Astrocyte D-serine modulates the activation of neuronal NOS leading to the development of mechanical allodynia in peripheral neuropathy.

Spinal D-serine plays an important role in nociception via an increase in phosphorylation of the N-Methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). However, the cellular mechanisms underlying this process have not been elucidated. Here, we investigate the possible role of neuronal nitric oxide synthase (nNOS) in the D-serine-induced potentiation of NMDA receptor function and the induction of neuropathic pain in a chronic constriction injury (CCI) model. Intrathecal administration of the serine racemase inhibitor, L-serine O-sulfate potassium salt (LSOS) or the D-serine degrading enzyme, D-amino acid oxidase (DAAO) on post-operative days 0-3 significantly reduced the CCI-induced increase in nitric oxide (NO) levels and nicotinamide adenine dinucleotide phosphate-diaphorase staining in lumbar dorsal horn neurons, as well as the CCI-induced decrease in phosphorylation (Ser847) of nNOS (pnNOS) on day 3 post-CCI surgery. LSOS or DAAO administration suppressed the CCI-induced development of mechanical allodynia and protein kinase C (PKC)-dependent (Ser896) phosphorylation of GluN1 on day 3 post-surgery, which were reversed by the co-administration of the NO donor, 3-morpholinosydnonimine hydrochloride (SIN-1). In naïve mice, exogenous D-serine increased NO levels via decreases in pnNOS. D-serine-induced increases in mechanical hypersensitivity, NO levels, PKC-dependent pGluN1, and NMDA-induced spontaneous nociception were reduced by pretreatment with the nNOS inhibitor, 7-nitroindazole or with the NMDA receptor antagonists, 7-chlorokynurenic acid and MK-801. Collectively, we show that spinal D-serine modulates nNOS activity and concomitant NO production leading to increases in PKC-dependent pGluN1 and ultimately contributing to the induction of mechanical allodynia following peripheral nerve injury.

Neuronal NOS Activates Spinal NADPH Oxidase 2 Contributing to Central Sigma-1 Receptor-Induced Pain Hypersensitivity in Mice.

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.

Spinal sigma-1 receptor activation increases the production of D-serine in astrocytes which contributes to the development of mechanical allodynia in a mouse model of neuropathic pain.

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.

Acid evoked thermal hyperalgesia involves peripheral P2Y1 receptor mediated TRPV1 phosphorylation in a rodent model of thrombus induced ischemic pain.

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.

Spinal sigma-1 receptors activate NADPH oxidase 2 leading to the induction of pain hypersensitivity in mice and mechanical allodynia in neuropathic rats.

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.

NAG-1/GDF-15 Transgenic Female Mouse Shows Delayed Peak Period of the Second Phase Nociception in Formalin-induced Inflammatory Pain.

Non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1), also known as growth differentiation factor-15 (GDF-15), is associated with cancer, diabetes, and inflammation, while there is limited understanding of the role of NAG-1 in nociception. Here, we examined the nociceptive behaviors of NAG-1 transgenic (TG) mice and wild-type (WT) littermates. Mechanical sensitivity was evaluated by using the von Frey filament test, and thermal sensitivity was assessed by the hot-plate, Hargreaves, and acetone tests. c-Fos, glial fibrillary acidic protein (GFAP), and ionized calcium binding adaptor molecule-1 (Iba-1) immunoreactivity was examined in the spinal cord following observation of the formalin-induced nociceptive behaviors. There was no difference in mechanical or thermal sensitivity for NAG-1 TG and WT mice. Intraplantar formalin injection induced nociceptive behaviors in both male and female NAG-1 TG and WT mice. The peak period in the second phase was delayed in NAG-1 TG female mice compared with that of WT female mice, while there was no difference in the cumulative time of nociceptive behaviors between the two groups of mice. Formalin increased spinal c-Fos immunoreactivity in both TG and WT female mice. Neither GFAP nor Iba-1 immunoreactivity was increased in the spinal cord of TG and WT female mice. These findings indicate that NAG-1 TG mice have comparable baseline sensitivity to mechanical and thermal stimulation as WT mice and that NAG-1 in female mice may have an inhibitory effect on the second phase of inflammatory pain. Therefore, it could be a novel target to inhibit central nervous system response in pain.

Phase-specific differential regulation of mechanical allodynia in a murine model of neuropathic pain by progesterone.

Progesterone has been shown to have neuroprotective capabilities against a wide range of nervous system injuries, however there are negative clinical studies that have failed to demonstrate positive effects of progesterone therapy. Specifically, we looked into whether progesterone receptors or its metabolizing enzymes, cytochrome P450c17 and 5α-reductase, are involved in the effects of progesterone on neuropathic pain after chronic constriction injury (CCI) of the sciatic nerve in mice. Intrathecal progesterone administration during the induction phase of chronic pain enhanced mechanical allodynia development and spinal glial fibrillary acidic protein (GFAP) expression, and this enhancement was inhibited by administration of ketoconazole, a P450c17 inhibitor, but not finasteride, a 5α-reductase inhibitor. Furthermore, phospho-serine levels of P450c17 in the spinal cord were elevated on day 1 after CCI operation, but not on day 17. In contrast, intrathecal progesterone administration during the maintenance phase of chronic pain decreased the acquired pain and elevated GFAP expression; this inhibition was restored by finasteride administration, but not by ketoconazole. The modification of mechanical allodynia brought on by progesterone in CCI mice was unaffected by the administration of mifepristone, a progesterone receptor antagonist. Collectively, these findings imply that progesterone suppresses spinal astrocyte activation via 5α-reductase activity during the maintenance phase of chronic pain and has an analgesic impact on the mechanical allodynia associated with the growing neuropathy. Progesterone, however, stimulates spinal astrocytes during the induction stage of peripheral neuropathy and boosts the allodynic impact caused by CCI through early spinal P450c17 activation.

Antinociceptive effect of intermittent fasting via the orexin pathway on formalin-induced acute pain in mice.

It has been suggested that stress responses induced by fasting have analgesic effects on nociception by elevating the levels of stress-related hormones, while there is limited understanding of pain control mechanisms. Here, we investigated whether acute or intermittent fasting alleviates formalin-induced pain in mice and whether spinal orexin A (OXA) plays a role in this process. 6, 12, or 24 h acute fasting (AF) and 12 or 24 h intermittent fasting (IF) decreased the second phase of pain after intraplantar formalin administration. There was no difference in walking time in the rota-rod test and distance traveld in the open field test in all groups. Plasma corticosterone level and immobility time in the forced swim test were increased after 12 h AF, but not after 12 h IF. 12 h AF and IF increased not only the activation of OXA neurons in the lateral hypothalamus but also the expression of OXA in the lateral hypothalamus and spinal cord. Blockade of spinal orexin 1 receptor with SB334867 restored formalin-induced pain and spinal c-Fos immunoreactivity that were decreased after 12 h IF. These results suggest that 12 h IF produces antinociceptive effects on formalin-induced pain not by corticosterone elevation but by OXA-mediated pathway.

Inhibition of angiotensin converting enzyme increases PKCßI isoform expression via activation of substance P and bradykinin receptors in cultured astrocytes of mice.

BACKGROUND: Angiotensin-converting enzyme inhibitor (ACEi) inhibits the catalysis of angiotensin I to angiotensin II and the degradation of substance P (SP) and bradykinin (BK). While the possible relationship between ACEi and SP in nociceptive mice was recently suggested, the effect of ACEi on signal transduction in astrocytes remains unclear. OBJECTIVES: This study examined whether ACE inhibition with captopril or enalapril modulates the levels of SP and BK in primary cultured astrocytes and whether this change modulates PKC isoforms (PKCα, PKCßI, and PKCε) expression in cultured astrocytes. METHODS: Immunocytochemistry and Western blot analysis were performed to examine the changes in the levels of SP and BK and the expression of the PKC isoforms in primary cultured astrocytes, respectively. RESULTS: The treatment of captopril or enalapril increased the immunoreactivity of SP and BK significantly in glial fibrillary acidic protein-positive cultured astrocytes. These increases were suppressed by a pretreatment with an angiotensin-converting enzyme. In addition, treatment with captopril increased the expression of the PKCßI isoform in cultured astrocytes, while there were no changes in the expression of the PKCα and PKCε isoforms after the captopril treatment. The captopril-induced increased expression of the PKCßI isoform was inhibited by a pretreatment with the neurokinin-1 receptor antagonist, L-733,060, the BK B1 receptor antagonist, R 715, or the BK B2 receptor antagonist, HOE 140. CONCLUSIONS: These results suggest that ACE inhibition with captopril or enalapril increases the levels of SP and BK in cultured astrocytes and that the activation of SP and BK receptors mediates the captopril-induced increase in the expression of the PKCßI isoform.

Sigma-1 receptor increases intracellular calcium in cultured astrocytes and contributes to mechanical allodynia in a model of neuropathic pain.

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 naïve 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 naïve 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.

Intrathecal interleukin-1ß decreases sigma-1 receptor expression in spinal astrocytes in a murine model of neuropathic pain.

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-1ß (IL-1ß) 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-1ß 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-1ß. 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-1ß. Agonistic stimulation of the Sig-1R with PRE084 restored pGluN1 expression and the development of mechanical allodynia that were originally suppressed by IL-1ß in CCI mice. Collectively these results demonstrate that IL-1ß 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.

Burn Injury-Induced Pain and Depression-Like Behavior in Mice.

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.

Inhibition of angiotensin converting enzyme induces mechanical allodynia through increasing substance P expression in mice.

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.

Inhibition of cytochrome P450c17 reduces spinal astrocyte activation in a mouse model of neuropathic pain via regulation of p38 MAPK phosphorylation.

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.

Inhibition of Cytochrome P450 Side-Chain Cleavage Attenuates the Development of Mechanical Allodynia by Reducing Spinal D-Serine Production in a Murine Model of Neuropathic Pain.

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.

Spinal Nitric Oxide Synthase Type II Increases Neurosteroid-metabolizing Cytochrome P450c17 Expression in a Rodent Model of Neuropathic Pain.

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 PKCand 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.

nNOS-PSD95 interactions activate the PKC-ε isoform leading to increased GluN1 phosphorylation and the development of neuropathic mechanical allodynia in mice.

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-ε 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-ε, 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-ε inhibitor, εV1-2 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-ε isoform, and ultimately contributes to the development of MA in peripheral neuropathy.

Spinal Interleukin-1ß Inhibits Astrocyte Cytochrome P450c17 Expression Which Controls the Development of Mechanical Allodynia in a Mouse Model of Neuropathic Pain.

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-1ß (IL-1ß) 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-1ß, interleukin-1 receptor type 1 (IL-1R1), P450c17, and GFAP. Spinal IL-1ß 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-1ß (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-1ß 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-1ß 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.

Spinal cytochrome P450c17 plays a key role in the development of neuropathic mechanical allodynia: Involvement of astrocyte sigma-1 receptors.

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 3ß-hydroxysteroid dehydrogenase (3ß-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 3ß-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 3ß-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.

Bee venom stimulation of a lung meridian acupoint reduces inflammation in carrageenan-induced pleurisy: an alternative therapeutic approach for respiratory inflammation.

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.