Descending Control of Nociception Poorly Predicts the Development of Persistent Postsurgical Pain-like Behavior in Consomic Dahl S Rat Strains.

BACKGROUND: Chronic postsurgical pain is a poorly recognized outcome of surgery where patients experience pain long after healing from the surgical insult. Descending control of nociception, a phenomenon whereby application of a strong nociceptive stimulus to one part of the body of animals inhibits pain in remote body regions, offers one strategy to identify a propensity to develop chronic postsurgical pain-like behavior. Here, consomic rat panel was used to test the hypothesis that pain persistence is mechanistically linked to ineffective descending control of nociception. METHODS: Male and female Brown Norway, Dahl S, and eight consomic strains (SS-xBN) were used to determine the presence of chronic postsurgical pain-like behaviors by using paw-withdrawal threshold evaluation (von Frey method) in the area adjacent to a hind paw plantar incision. Descending control of nociception was assessed by measuring hind paw-withdrawal thresholds (Randall-Selitto method) after capsaicin (125 µg) injection into a forepaw. Consomic rats were developed by introgressing individual Brown Norway chromosomes on the Dahl S rat genetic background, as Dahl S rats lack preoperative descending control of nociception. RESULTS: Substitution of several chromosomes from the "pain-resistant" Brown Norway to the "pain-prone" Dahl S/Medical College of Wisconsin reduced mechanical nociceptive sensitivity and increased endogenous pain modulation capacity by differing degrees. Statistical modeling of these data revealed that descending control of nociception is a poor general predictor of the propensity to develop chronic postsurgical pain-like behavior (poor fit for model 1). However, a significant strain-by-descending control of nociception interaction was revealed (model 3, -2*log likelihood; 550.668, -2ll change; 18.093, P = 0.034) with SS-13BN and SS-15BN strains showing a negative descending control of nociception relationship with chronic postsurgical pain-like behavior. CONCLUSIONS: Descending control of nociception poorly predicted which rat strains developed chronic postsurgical pain-like behavior despite controlling for genetic, environmental, and sex differences. Two consomic strains that mimic clinical chronic postsurgical pain criteria and display a strong negative correlation with descending control of nociception were identified, offering novel candidates for future experiments exploring mechanisms that lead to chronic postsurgical pain.

Characterization of the Dahl salt-sensitive rat as a rodent model of inherited, widespread, persistent pain.

Animal models are essential for studying the pathophysiology of chronic pain disorders and as screening tools for new therapies. However, most models available do not reproduce key characteristics of clinical persistent pain. This has limited their ability to accurately predict which new medicines will be clinically effective. Here, we characterize the Dahl salt-sensitive (SS) rat strain as the first rodent model of inherited widespread hyperalgesia. We show that this strain exhibits physiological phenotypes known to contribute to chronic pain, such as neuroinflammation, defective endogenous pain modulation, dysfunctional hypothalamic-pituitary-adrenal axis, increased oxidative stress and immune cell activation. When compared with Sprague Dawley and Brown Norway rats, SS rats have lower nociceptive thresholds due to increased inflammatory mediator concentrations, lower corticosterone levels, and high oxidative stress. Treatment with dexamethasone, the reactive oxygen species scavenger tempol, or the glial inhibitor minocycline attenuated the pain sensitivity in SS rats without affecting the other strains while indomethacin and gabapentin provided less robust pain relief. Moreover, SS rats presented impaired diffuse noxious inhibitory controls and an exacerbated response to the proalgesic mediator PGE2, features of generalized pain conditions. These data establish this strain as a novel model of spontaneous, widespread hyperalgesia that can be used to identify biomarkers for chronic pain diagnosis and treatment.

A novel rat model of temporomandibular disorder with improved face and construct validities.

AIMS: Temporomandibular disorders are a cluster of orofacial conditions that are characterized by pain in the temporomandibular joint (TMJ) and surrounding muscles/tissues. Animal models of painful temporomandibular dysfunction (TMD) are valuable tools to investigate the mechanisms responsible for symptomatic temporomandibular joint and associated structures disorders. We tested the hypothesis that a predisposing and a precipitating factor are required to produce painful TMD in rats, using the ratgnawmeter, a device that determines temporomandibular pain based on the time taken for the rat to chew through two obstacles. MATERIALS AND METHODS: Increased time in the ratgnawmeter correlated with nociceptive behaviors produced by TMJ injection of formalin (2.5%), confirming chewing time as an index of painful TMD. Rats exposed only to predisposing factors, carrageenan-induced TMJ inflammation or sustained inhibition of the catechol-O-methyltransferase (COMT) enzyme by OR-486, showed no changes in chewing time. However, when combined with a precipitating event, i.e., exaggerated mouth opening produced by daily 1-h jaw extension for 7 consecutive days, robust function impairment was produced. KEY FINDINGS: These results validate the ratgnawmeter as an efficient method to evaluate functional TMD pain by evaluating chewing time, and this protocol as a model with face and construct validities to investigate symptomatic TMD mechanisms. SIGNIFICANCE: This study suggests that a predisposition factor must be present in order for an insult to the temporomandibular system to produce painful dysfunction. The need for a combined contribution of these factors might explain why not all patients experiencing traumatic events, such as exaggerated mouth opening, develop TMDs.

D2 Receptors in the Periaqueductal Gray/Dorsal Raphe Modulate Peripheral Inflammatory Hyperalgesia via the Rostral Ventral Medulla.

Dopamine neurons in the periaqueductal gray (PAG)/dorsal raphe are key modulators of antinociception with known supraspinal targets. However, no study has directly tested whether these neurons contribute to descending pain inhibition. We hypothesized that PAG dopamine neurons contribute to the analgesic effect of D-amphetamine via a mechanism that involves descending modulation via the rostral ventral medulla (RVM). Male C57BL/6 mice showed increased c-FOS expression in PAG dopamine neurons and a significant increase in paw withdrawal latency to thermal stimulation after receiving a systemic injection of D-amphetamine. Targeted microinfusion of D-amphetamine, L-DOPA, or the selective D2 agonist quinpirole into the PAG produced analgesia, while a D1 agonist, chloro APB, had no effect. In addition, inhibition of D2 receptors in the PAG by eticlopride prevented the systemic D-amphetamine analgesic effect. D-amphetamine and PAG D2 receptor-mediated analgesia were inhibited by intra-RVM injection of lidocaine or the GABAA receptor agonist muscimol, indicating a PAG-RVM signaling pathway in this model of analgesia. Finally, both systemic D-amphetamine and local PAG microinjection of quinpirole, inhibited inflammatory hyperalgesia induced by carrageenan. This hyperalgesia was transiently restored by intra-PAG injection of eticlopride, as well as RVM microinjection of muscimol. We conclude that D-amphetamine analgesia is partially mediated by descending inhibition and that D2 receptors in the PAG are responsible for this effect via modulating neurons that project to the RVM. These results further our understanding of the antinociceptive effects of dopamine and elucidate a mechanism by which clinically available dopamine modulators produce analgesia.

Sexual dimorphism in the contribution of neuroendocrine stress axes to oxaliplatin-induced painful peripheral neuropathy.

ABSTRACT: Although 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 ß2-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.

Marked sexual dimorphism in neuroendocrine mechanisms for the exacerbation of paclitaxel-induced painful peripheral neuropathy by stress.

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 ß2-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.

Systemic Morphine Produces Dose-dependent Nociceptor-mediated Biphasic Changes in Nociceptive Threshold and Neuroplasticity.

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.

Mu-opioid Receptor (MOR) Biased Agonists Induce Biphasic Dose-dependent Hyperalgesia and Analgesia, and Hyperalgesic Priming in the Rat.

Stimulation of the mu-opioid receptor (MOR) on nociceptors with fentanyl can produce hyperalgesia (opioid-induced hyperalgesia, OIH) and hyperalgesic priming, a model of transition to chronic pain. We investigated if local and systemic administration of biased MOR agonists (PZM21 and TRV130 [oliceridine]), which preferentially activate G-protein over ß-arrestin translocation, and have been reported to minimize some opioid side effects, also produces OIH and priming. Injected intradermally (100 ng), both biased agonists induced mechanical hyperalgesia and, when injected at the same site, 5 days later, prostaglandin E2 (PGE2) produced prolonged hyperalgesia (priming). OIH and priming were both prevented by intrathecal treatment with an oligodeoxynucleotide (ODN) antisense (AS) for MOR mRNA. Agents that reverse Type I (the protein translation inhibitor cordycepin) and Type II (combination of Src and mitogen-activated protein kinase [MAPK] inhibitors) priming, or their combination, did not reverse priming induced by local administration of PZM21 or TRV130. While systemic PZM21 at higher doses (1 and 10 mg/kg) induced analgesia, lower doses (0.001, 0.01, 0.1, and 0.3 mg/kg) induced hyperalgesia; all doses induced priming. Hyperalgesia, analgesia and priming induced by systemic administration of PZM21 were also prevented by MOR AS-ODN. And, priming induced by systemic PZM21 was also not reversed by intradermal cordycepin or the combination of Src and MAPK inhibitors. Thus, maintenance of priming induced by biased MOR agonists, in the peripheral terminal of nociceptors, has a novel mechanism.

Swedish Nerve Growth Factor Mutation (NGFR100W) Defines a Role for TrkA and p75NTR in Nociception.

Nerve growth factor (NGF) exerts multiple functions on target neurons throughout development. The recent discovery of a point mutation leading to a change from arginine to tryptophan at residue 100 in the mature NGFß sequence (NGFR100W) in patients with hereditary sensory and autonomic neuropathy type V (HSAN V) made it possible to distinguish the signaling mechanisms that lead to two functionally different outcomes of NGF: trophic versus nociceptive. We performed extensive biochemical, cellular, and live-imaging experiments to examine the binding and signaling properties of NGFR100W Our results show that, similar to the wild-type NGF (wtNGF), the naturally occurring NGFR100W mutant was capable of binding to and activating the TrkA receptor and its downstream signaling pathways to support neuronal survival and differentiation. However, NGFR100W failed to bind and stimulate the 75 kDa neurotrophic factor receptor (p75NTR)-mediated signaling cascades (i.e., the RhoA-Cofilin pathway). Intraplantar injection of NGFR100W into adult rats induced neither TrkA-mediated thermal nor mechanical acute hyperalgesia, but retained the ability to induce chronic hyperalgesia based on agonism for TrkA signaling. Together, our studies provide evidence that NGFR100W retains trophic support capability through TrkA and one aspect of its nociceptive signaling, but fails to engage p75NTR signaling pathways. Our findings suggest that wtNGF acts via TrkA to regulate the delayed priming of nociceptive responses. The integration of both TrkA and p75NTR signaling thus appears to regulate neuroplastic effects of NGF in peripheral nociception.SIGNIFICANCE STATEMENT In the present study, we characterized the naturally occurring nerve growth factor NGFR100W mutant that is associated with hereditary sensory and autonomic neuropathy type V. We have demonstrated for the first time that NGFR100W retains trophic support capability through TrkA, but fails to engage p75NTR signaling pathways. Furthermore, after intraplantar injection into adult rats, NGFR100W induced neither thermal nor mechanical acute hyperalgesia, but retained the ability to induce chronic hyperalgesia. We have also provided evidence that the integration of both TrkA- and p75NTR-mediated signaling appears to regulate neuroplastic effects of NGF in peripheral nociception. Our study with NGFR100W suggests that it is possible to uncouple trophic effect from nociceptive function, both induced by wild-type NGF.

Fentanyl Induces Rapid Onset Hyperalgesic Priming: Type I at Peripheral and Type II at Central Nociceptor Terminals.

Systemic fentanyl induces hyperalgesic priming, long-lasting neuroplasticity in nociceptor function characterized by prolongation of inflammatory mediator hyperalgesia. To evaluate priming at both nociceptor terminals, we studied, in male Sprague Dawley rats, the effect of local administration of agents that reverse type I (protein translation) or type II [combination of Src and mitogen-activated protein kinase (MAPK)] priming. At the central terminal, priming induced by systemic, intradermal, or intrathecal fentanyl was reversed by the combination of Src and MAPK inhibitors, but at the peripheral terminal, it was reversed by the protein translation inhibitor. Mu-opioid receptor (MOR) antisense prevented fentanyl hyperalgesia and priming. To determine whether type I and II priming occur in the same population of neurons, we used isolectin B4-saporin or [Sar9, Met(O2)11]-substance P-saporin to deplete nonpeptidergic or peptidergic nociceptors, respectively. Following intrathecal fentanyl, central terminal priming was prevented by both saporins, whereas that in peripheral terminal was not attenuated even by their combination. However, after intradermal fentanyl, priming in the peripheral terminal requires both peptidergic and nonpeptidergic nociceptors, whereas that in the central terminal is dependent only on peptidergic nociceptors. Pretreatment with dantrolene at either terminal prevented fentanyl-induced priming in both terminals, suggesting communication between central and peripheral terminals mediated by intracellular Ca2+ signaling. In vitro application of fentanyl increased cytoplasmic Ca2+ concentration in dorsal root ganglion neurons, which was prevented by pretreatment with dantrolene and naloxone. Therefore, acting at MOR in the nociceptor, fentanyl induces hyperalgesia and priming rapidly at both the central (type II) and peripheral (type I) terminal and this is mediated by Ca2+ signaling.SIGNIFICANCE STATEMENT Fentanyl, acting at the µ-opioid receptor (MOR), induces hyperalgesia and hyperalgesic priming at both the central and peripheral terminal of nociceptors and this is mediated by endoplasmic reticulum Ca2+ signaling. Priming in the central terminal is type II, whereas that in the peripheral terminal is type I. Our findings may provide useful information for the design of drugs with improved therapeutic profiles, selectively disrupting individual MOR signaling pathways, to maintain an adequate long-lasting control of pain.

Role of GPCR (mu-opioid)-receptor tyrosine kinase (epidermal growth factor) crosstalk in opioid-induced hyperalgesic priming (type II).

Repeated stimulation of mu-opioid receptors (MORs), by an MOR-selective agonist DAMGO induces type II priming, a form of nociceptor neuroplasticity, which has 2 components: opioid-induced hyperalgesia (OIH) and prolongation of prostaglandin-E2 (PGE2)-induced hyperalgesia. We report that intrathecal antisense knockdown of the MOR in nociceptors, prevented the induction of both components of type II priming. Type II priming was also eliminated by SSP-saporin, which destroys the peptidergic class of nociceptors. Because the epidermal growth factor receptor (EGFR) participates in MOR signaling, we tested its role in type II priming. The EGFR inhibitor, tyrphostin AG 1478, prevented the induction of prolonged PGE2-induced hyperalgesia, but not OIH, when tested out to 30 days after DAMGO. However, even when repeatedly injected, an EGFR agonist did not induce hyperalgesia or priming. A phosphopeptide, which blocks the interaction of Src, focal adhesion kinase (FAK), and EGFR, also prevented DAMGO-induced prolongation of PGE2 hyperalgesia, but only partially attenuated the induction of OIH. Inhibitors of Src and mitogen-activated protein kinase (MAPK) also only attenuated OIH. Inhibitors of matrix metalloproteinase, which cleaves EGF from membrane protein, markedly attenuated the expression, but did not prevent the induction, of prolongation of PGE2 hyperalgesia. Thus, although the induction of prolongation of PGE2-induced hyperalgesia at the peripheral terminal of peptidergic nociceptor is dependent on Src, FAK, EGFR, and MAPK signaling, Src, FAK, and MAPK signaling is only partially involved in the induction of OIH.

CD44 Signaling Mediates High Molecular Weight Hyaluronan-Induced Antihyperalgesia.

We studied, in male Sprague Dawley rats, the role of the cognate hyaluronan receptor, CD44 signaling in the antihyperalgesia induced by high molecular weight hyaluronan (HMWH). Low molecular weight hyaluronan (LMWH) acts at both peptidergic and nonpeptidergic nociceptors to induce mechanical hyperalgesia that is prevented by intrathecal oligodeoxynucleotide antisense to CD44 mRNA, which also prevents hyperalgesia induced by a CD44 receptor agonist, A6. Ongoing LMWH and A6 hyperalgesia are reversed by HMWH. HMWH also reverses the hyperalgesia induced by diverse pronociceptive mediators, prostaglandin E2, epinephrine, TNFα, and interleukin-6, and the neuropathic pain induced by the cancer chemotherapy paclitaxel. Although CD44 antisense has no effect on the hyperalgesia induced by inflammatory mediators or paclitaxel, it eliminates the antihyperalgesic effect of HMWH. HMWH also reverses the hyperalgesia induced by activation of intracellular second messengers, PKA and PKCε, indicating that HMWH-induced antihyperalgesia, although dependent on CD44, is mediated by an intracellular signaling pathway rather than as a competitive receptor antagonist. Sensitization of cultured small-diameter DRG neurons by prostaglandin E2 is also prevented and reversed by HMWH. These results demonstrate the central role of CD44 signaling in HMWH-induced antihyperalgesia, and establish it as a therapeutic target against inflammatory and neuropathic pain.SIGNIFICANCE STATEMENT We demonstrate that hyaluronan (HA) with different molecular weights produces opposing nociceptive effects. While low molecular weight HA increases sensitivity to mechanical stimulation, high molecular weight HA reduces sensitization, attenuating inflammatory and neuropathic hyperalgesia. Both pronociceptive and antinociceptive effects of HA are mediated by activation of signaling pathways downstream CD44, the cognate HA receptor, in nociceptors. These results contribute to our understanding of the role of the extracellular matrix in pain, and indicate CD44 as a potential therapeutic target to alleviate inflammatory and neuropathic pain.

Age-Dependent Sexual Dimorphism in Susceptibility to Develop Chronic Pain in the Rat.

Neonatal pain has been suggested to contribute to the development and/or persistence of adult pain. Observations from animal models have shown that neonatal inflammation produces long-term changes in sensory neuron function, which can affect the susceptibility of adults to develop persistent pain. We used a preclinical model of transition to chronic pain, hyperalgesic priming, in which a previous inflammatory stimulus triggers a long-lasting increase in responsiveness to pro-algesic mediators, prototypically prostaglandin E2 (PGE2), to investigate if post-natal age influences susceptibility of adult rats to develop chronic pain. Priming was induced by tumor necrosis factor alpha (TNFα), in male and female rats, 1, 2, 3, 4, 5 or 7weeks after birth. When adults (8weeks after birth), to evaluate for the presence of priming, PGE2 was injected at the same site as TNFα. In males that had received TNFα at post-natal weeks 1, 2 or 3, priming was attenuated compared to the 4-, 5- and 7-week-old treated groups, in which robust priming developed. In contrast, in females treated with TNFα at post-natal week 1, 2, 3, or 4, but not at 5 or 7, priming was present. This age and sex difference in the susceptibility to priming was estrogen-dependent, since injection of TNFα in 3-week-old males and 5-week-old females, in the presence of the estrogen receptor antagonist ICI 182,780, did produce priming. These results suggest that estrogen levels, which vary differently in males and females over the post-natal period, until they stabilize after puberty, impact pain as an adult.

Hyperalgesic priming (type II) induced by repeated opioid exposure: maintenance mechanisms.

We previously developed a model of opioid-induced neuroplasticity in the peripheral terminal of the nociceptor that could contribute to opioid-induced hyperalgesia, type II hyperalgesic priming. Repeated administration of mu-opioid receptor (MOR) agonists, such as DAMGO, at the peripheral terminal of the nociceptor, induces long-lasting plasticity expressed, prototypically as opioid-induced hyperalgesia and prolongation of prostaglandin E2-induced hyperalgesia. In this study, we evaluated the mechanisms involved in the maintenance of type II priming. Opioid receptor antagonist, naloxone, induced hyperalgesia in DAMGO-primed paws. When repeatedly injected, naloxone-induced hyperalgesia, and hyperalgesic priming, supporting the suggestion that maintenance of priming involves changes in MOR signaling. However, the knockdown of MOR with oligodeoxynucleotide antisense did not reverse priming. Mitogen-activated protein kinase and focal adhesion kinase, which are involved in the Src signaling pathway, previously implicated in type II priming, also inhibited the expression, but not maintenance of priming. However, when Src and mitogen-activated protein kinase inhibitors were coadministered, type II priming was reversed, in male rats. A second model of priming, latent sensitization, induced by complete Freund's adjuvant was also reversed, in males. In females, the inhibitor combination was only able to inhibit the expression and maintenance of DAMGO-induced priming when knockdown of G-protein-coupled estrogen receptor 30 (GPR30) in the nociceptor was performed. These findings demonstrate that the maintenance of DAMGO-induced type II priming, and latent sensitization is mediated by an interaction between, Src and MAP kinases, which in females is GPR30 dependent.

Sexual Dimorphism in a Reciprocal Interaction of Ryanodine and IP3 Receptors in the Induction of Hyperalgesic Priming.

Hyperalgesic priming, a model of pain chronification in the rat, is mediated by ryanodine receptor-dependent calcium release. Although ryanodine induces priming in both sexes, females are 5 orders of magnitude more sensitive, by an estrogen receptor α (EsRα)-dependent mechanism. An inositol 1,4,5-triphosphate (IP3) receptor inhibitor prevented the induction of priming by ryanodine. For IP3 induced priming, females were also more sensitive. IP3-induced priming was prevented by pretreatment with inhibitors of the sarcoendoplasmic reticulum calcium ATPase and ryanodine receptor. Antisense to EsRα prevented the induction of priming by low-dose IP3 in females. The induction of priming by an EsRα agonist was ryanodine receptor-dependent and prevented by the IP3 antagonist. Thus, an EsRα-dependent bidirectional interaction between endoplasmic reticulum IP3 and ryanodine receptor-mediated calcium signaling is present in the induction of hyperalgesic priming, in females. In cultured male DRG neurons, IP3 (100 µm) potentiated depolarization-induced transients produced by extracellular application of high-potassium solution (20 mm, K20), in nociceptors incubated with ß-estradiol. This potentiation of depolarization-induced calcium transients was blocked by the IP3 antagonist, and not observed in the absence of IP3 IP3 potentiation was also blocked by ryanodine receptor antagonist. The application of ryanodine (2 nm), instead of IP3, also potentiated K20-induced calcium transients in the presence of ß-estradiol, in an IP3 receptor-dependent manner. Our results point to an EsRα-dependent, reciprocal interaction between IP3 and ryanodine receptors that contributes to sex differences in hyperalgesic priming.SIGNIFICANCE STATEMENT The present study demonstrates a mechanism that plays a role in the marked sexual dimorphism observed in a model of the transition to chronic pain, hyperalgesic priming. This mechanism involves a reciprocal interaction between the endoplasmic reticulum receptors, IP3 and ryanodine, in the induction of priming, regulated by estrogen receptor α in the nociceptor of female rats. The presence of this signaling pathway modulating the susceptibility of nociceptors to develop plasticity may contribute to our understanding of sex differences observed clinically in chronic pain syndromes.

Regulation of Expression of Hyperalgesic Priming by Estrogen Receptor α in the Rat.

Hyperalgesic priming, a sexually dimorphic model of transition to chronic pain, is expressed as prolongation of prostaglandin E2-induced hyperalgesia by the activation of an additional pathway including an autocrine mechanism at the plasma membrane. The autocrine mechanism involves the transport of cyclic adenosine monophosphate (AMP) to the extracellular space, and its conversion to AMP and adenosine, by ecto-5'phosphodiesterase and ecto-5'nucleotidase, respectively. The end product, adenosine, activates A1 receptors, producing delayed onset prolongation of prostaglandin E2 hyperalgesia. We tested the hypothesis that the previously reported, estrogen-dependent, sexual dimorphism observed in the induction of priming is present in the mechanisms involved in its expression, as a regulatory effect on ecto-5'nucleotidase by estrogen receptor α (EsRα), in female rats. In the primed paw AMP hyperalgesia was dependent on conversion to adenosine, being prevented by ecto-5'nucleotidase inhibitor α,ß-methyleneadenosine 5'-diphosphate sodium salt and A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. To investigate an interaction between EsRα and ecto-5'nucleotidase, we treated primed female rats with oligodeoxynucleotide antisense or mismatch against EsRα messenger RNA. Whereas in rats treated with antisense AMP-induced hyperalgesia was abolished, the A1 receptor agonist N6-cyclopentiladenosine still produced hyperalgesia. Thus, EsRα interacts with this autocrine pathway at the level of ecto-5'nucleotidase. These results demonstrate a sexually dimorphic mechanism for the expression of priming. PERSPECTIVE: This study presents evidence of an estrogen-dependent mechanism of expression of chronic pain in female rats, supporting the suggestion that differential targets must be considered when establishing protocols for the treatment of painful conditions in men and women.

Marked sexual dimorphism in 5-HT1 receptors mediating pronociceptive effects of sumatriptan.

Amongst the side effects of triptans, a substantial percentage of patients experience injection site pain and tenderness, the underlying mechanism of which is unknown. We found that the dose range from 10fg to 1000ng (intradermal) of sumatriptan induced a complex dose-dependent mechanical hyperalgesia in male rats, with distinct peaks, at 1pg and 10ng, but no hyperalgesia at 1ng. In contrast, in females, there was 1 broad peak. The highest dose (1000ng) did not produce hyperalgesia in either sex. We evaluated the receptors mediating sumatriptan hyperalgesia (1pg, 1 and 10ng). In males, the injection of an antagonist for the serotonin (5-HT) receptor subtype 1B (5-HT1B), but not 5-HT1D, markedly inhibited sumatriptan (1pg)-induced hyperalgesia, at 10ng a 5-HT1D receptor antagonist completely eliminated hyperalgesia. In contrast, in females, the 5-HT1D, but not 5-HT1B, receptor antagonist completely blocked sumatriptan (1pg and 10ng) hyperalgesia and both 5-HT1B and 5-HT1D receptor antagonists attenuated hyperalgesia (1ng) in females, which is GPR30 estrogen receptor dependent. While selective 5-HT1D or 5-HT1B, agonists produce robust hyperalgesia in female and male rats, respectively, when co-injected the hyperalgesia induced in both sexes was attenuated. Mechanical hyperalgesia induced by sumatriptan (1pg and 10ng) is dependent on the G-protein αi subunit and protein kinase A (PKA), in IB4-positive and negative nociceptors. Understanding the mechanisms responsible for the complex dose dependence for triptan hyperalgesia may provide useful information for the design of anti-migraine drugs with improved therapeutic profiles.

Marked Sexual Dimorphism in the Role of the Ryanodine Receptor in a Model of Pain Chronification in the Rat.

Hyperalgesic priming, an estrogen dependent model of the transition to chronic pain, produced by agonists at receptors that activate protein kinase C epsilon (PKCε), occurs in male but not in female rats. However, activation of second messengers downstream of PKCε, such as the ryanodine receptor, induces priming in both sexes. Since estrogen regulates intracellular calcium, we investigated the interaction between estrogen and ryanodine in the susceptibility to develop priming in females. The lowest dose of ryanodine able to induce priming in females (1 pg) is 1/100,000(th) that needed in males (100 ng), an effect dependent on the activation of ryanodine receptors. Treatment of female rats with antisense to estrogen receptor alpha (ERα), but not beta (ERß), mRNA, prevented the induction of priming by low dose ryanodine, and the ERα agonist, PPT, induced ryanodine receptor-dependent priming. In vitro application of ryanodine in low concentration (2 nM) to small DRG neurons cultured from females, significantly potentiated calcium release via ryanodine receptors induced by caffeine. This effect was only observed in IB4+ neurons, cultured in the presence of ß-estradiol or PPT. Our results demonstrate a profound regulatory role of ERα in ryanodine receptor-dependent transition to chronic pain.

Gi-protein-coupled 5-HT1B/D receptor agonist sumatriptan induces type I hyperalgesic priming.

We have recently described a novel form of hyperalgesic priming (type II) induced by agonists at two clinically important Gi-protein-coupled receptors (Gi-GPCRs), mu-opioid and A1-adenosine. Like mu-opioids, the antimigraine triptans, which act at 5-HT1B/D Gi-GPCRs, have been implicated in pain chronification. We determined whether sumatriptan, a prototypical 5-HT1B/D agonist, produces type II priming. Characteristic of hyperalgesic priming, intradermal injection of sumatriptan (10 ng) induced a change in nociceptor function such that a subsequent injection of prostaglandin-E2 (PGE2) induces prolonged mechanical hyperalgesia. However, onset to priming was delayed 3 days, characteristic of type I priming. Also characteristic of type I priming, a protein kinase Cε, but not a protein kinase A inhibitor attenuated the prolongation phase of PGE2 hyperalgesia. The prolongation of PGE2 hyperalgesia was also permanently reversed by intradermal injection of cordycepin, a protein translation inhibitor. Also, hyperalgesic priming did not occur in animals pretreated with pertussis toxin or isolectin B4-positive nociceptor toxin, IB4-saporin. Finally, as observed for other agonists that induce type I priming, sumatriptan did not induce priming in female rats. The prolongation of PGE2 hyperalgesia induced by sumatriptan was partially prevented by coinjection of antagonists for the 5-HT1B and 5-HT1D, but not 5-HT7, serotonin receptors and completely prevented by coadministration of a combination of the 5-HT1B and 5-HT1D antagonists. Moreover, the injection of selective agonists, for 5-HT1B and 5-HT1D receptors, also induced hyperalgesic priming. Our results suggest that sumatriptan, which signals through Gi-GPCRs, induces type I hyperalgesic priming, unlike agonists at other Gi-GPCRs, which induce type II priming.

Adenosine-A1 receptor agonist induced hyperalgesic priming type II.

We have recently shown that repeated exposure of the peripheral terminal of the primary afferent nociceptor to the mu-opioid receptor (MOR) agonist DAMGO ([D-Ala, N-Me-Phe, Gly-ol]-enkephalin acetate salt) induces a model of transition to chronic pain that we have termed type II hyperalgesic priming. Similar to type I hyperalgesic priming, there is a markedly prolonged response to subsequent administration of proalgesic cytokines, prototypically prostaglandin E2 (PGE2). However, type II hyperalgesic priming differs from type I in being rapidly induced, protein kinase A (PKA), rather than PKCε dependent, not reversed by a protein translation inhibitor, occurring in female as well as in male rats, and isolectin B4-negative neuron dependent. We report that, as with the repeated injection of a MOR agonist, the repeated administration of an agonist at the A1-adenosine receptor, also a Gi-protein coupled receptor, N-cyclopentyladenosine (CPA), also produces priming similar to DAMGO-induced type II hyperalgesic priming. In this study, we demonstrate that priming induced by repeated exposure to this A1-adenosine receptor agonist shares the same mechanisms, as MOR-agonist induced priming. However, the prolongation of PGE2 hyperalgesia induced by repeated administration of CPA depends on G-protein αi subunit activation, differently from DAMGO-induced type II priming, in which it depends on the ß/γ subunit. These data implicate a novel form of Gi-protein signaling pathway in the type II hyperalgesic priming induced by repeated administration of an agonist at A1-adenosine receptor to the peripheral terminal of the nociceptor.