Investigating The Role of AEG-1 in Mouse Models of Pain

Background: Astrocyte Elevated Gene 1 (AEG-1) is a multifunctional protein shown to be a regulator of transcription and multiple intracellular signaling pathways. The role of AEG-1 in cellular inflammation appears to be primarily facilitated by its direct interaction with the transcription factor NFκB, transcriptional regulator of inflammatory cytokines. May be have a potential role in models of pain, particularly chronic inflammatory and chemotherapy induced peripheral neuropathy (CIPN). Methods: C57BL6/J male and female mice, 8-14 weeks old. AEG-1 wild type (WT) and global knockout (KO) male and female mice, 8-14 weeks old. Chronic Inflammatory Pain induced via i.pl. injection of 50% Freund\u27s Complete Adjuvant (CFA) or vehicle into mouse right hind paw. CIPN induced via four 8 mg/kg, i.p. injections of Paclitaxel or vehicle (Toma, et. al). Mechanical hypersensitivity assessed via von frey filaments. Acetone Test was used to assess cold sensitivity. mRNA transcripts collected from tissues were measured via qRT-PCR. Results: AEG-1 KO mice displayed protection from CFA induced mechanical hypersensitivity, thermal sensitivity, and reduces paw edema compare to WT mice. AEG-1 KO mice displayed enhanced recovery from paclitaxel induced mechanical hypersensitivity and cold sensitivity compared to WT mice. AEG-1 expression levels in the periaqueductal grey, spinal cord, and L4-6 corresponding dorsal root ganglia collected from C57BL6/J mice treated with 8mg/Kg paclitaxel or 50% CFA (3 days post injection) showed no difference from control groups. Conclusions: Our data suggest that AEG-1 may be involved in inflammatory and CIPN related nociception in C57BL6/J mice.https://scholarscompass.vcu.edu/gradposters/1093/thumbnail.jp

The role of alpha5 nicotinic acetylcholine receptors in mouse models of chronic inflammatory and neuropathic pain

The aim of the present study was to determine the impact of as nicotinic acetylcholine receptor (nAChR) subunit deletion in the mouse on the development and intensity of nociceptive behavior in various chronic pain models. The role of as-containing nAChRs was explored in mouse models of chronic pain, including peripheral neuropathy (chronic constriction nerve injury, CCI), tonic inflammatory pain (the formalin test) and short and long-term inflammatory pain (complete Freund's adjuvant, CFA and carrageenan tests) in alpha(5) knock-out (1(0) and wild-type (WT) mice. The results showed that paw-licking time was decreased in the formalin test, and the hyperalgesic and allodynic responses to carrageenan and CFA injections were also reduced. In addition, paw edema in formalin-, carrageenan- or CFA-treated mice were attenuated in alpha(5)-K-O mice significantly. Furthermore, tumor necrosis factor-alpha (TNF-alpha) levels of carrageenan-treated paws were lower in alpha(5)-K-O mice. The antinociceptive effects of nicotine and sazetidine-A but not varenicline were alpha(5)-dependent in the formalin test. Both hyperalgesia and allodynia observed in the CCI test were reduced in alpha(5)-K-O mice. Nicotine reversal of mechanical allodynia in the CCI test was mediated through alpha(5)-nAChRs at spinal and peripheral sites. In summary, our results highlight the involvement of the et, nAChR subunit in the development of hyperalgesia, allodynia and inflammation associated with chronic neuropathic and inflammatory pain models. They also suggest the importance of alpha(5)-nAChRs as a target for the treatment of chronic pain.United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) (DA-12610)United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (R01DA032246)United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (R01DA012610

Effects of methoxsalen, a CYP2A5/6 inhibitor, on nicotine dependence behaviors in mice

Metabolism of nicotine to inactive cotinine by hepatic enzyme CYP2A6 is the principal pathway by which active nicotine is removed from circulation. We therefore hypothesized that inhibition of mouse CYP2A5, the ortolog of human CYP2A6, by methoxsalen (8-methoxypsoralen) alter dependence-related behaviors of nicotine in the mouse. Conditioned place preference (CPP) test was used to assess the appetitive reward-like properties and precipitated nicotine withdrawal to assess physical (somatic and hyperalgesia) and affective (anxiety-related behaviors) measures. The nicotine plasma levels were also measured with or without methoxsalen pretreatment. Methoxsalen (15 and 30 mg/kg, intraperitoneally) pretreatment enhanced nicotine-induced preference in mice (p < 0.05). However, there was a lack of enhancement of nicotine in the CPP test after the highest dose of the CYP-2A5 inhibitor. Similarly to the CPP results, repeated administration of methoxsalen increased the intensity of mecamylamine-precipitated withdrawal signs. The potentiation of nicotine preference and withdrawal intensity by methoxsalen was accompanied by significant increase in nicotine plasma levels in mice (p < 0.05). Finally, methoxsalen enhanced the ability of a very low dose of nicotine (0.05 mg/kg) to reverse withdrawal signs in mice undergoing spontaneous withdrawal after chronic nicotine infusion (p < 0.05). In conclusion, inhibition of nicotine metabolism by methoxsalen alters the behavioral effects of nicotine in the mouse. Combining CYP2A6 inhibitors with low dose nicotine replacement therapies may have a beneficial role in smoking cessation because it will decrease the drug elimination rate and maintain plasma and brain nicotine levels.United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (DA-05274)United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (R01DA032246) (P30DA033934) (P50DA005274

The analgesic-like properties of the alpha7 nAChR silent agonist NS6740 is associated with non-conducting conformations of the receptor

The alpha 7 nicotinic acetylcholine receptor (nAChR) is a promising drug target for a number of neurological disorders including chronic pain and inflammatory diseases. Since alpha 7 can function as a ligand-gated ion channel, drug development initially focused on ligands that were selective activators of the alpha 7 ion channel. However, the best alpha 7 drugs for chronic pain and inflammation indications may not be ion channel activators but rather "silent agonists", which bind to the receptor but preferentially induce non-conducting states that modulate signal transduction in non-neuronal cells. One such compound is NS6740. We show that NS6740 selectively induces prolonged desensitization of alpha 7 nAChRs. There are two forms of alpha 7 desensitization that can be distinguished by their sensitivity to the positive allosteric modulators (PAMs). At high concentrations, NS6740 preferentially induces PAM-insensitive desensitization, which over the course of several minutes reverts to the sensitive form. NS6740 was tested in several pain models after in vivo administration in the mouse. Although it had no effects in acute thermal pain, NS6740 induced significant dose- and time-dependent antinociceptive activity in formalin- and acetic acid-induced nociceptive behaviors as well as in the chronic constrictive nerve injury (CCI) model for neuropathic pain. The antinociceptive activity of NS6740 in these models was alpha 7-dependent. In addition, NS6740 administration reversed pain-induced aversion, an important affective component of pain. The time and concentration dependence of the effects were consistent with NS6740 induction of PAM-insensitive non-conducting states, suggesting that signal transduction required for analgesia is accomplished by alpha 7 receptors in that conformation.VCU Massey Cancer Center (A-35337)United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (DA032246)United States Department of Health & Human Services National Institutes of Health (NIH) - USA (GM57481)United States Department of Health & Human Services National Institutes of Health (NIH) - USA (DA027113)United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of General Medical Sciences (NIGMS) (R01GM057481)United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (R01DA032246)United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (R01DA012610)United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission (R03DA027113

The α7 nicotinic receptor dual allosteric agonist and positive allosteric modulator GAT107 reverses nociception in mouse models of inflammatory and neuropathic pain

Background and PurposeOrthosteric agonists and positive allosteric modulators (PAMs) of the 7 nicotinic ACh receptor (nAChR) represent novel therapeutic approaches for pain modulation. Moreover, compounds with dual function as allosteric agonists and PAMs, known as ago-PAMs, add further regulation of receptor function. Experimental ApproachInitial studies examined the 7 ago-PAM, GAT107, in the formalin, complete Freund's adjuvant (CFA), LPS inflammatory pain models, the chronic constriction injury neuropathic pain model and the tail flick and hot plate acute thermal nociceptive assays. Additional studies examined the locus of action of GAT107 and immunohistochemical markers in the dorsal horn of the spinal cord in the CFA model. Key ResultsComplementary pharmacological and genetic approaches confirmed that the dose-dependent antinociceptive effects of GAT107 were mediated through 7 nAChR. However, GAT107 was inactive in the tail flick and hot plate assays. In addition, GAT107 blocked conditioned place aversion elicited by acetic acid injection. Furthermore, intrathecal, but not intraplantar, injections of GAT107 reversed nociception in the CFA model, suggesting a spinal component of action. Immunohistochemical evaluation revealed an increase in the expression of astrocyte-specific glial fibrillary acidic protein and phosphorylated p38MAPK within the spinal cords of mice treated with CFA, which was attenuated by intrathecal GAT107 treatment. Importantly, GAT107 did not elicit motor impairment and continued to produce antinociceptive effects after subchronic administration in both phases of the formalin test. Conclusions and ImplicationsCollectively, these results provide the first proof of principle that 7 ago-PAMs represent an effective pharmacological strategy for treating inflammatory and neuropathic pain.Pilot Project from VCU Massey Cancer CenterUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA - DA032246NIH National Institute on Drug Abuse (NIDA)European CommissionUnited States Department of Health & Human Services National Institutes of Health (NIH) - USA - GM57481 - EY024717 - DA038493-01A1R01DA032246P30DA033934F32DA03849

The interaction between alpha 7 nicotinic acetylcholine receptor and nuclear peroxisome proliferator-activated receptor-alpha represents a new antinociceptive signaling pathway in mice

Recently, alpha 7 nicotinic acetylcholine receptors (nAChRs), primarily activated by binding of orthosteric agonists, represent a target for anti-inflammatory and analgesic drug development. These receptors may also be modulated by positive allosteric modulators (PAMs), ago-allosteric ligands (ago-PAMs), and alpha 7-silent agonists. Activation of 00 nAChRs has been reported to increase the brain levels of endogenous ligands for nuclear peroxisome proliferator-activated receptors type-alpha (PPAR-alpha), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), in a Ca2+-dependent manner. Here, we investigated potential crosstalk between alpha 7 nAChR and PPAR-alpha, using the formalin test, a mouse model of tonic pain. Using pharmacological and genetic approaches, we found that PNU282987, a full alpha 7 agonist, attenuated formalin-induced nociceptive behavior in alpha 7 -dependent manner. Interestingly, the selective PPAR-alpha antagonist GW6471 blocked the antinociceptive effects of PNU282987, but did not alter the antinociceptive responses evoked by the alpha 7 nAChR PAM PNU120596, ago-PAM GAT107, and silent agonist NS6740. Moreover, GW6471 administered systemically or spinally, but not via the intraplantar surface of the formalin-injected paw blocked PNU282987-induced antinociception. Conversely, exogenous administration of the naturally occurring PPAR-alpha agonist PEA potentiated the antinociceptive effects of PNU282987. In contrast, the cannabinoid 031 antagonist rimonabant and the CB2 antagonist SR144528 failed to reverse the antinociceptive effects of PNU282987. These findings suggest that PPAR-alpha plays a key role in a putative antinociceptive alpha 7 nicotinic signaling pathway.United States Department of Health & Human Services National Institutes of Health (NIH) - USA - GM57481 - R01 CA206028United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Cancer Institute (NCI) - R01CA206028United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute of General Medical Sciences (NIGMS) - R01GM057481United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission - T32DA00702

Neuropathic insult increases the responsiveness to acetic acid in mice

Chronic neuropathic pain is a burden to millions of patients every day. Patients with neuropathic pain will also experience acute pain throughout their everyday lives adding to their nociceptive burden. Using nociceptive models in mice this study aimed to investigate the relationship between acute visceral pain and chronic neuropathic pain in spontaneous and affective behaviors. Neuropathic pain was induced by chronic constriction injury (CCI) of the sciatic nerve of C57BL/6J male mice and examined in assays of acetic acid (AA)-induced stretching or conditioned place aversion to assess nociceptive and aversive behaviors. Stretching induced by a low concentration (0.32%) of AA given intraperitoneally was significantly increased in CCI and paclitaxel-treated animals compared to control animals. A higher concentration (1.2%) of AA was able to induce stretching equally in both neuropathic and control mice. In the conditioned place aversion test, an AA concentration of 0.32% did not induce place aversion in either sham or CCI animals. However, the 1.2% concentration of AA-induced higher place aversion scores in CCI mice compared to sham mice. No difference in place conditioning was observed between paclitaxel and vehicle-treated mice. Overall, our results show that peripheral nerve injury and paclitaxel treatment induces hypersensitivity to AA-induced nociception and place aversion. Copyright (C) 2019 Wolters Kluwer Health, Inc. All rights reserved

The antinociceptive and antiinflammatory properties of 3-furan-2-yl-N-p-tolyl-acrylamide, a positive allosteric modulator of α7 nicotinic acetylcholine receptors in mice

BACKGROUND: Positive allosteric modulators (PAMs) facilitate endogenous neurotransmission and/or enhance the efficacy of agonists without directly acting on the orthosteric binding sites. In this regard, selective 7 nicotinic acetylcholine receptor type II PAMs display antinociceptive activity in rodent chronic inflammatory and neuropathic pain models. This study investigates whether 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a new putative 7-selective type II PAM, attenuates experimental inflammatory and neuropathic pains in mice. METHODS: We tested the activity of PAM-2 after intraperitoneal administration in 3 pain assays: the carrageenan-induced inflammatory pain, the complete Freund adjuvant-induced inflammatory pain, and the chronic constriction injury-induced neuropathic pain in mice. We also tested whether PAM-2 enhanced the effects of the selective 7 agonist choline in the mouse carrageenan test given intrathecally. Because the experience of pain has both sensory and affective dimensions, we also evaluated the effects of PAM-2 on acetic acid-induced aversion by using the conditioned place aversion test. RESULTS: We observed that systemic administration of PAM-2 significantly reversed mechanical allodynia and thermal hyperalgesia in inflammatory and neuropathic pain models in a dose- and time-dependent manner without motor impairment. In addition, by attenuating the paw edema in inflammatory models, PAM-2 showed antiinflammatory properties. The antinociceptive effect of PAM-2 was inhibited by the selective competitive antagonist methyllycaconitine, indicating that the effect is mediated by 7 nicotinic acetylcholine receptors. Furthermore, PAM-2 enhanced the antiallodynic and antiinflammatory effects of choline, a selective 7 agonist, in the mouse carrageenan test. PAM-2 was also effective in reducing acetic acid-induced aversion in the conditioned place aversion assay. CONCLUSIONS: These findings suggest that the administration of PAM-2, a new 7-selective type II PAM, reduces the neuropathic and inflammatory pain sensory and affective behaviors in the mouse. Thus, this drug may have therapeutic applications in the treatment and management of chronic pain.Massey Cancer Center at VCUComision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) (1130079)Millennium Scientific Initiative (P10-035-F)Polish National Science Center (UMO-2013/09/D/NZ7/04549

In vivo interactions between alpha 7 nicotinic acetylcholine receptor and nuclear peroxisome proliferator-activated receptor-alpha: Implication for nicotine dependence

United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) - R01 DA12610 - R01 DA032246 - T32 DA007027-41United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Alcohol Abuse & Alcoholism (NIAAA) - P50AA022537United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Institute on Drug Abuse (NIDA) European Commission - T32DA007027 - R01DA032246 - R01DA012610Chronic tobacco use dramatically increases health burdens and financial costs. Limitations of current smoking cessation therapies indicate the need for improved molecular targets. The main addictive component of tobacco, nicotine, exerts its dependency effects via nicotinic acetylcholine receptors (nAChRs). Activation of the homomeric alpha 7 nAChR reduces nicotine's rewarding properties in conditioned place preference (CPP) test and i.v. self-administration models, but the mechanism underlying these effects is unknown. Recently, the nuclear receptor peroxisome proliferator-activated receptor type-alpha (PPAR alpha) has been implicated as a downstream signaling target of the alpha 7 nAChR in ventral tegmental area dopamine cells. The present study investigated PPAR alpha as a possible mediator of the effect of alpha 7 nAChR activation in nicotine dependence. Our results demonstrate the PPAR alpha antagonist GW6471 blocks actions of the alpha 7 nAChR agonist PNU282987 on nicotine reward in an unbiased CPP test in male ICR adult mice. These findings suggests that alpha 7 nAChR activation attenuates nicotine CPP in a PPAR alpha-dependent manner. To evaluate PPAR alpha activation in nicotine dependence we used the selective and potent PPAR alpha agonist, WY-14643 and the clinically used PPAR alpha activator, fenofibrate, in nicotine CPP and we observed attenuation of nicotine preference, but fenofibrate was less potent. We also studied PPAR alpha in nicotine dependence by evaluating its activation in nicotine withdrawal. WY-14643 reversed nicotine withdrawal signs whereas fenofibrate had modest efficacy. This suggests that PPAR alpha plays a role in nicotine reward and withdrawal and that further studies are warranted to elucidate its function in mediating the effects of alpha 7 nAChRs in nicotine dependence