at µ-opioid receptors. We demonstrate that OIH and priming induced by systemic low-dose morphine (LDM) share action at Toll-like receptor 4 (TLR4) and signaling via protein kinase C ε (PKCε) in common, whereas systemic high-dose morphine (HDM)-induced analgesia and priming are neither TLR4 nor PKCε dependent. OIH produced by systemic LDM is mediated by isolectin B4-positive (IB4+) and peptidergic nociceptors, whereas priming is dependent on a different class of nociceptors. Priming induced by systemic HDM is, however, mediated by both IB4+ and peptidergic nociceptors. Our findings may provide useful information for the use of low-dose opioids combined with nonopioid analgesics to treat pain and opioid use disorders.

Exogenous Application of Proteoglycan to the Cell Surface Microenvironment Facilitates to Chondrogenic Differentiation and Maintenance.

Osteoarthritis (OA), a disease that greatly impacts quality of life, has increasing worldwide prevalence as the population ages. However, its pathogenic mechanisms have not been fully elucidated and current therapeutic treatment strategies are inadequate. In recent years, abnormal endochondral ossification in articular cartilage has received attention as a pathophysiological mechanism in OA. Cartilage is composed of abundant extracellular matrix components, which are involved in tissue maintenance and regeneration, but how these factors affect endochondral ossification is not clear. Here, we show that the application of aggrecan-type proteoglycan from salmon nasal cartilage (sPG) exhibited marked proliferative capacity through receptor tyrosine kinases in chondroprogenitor cells, and also exhibited differentiation and three-dimensional structure formation via phosphorylation of Insulin-like Growth Factor-1 Receptor and Growth Differentiation Factor 5 expression. Furthermore, sPG inhibited calcification via expression of Runx2 and Col10 (factors related to induction of calcification), while increasing Mgp, a mineralization inhibitory factor. As a result of analyzing the localization of sPG applied to the cells, it was localized on the surface of the cell membrane. In this study, we found that sPG, as a biomaterial, could regulate cell proliferation, differentiation and calcification inhibition by acting on the cell surface microenvironment. Therefore, sPG may be the foundation for a novel therapeutic approach for cartilage maintenance and for improved symptoms in OA.

Retinal Cell Degeneration in Animal Models.

The aim of this review is to provide an overview of various retinal cell degeneration models in animal induced by chemicals (N-methyl-D-aspartate- and CoCl2-induced), autoimmune (experimental autoimmune encephalomyelitis), mechanical stress (optic nerve crush-induced, light-induced) and ischemia (transient retinal ischemia-induced). The target regions, pathology and proposed mechanism of each model are described in a comparative fashion. Animal models of retinal cell degeneration provide insight into the underlying mechanisms of the disease, and will facilitate the development of novel effective therapeutic drugs to treat retinal cell damage.

Vascular endothelial cells mediate mechanical stimulation-induced enhancement of endothelin hyperalgesia via activation of P2X2/3 receptors on nociceptors.

Endothelin-1 (ET-1) is unique among a broad range of hyperalgesic agents in that it induces hyperalgesia in rats that is markedly enhanced by repeated mechanical stimulation at the site of administration. Antagonists to the ET-1 receptors, ET(A) and ET(B), attenuated both initial as well as stimulation-induced enhancement of hyperalgesia (SIEH) by endothelin. However, administering antisense oligodeoxynucleotide to attenuate ET(A) receptor expression on nociceptors attenuated ET-1 hyperalgesia but had no effect on SIEH, suggesting that this is mediated via a non-neuronal cell. Because vascular endothelial cells are both stretch sensitive and express ET(A) and ET(B) receptors, we tested the hypothesis that SIEH is dependent on endothelial cells by impairing vascular endothelial function with octoxynol-9 administration; this procedure eliminated SIEH without attenuating ET-1 hyperalgesia. A role for protein kinase Cε (PKCε), a second messenger implicated in the induction and maintenance of chronic pain, was explored. Intrathecal antisense for PKCε did not inhibit either ET-1 hyperalgesia or SIEH, suggesting no role for neuronal PKCε; however, administration of a PKCε inhibitor at the site of testing selectively attenuated SIEH. Compatible with endothelial cells releasing ATP in response to mechanical stimulation, P2X(2/3) receptor antagonists eliminated SIEH. The endothelium also appears to contribute to hyperalgesia in two ergonomic pain models (eccentric exercise and hindlimb vibration) and in a model of endometriosis. We propose that SIEH is produced by an effect of ET-1 on vascular endothelial cells, sensitizing its release of ATP in response to mechanical stimulation; ATP in turn acts at the nociceptor P2X(2/3) receptor.

Does the antihyperalgesic disruptor of endothelial cells, octoxynol-9, alter nociceptor function?

The vasoactive mediator, endothelin-1, elicits a novel form of hyperalgesia, stimulation-dependent hyperalgesia. Acting on its cognate receptor on the vascular endothelial cell, endothelin-1 produces a state in which mechanical stimulation now elicits release of pronociceptive mediators from endothelium that, in turn, acts at receptors on sensory neurons. The only evidence that octoxynol-9, a surface-active agent that attenuates both endothelial cell function and stimulus-dependent hyperalgesia, does not affect nociceptors is indirect (i.e., octoxynol-9 treatment did not affect behavioral nociceptive threshold or hyperalgesia induced by agents that act directly on nociceptors). To help address the question of whether the attenuation of stimulation-dependent hyperalgesia by octoxynol-9 treatment is due to alteration of nociceptor function, we used in vivo single-fiber electrophysiological recordings. Consistent with our previous behavioral observations, we observed no significant effect of octoxynol-9 on mechanical threshold in nociceptors, their response to sustained suprathreshold mechanical stimulation, conduction velocity, and change in mechanical threshold in response to the direct-acting hyperalgesic agent, PGE2. Although octoxynol-9 did not produce a biologically meaningful change in parameters of nociceptor function, we cannot exclude the possibility of a type II error. However, our data provide preliminary evidence of no effect of octoxynol-9 on nociceptors and are consistent with the suggestion that the primary action of octoxynol-9 in our studies is due to its action on the endothelium.

Neuroendocrine mechanisms in oxaliplatin-induced hyperalgesic priming.

ABSTRACT: Stress plays a major role in the symptom burden of oncology patients and can exacerbate cancer chemotherapy-induced peripheral neuropathy (CIPN), a major adverse effect of many classes of chemotherapy. We explored the role of stress in the persistent phase of the pain induced by oxaliplatin. Oxaliplatin induced hyperalgesic priming, a model of the transition to chronic pain, as indicated by prolongation of hyperalgesia produced by prostaglandin E 2 , in male rats, which was markedly attenuated in adrenalectomized rats. A neonatal handling protocol that induces stress resilience in adult rats prevented oxaliplatin-induced hyperalgesic priming. To elucidate the role of the hypothalamic-pituitary-adrenal and sympathoadrenal neuroendocrine stress axes in oxaliplatin CIPN, we used intrathecally administered antisense oligodeoxynucleotides (ODNs) directed against mRNA for receptors mediating the effects of catecholamines and glucocorticoids, and their second messengers, to reduce their expression in nociceptors. Although oxaliplatin-induced hyperalgesic priming was attenuated by intrathecal administration of ß 2 -adrenergic and glucocorticoid receptor antisense ODNs, oxaliplatin-induced hyperalgesia was only attenuated by ß 2 -adrenergic receptor antisense. Administration of pertussis toxin, a nonselective inhibitor of Gα i/o proteins, attenuated hyperalgesic priming. Antisense ODNs for Gα i 1 and Gα o also attenuated hyperalgesic priming. Furthermore, antisense for protein kinase C epsilon, a second messenger involved in type I hyperalgesic priming, also attenuated oxaliplatin-induced hyperalgesic priming. Inhibitors of second messengers involved in the maintenance of type I (cordycepin) and type II (SSU6656 and U0126) hyperalgesic priming both attenuated hyperalgesic priming. These experiments support a role for neuroendocrine stress axes in hyperalgesic priming, in male rats with oxaliplatin CIPN.

Topical coapplication of hyaluronan with transdermal drug delivery enhancers attenuates inflammatory and neuropathic pain.

ABSTRACT: We have previously shown that intradermal injection of high-molecular-weight hyaluronan (500-1200 kDa) produces localized antihyperalgesia in preclinical models of inflammatory and neuropathic pain. In the present experiments, we studied the therapeutic effect of topical hyaluronan, when combined with each of 3 transdermal drug delivery enhancers (dimethyl sulfoxide [DMSO], protamine or terpene), in preclinical models of inflammatory and neuropathic pain. Topical application of 500 to 1200 kDa hyaluronan (the molecular weight range used in our previous studies employing intradermal administration), dissolved in 75% DMSO in saline, markedly reduced prostaglandin E 2 (PGE 2 ) hyperalgesia, in male and female rats. Although topical 500- to 1200-kDa hyaluronan in DMSO vehicle dose dependently, also markedly, attenuated oxaliplatin chemotherapy-and paclitaxel chemotherapy-induced painful peripheral neuropathy (CIPN) in male rats, it lacked efficacy in female rats. However, following ovariectomy or intrathecal administration of an oligodeoxynucleotide antisense to G-protein-coupled estrogen receptor (GPR30) mRNA, CIPN in female rats was now attenuated by topical hyaluronan. Although topical coadministration of 150 to 300, 300 to 500, or 1500 to 1750 kDa hyaluronan with DMSO also attenuated CIPN, a slightly lower-molecular-weight hyaluronan (70-120 kDa) did not. The topical administration of a combination of hyaluronan with 2 other transdermal drug delivery enhancers, protamine and terpene, also attenuated CIPN hyperalgesia, an effect that was more prolonged than with DMSO vehicle. Repeated administration of topical hyaluronan prolonged the duration of antihyperalgesia. Our results support the use of topical hyaluronan, combined with chemically diverse nontoxic skin penetration enhancers, to induce marked antihyperalgesia in preclinical models of inflammatory and neuropathic pain.

IB4(+) nociceptors mediate persistent muscle pain induced by GDNF.

Skeletal muscle is a well-known source of glial cell line-derived neurotrophic factor (GDNF), which can produce mechanical hyperalgesia. Since some neuromuscular diseases are associated with both increased release of GDNF and intense muscle pain, we explored the role of GDNF as an endogenous mediator in muscle pain. Intramuscularly injected GDNF induced a dose-dependent (0.1-10 ng/20 µl) persistent (up to 3 wk) mechanical hyperalgesia in the rat. Once hyperalgesia subsided, injection of prostaglandin E(2) at the site induced a prolonged mechanical hyperalgesia (>72 h) compared with naïve rats (<4 h; hyperalgesic priming). Selective neurotoxic destruction of IB4(+) nociceptors attenuated both GDNF hyperalgesia and hyperalgesic priming. Ergonomic muscular injury induced by eccentric exercise or mechanical vibration increased muscle GDNF levels at 24 h, a time point where rats also exhibited marked muscle hyperalgesia. Intrathecal antisense oligodeoxynucleotides to mRNA encoding GFRα1, the canonical binding receptor for GDNF, reversibly inhibited eccentric exercise- and mechanical vibration-induced muscle hyperalgesia. Finally, electrophysiological recordings from nociceptors innervating the gastrocnemius muscle in anesthetized rats, revealed significant increase in response to sustained mechanical stimulation after local GDNF injection. In conclusion, these data indicate that GDNF plays a role as an endogenous mediator in acute and induction of chronic muscle pain, an effect likely to be produced by GDNF action at GFRα1 receptors located in IB4(+) nociceptors.

Second messengers mediating high-molecular-weight hyaluronan-induced antihyperalgesia in rats with chemotherapy-induced peripheral neuropathy.

ABSTRACT: High-molecular-weight hyaluronan (HMWH) is an agonist at cluster of differentiation (CD)44, the cognate hyaluronan receptor, on nociceptors, where it acts to induce antihyperalgesia in preclinical models of inflammatory and neuropathic pain. In the present experiments, we studied the CD44 second messengers that mediate HMWH-induced attenuation of pain associated with oxaliplatin and paclitaxel chemotherapy-induced peripheral neuropathy (CIPN). While HMWH attenuated CIPN only in male rats, after ovariectomy or intrathecal administration of an oligodeoxynucleotide (ODN) antisense to G protein-coupled estrogen receptor (GPR30) mRNA, female rats were also sensitive to HMWH. Intrathecal administration of an ODN antisense to CD44 mRNA markedly attenuated HMWH-induced antihyperalgesia in male rats with CIPN induced by oxaliplatin or paclitaxel. Intradermal administration of inhibitors of CD44 second messengers, RhoA (member of the Rho family of GTPases), phospholipase C, and phosphatidylinositol (PI) 3-kinase gamma (PI3Kγ), attenuated HMWH-induced antihyperalgesia as does intrathecal administration of an ODN antisense to PI3Kγ. Our results demonstrated that HMWH induced antihyperalgesia in CIPN, mediated by its action at CD44 and downstream signaling by RhoA, phospholipase C, and PI3Kγ.

Abnormal muscle afferent function in a model of Taxol chemotherapy-induced painful neuropathy.

Despite muscle pain being a well-described symptom in patients with diverse forms of peripheral neuropathy, the role of neuropathic mechanisms in muscle pain have received remarkably little attention. We have recently demonstrated in a well-established model of chemotherapy-induced painful neuropathy (CIPN) that the anti-tumor drug paclitaxel (Taxol) produces mechanical hyperalgesia in skeletal muscle, of similar time course to and with shared mechanism with cutaneous symptoms. In the present study, we evaluated muscle afferent neuron function in this rat model of CIPN. The mechanical threshold of muscle afferents in rats exposed to paclitaxel was not significantly different from the mechanical threshold of muscle afferents in control animals (P = 0.07). However, paclitaxel did produce a marked increase in the number of action potentials elicited by prolonged suprathreshold fixed intensity mechanical stimulation and a marked increase in the conduction velocity. In addition, the interspike interval (ISI) analysis (to evaluate the temporal characteristics of the response of afferents to sustained mechanical stimulation) showed a significant difference in rats treated with paclitaxel; there was a significantly greater ISI percentage of paclitaxel-treated muscle afferents with 0.01- and 0.02-s ISI. In contrast, an analysis of variability of neuronal firing over time (CV2 analysis) showed no effect of paclitaxel administration. These effects of paclitaxel on muscle afferent function contrast with the previously reported effects of paclitaxel on the function of cutaneous nociceptors.