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

최스란,Alvin J Beitz,이장헌 한국뇌신경과학회 2019 Experimental Neurobiology Vol.28 No.4

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

General pharmacological profiles of bee venom and its water soluble fractions in rodent models

김현우,양일석,한호재,이혜정,Alvin J. Beitz,이장헌,권영배,함태원,노대현,윤서연,강석윤 대한수의학회 2004 Journal of Veterinary Science Vol.5 No.4

Recently, the antinociceptive and anti-inflammatory efficacy of bee venom (BV, Apis mellifera) has been confirmed in rodent models of inflammation and arthritis. Interestingly, the antinociceptive and anti-inflammatory effect of whole BV can be reproduced by two water-soluble fractions of BV (>20 kDa:BVAF1 and <10 kDa: BVAF3). Based on these scientific findings, BV and its effective water-soluble fractions have been proposed as potential anti-inflammatory and antinociceptive pharmaceuticals. While BV’s anti-inflammatory and antinociceptive properties have been well documented, there have been no careful studies of potential, side effects of BV and its fractions when administered in the therapeutic range (BV, 5 μg/kg; BVAF1, 0.2 μg/kg: BVAF3, 3 μg/kg; subcutaneous or intradermal). Such information is critical for future clinical use of BV in humans. Because of this paucity of information, the present study was designed to determine the general pharmacological/physiological effects of BV and its fractions administration on the rodent central nervous, cardiovascular, respiratory and gastrointestinal system. Subcutaneous BV and its fractions treatment did not produce any significant effects on general physiological functions at the highest dose tested (200-fold and 100-fold doses higher than that used clinically, respectively) except writhing test. These results demonstrate that doses of BV or BV subfractions in the therapeutic range or higher can be used as safe antinociceptive and anti-inflammatory agents.

Bee Venom Acupoint Stimulation Increases Fos Expression in Catecholaminergic Neurons in the Rat Brain

권영배,이장헌,한호재,Alvin J Beitz 한국분자세포생물학회 2004 Molecules and cells Vol.17 No.2

Fos immunocytochemistry was combined with tyrosine hydroxylase (TH) or dopamine-beta-hydroxylase (DBH) immunolabeling to examine brainstem catecholaminer-gic neuronal activation resulting from bee venom (BV) stimulation of the Zusanli acupoint (ST36) in Sprague-Dawley rats. BV injection into the Zusanli acupoint caused increased Fos expression in catecholaminergic neurons located in the hypothalamic arcuate nucleus (Arc), the dorsal raphe (DR), the A5 cell group (A5) and the locus coeruleus (LC). BV acupoint stimulation significantly increased Fos-TH double-labeled neurons in the Arc, LC and DR. Fos-DBH positive neurons were also increased by BV acupoint stimulation in the LC and A5. In contrast BV stimulation of a non-acupoint only increased Fos expression and Fos-TH double-labeled neurons in the Arc. These data indicate that BV acupoint stimulation activates brainstem cate-cholaminergic neurons and that this activation under-lies BV acupoint-induced antinociception.

Morphine을 전신투여한 랫드의 뇌에서 분비되는 amino acid 성 신경전달물질 측정을 위한 미세투석법의 개선에 관한 연구

이장헌,Lee, Jang-hern,Beitz, Alvin J 대한수의학회 1998 大韓獸醫學會誌 Vol.38 No.3

In the present study, we designed and constructed new microdialysis probe in order to improve the efficacy and accuracy of microdialysis method. In addition, extracellular concentrations of GABA, glutamate, aspartate and glycine were monitored with new designed probe in the lateral portion of the ventrocaudal periaqueductal gray using unanesthetized and unrestrained rats. Furthermore, the effect of opiates on release of these amino acids, especially GABA, was analyzed by measuring their concentration in PAG dialysates following veratridine administration in the presence of systemic morphine. The results were summerized as follow : 1. The damaging rates of 1.0mm or 1.5mm window probe were 12.5% or 42.8%, respectively. In the group using 1.5mm window probe, the damaging area was extended into mesencephalic aqueduct because of microdialyzing pressure. 2. Because of the unique design of our probes with an opening facing one side, dialysis occurs in a hemisphere($600{\mu}m$ in mediolateral direction and $100{\mu}m$ in opposite side of the dialysis probe) around the opening rather than in a spherical shaped configuration which is typical of most commercially available probe designs. 3. Glutamate, taurine and glycine were present in the highest concentration in the dialysate sample obtained before treatment with veratridine, whereas, aspartate and GABA were present in the lowest concentration. 4. The concentration of all 5 amino acids increased significantly following $75{\mu}m$ veratridine perfusion into lateral ventrocaudal PAG. 5. There was no significant difference between basal and peak amino acid concentrations according to window sizes. 6. Morphine had no effect on baseline concentrations of amino acids in dialysates obtained from the lateral PAG as compared to saline treated controls. However, following veratridine treatment, morphine selectively affected GABA release in the lateral ventrocaudal PAG as compared to saline treated controls. These results suggest that GABAergic interneurons in the PAG are inhibited by opioids. Therefore, endogenous enkephalins or endorphins may directly inhibit intrinsic GABAergic intemeurons and block their tonic inhibition of PAG-NMR projection neurons. Moreover, new designed probes demonstrate improved efficiency and accuracy in collecting samples as compared to commercial types of microdialysis probes.

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

Choi, Sheu-Ran,Beitz, Alvin J.,Lee, Jang-Hern Elsevier 2019 BIOMEDICINE AND PHARMACOTHERAPY Vol.118 No.-

<P><B>Abstract</B></P> <P>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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> P450c17 inhibition suppresses the activation of spinal astrocytes in CCI mice. </LI> <LI> P450c17 inhibition has no effect on the activation of spinal microglial cells in CCI mice. </LI> <LI> Spinal P450c17 increases p38 MAPK phosphorylation in the spinal cord of CCI mice. </LI> <LI> SB203580 potentiates the pharmacological effects of ketoconazole on neuropathic pain and astrocyte activation. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Influence of social interaction on nociceptive-induced changes in locomotor activity in a mouse model of acute inflammatory pain: Use of novel thermal assays

Smeester, Branden A.,Lee, Jang-Hern,Beitz, Alvin J. Elsevier 2017 Brain research bulletin Vol.134 No.-

<P><B>Abstract</B></P> <P>Most acute and chronic animal models of pain rely heavily on reflexive assays for evaluating levels of nociception, which involves removing the animal from its normal social environment. Here, we examine and characterize the influence of social interactions on inflammatory pain-evoked changes in movement in two different mouse strains. To produce inflammatory nociception, we injected CFA bilaterally into the hind paws of Balb/c and C3H mice and then recorded exploratory locomotor activity using an automated detector system to first evaluate the effects of social behavior on nociception. Secondly, we determined if carprofen administration altered the effects of social behavior on nociceptive-evoked movement. This methodology was expanded to create a novel thermal activity assay to objectively measure the effect of heat and cold on CFA-evoked animal movement in paired animals. Paired Balb/c and C3H mice exhibited significant hyper-locomotion that lasted for 3h post-injection in Balb/c, but only 1h post-injection in C3H. Single Balb/c mice only showed increased activity for 1h post-injection, while single C3H mice showed no increase. This CFA-induced increase in activity in paired animals was highly inversely correlated with mechanical allodynia as measured using standard Von Frey filaments. Carprofen administration completely blocked this CFA-induced hyperlocomotor activity. Both heat and cold induced a significant increase in locomotor activity in paired mice injected with CFA, while having no effect on activity in control mice injected with saline. The results presented here indicate that social interactions greatly influence inflammatory pain-induced changes in locomotor activity and indicate that the use of movement-based assays to evaluate nociception in paired mice may provide an alternative and more sensitive method to quantify nociception and characterize novel analgesic effects over time in the context of social interactions in rodent models of pain.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Normal locomotion is affected by social interactions in mice. </LI> <LI> Social interactions alter nociceptive-induced changes in locomotion. </LI> <LI> Carprofen blocks the effect of social interaction on nociception. </LI> <LI> A novel thermal locomotion assay allows detection of nociception in paired mice. </LI> </UL> </P>

Spinal Sigma-1 Receptor-mediated Dephosphorylation of Astrocytic Aromatase Plays a Key Role in Formalin-induced Inflammatory Nociception

Choi, Hoon-Seong,Lee, Mi-Ji,Choi, Sheu-Ran,Smeester, Branden A.,Beitz, Alvin J.,Lee, Jang-Hern Elsevier 2018 NEUROSCIENCE Vol.372 No.-

<P><B>Abstract</B></P> <P>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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Dephosphorylated aromatase in astrocyte is involved in the process of formalin-induced acute nociception. </LI> <LI> Activation of astrocyte sigma-1 receptors is associated with the dephosphorylation of aromatase. </LI> <LI> Calcineurin signaling pathway is involved in the sigma-1 receptor-mediated dephosphorylation of aromatase. </LI> </UL> </P>

Blockade of Adrenal Medulla-Derived Epinephrine Potentiates Bee Venom-Induced Antinociception in the Mouse Formalin Test: Involvement of Peripheral <b><i><i>β</i></i></b> -Adrenoceptors

Kang, Suk-Yun,Roh, Dae-Hyun,Kim, Hyun-Woo,Han, Ho-Jae,Beitz, Alvin J.,Lee, Jang-Hern Hindawi Publishing Corporation 2013 Evidence-based Complementary and Alternative Medic Vol.2013 No.-

<P>The injection of diluted bee venom (DBV) into an acupoint has been used traditionally in eastern medicine to treat a variety of inflammatory chronic pain conditions. We have previously shown that DBV had a potent antinociceptive efficacy in several rodent pain models. However, the peripheral mechanisms underlying DBV-induced antinociception remain unclear. The present study was designed to investigate the role of peripheral epinephrine on the DBV-induced antinociceptive effect in the mouse formalin assay. Adrenalectomy significantly enhanced the antinociceptive effect of DBV during the late phase of the formalin test, while chemical sympathectomy had no effect. Intraperitoneal injection of epinephrine blocked this adrenalectomy-induced enhancement of the DBV-induced antinociceptive effect. Moreover, injection of a phenylethanolamine N-methyltransferase (PNMT) inhibitor enhanced the DBV-induced antinociceptive effect. Administration of nonselective <I><I>β</I></I>-adrenergic antagonists also significantly potentiated this DBV-induced antinociception, in a manner similar to adrenalectomy. These results demonstrate that the antinociceptive effect of DBV treatment can be significantly enhanced by modulation of adrenal medulla-derived epinephrine and this effect is mediated by peripheral <I><I>β</I></I>-adrenoceptors. Thus, DBV acupoint stimulation in combination with inhibition of peripheral <I><I>β</I></I>-adrenoceptors could be a potentially novel strategy for the management of inflammatory pain.</P>

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

Choi, Sheu-Ran,Roh, Dae-Hyun,Yoon, Seo-Yeon,Choi, Hoon-Seong,Kang, Suk-Yun,Han, Ho-Jae,Beitz, Alvin J.,Lee, Jang-Hern Elsevier 2019 NEUROPHARMACOLOGY - Vol.149 No.-

<P><B>Abstract</B></P> <P>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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Sciatic nerve injury increases P450c17 expression in astrocytes of the ipsilateral lumbar spinal cord dorsal horn. </LI> <LI> Spinal P450c17 contributes to the CCI-induced development of neuropathic pain. </LI> <LI> Spinal P450c17 modulates the CCI-induced increase in astrocyte Sig-1R expression. </LI> <LI> P450c17 increases the dissociation of Sig-1Rs from BiP in the spinal cord dorsal horn of CCI mice. </LI> <LI> Sig-1R plays an important role in the P450c17-induced development of mechanical allodynia in neuropathic mice. </LI> </UL> </P>

Intrathecal injection of the neurosteroid, DHEAS, produces mechanical allodynia in mice: involvement of spinal sigma-1 and GABAA receptors : The role of spinal DHEAS in nociception

Yoon, Seo-Yeon,Roh, Dae-Hyun,Seo, Hyoung-Sig,Kang, Suk-Yun,Han, Ho-Jae,Beitz, Alvin J,Lee, Jang-Hern Wiley (Blackwell Publishing) 2009 British journal of pharmacology Vol.157 No.4

<P>BACKGROUND AND PURPOSE: The neurosteroid, dehydroepiandrosterone sulphate (DHEAS) and its non-sulphated form, DHEA, are considered as crucial endogenous modulators of a number of important physiological events. Evidence suggests that DHEAS and DHEA modulate central nervous system-related functions by activating sigma-1 receptors and/or allosterically inhibiting gamma-aminobutyric acid receptor type A (GABA(A)) receptors. As both the sigma-1 receptor and the GABA(A) receptor play important roles in spinal pain transmission, the present study was designed to examine whether intrathecally injected DHEAS or DHEA affect nociceptive signalling at the spinal cord level. EXPERIMENTAL APPROACH: We first determined whether intrathecal (i.t.) DHEA or DHEAS injection was able to affect nociceptive thresholds to peripheral mechanical stimulation and subsequently examined whether this effect was mediated by sigma-1 or the GABA(A) receptors. KEY RESULTS: The i.t. DHEAS injection dose-dependently decreased the nociceptive threshold to mechanical stimulation, thus producing mechanical allodynia. Moreover, this DHEAS-induced mechanical allodynia was significantly reduced by administration of the sigma-1 receptor antagonist, BD-1047 or the GABA(A) receptor agonist, muscimol. Conversely, i.t. DHEA had no effect on mechanical sensitivity. However, when i.t. DHEA was combined with the GABA(A) receptor antagonist bicuculline, DHEA dose-dependently produced mechanical allodynia similar to that of DHEAS. This effect was blocked by BD-1047 and by muscimol. CONCLUSIONS AND IMPLICATIONS: These findings indicate that i.t. injection of DHEAS produces mechanical allodynia and that the development of this mechanical allodynia is mediated by sigma-1 and GABA(A) receptors. The findings of this study raise several interesting questions for further investigations into the mechanisms underlying neurosteroid modulation of spinal pain transmission.</P>