Search

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 β-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 β-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.

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.

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.

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.

High molecular weight hyaluronan (HMWH), a prominent component of the extracellular matrix binds to and signals via multiple receptors, including cluster of differentiation 44 (CD44) and toll-like receptor 4 (TLR4). We tested the hypothesis that, in the setting of inflammation, HMWH acts at TLR4 to attenuate hyperalgesia. We found that the attenuation of prostaglandin E2 (PGE2)-induced hyperalgesia by HMWH was attenuated by a TLR4 antagonist (NBP2-26245), but only in male and ovariectomized female rats. In this study we sought to evaluated the role of the TLR4 signaling pathway in anti-hyperalgesia induced by HMWH in male rats. Decreasing expression of TLR4 in nociceptors, by intrathecal administration of an oligodeoxynucleotide (ODN) antisense to TLR4 mRNA, also attenuated HMWH-induced anti-hyperalgesia, in male and ovariectomized female rats. Estrogen replacement in ovariectomized females reconstituted the gonad-intact phenotype. The administration of an inhibitor of myeloid differentiation factor 88 (MyD88), a TLR4 second messenger, attenuated HMWH-induced anti-hyperalgesia, while an inhibitor of the MyD88-independent TLR4 signaling pathway did not. Since it has previously been shown that HMWH-induced anti-hyperalgesia is also mediated, in part by CD44 we evaluated the effect of the combination of ODN antisense to TLR4 and CD44 mRNA. This treatment completely reversed HMWH-induced anti-hyperalgesia in male rats. Our results demonstrate a sex hormone-dependent, sexually dimorphic involvement of TLR4 in HMWH-induced anti-hyperalgesia, that is MyD88 dependent. PERSPECTIVE: The role of TLR4 in anti-hyperalgesia induced by HMWH is a sexually dimorphic, TLR4 dependent inhibition of inflammatory hyperalgesia that provides a novel molecular target for the treatment of inflammatory pain.

Adult rats previously submitted to neonatal limited bedding (NLB), a model of early-life stress, display muscle mechanical hyperalgesia and nociceptor hyperexcitability, the underlying mechanism for which is unknown. Since voltage-gated sodium channel subtype 7 (NaV1.7) contributes to mechanical hyperalgesia in several preclinical pain models and is critical for nociceptor excitability, we explored its role in the muscle hyperalgesia exhibited by adult NLB rats. Western blot analyses demonstrated increased NaV1.7 protein expression in L4-L5 dorsal root ganglia (DRG) from adult NLB rats, and antisense oligodeoxynucleotide (AS ODN) targeting NaV1.7 alpha subunit mRNA attenuated the expression of NaV1.7 in DRG extracts. While this AS ODN did not affect nociceptive threshold in normal rats it significantly attenuated hyperalgesia in NLB rats. The selective NaV1.7 activator OD1 produced dose-dependent mechanical hyperalgesia that was enhanced in NLB rats, whereas the NaV1.7 blocker ProTx-II prevented OD1-induced hyperalgesia in control rats and ongoing hyperalgesia in NLB rats. AS ODN knockdown of extracellular signal-regulated kinase 1/2, which enhances NaV1.7 function, also inhibited mechanical hyperalgesia in NLB rats. Our results support the hypothesis that overexpression of NaV1.7 in muscle nociceptors play a role in chronic muscle pain induced by early-life stress, suggesting that NaV1.7 is a target for the treatment of chronic muscle pain. PERSPECTIVE: We demonstrate that early-life adversity, induced by exposure to inconsistent maternal care, produces chronic muscle hyperalgesia, which depends, at least in part, on increased expression of NaV1.7 in nociceptors.

AbstractIntradermal administration of low-molecular-weight hyaluronan (LMWH) in the hind paw induced dose-dependent (0.1, 1, or 10 µg) mechanical hyperalgesia of similar magnitude in male and female rats. However, the duration of LMWH hyperalgesia was greater in females. This sexual dimorphism was eliminated by bilateral ovariectomy and by intrathecal administration of an oligodeoxynucleotide (ODN) antisense to the G-protein-coupled estrogen receptor (GPR30) mRNA in females, indicating estrogen dependence. To assess the receptors at which LMWH acts to induce hyperalgesia, LMWH was administered to groups of male and female rats that had been pretreated with ODN antisense (or mismatch) to the mRNA for 1 of 3 hyaluronan receptors, cluster of differentiation 44 (CD44), toll-like receptor 4, or receptor for hyaluronan-mediated motility (RHAMM). Although LMWH-induced hyperalgesia was attenuated in both male and female rats pretreated with ODN antisense for CD44 and toll-like receptor 4 mRNA, RHAMM antisense pretreatment only attenuated LMWH-induced hyperalgesia in males. Oligodeoxynucleotide antisense for RHAMM, however, attenuated LMWH-induced hyperalgesia in female rats treated with ODN antisense to GPR30, as well as in ovariectomized females. Low-molecular-weight hyaluronan-induced hyperalgesia was significantly attenuated by pretreatment with high-molecular-weight hyaluronan (HMWH) in male, but not in female rats. After gonadectomy or treatment with ODN antisense to GPR30 expression in females, HMWH produced similar attenuation of LMWH-induced hyperalgesia to that seen in males. These experiments identify nociceptors at which LMWH acts to produce mechanical hyperalgesia, establishes estrogen dependence in the role of RHAMM in female rats, and establishes estrogen dependence in the inhibition of LMWH-induced hyperalgesia by HMWH.

AbstractAlthough clinical studies support the suggestion that stress is a risk factor for painful chemotherapy-induced peripheral neuropathy (CIPN), there is little scientific validation to support this link. Here, we evaluated the impact of stress on CIPN induced by oxaliplatin, and its underlying mechanisms, in male and female rats. A single dose of oxaliplatin produced mechanical hyperalgesia of similar magnitude in both sexes, still present at similar magnitude in both sexes, on day 28. Adrenalectomy mitigated oxaliplatin-induced hyperalgesia, in both sexes. To confirm the role of neuroendocrine stress axes in CIPN, intrathecal administration of antisense oligodeoxynucleotide targeting β₂-adrenergic receptor mRNA both prevented and reversed oxaliplatin-induced hyperalgesia, only in males. By contrast, glucocorticoid receptor antisense oligodeoxynucleotide prevented and reversed oxaliplatin-induced hyperalgesia in both sexes. Unpredictable sound stress enhanced CIPN, in both sexes. The administration of stress hormones, epinephrine, corticosterone, and their combination, at stress levels, mimicked the effects of sound stress on CIPN, in males. In females, only corticosterone mimicked the effect of sound stress. Also, a risk factor for CIPN, early-life stress, was evaluated by producing both stress-sensitive (produced by neonatal limited bedding) and stress-resilient (produced by neonatal handling) phenotypes in adults. Although neonatal limited bedding significantly enhanced CIPN only in female adults, neonatal handling significantly attenuated CIPN, in both sexes. Our study demonstrates a sexually dimorphic role of the 2 major neuroendocrine stress axes in oxaliplatin-induced neuropathic pain.

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.

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.

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-β-cyclodextrin (MβCD), which disrupts lipid rafts. MβCD 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.

Chemotherapy-induced neuropathic pain is a serious adverse effect of chemotherapeutic agents. Clinical evidence suggests that stress is a risk factor for development and/or worsening of chemotherapy-induced peripheral neuropathy (CIPN). We evaluated the impact of stress and stress axis mediators on paclitaxel CIPN in male and female rats. Paclitaxel produced mechanical hyperalgesia, over the 4-day course of administration, peaking by day 7, and still present by day 28, with no significant difference between male and female rats. Paclitaxel hyperalgesia was enhanced in male and female rats previously exposed to unpredictable sound stress, but not in rats that were exposed to sound stress after developing paclitaxel CIPN. We evaluated the role of the neuroendocrine stress axes: in adrenalectomized rats, paclitaxel did not produce hyperalgesia. Intrathecal administration of antisense oligodeoxynucleotides (ODN) reduced expression of β2-adrenergic receptors on nociceptors, and paclitaxel-induced hyperalgesia was slightly attenuated in males, but markedly attenuated in females. By contrast, after intrathecal administration of antisense ODN to decrease expression of glucocorticoid receptors, hyperalgesia was markedly attenuated in males, but unaffected in females. Both ODNs together markedly attenuated paclitaxel-induced hyperalgesia in both males and females. We evaluated paclitaxel-induced CIPN in stress-resilient (produced by neonatal handling) and stress-sensitive (produced by neonatal limited bedding). Neonatal handling significantly attenuated paclitaxel-induced CIPN in adult male, but not in adult female rats. Neonatal limited bedding did not affect the magnitude of paclitaxel-induced CIPN in either male or female. This study provides evidence that neuroendocrine stress axis activity has a marked, sexually dimorphic, effect on paclitaxel-induced painful CIPN.

IntroductionAlthough stress is a well-establish risk factor for the development of chronic musculoskeletal pain, the underlying mechanisms, specifically the contribution of neuroendocrine stress axes, remain poorly understood.ObjectiveTo evaluate the hypothesis that psychological stress-induced activation of the sympathoadrenal stress axis prolongs the muscle pain observed after strenuous exercise.MethodsAdult male Sprague-Dawley rats were exposed to unpredictable sound stress and eccentric exercise. The involvement of the sympathoadrenal stress axis was evaluated by means of surgical interventions, systemic administration of epinephrine, and intrathecal β2-adrenergic receptor antisense.ResultsAlthough sound stress alone did not modify nociceptive threshold, it prolonged eccentric exercise-induced mechanical hyperalgesia. Adrenal medullectomy (ADMdX) attenuated, and administration of stress levels of epinephrine to ADMdX rats mimicked this effect of sound stress. Knockdown of β2-adrenergic receptors by intrathecal antisense also attenuated sound stress-induced prolongation of eccentric exercise-induced hyperalgesia.ConclusionTogether, these results indicate that sympathoadrenal activation, by unpredictable sound stress, disrupts the capacity of nociceptors to sense recovery from eccentric exercise, leading to the prolongation of muscle hyperalgesia. This prolonged recovery from ergonomic pain is due, at least in part, to the activation of β2-adrenergic receptors on muscle nociceptors.

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.

We investigated the dose dependence of the role of nociceptors in opioid-induced side-effects, hyperalgesia and pain chronification, in the rat. Systemic morphine produced a dose-dependent biphasic change in mechanical nociceptive threshold. At lower doses (0.003-0.03 mg/kg, s.c.) morphine induced mechanical hyperalgesia, while higher doses (1-10 mg/kg, s.c.) induced analgesia. Intrathecal (i.t.) oligodeoxynucleotide (ODN) antisense to mu-opioid receptor (MOR) mRNA, attenuated both hyperalgesia and analgesia. 5 days after systemic morphine (0.03-10 mg/kg s.c.), mechanical hyperalgesia produced by intradermal (i.d.) prostaglandin E2 (PGE2) was prolonged, indicating hyperalgesic priming at the peripheral terminal of the nociceptor. The hyperalgesia induced by i.t. PGE2 (400 ng/10 µl), in groups that received 0.03 (that induced hyperalgesia) or 3 mg/kg (that induced analgesia) morphine, was also prolonged, indicating priming at the central terminal of the nociceptor. The prolongation of the hyperalgesia induced by i.d. or i.t. PGE2, in rats previously treated with either a hyperalgesic (0.03 mg/kg, s.c.) or analgesic (3 mg/kg, s.c.) dose, was reversed by i.d. or i.t. injection of the protein translation inhibitor cordycepin (1 µg), indicative of Type I priming at both terminals. Although pretreatment with MOR antisense had no effect on priming induced by 0.03 mg/kg morphine, it completely prevented priming by 3 mg/kg morphine, in both terminals. Thus, the induction of hyperalgesia, but not priming, by low-dose morphine, is MOR-dependent. In contrast, induction of both hyperalgesia and priming by high-dose morphine is MOR-dependent. The receptor at which low-dose morphine acts to produce priming remains to be established.

We recently demonstrated that transient attenuation of Toll-like receptor 4 (TLR4) in dorsal root ganglion (DRG) neurons, can both prevent and reverse pain associated with chemotherapy-induced peripheral neuropathy (CIPN), a severe side effect of cancer chemotherapy, for which treatment options are limited. Given the reduced efficacy of opioid analgesics to treat neuropathic, compared with inflammatory pain, the cross talk between nociceptor TLR4 and mu-opioid receptors (MORs), and that MOR and TLR4 agonists induce hyperalgesic priming (priming), which also occurs in CIPN, we determined, using male rats, whether (1) antisense knockdown of nociceptor MOR attenuates CIPN, (2) and attenuates the priming associated with CIPN, and (3) CIPN also produces opioid-induced hyperalgesia (OIH). We found that intrathecal MOR antisense prevents and reverses hyperalgesia induced by oxaliplatin and paclitaxel, two common clinical chemotherapy agents. Oxaliplatin-induced priming was also markedly attenuated by MOR antisense. Additionally, intradermal morphine, at a dose that does not affect nociceptive threshold in controls, exacerbates mechanical hyperalgesia (OIH) in rats with CIPN, suggesting the presence of OIH. This OIH associated with CIPN is inhibited by interventions that reverse Type II priming [the combination of an inhibitor of Src and mitogen-activated protein kinase (MAPK)], an MOR antagonist, as well as a TLR4 antagonist. Our findings support a role of nociceptor MOR in oxaliplatin-induced pain and priming. We propose that priming and OIH are central to the symptom burden in CIPN, contributing to its chronicity and the limited efficacy of opioid analgesics to treat neuropathic pain. [ABSTRACT FROM AUTHOR]

Neonatal handling (NH) of male rat pups strongly attenuates stress response and stress-induced persistent muscle hyperalgesia in adults. Because female sex is a well established risk factor for stress-induced chronic muscle pain, we explored whether NH provides resilience to stress-induced hyperalgesia in adult female rats. Rat pups underwent NH, or standard (control) care. Muscle mechanical nociceptive threshold was assessed before and after water avoidance (WA) stress, when they were adults. In contrast to male rats, NH produced only a modest protection against WA stress-induced muscle hyperalgesia in female rats. Gonadectomy completely abolished NH-induced resilience in male rats but produced only a small increase in this protective effect in female rats. The administration of the antiestrogen drug fulvestrant, in addition to gonadectomy, did not enhance the protective effect of NH in female rats. Finally, knockdown of the androgen receptor by intrathecal antisense treatment attenuated the protective effect of NH in intact male rats. Together, these data indicate that androgens play a key role in NH-induced resilience to WA stress-induced muscle hyperalgesia. PERSPECTIVE:NH induces androgen-dependent resilience to stress-induced muscle pain. Therefore, androgens may contribute to sex differences observed in chronic musculoskeletal pain and its enhancement by stress.

Delayed-onset muscle soreness is typically observed after strenuous or unaccustomed eccentric exercise. Soon after recovery, blunted muscle soreness is observed on repeated eccentric exercise, a phenomenon known as repeated bout effect (RBE). Although regular physical activity decreases muscle hyperalgesia, likely because of increased production of the anti-inflammatory cytokine interleukin-10 (IL-10) in the skeletal muscle, whether IL-10 also contributes to the antinociceptive effect of RBE is unknown. Furthermore, whether IL-10 attenuates muscle hyperalgesia by acting on muscle nociceptors remains to be established. Here, we explored the hypothesis that blunted muscle nociception observed in RBE depends on a local effect of IL-10, acting on IL-10 receptor 1 (IL-10R1) expressed by muscle nociceptors. Results show that after a second bout of eccentric exercise, rats exhibited decreased muscle hyperalgesia, indicative of RBE, and increased expression of IL-10 in the exercised gastrocnemius muscle. Although knockdown of IL-10R1 protein in nociceptors innervating the gastrocnemius muscle by intrathecal antisense oligodeoxynucleotide did not change nociceptive threshold in naive rats, it unveiled latent muscle hyperalgesia in rats submitted to eccentric exercise 12 days ago. Furthermore, antisense also prevented the reduction of muscle hyperalgesia observed after a second bout of eccentric exercise. These data indicate that recovery of nociceptive threshold after eccentric exercise and RBE-induced analgesia depend on a local effect of IL-10, acting on its canonical receptor in muscle nociceptors.

Corneal injury can produce photophobia, an aversive sensitivity to light. Using topical application of lidocaine, a local anesthetic, and tetrodotoxin (TTX), a selective voltage-sensitive sodium channel blocker, we assessed whether enhanced aversiveness to light induced by corneal injury in rats was caused by enhanced activity in corneal afferents. Eye closure induced by 30 seconds of exposure to bright light (460-485 nm) was increased 24 hours after corneal injury induced by de-epithelialization. Although the topical application of lidocaine did not affect the baseline eye closure response to bright light in control rats, it eliminated the enhancement of the response to the light stimulus after corneal injury (photophobia). Similarly, topical application of TTX had no effect on the eye closure response to bright light in rats with intact corneas, but it markedly attenuated photophobia in rats with corneal injury. Given the well-established corneal toxicity of local anesthetics, we suggest TTX as a therapeutic option to treat photophobia and possibly other symptoms that occur in clinical diseases that involve corneal nociceptor sensitization. Perspective: We show that lidocaine and TTX attenuate photophobia induced by corneal injury. Although corneal toxicity limits use of local anesthetics, TTX may be a safer therapeutic option to reduce the symptom of photophobia associated with corneal injury.

Chronic stress is well known to exacerbate pain. We tested the hypothesis that neonatal handling, which induces resilience to the negative impact of stress by increasing the quality and quantity of maternal care, attenuates the mechanical hyperalgesia produced by water-avoidance stress in the adult rat.Neonatal male rats underwent the handling protocol on postnatal days 2-9, weaned at 21 days and tested for muscle mechanical nociceptive threshold at postnatal days 50-75.Decrease in mechanical nociceptive threshold in skeletal muscle in adult rats, produced by exposure to water-avoidance stress, was significantly attenuated by neonatal handling. Neonatal handling also attenuated the mechanical hyperalgesia produced by intramuscular administration of the pronociceptive inflammatory mediator, prostaglandin E2 in rats exposed as adults to water-avoidance stress.Neonatal handling, which induces a smaller corticosterone response in adult rats exposed to a stressor as well as changes in central nervous system neurotransmitter systems, attenuates mechanical hyperalgesia produced by water-avoidance stress and enhanced prostaglandin hyperalgesia in adult animals.

UnlabelledEndothelin-1 (ET-1) acts on endothelial cells to enhance mechanical stimulation-induced release of adenosine triphosphate (ATP), which in turn can act on sensory neurons innervating blood vessels to contribute to vascular pain, a phenomenon we have referred to as stimulus-dependent hyperalgesia (SDH). In the present study, we evaluated the role of the major classes of ATP release mechanisms to SDH: vesicular exocytosis, plasma membrane-associated ATP synthase, ATP-binding cassette transporters, and ion channels. Inhibitors of vesicular exocytosis (ie, monensin, brefeldin A, and bafilomycin), plasma membrane-associated ATPase (ie, oligomycin and pigment epithelium-derived factor peptide 34-mer), and connexin ion channels (carbenoxolone and flufenamic acid) but not ATP-binding cassette transporter (ie, dipyridamole, nicardipine, or CFTRinh-172) attenuated SDH. This study reports a role of ATP in SDH and suggests novel targets for the treatment of vascular pain syndromes.PerspectiveET-1 acts on endothelial cells to produce mechanical stimulation-induced hyperalgesia. Inhibitors of 3 different ATP release mechanisms attenuated this SDH. This study provides support for a role of ATP in SDH and suggests novel targets for the treatment of vascular pain syndromes.

While raised levels of monocyte chemoattractant protein 1 (MCP-1) have been observed in patients with chronic muscle pain, direct evidence for its role as an algogen in skeletal muscle is still lacking. In the rat, MCP-1 induces a dose-dependent mechanical hyperalgesia lasting for up to 6weeks. Following recovery, rats exhibited a markedly prolonged hyperalgesia to an intramuscular injection of prostaglandin E2, hyperalgesic priming. Intrathecal pretreatment with isolectin B4 (IB4)-saporin, which selectively destroys IB4-positive (IB4+) nociceptors, markedly decreased MCP-1-induced hyperalgesia and prevented the subsequent development of priming. To evaluate the involvement of MCP-1 in stress-induced chronic pain we administered, intrathecally, antisense (AS) or mismatch oligodeoxynucleotides directed against CCR2 (the canonical receptor for MCP-1) mRNA, during the exposure to water-avoidance stress, a model of stress-induced persistent muscle pain. The AS treatment attenuated this hyperalgesia, whereas IB4-saporin abolished water-avoidance stress-induced muscle hyperalgesia and prevented stress-induced hyperalgesic priming. These results indicate that MCP-1 induces persistent muscle hyperalgesia and a state of latent chronic sensitization to other algogens, by action on its cognate receptor on IB4+ nociceptors. Because MCP-1 also contributes to stress-induced widespread chronic muscle pain, it should be considered as a player in chronic musculoskeletal pain syndromes.

BackgroundEarly-life stress and exposure to stressful stimuli play a major role in the development of chronic widespread pain in adults. However, how they interact in chronic pain syndromes remains unclear.MethodsDams and neonatal litters were submitted to a restriction of nesting material (neonatal limited bedding [NLB]) for 1 week. As adults, these rats were exposed to a painless sound stress protocol. The involvement of sympathoadrenal catecholamines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFα) in nociception was evaluated through behavioral and enzyme-linked immunosorbent assays, surgical interventions, and intrathecal antisense treatments.ResultsAdult NLB rats exhibited mild muscle hyperalgesia, which was markedly aggravated by sound stress (peaking 15 days after exposure). Adrenal medullectomy did not modify hyperalgesia in NLB rats but prevented its aggravation by sound stress. Sustained administration of epinephrine to NLB rats mimicked sound stress effect. Intrathecal treatment with antisense directed to IL-6 receptor subunit gp130 (gp130), but not to tumor necrosis factor receptor type 1 (TNFR1), inhibited hyperalgesia in NLB rats. However, antisense against either gp130 or TNFR1 inhibited sound stress-induced enhancement of hyperalgesia. Compared with control rats, NLB rats exhibit increased plasma levels of IL-6 but decreased levels of TNFα, whereas sound stress increases IL-6 plasma levels in control rats but not in NLB rats.ConclusionsEarly-life stress induces a persistent elevation of IL-6, hyperalgesia, and susceptibility to chronic muscle pain, which is unveiled by exposure to stress in adults. This probably depends on an interaction between adrenal catecholamines and proinflammatory cytokines acting at muscle nociceptor level.

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.

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. [ABSTRACT FROM AUTHOR]