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Recent studies have revealed that glial sigma-1 receptor (Sig-1R) in the spinal cord may be a critical factor to mediate sensory function. However, the functional role of Sig-1R in astrocyte has not been clearly elucidated. Here, we determined whether Sig-1Rs modulate calcium responses in primary cultured astrocytes and pathological changes in spinal astrocytes, and whether they contribute to pain hypersensitivity in naive mice and neuropathic pain following chronic constriction injury (CCI) of the sciatic nerve in mice. Sig-1R was expressed in glial fibrillary acidic protein (GFAP)-positive cultured astrocytes. Treatment with the Sig-1R agonist, PRE-084 or neurosteroid dehydroepiandrosterone (DHEA) increased intracellular calcium responses in cultured astrocytes, and this increase was blocked by the pretreatment with the Sig-1R antagonist, BD-1047 or neurosteroid progesterone. Intrathecal administration of PRE-084 or DHEA for 10 days induced mechanical and thermal hypersensitivity and increased the number of Sig-1R-immunostained GFAP-positive cells in the superficial dorsal horn (SDH) region of the spinal cord in naive mice, and these changes were inhibited by administration with BD-1047 or progesterone. In CCI mice, intrathecal administration of BD-1047 or progesterone at post-operative day 14 suppressed the developed mechanical allodynia and the number of Sig-1R-immunostained GFAP-positive cells that were increased in the SDH region of the spinal cord following CCI of the sciatic nerve. These results demonstrate that Sig-1Rs play an important role in the modulation of intracellular calcium responses in cultured astrocytes and pathological changes in spinal astrocytes and that administration of BD-1047 or progesterone alleviates the Sig-1R-induced pain hypersensitivity and the peripheral nerve injury-induced mechanical allodynia.

The sigma-1 receptor (Sig-1R) plays an important role in spinal pain transmission by increasing phosphorylation of the N-methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). As a result Sig-1R has been suggested as a novel therapeutic target for prevention of chronic pain. Here we investigated whether interleukin-1β (IL-1β) modulates the expression of the Sig-1R in spinal astrocytes during the early phase of nerve injury, and whether this modulation affects spinal pGluN1 expression and the development of neuropathic pain following chronic constriction injury (CCI) of the sciatic nerve. Repeated intrathecal (i.t.) administration of IL-1β from days 0-3 post-surgery significantly reduced the increased pGluN1 expression at the Ser896 and Ser897 sites in the ipsilateral spinal cord, as well as, the development of mechanical allodynia and thermal hyperalgesia in the ipsilateral hind paw of CCI mice, which were restored by co-administration of IL-1 receptor antagonist with IL-1β. Sciatic nerve injury increased the expression of Sig-1R in astrocytes of the ipsilateral spinal cord, and this increase was suppressed by i.t. administration of IL-1β. Agonistic stimulation of the Sig-1R with PRE084 restored pGluN1 expression and the development of mechanical allodynia that were originally suppressed by IL-1β in CCI mice. Collectively these results demonstrate that IL-1β administration during the induction phase of neuropathic pain produces an analgesic effect on neuropathic pain development by controlling the expression of Sig-1R in spinal astrocytes.

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the high mortality and sequelae in low- and middle-income countries. Burns frequently induce intense spontaneous pain and persistent allodynia as well as life-threatening problem. More importantly, excessive pain is often accompanied by depression, which may significantly decrease the quality of life. This article shows how to develop an animal model for the study of burn-induced pain and depression-like behavior. After anesthesia, burn injury was induced by dipping one hind paw of the mouse into hot water (65 °C ± 0.5 °C) for 3 s. The von Frey test and automated gait analysis were performed every 2 days after burn injury. In addition, depression-like behavior was examined using the forced swimming test, and the rota-rod test was performed to differentiate the abnormal motor function after burn injury. The main purpose of this study is to describe the development of an animal model for the study of burn injury-induced pain and depression-like behavior in mice.

While evidence indicates that sigma-1 receptors (Sig-1Rs) play an important role in the induction of peripheral neuropathic pain, there is limited understanding of the role that the neurosteroidogenic enzymes, which produce Sig-1R endogenous ligands, play during the development of neuropathic pain. We examined whether sciatic nerve injury upregulates the neurosteroidogenic enzymes, cytochrome P450c17 and 3β-hydroxysteroid dehydrogenase (3β-HSD), which modulate the expression and/or activation of Sig-1Rs leading to the development of peripheral neuropathic pain. Chronic constriction injury (CCI) of the sciatic nerve induced a significant increase in the expression of P450c17, but not 3β-HSD, in the ipsilateral lumbar spinal cord dorsal horn at postoperative day 3. Intrathecal administration of the P450c17 inhibitor, ketoconazole during the induction phase of neuropathic pain (day 0 to day 3 post-surgery) significantly reduced the development of mechanical allodynia and thermal hyperalgesia in the ipsilateral hind paw. However, administration of the 3β-HSD inhibitor, trilostane had no effect on the development of neuropathic pain. Sciatic nerve injury increased astrocyte Sig-1R expression as well as dissociation of Sig-1Rs from BiP in the spinal cord. These increases were suppressed by administration of ketoconazole, but not by administration of trilostane. Co-administration of the Sig-1R agonist, PRE084 restored the development of mechanical allodynia originally suppressed by the ketoconazole administration. However, ketoconazole-induced inhibition of thermal hyperalgesia was not affected by co-administration of PRE084. Collectively these results demonstrate that early activation of P450c17 modulates the expression and activation of astrocyte Sig-1Rs, ultimately contributing to the development of mechanical allodynia induced by peripheral nerve injury.

Although emerging evidence shows that angiotensin converting enzyme (ACE) is associated with pain, it is not clear whether inhibition of ACE could affect to nociceptive transmission and which mediators are involved in this process. Here we investigated whether administration of the ACE inhibitors, captopril and enalapril increases the expression of substance P (SP) and whether this increase contributes to the induction of mechanical allodynia in mice. ACE was expressed in the lumbar dorsal root ganglion (DRG) and the superficial dorsal horn (SDH) region of the spinal cord in mice. Either intraperitoneal or intrathecal administration of the ACE inhibitors, captopril and enalapril for 10 days significantly increased the paw withdrawal frequency to innocuous mechanical stimuli and the levels of SP in both the lumbar DRG and the SDH region of the spinal cord dorsal horn. In addition, intraperitoneal administration of the SP receptor (neurokinin-1 receptor) antagonist, L-733,060 suppressed mechanical allodynia that was induced by pretreatment of captopril and enalapril. Intraplantar administration of SP for 3 days induces mechanical allodynia, and this effect was reduced by exogenous ACE administration. These findings demonstrate that inhibition of ACE increases the levels of SP in both the lumbar DRG and spinal cord dorsal horn, ultimately contributing to the induction of mechanical allodynia in mice.

Research indicates that neurosteroids are locally synthesized in the central nervous system and play an important modulatory role in nociception. While the neurosteroidogenic enzyme, cytochrome P450 side-chain cleavage enzyme (P450scc), is the initiating enzyme of steroidogenesis, P450scc has not been examined under the pathophysiological conditions associated with peripheral neuropathy. Thus, we investigated whether chronic constriction injury (CCI) of the sciatic nerve increases the expression of P450scc in the spinal cord and whether this increase modulates serine racemase (Srr) expression and D-serine production contributing to the development of neuropathic pain. CCI increased the immunoreactivity of P450scc in astrocytes of the ipsilateral lumbar spinal cord dorsal horn. Intrathecal administration of the P450scc inhibitor, aminoglutethimide, during the induction phase of neuropathic pain (days 0 to 3 post-surgery) significantly suppressed the CCI-induced development of mechanical allodynia and thermal hyperalgesia, the increased expression of astrocyte Srr in both the total and cytosol levels, and the increases in D-serine immunoreactivity at day 3 post-surgery. By contrast, intrathecal administration of aminoglutethimide during the maintenance phase of pain (days 14 to 17 post-surgery) had no effect on the developed neuropathic pain nor the expression of spinal Srr and D-serine immunoreactivity at day 17 post-surgery. Intrathecal administration of exogenous D-serine during the induction phase of neuropathic pain (days 0 to 3 post-surgery) restored the development of mechanical allodynia, but not the thermal hyperalgesia, that were suppressed by aminoglutethimide administration. Collectively, these results demonstrate that spinal P450scc increases the expression of astrocyte Srr and D-serine production, ultimately contributing to the development of mechanical allodynia induced by peripheral nerve injury.

Aromatase is a key enzyme responsible for the biosynthesis of estrogen from testosterone. Although recent evidence indicates that spinal cord aromatase participates in nociceptive processing, the mechanisms underlying its regulation and its involvement in nociception remain unclear. The present study focuses on the potential role of astrocyte aromatase in formalin-induced acute pain and begins to uncover one mechanism by which spinal aromatase activation is controlled. Following intraplantar formalin injection, nociceptive responses were quantified and immunohistochemistry/co-immunoprecipitation assays were used to investigate the changes in spinal Fos expression and the phospho-serine levels of spinal aromatase. Intrathecal (i.t.) injection of letrozole (an aromatase inhibitor) mitigated both the late phase formalin-induced nociceptive responses and formalin-induced spinal Fos expression. Furthermore, formalin-injected mice showed significantly reduced phospho-serine levels of aromatase, which is associated with the rapid activation of this enzyme. However, sigma-1 receptor inhibition with i.t. BD1047 blocked the dephosphorylation of aromatase and potentiated the pharmacological effect of letrozole on formalin-induced nociceptive responses. In addition, i.t. administration of a sub-effective dose of BD1047 potentiated the pharmacological effect of cyclosporin A (a calcineurin inhibitor) on both the formalin-induced reduction in phospho-serine levels of aromatase and nociceptive behavior. These results suggest that dephosphorylation is an important regulatory mechanism involved in the rapid activation of aromatase and that spinal sigma-1 receptors mediate this dephosphorylation of aromatase through an intrinsic calcineurin pathway.

Background and purpose Although we have recently demonstrated that spinal astrocyte gap junctions mediate the development of mirror-image pain (MIP), it is still unclear which astrocyte-derived factor is responsible for the development of MIP and how its production is controlled. In the present study, we focused on the role of ipsilateral versus contralateral D-serine in the development of MIP and investigated the possible involvement of σ1 receptors and gap junctions in astrocyte D-serine production. Experimental approach Following carrageenan injection, mechanical allodynia was tested at various time points to examine the effect of individual drugs. Immunohistochemistry and Western blot analyses were performed to clarify the expression levels of spinal D-serine, serine racemase, σ1 receptors and connexin 43. Key results The expression of ipsilateral D-serine was up-regulated during the early phase of inflammation, while contralateral D-serine increased during the later phase of inflammation. The pharmacological inhibition of D-serine during the early phase blocked the development of both ipsilateral and contralateral mechanical allodynia. However, the inhibition of D-serine during the later phase of inflammation blocked contralateral, but not ipsilateral mechanical allodynia. Furthermore, the inhibition of σ1 receptors during the earlier phase of inflammation inhibited the increase in ipsilateral D-serine. Conversely, the blockade of astrocyte gap junctions suppressed the up-regulation of contralateral D-serine during the later phase of inflammation. Conclusion and implications Spinal astrocyte D-serine plays an important role in the development of mirror-image pain. Furthermore, σ1 receptors and astrocyte gap junction signalling mediate ipsilateral and contralateral D-serine production respectively.

Although we have recently demonstrated that carrageenan-induced inflammation upregulates the expression of spinal interleukin (IL)-1β, which inhibits spinal astrocyte activation and results in the delayed development of Mirror-Image Pain (MIP), little is known regarding the mechanisms that underlie how spinal IL-1β inhibits the astrocyte activation. In this study, we examined the effect of spinal IL-1β on astrocyte gap junctions (GJ) and the development of MIP. Following unilateral carrageenan (CA) injection, mechanical allodynia (MA) was evaluated at various time points. Immunohistochemistry and Western blot analysis were used to determine changes in the expression of GFAP and connexins (Cx) in the spinal cord dorsal horn. Carrageenan rats showed a delayed onset of contralateral MA, which mimicked the temporal expression pattern of spinal Cx43 (an astrocyte gap junctional protein) and GFAP. Intrathecal administration of an interleukin-1 receptor antagonist (IL-1ra) twice-a-day on post-carrageenan injection days 0 to 3 caused a significant increase in contralateral MA and spinal Cx43 and GFAP expression. In addition, co-administration of IL-1β with IL-1ra blocked the IL-1ra-induced increase in contralateral MA and the upregulated expression of spinal Cx43 and GFAP. Finally, co-administration of carbenoxolone (CBX; a GJ decoupler) or Gap26 (a specific Cx43 mimetic blocking peptide) with IL-1ra significantly blocked the IL-1ra-induced early development of contralateral MA and the associated upregulation of spinal Cx43 and GFAP expression. These results demonstrate that spinal IL-1β suppresses Cx43 expression and astrocyte activation during the early phase of CA-induced inflammation resulting in the delayed onset of contralateral MA. These findings imply that spinal IL-1β can inhibit astrocyte activation and regulate the time of induction of contralateral MA through modulation of spinal Cx43 expression.

We have recently demonstrated that the neurosteroid-metabolizing enzyme, cytochrome P450c17 is increased in spinal astrocytes contributing to the development of mechanical allodynia in chronic constriction injury (CCI)-induced neuropathic mice. However, the mechanisms by which spinal P450c17 modulates pathological changes in astrocytes remain unclear. In this study we investigated whether P450c17 modulates astrocyte activation and whether this process is mediated by spinal p38 mitogen-activated protein kinase phosphorylation ultimately leading to the development of mechanical allodynia in CCI mice. Sciatic nerve injury induced a significant increase in glial fibrillary acidic protein (GFAP) expression in the superficial dorsal horn (SDH, laminae I-II) and nucleus proprius (NP, laminae III-IV) regions of the spinal cord dorsal horn. Repeated daily (from days 0–3 post-surgery) intrathecal administration of the P450c17 inhibitor, ketoconazole (10 nmol) significantly inhibited the CCI-induced increase in GFAP-immunoreactivity, but had no effect on the CCI-induced increase in Iba-1-immunoreactivity. In addition, intrathecal administration of ketoconazole significantly inhibited the CCI-induced increase in p38 phosphorylation, while the levels of ERK and JNK phosphorylation remained unchanged. The CCI-induced development of mechanical allodynia was attenuated by administration of either ketoconazole (10 nmol) or the p38 MAPK inhibitor, SB203580 (5 nmol). Administration of a sub-effective dose of SB203580 (0.5 nmol) potentiated the pharmacological effect of ketoconazole (1 nmol) on spinal GFAP-immunostaining, as well as, the development of mechanical allodynia following CCI. Collectively these data suggest that spinal P450c17 activates astrocytes via p38 phosphorylation, ultimately leading to the development of mechanical allodynia in a model of peripheral neuropathy.

We have recently demonstrated that sciatic nerve injury increases the expression of spinal cytochrome P450c17, a key neurosteroidogenic enzyme, which plays a critical role in the development of peripheral neuropathic pain. However, the modulatory mechanisms responsible for the expression of spinal P450c17 have yet to be examined. Here we investigated the possible involvement of interleukin-1β (IL-1β) in altering P450c17 expression during the induction phase of neuropathic pain. Neuropathic pain was produced by chronic constriction injury (CCI) of the right sciatic nerve in mice and mechanical allodynia was evaluated in the hind paws using a von-Frey filament (0.16 g). Western blotting and immunohistochemistry were performed to assess the expression of spinal IL-1β, interleukin-1 receptor type 1 (IL-1R1), P450c17, and GFAP. Spinal IL-1β was significantly increased on day 1 post-surgery and its receptor, IL-1R1 was expressed in GFAP-positive astrocytes. Intrathecal administration of the recombinant interleukin-1 receptor antagonist (IL-1ra, 20 ng) on days 0 and 1 post-surgery enhanced GFAP expression on day 1 post-surgery and induced an early increase in P450c17 expression in astrocytes, but not in neurons. Administration of IL-1β (10 ng) on days 0 and 1 post-surgery blocked the enhancement of both spinal P450c17 and GFAP expression induced by IL-1ra (20 ng) administration. Intrathecal administration of IL-1ra (20 ng) on days 0 to 3 post-surgery also facilitated the CCI-induced development of mechanical allodynia, and this early developed pain was dose-dependently attenuated by the administration of the P450c17 inhibitor, ketoconazole (1, 3, or 10 nmol) or the astrocyte metabolic inhibitor, fluorocitrate (0.01, 0.03, or 0.1 nmol). These results demonstrate that early increases in spinal IL-1β temporally inhibit astrocyte P450c17 expression and astrocyte activation ultimately controlling the development of mechanical allodynia induced by peripheral nerve injury. These findings imply that spinal IL-1β plays an important role as an early, but transient, control mechanism in the development of peripheral neuropathic pain via the inhibition of astrocyte P450c17 expression and astrocyte activation.

We have previously demonstrated that the neurosteroid dehydroepiandrosterone sulfate (DHEAS) induces functional potentiation of N-methyl-D-aspartate (NMDA) receptors via increases in phosphorylation of NMDA receptor GluN1 subunit (pGluN1). However, the modulatory mechanisms responsible for the expression of the DHEA-synthesizing enzyme, cytochrome P450c17 following peripheral nerve injury have yet to be examined. Here we determined whether oxidative stress induced by the spinal activation of nitric oxide synthase type II (NOS-II) modulates the expression of P450c17 and whether this process contributes to the development of neuropathic pain in rats. Chronic constriction injury (CCI) of the sciatic nerve induced a significant increase in the expression of NOS-II in microglial cells and NO levels in the lumbar spinal cord dorsal horn at postoperative day 5. Intrathecal administration of the NOS-II inhibitor, L-NIL during the induction phase of neuropathic pain (postoperative days 0~5) significantly reduced the CCI-induced development of mechanical allodynia and thermal hyperalgesia. Sciatic nerve injury increased the expression of PKC- and PKA-dependent pGluN1 as well as the mRNA and protein levels of P450c17 in the spinal cord at postoperative day 5, and these increases were suppressed by repeated administration of L-NIL. Co-administration of DHEAS together with L-NIL restored the development of neuropathic pain and pGluN1 that were originally inhibited by L-NIL administration alone. Collectively these results provide strong support for the hypothesis that activation of NOS-II increases the mRNA and protein levels of P450c17 in the spinal cord, ultimately leading to the development of central sensitization and neuropathic pain induced by peripheral nerve injury.

It has been suggested that interactions of neuronal nitric oxide synthase (nNOS) with postsynaptic density 95 (PSD95) play important roles in the development of chronic neuropathic pain. Here we examine the possible role of nNOS-PSD95 interactions in central sensitization as represented by phosphorylation of the NMDA receptor GluN1 subunit (pGluN1) in mice with chronic constriction injury (CCI) of the sciatic nerve. Intrathecal administration of the nNOS-PSD95 interactions inhibitor, IC87201 on post-operative days 0-3 significantly reduced the CCI-induced increase in total NO levels in the lumbar spinal cord dorsal horn. IC87201 administration on post-operative days 0-3 also attenuated the CCI-induced development of mechanical allodynia (MA) and PKC-dependent (Ser896) pGluN1. Sciatic nerve injury elicited a significant translocation of the PKC-e isoform from the cytosol to the membrane fraction in the lumbar spinal cord dorsal horn on day 3 post-CCI surgery. Administration of IC87201 significantly inhibited this translocation of PKC-e, while the expression of PKC-α and -ξ in the cytosol and membrane fractions was unaffected by sciatic nerve injury or injection of IC87201. Furthermore, administration of the PKC-e inhibitor, eV1-2 on post-operative days 0-3 attenuated the CCI-induced development of MA and pGluN1. Collectively these results demonstrate that spinal nNOS-PSD95 interactions play an important role in PKC-dependent GluN1 phosphorylation via activation of the PKC-e isoform, and ultimately contributes to the development of MA in peripheral neuropathy.

We recently demonstrated that activation of spinal sigma-1 receptors (Sig-1Rs) induces pain hypersensitivity via the activation of neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (Nox2). However, the potential direct interaction between nNOS-derived nitric oxide (NO) and Nox2-derived reactive oxygen species (ROS) is poorly understood, particularly with respect to the potentiation of N-methyl-D-aspartate (NMDA) receptor activity in the spinal cord associated with the development of central sensitization. Thus, the main purpose of this study was to investigate whether Sig-1R-induced and nNOS-derived NO modulates spinal Nox2 activation leading to an increase in ROS production and ultimately to the potentiation of NMDA receptor activity and pain hypersensitivity. Intrathecal pretreatment with the nNOS inhibitor, 7-nitroindazole or with the Nox inhibitor, apocynin significantly inhibited the mechanical and thermal hypersensitivity induced by intrathecal administration of the Sig-1R agonist, 2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride (PRE084). Conversely, pretreatment with 5,10,15,20-tetrakis-(4-sulphonatophenyl)-porphyrinato iron(III) (FeTPPS; a scavenger of peroxynitrite, a toxic reaction product of NO and superoxide) had no effect on the PRE084-induced pain hypersensitivity. Pretreatment with 7-nitroindazole significantly reduced the PRE084-induced increase in Nox2 activity and concomitant ROS production in the lumbar spinal cord dorsal horn, whereas apocynin did not alter the PRE084-induced changes in nNOS phosphorylation. On the other hand pretreatment with apocynin suppressed the PRE084-induced increase in the protein kinase C (PKC)-dependent phosphorylation of NMDA receptor GluN1 subunit (pGluN1) at Ser896 site in the dorsal horn. These findings demonstrate that spinal Sig-1R-induced pain hypersensitivity is mediated by nNOS activation, which leads to an increase in Nox2 activity ultimately resulting in a ROS-induced increase in PKC-dependent pGluN1 expression.

Background The role of peripheral sigma-1 receptors (Sig-1Rs) in normal nociception and in pathologically induced pain conditions has not been thoroughly investigated. Since there is mounting evidence that Sig-1Rs modulate ischaemia-induced pathological conditions, we investigated the role of Sig-1Rs in ischaemia-induced mechanical allodynia (MA) and addressed their possible interaction with acid-sensing ion channels (ASICs) and P2X receptors at the ischaemic site. Methods We used a rodent model of hindlimb thrombus-induced ischaemic pain (TIIP) to investigate their role. Western blot was performed to observe changes in Sig-1R expression in peripheral nervous tissues. MA was measured after intraplantar (i.pl.) injections of antagonists for the Sig-1, ASIC and P2X receptors in TIIP rats or agonists of each receptor in naive rats. Results Sig-1R expression significantly increased in skin, sciatic nerve and dorsal root ganglia at 3 days post-TIIP surgery. I.pl. injections of the Sig-1R antagonist, BD-1047 on post-operative days 0–3 significantly attenuated the development of MA during the induction phase, but had no effect on MA when given during the maintenance phase (days 3–6 post-surgery). BD-1047 synergistically increased amiloride (an ASICs blocker)- and TNP-ATP (a P2X antagonist)-induced analgesic effects in TIIP rats. In naive rats, i.pl. injection of Sig-1R agonist PRE-084 alone did not produce MA; but it did induce MA when co-administered with either an acidic pH solution or a sub-effective dose of αβmeATP. Conclusion Peripheral Sig-1Rs contribute to the induction of ischaemia-induced MA via facilitation of ASICs and P2X receptors. Thus, peripheral Sig-1Rs represent a novel therapeutic target for the treatment of ischaemic pain.

Background and Purpose Spinal astrocytes have emerged as important mechanistic contributors to the genesis of mechanical allodynia (MA) in neuropathic pain. We recently demonstrated that the spinal sigma non-opioid intracellular receptor 1 (σ1 receptor) modulates p38 MAPK phosphorylation (p-p38), which plays a critical role in the induction of MA in neuropathic rats. However, the histological and physiological relationships among σ1, p-p38 and astrocyte activation is unclear.

Background We previously developed a thrombus-induced ischemic pain (TIIP) animal model, which was characterized by chronic bilateral mechanical allodynia without thermal hyperalgesia (TH). On the other hand we had shown that intraplantar injection of acidic saline facilitated ATP-induced pain, which did result in the induction of TH in normal rats. Because acidic pH and increased ATP are closely associated with ischemic conditions, this study is designed to: (1) examine whether acidic saline injection into the hind paw causes the development of TH in TIIP, but not control, animals; and (2) determine which peripheral mechanisms are involved in the development of this TH. Results Repeated intraplantar injection of pH 4.0 saline, but not pH 5.5 and 7.0 saline, for 3 days following TIIP surgery resulted in the development of TH. After pH 4.0 saline injections, protein levels of hypoxia inducible factor-1α (HIF-1α) and carbonic anhydrase II (CA II) were elevated in the plantar muscle indicating that acidic stimulation intensified ischemic insults with decreased tissue acidity. At the same time point, there were no changes in the expression of TRPV1 in hind paw skin, whereas a significant increase in TRPV1 phosphorylation (pTRPV1) was shown in acidic saline (pH 4.0) injected TIIP (AS-TIIP) animals. Moreover, intraplantar injection of chelerythrine (a PKC inhibitor) and AMG9810 (a TRPV1 antagonist) effectively alleviated the established TH. In order to investigate which proton- or ATP-sensing receptors contributed to the development of TH, amiloride (an ASICs blocker), AMG9810, TNP-ATP (a P2Xs antagonist) or MRS2179 (a P2Y1 antagonist) were pre-injected before the pH 4.0 saline. Only MRS2179 significantly prevented the induction of TH, and the increased pTRPV1 ratio was also blocked in MRS2179 injected animals. Conclusion Collectively these data show that maintenance of an acidic environment in the ischemic hind paw of TIIP rats results in the phosphorylation of TRPV1 receptors via a PKC-dependent pathway, which leads to the development of TH mimicking what occurs in chronic ischemic patients with severe acidosis. More importantly, peripheral P2Y1 receptors play a pivotal role in this process, suggesting a novel peripheral mechanism underlying the development of TH in these patients.

The direct activation of the spinal sigma-1 receptor (Sig-1R) produces mechanical allodynia (MA) and thermal hyperalgesia (TH) in mice. In addition, the blockade of the spinal Sig-1R prevents the induction of MA, but not TH in chronic constriction injury (CCI)-induced neuropathic rats. The present study was designed to investigate whether the increase in spinal p38 MAPK phosphorylation (p-p38 MAPK) mediates Sig-1R-induced MA or TH in mice and the induction of MA in neuropathic rats. MA and TH were evaluated using von Frey filaments and a hot-plate apparatus, respectively. Neuropathic pain was produced by CCI of the right sciatic nerve in rats. Western blot assay and immunohistochemistry were performed to determine the changes of p-p38 MAPK expression in the spinal cord. Intrathecal (i.t.) injection of PRE084, a selective Sig-1R agonist, into naïve mice time-dependently increased the expression of p-p38 MAPK, which was blocked by pretreatment with BD1047, a Sig-1R antagonist. I.t. pretreatment with SB203580, a p38 MAPK inhibitor also dose-dependently inhibited PRE084-induced MA, whereas TH induction was not affected. In CCI rats, i.t. injection of BD1047 during the induction phase (postoperative days 0 to 5) reduced the CCI-induced increase in p-p38 MAPK. In addition, i.t. SB203580 treatment during the induction phase also suppressed the development of CCI-induced MA, but not TH. Conversely, i.t. SB203580 treatment during the maintenance phase (postoperative days 15 to 20) had no effect on CCI-induced MA or TH. These results demonstrate that the increase in spinal p-p38 MAPK is closely associated with the induction of Sig-1R mediated MA, but not TH. Sigma-1 receptor modulation of p-p38 MAPK also plays an important role in the induction, but not the maintenance, of MA in neuropathic pain.

We have recently demonstrated that spinal sigma-1 receptors (Sig-1Rs) mediate pain hypersensitivity in mice and neuropathic pain in rats. In this study, we examine the role of NADPH oxidase 2 (Nox2)-induced reactive oxygen species (ROS) on Sig-1R-induced pain hypersensitivity and the induction of chronic neuropathic pain. Neuropathic pain was produced by chronic constriction injury (CCI) of the right sciatic nerve in rats. Mechanical allodynia and thermal hyperalgesia were evaluated in mice and CCI-rats. Western blotting and dihydroethidium (DHE) staining were performed to assess the changes in Nox2 activation and ROS production in spinal cord, respectively. Direct activation of spinal Sig-1Rs with the Sig-1R agonist, PRE084 induced mechanical allodynia and thermal hyperalgesia, which were dose-dependently attenuated by pretreatment with the ROS scavenger, NAC or the Nox inhibitor, apocynin. PRE084 also induced an increase in Nox2 activation and ROS production, which were attenuated by pretreatment with the Sig-1R antagonist, BD1047 or apocynin. CCI-induced nerve injury produced an increase in Nox2 activation and ROS production in the spinal cord, all of which were attenuated by intrathecal administration with BD1047 during the induction phase of neuropathic pain. Furthermore, administration with BD1047 or apocynin reversed CCI-induced mechanical allodynia during the induction phase, but not the maintenance phase. These findings demonstrate that spinal Sig-1Rs modulate Nox2 activation and ROS production in the spinal cord, and ultimately contribute to the Sig-1R-induced pain hypersensitivity and the peripheral nerve injury-induced induction of chronic neuropathic pain.

Although interleukin-1β (IL-1β) is a prototypical pro-inflammatory cytokine, the specific mechanisms underlying the role of its cognate receptor, the interleukin-1 receptor (IL-1R) in peripheral sensitization remain to be investigated. Since emerging evidence in the literature indicates that IL-1β can modulate membrane-bound receptors, we decided to examine the involvement of P2Y1 receptor (P2Y1R) in IL-1β induced pain and the potential interaction of P2Y1Rs and IL-1Rs in both naive and carrageenan injected rats. Intraplantar (i.pl) injection of IL-1β dose-dependently produced mechanical and thermal hypersensitivity in naive rats. Pre-treatment with IL-1ra (i.pl, 30 and 100ng), an endogenous IL-1R antagonist, prevented the IL-1β induced mechanical and thermal hypersensitivity. Pre-treatment with MRS2500 (i.pl, 1 and 3nmol), a specific P2Y1R antagonist, dose-dependently reduced IL-1β induced thermal hypersensitivity, but did not affect the development of mechanical hypersensitivity. Conversely coadministration of MRS2500 (i.pl, 0.1nmol, sub-effective dose) together with IL-1ra (10nmol, sub-effective dose) significantly reduced IL-1β induced thermal, but not mechanical hypersensitivity. We next used immunohistochemistry to demonstrate that P2Y1 and IL-1 type I receptors co-localize predominantly in small diameter neurons in the dorsal root ganglion. We also performed experiments to examine the interaction of P2Y1Rs and IL-1Rs under the inflammatory conditions induced by 2% carrageenan. Intraplantar coadministration of MRS2500 (3nmol, sub-effective dose) and IL-1ra (30ng, sub-effective dose) significantly reduced inflammatory thermal, but not mechanical, hypersensitivity. These data indicate the involvement of P2Y1Rs in IL-1β mediated pain in both naive and carrageenan injected rats. There is a positive interaction between peripheral P2Y1Rs and IL-1Rs in both IL-1β and carrageenan-induced thermal hypersensitivity.

We have recently shown that spinal sigma-1 receptor (Sig-1R) activation facilitates nociception via an increase in phosphorylation of the N -methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). The present study was designed to examine whether the Sig-1R-induced facilitative effect on NMDA-induced nociception is mediated by D-serine, and whether D-serine modulates spinal pGluN1 expression and the development of neuropathic pain after chronic constriction injury (CCI) of the sciatic nerve. Intrathecal administration of the D-serine degrading enzyme, D-amino acid oxidase attenuated the facilitation of NMDA-induced nociception induced by the Sig-1R agonist, 2-(4-morpholinethyl)1-phenylcyclohexane carboxylate. Exogenous D-serine increased protein kinase C (PKC)-dependent (Ser896) pGluN1 expression and facilitated NMDA-induced nociception, which was attenuated by preteatment with the PKC inhibitor, chelerythrine. In CCI mice, administration of the serine racemase inhibitor, L-serine O-sulfate potassium salt or D-amino acid oxidase on postoperative days 0 to 3 suppressed CCI-induced mechanical allodynia (MA) and pGluN1 expression on day 3 after CCI surgery. Intrathecal administration of D-serine restored MA as well as the GluN1 phosphorylation on day 3 after surgery that was suppressed by the Sig-1R antagonist, N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide or the astrocyte inhibitor, fluorocitrate. In contrast, D-serine had no effect on CCI-induced thermal hyperalgesia or GluN1 expression. These results indicate that spinal D-serine: 1) mediates the facilitative effect of Sig-1R on NMDA-induced nociception, 2) modulates PKC-dependent pGluN1 expression, and 3) ultimately contributes to the induction of MA after peripheral nerve injury. Perspective This report shows that reducing D-serine suppresses central sensitization and significantly alleviates peripheral nerve injury-induced chronic neuropathic pain and that this process is modulated by spinal Sig-1Rs. This preclinical evidence provides a strong rationale for using D-serine antagonists to treat peripheral nerve injury-induced neuropathy.

We have previously shown using a spinal cord injury (SCI) model that gap junctions contribute to the early spread of astrocyte activation in the lumbar spinal cord and that this astrocyte communication plays critical role in the induction of central neuropathic pain. Sigma-1 receptors (Sig-1Rs) have been implicated in spinal astrocyte activation and the development of peripheral neuropathic pain, yet their contribution to central neuropathic pain remains unknown. Thus, we investigated whether SCI upregulates spinal Sig-1Rs, which in turn increase the expression of the astrocytic gap junction protein, connexin 43 (Cx43) leading to the induction of central neuropathic pain. A thoracic spinal cord hemisection significantly increased both astrocyte activation and Cx43 expression in lumbar dorsal horn. Sig-1Rs were also increased in lumbar dorsal horn astrocytes, but not neurons or microglia. Intrathecal injection of an astrocyte metabolic inhibitor (fluorocitrate); a gap junction/hemichannel blocker (carbenoxolone); or a Cx43 mimetic peptide (43Gap26) significantly reduced SCI-induced bilateral below-level mechanical allodynia. Blockade of Sig-1Rs with BD1047 during the induction phase of pain significantly suppressed the SCI-induced development of mechanical allodynia, astrocyte activation, increased expression of Cx43 in both total and membrane levels, and increased association of Cx43 with Sig-1R. However, SCI did not change the expression of oligodendrocyte (Cx32) or neuronal (Cx36) gap junction proteins. These findings demonstrate that SCI activates astrocyte Sig-1Rs leading to increases in the expression of the gap junction protein, Cx43 and astrocyte activation in the lumbar dorsal horn, and ultimately contribute to the induction of bilateral below-level mechanical allodynia.

BACKGROUND AND PURPOSE We recently demonstrated that activation of the spinal sigma-1 receptor induces mechanical and thermal hypersensitivity via calcium-dependent second messenger cascades and phosphorylation of the spinal NMDA receptor GluN1 subunit (pGluN1). Here we examined the role of NO in this process, as it plays a critical role in PKC-mediated calcium signalling and the potentiation of NMDA receptor function.

Peripheral ischemia is commonly associated with an increase in tissue ATP concentration and a decrease in tissue pH. Although in vitro data suggest that low tissue pH can affect ATP-binding affinities to P2 receptors, the mechanistic relationship between ATP and low pH on peripheral nociception has not been fully examined. This study was designed to investigate the potential role of an acidified environment on intraplantar αβmeATP-induced peripheral pain responses in rats. The mechanical allodynia (MA) produced by injection of αβmeATP was significantly increased in animals that received the drug diluted in pH 4.0 saline compared to those that received the drug diluted in pH 7.0 saline. Moreover, animals injected with αβmeATP (100 nmol) in pH 4.0 saline developed thermal hyperalgesia (TH), which did not occur in animals treated with αβmeATP diluted in pH 7.0 saline. To elucidate which receptors were involved in this pH-related facilitation of αβmeATP-induced MA and TH, rats were pretreated with PPADS (P2 antagonist), TNP–ATP (P2X antagonist), MRS2179 (P2Y1 antagonist), AMG9810 (TRPV1 antagonist) or amiloride (ASIC blocker). Both PPADS and TNP–ATP dose-dependently blocked pH-facilitated MA, while TH was significantly reduced by pre-treatment with MRS2179 or AMG9810. Moreover, amiloride injection significantly reduced low pH-induced facilitation of αβmeATP-mediated MA, but not TH. These results demonstrate that low tissue pH facilitates ATP-mediated MA via the activation of P2X receptors and ASICs, whereas TH induced by ATP under low pH conditions is mediated by the P2Y1 receptor and TRPV1, but not ASIC. Thus distinct mechanisms are responsible for the development of MA and TH under conditions of tissue acidosis and increased ATP.

Respiratory inflammation is a frequent and fatal pathologic state encountered in veterinary medicine. Although diluted bee venom (dBV) has potent anti-inflammatory effects, the clinical use of dBV is limited to several chronic inflammatory diseases. The present study was designed to propose an acupoint dBV treatment as a novel therapeutic strategy for respiratory inflammatory disease. Experimental pleurisy was induced by injection of carrageenan into the left pleural space in mouse. The dBV was injected into a specific lung meridian acupoint (LU-5) or into an arbitrary non-acupoint located near the midline of the back in mouse. The inflammatory responses were evaluated by analyzing inflammatory indicators in pleural exudate. The dBV injection into the LU-5 acupoint significantly suppressed the carrageenan-induced increase of pleural exudate volume, leukocyte accumulation, and myeloperoxidase activity. Moreover, dBV acupoint treatment effectively inhibited the production of interleukin 1 beta, but not tumor necrosis factor alpha in the pleural exudate. On the other hand, dBV treatment at non-acupoint did not inhibit the inflammatory responses in carrageenan-induced pleurisy. The present results demonstrate that dBV stimulation in the LU-5 lung meridian acupoint can produce significant anti-inflammatory effects on carrageenan-induced pleurisy suggesting that dBV acupuncture may be a promising alternative medicine therapy for respiratory inflammatory diseases.

The role of peripheral neurosteroids and their related mechanisms on nociception have not been thoroughly investigated. Based on emerging evidence in the literature indicating that neurosteroids and their main target receptors, i.e., sigma-1, GABAA and NMDA, affect P2X-induced changes in neuronal activity, this study was designed to investigate the effect of peripherally injected dehydroepiandrosterone sulphate (DHEAS) and pregnenolone sulfate (PREGS) on P2X receptor-mediated mechanical allodynia in rats. Intraplantar injection of either neurosteroids alone did not produced any detectable changes in paw withdrawal frequency to the innocuous mechanical stimulation in naive rats. However, When DHEAS or PREGS were co-injected with a sub-effective dose of αβmeATP, mechanical allodynia was developed and this was dose dependently blocked by pre-injection of the P2X antagonist, TNP-ATP. These results demonstrates that DHEAS and PREGS potentiate the activity of P2X receptors which results in the enhancement of αβmeATP-induced mechanical allodynia. In order to investigate the potential role of peripheral sigma-1, GABAA and NMDA receptors in this facilitatory action, we pretreated animals with BD-1047 (a sigma-1 antagonist), muscimol (a GABAA agonist) or MK-801 (a NMDA antagonist) prior to DHEAS or PREGS+αβmeATP injection. Only BD-1047 effectively prevented the facilitatory effects induced by neurosteroids on αβmeATP-induced mechanical allodynia. Collectively, we have shown that peripheral neurosteroids potentiate P2X-induced mechanical allodynia and that this action is mediated by sigma-1, but not by GABAA nor NMDA, receptors.

Damage on one side of the body can also result in pain on the contralateral unaffected side, called mirror-image pain (MIP). Currently, the mechanisms responsible for the development of MIP are unknown. In this study, we investigated the involvement of spinal microglia and interleukin-1β (IL-1β) in the development of MIP using a peripheral inflammatory pain model. After unilateral carrageenan injection, mechanical allodynia (MA) in both hind paws and the expression levels of spinal Iba-1, IL-1β, and GFAP were evaluated. Ipsilateral MA was induced beginning at 3 hours after carrageenan injection, whereas contralateral MA showed a delayed onset occurring 5 days after injection. A single intrathecal (i.t.) injection of minocycline, a tetracycline derivative that displays selective inhibition of microglial activation, or an interleukin-1 receptor antagonist (IL-1ra) on the day of carrageenan injection caused an early temporary induction of contralateral MA, whereas repeated i.t. treatment with these drugs from days 0 to 3 resulted in a long-lasting contralateral MA, which was evident in its advanced development. We further showed that IL-1β was localized to microglia and that minocycline inhibited the carrageenan-induced increases in spinal Iba-1 and IL-1β expression. Conversely, minocycline or IL-1ra pretreatment increased GFAP expression as compared with that of control rats. However, i.t. pretreatment with fluorocitrate, an astrocyte inhibitor, restored minocycline- or IL-1ra-induced contralateral MA. These results suggest that spinal IL-1β derived from activated microglia temporarily suppresses astrocyte activation, which can ultimately prevent the development of contralateral MA under inflammatory conditions. These findings imply that microglial IL-1β plays an important role in regulating the induction of inflammatory MIP.

We have previously demonstrated that activation of the spinal sigma-1 receptor (Sig-1R) plays an important role in the development of mechanical allodynia (MA) via secondary activation of the N-methyl-d-aspartate (NMDA) receptor. Sig-1Rs have been shown to localize to astrocytes, and blockade of Sig-1Rs inhibits the pathologic activation of astrocytes in neuropathic mice. However, the mechanism by which Sig-1R activation in astrocytes modulates NMDA receptors in neurons is currently unknown. d-serine, synthesized from l-serine by serine racemase (Srr) in astrocytes, is an endogenous co-agonist for the NMDA receptor glycine site and can control NMDA receptor activity. Here, we investigated the role of d-serine in the development of MA induced by spinal Sig-1R activation in chronic constriction injury (CCI) mice. The production of d-serine and Srr expression were both significantly increased in the spinal cord dorsal horn post-CCI surgery. Srr and d-serine were only localized to astrocytes in the superficial dorsal horn, while d-serine was also localized to neurons in the deep dorsal horn. Moreover, we found that Srr exists in astrocytes that express Sig-1Rs. The CCI-induced increase in the levels of d-serine and Srr was attenuated by sustained intrathecal treatment with the Sig-1R antagonist, BD-1047 during the induction phase of neuropathic pain. In behavioral experiments, degradation of endogenous d-serine with DAAO, or selective blockade of Srr by LSOS, effectively reduced the development of MA, but not thermal hyperalgesia in CCI mice. Finally, BD-1047 administration inhibited the development of MA and this inhibition was reversed by intrathecal treatment with exogenous d-serine. These findings demonstrate for the first time that the activation of Sig-1Rs increases the expression of Srr and d-serine in astrocytes. The increased production of d-serine induced by CCI ultimately affects dorsal horn neurons that are involved in the development of MA in neuropathic mice.

Although previous reports have suggested that P2Y1 receptors (P2Y1Rs) are involved in cutaneous nociceptive signaling, it remains unclear how P2Y1Rs contribute to peripheral sensitization. The current study was designed to delineate the role of peripheral P2Y1Rs in pain and to investigate potential linkages to mitogen-activated protein kinase (MAPK) in DRGs and Transient Receptor Potential Vanilloid 1 (TRPV1) expression in a rodent inflammatory pain model. Following injection of 2% carrageenan into the hind paw, expressions of P2Y1 and TRPV1 and the phosphorylation rates of both p38 MAPK and ERK but not JNK were increased and peaked at day 2 post-injection. Blockade of peripheral P2Y1Rs by the P2Y1R antagonist, MRS2500 injection (i.pl, D0 to D2) significantly reduced the induction of thermal hyperalgesia, but not mechanical allodynia. Simultaneously, MRS2500 injections suppressed upregulated TRPV1 expression and DRG p38 phosphorylation, while pERK signaling was not affected. Furthermore, inhibition of p38 activation in the DRGs by SB203580 (a p38 inhibitor, i.t, D0 to D2) prevented the upregulation of TRPV1 and a single i.t injection of SB203580 reversed the established thermal hyperalgesia, but not mechanical allodynia. Lastly, to identify the mechanism of action of P2Y1Rs, we repeatedly injected the P2Y1 agonist, MRS2365 into the naive rat's hind paw and observed a dose-dependent increase in TRPV1 expression and p38 MAPK phosphorylation. These data demonstrate a sequential role for P2Y1R, p38 MAPK and TRPV1 in inflammation-induced thermal hyperalgesia; thus, peripheral P2Y1Rs activation modulates p38 MAPK signaling and TRPV1 expression, which ultimately leads to the induction of thermal hyperalgesia.

BACKGROUND AND PURPOSE We recently demonstrated that activation of the spinal sigma-1 receptor induces mechanical and thermal hypersensitivity via calcium-dependent second messenger cascades and phosphorylation of the spinal NMDA receptor GluN1 subunit (pGluN1). Here we examined the role of NO in this process, as it plays a critical role in PKC-mediated calcium signalling and the potentiation of NMDA receptor function. EXPERIMENTAL APPROACH The effects of intrathecal (i.t.) pretreatment with nNOS inhibitors on PRE084 (sigma-1 receptor agonist)-induced pain were assessed in mice by use of mechanical allodynia and thermal hyperalgesia tests. Western blot analysis, immunoprecipitation and immunohistochemical techniques were used to determine effects of these treatments on spinal pGluN1-immunoreactive (ir) cells, whether PRE084 induces a time-dependent modification of nNOS activity in the dorsal horn, and if any changes in nNOS activity can be blocked by sigma-1 receptor, calcineurin or soluble guanylyl cyclase (sGC) inhibitors. KEY RESULTS PRE084, injected i.t., induced mechanical and thermal hypersensitivity, and increased the number of PKC- and PKA-dependent pGluN1-ir cells in spinal cord. This PRE084-induced hypersensitivity and increase in PKC-dependent pGluN1 expression were blocked by pretreatment with N(G) -nitro-L-arginine methyl ester (L-NAME) or 7-nitroindazole (7-NI). PRE084 also time-dependently decreased the ratio of phosphorylated nNOS (pnNOS) to nNOS expression and the number of spinal pnNOS-ir cells. This decrease in pnNOS was prevented by BD1047, a sigma-1 receptor antagonist and cyclosporin A, a calcineurin inhibitor, but not by a sGC inhibitor. CONCLUSIONS AND IMPLICATIONS Spinal sigma-1 receptor-induced sensitization is mediated by an increase in nNOS activity, which is associated with an NO-induced increase in PKC-dependent pGluN1 expression.