Nociceptive Assays Research Articles

Open source timed pressure control hardware and software for delivery of air mediated distensions in animal models

Studying the visceral sensory component of peripheral nervous systems can be challenging due to limited options for consistent and controlled stimulation. One method for mechanical stimulation of hollow organs, including the colon and bladder, is controlled distensions mediated by compressed air. For example, distension of the bladder can be used as an assay for bladder nociception. Bladder distension causes a corresponding increase in abdominal electromyography, which increases with distension pressure and is attenuated with analgesics. However, the hardware used to control these distensions are primarily all one-off custom builds, without clear directions on how to build your own. This has made it difficult for these methods to be fully utilized and replicated as not everyone has the access, knowledge, and resources required to build this controller. Here we show an open-source Arduino-based system for controlling a solenoid valve to deliver timed pressure distensions in the experimental model. This device can be controlled by one of two methods through direct TTL pulses from the experimenter’s data acquisition software (ex. CED Spike2) or by a graphical user interface, where the user can set the time before, during, and after distension as well as the number of cycles. This system’s low cost and relative ease to build will allow more groups to utilize timed pressure distensions in their experiments.

Celecoxib in the treatment of orofacial pain and discomfort in rats subjected to a dental occlusal interference model.

ABSTRACTPurpose:To evaluate the effect of a selective cyclooxygenase 2 (COX-2) inhibitor on trigeminal ganglion changes and orofacial discomfort/nociception in rats submitted to an experimental model of dental occlusal interference (DOI).Methods:Female Wistar rats (180-200 g) were divided into five groups: a sham group (without DOI) (n=15); and four experimental groups with DOI treated daily with 0.1 mL/kg saline (DOI+SAL), 8, 16, or 32 mg/kg celecoxib (DOI+cel -8, -16, -32) (n=30/group). The animals were euthanized after one, three, and seven days. The bilateral trigeminal ganglia were analyzed histomorphometrically (neuron cell body area) and immunohistochemically (COX-2, nuclear factor-kappa B [NFkB], and peroxisome proliferator-activated receptor-y [PPARy]). A bilateral nociception assay of the masseter muscle was performed. The number of bites/scratches, weight, and grimace scale scores were determined daily. One-way/two-way analysis of variance (ANOVA)/Bonferroni post hoc tests were used (P < .05, GraphPad Prism 5.0).Results:DOI+SAL showed a reduction in neuron cell body area bilaterally, whereas DOI+cel-32 exhibited a significative increase in neuron cell body area compared with DOI+SAL group (P < 0.05). The ipsilateral (P=0.007 and P=0.039) and contralateral (P < 0.001 and P=0.005) overexpression of COX-2 and NFkB and downregulation of PPARy (P=0.016 and P < 0.001) occurred in DOI+SAL, but DOI+cel-32 reverted this alteration. DOI+SAL showed increase in isplateral (P < 0.001) and contralateral (P < 0.001) nociception, an increased number of bites (P=0.010), scratches (P < 0.001), and grimace scores (P=0.032). In the group of DOI+cel-32, these parameters were reduced.Conclusions:Celecoxib attenuated DOI-induced transitory nociception/orofacial discomfort resulting from trigeminal COX-2 overexpression.

Antinociceptive Activity of Syzygium Aromaticum Linn. Flower Buds (Clove) Extract and its Fractions

Syzygium aromaticum (Myrtaceae) flower buds (clove) are traditionally known to possess antimicrobial, antiprotozoal, antiviral and other activities, including the antinociceptive action. Scientific evidence also suggests the antileishmanial, antiherpetic and anti-HIV activities of the buds. This paper reports the antinociceptive activity of S. aromaticum flower buds, reinforcing its use in decreasing the pain. The present study was designed to confirm the analgesic activity of S. aromaticum extract and seven of its fractions to reveal the common belief in its painkilling effects. We chose two thermal nociception assays (i) hot–plate test (ii) and tail-flick method as our experimental techniques. Both of these methods are well established to screen anti-nociceptive activities in new molecules. The standard drug indomethacin (5 mg/kg) given by intra-peritoneal route was used in the study for comparison. The study has shown that the methanolic extract (SA-EXT) and its active fractions possess anti-nociceptive activity (pandlt;0.05) in the models of nociception used.

Differential Effects of Cannabidiol and a Novel Cannabidiol Analog on Oxycodone Place Preference and Analgesia in Mice: an Opioid Abuse Deterrent with Analgesic Properties.

Background and Purpose: This study sought to determine whether cannabidiol (CBD) or a CBD derivative, CBD monovalinate monohemisuccinate (CBD-val-HS), could attenuate the development of oxycodone reward while retaining its analgesic effects. Experimental Approach: To determine the effect on oxycodone reward, animals were enrolled in the conditioned place preference paradigm and received either saline or oxycodone (3.0 mg/kg) in combination with either CBD or CBD-val-HS utilizing three sets of drug-/no drug-conditioning trials. To determine if the doses of CBD or CBD-val-HS that blocked opioid reward would affect nociceptive processes, animals were enrolled in the hot plate and abdominal writhing assays when administered alone or in combination with a subanalgesic (1.0 mg/kg) or analgesic (3.0 mg/kg) dose of oxycodone. Key Results: Results from condition place preference demonstrated CBD was not able attenuate oxycodone place preference while CBD-val-HS attenuated these rewarding effects at 8.0 mg/kg and was void of rewarding or aversive properties. In contrast to CBD, CBD-val-HS alone produced analgesic effects in both nociceptive assays but was most effective compared with oxycodone against thermal nociception. Of interest, there was a differential interaction of CBD and CBD-val-HS×oxycodone across the two nociceptive assays producing subadditive responses on the hot plate assay, whereas additive responses were observed in the abdominal writhing assay. Conclusion: These findings suggest CBD-val-HS alone, a nonrewarding analgesic compound, could be useful in pain management and addiction treatment settings.

Blocking Neuropilin-1 in Drosophila melanogaster as a Possible Treatment for Pain and SARS-CoV-2

This study aims to validate a method of neuropilin-1 blocking in Drosophila melanogaster to aid the development of chronic pain treatment as well as increase scientific understanding of SARS-CoV-2 cell entry. If Drosophila melanogaster are exposed to monoclonal antibodies (mAbs) used for targeted chemotherapy against neuropilin-1, then this targeted protein blocking method may allow for the development of new pain treatment and possible SARS-CoV-2 treatment. mAbs were microinjected into mutant flies to block NRP-1 activity. Then, a thermal nociception and von frey assay were done to test drosophila nociception. Finally, an IHC assay was performed to quantify protein activity. Overall, the hypothesis was supported as both nociception assays showed significant data proving mutant flies had delayed or no reactions to stimuli. The Von Frey assay did show some varying results, but the data is still significant. However, the IHC did show that there was still some NRP-1 activity in the mutant flies. NRP-1 was able to be partially blocked by mAbs. The collected data may apply to the expansion of research in pain treatment as well as COVID-19 research. Chronic pain is a prevalent area of research that is still not completely solved today. The fight against the SARS-CoV-2 is an ongoing fight and further research is mandatory in finding treatments for this deadly virus.

Ultrastructural Evidence for Oxytocin and Oxytocin Receptor at the Spinal Dorsal Horn: Mechanism of Nociception Modulation

Oxytocin is a hypothalamic neuropeptide involved in the inhibition of nociception transmission at spinal dorsal horn (SDH) level (the first station where the incoming peripheral signals is modulated). Electrophysiological, behavioral, and pharmacological data strongly support the role of this neuropeptide and its receptor (the oxytocin receptor, OTR) as a key endogenous molecule with analgesic properties. Briefly, current data showed that oxytocin release from the hypothalamus induces OTR activation at the SDH, inducing selective inhibition of the nociceptive Aδ- and C-fibers (probably peptidergic) activity, but not the activity of proprioceptive fibers (i.e. Aβ-fibers). The above inhibition could be a direct presynaptic mechanism, or a mechanism mediated by GABAergic interneurons. However, the exact anatomical localization of oxytocin and OTR remains unclear. In this context, the present study set out to analyze the role of OTRs, GABAergic cells and CGRP fibers in the SDH in rats by using electron microscopy. Ultrastructural analyses of the SDH tissue show that: (i) oxytocin and OTR are found in asymmetrical synapsis; (ii) OTR is found in GABAergic interneurons (near unmyelinated fibers), CGRPergic fibers and glial cells; (iii) whereas oxytocin is present in supraspinal descending projection fibers. These anatomical data strongly support the notion that oxytocin released at the SDH could presynaptically inhibit the nociceptive input from the peripheral primary afferent fibers. This inhibitory action could be direct or use a GABA interneuron. Furthermore, our findings that OTR is exhibited in glial tissue at the SDH requires further exploration in nociception assays.

Comparison of Nociceptive Effects of Buprenorphine, Firocoxib, and Meloxicam in a Plantar Incision Model in Sprague–Dawley Rats

Due to their reduced frequency of dosing and ease of availability, NSAIDs are generally preferred over opioids for rodent analgesia. We evaluated the efficacy of the highly COX2-selective NSAID firocoxib as compared with meloxicam and buprenorphine for reducing allodynia and hyperalgesia in rats in a plantar incision model of surgical pain. After a preliminary pharmacokinetic study using firocoxib, Sprague-Dawley rats (n = 12 per group, 6 of each sex) were divided into 6 groups: no surgery (anesthesia only), saline (surgery but no analgesia), buprenorphine (0.05 mg/kg SC every 8 h), meloxicam (2 mg/kg SC every 24 h), and 2 dosages of firocoxib (10 and 20 mg/kg SC every 24 h). The nociception assays were performed by using von Frey and Hargreaves methodology to test mechanical allodynia and thermal hyperalgesia. These assays were performed at 24 h before and at 20, 28, 44, and 52 h after start of surgery. None of the analgesics used in this study produced significantly different responses in allodynia or hyperalgesia from those of saline-treated rats. In the Hargreaves assay, female saline-treated rats experienced significantly greater hyperalgesia than did males. These findings add to a growing body of literature suggesting that commonly used dosages of analgesics may not provide sufficient analgesia in rats experiencing incisional pain.

PD01-11 SENSORY GLIAL GQ-GPCR SIGNALING ALLEVIATES VISCERAL PAIN AND IMPROVES MICTURITION FUNCTION IN AN ANIMAL MODEL OF UROLOGICAL CHRONIC PELVIC PAIN SYNDROME

You have accessJournal of UrologyInfections/Inflammation/Cystic Disease of the Genitourinary Tract: Interstitial Cystitis (PD01)1 Sep 2021PD01-11 SENSORY GLIAL GQ-GPCR SIGNALING ALLEVIATES VISCERAL PAIN AND IMPROVES MICTURITION FUNCTION IN AN ANIMAL MODEL OF UROLOGICAL CHRONIC PELVIC PAIN SYNDROME Alison Xie, Randall Meacham, and Anna Malykhina Alison XieAlison Xie More articles by this author , Randall MeachamRandall Meacham More articles by this author , and Anna MalykhinaAnna Malykhina More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001965.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Patients with urological chronic pelvic pain syndrome (UCPPS) experience chronic pelvic pain (CPP) and lower urinary tract symptoms (LUTS). Afferent hypersensitivity in bladder-projecting sensory neurons plays an important role in the generation and maintenance of UCPPS symptoms, especially visceral pain and urinary frequency. We found that Gq-GPCR activation in satellite glial cells (SGCs) in the sensory ganglia decreases afferent sensitivity in lumbar nociceptive pathways, and could be a promising and highly innovative approach to alleviate the symptoms of bladder overactivity and pain. In this study, we aimed to test if activation of lumbosacral sensory glial Gq-GPCR signaling alleviates visceral pain, and improves LUTS in UCPPS mouse models. METHODS: A mouse model of UCPPS was induced by intravesical instillation of recombinant mouse vascular endothelial growth factor (VEGFA). VEGF levels were associated with CPP in UCPPS patients, and in animal models of bladder overactivity. In mice, VEGFA induces sensory nerve remodeling in the urinary bladder, visceral mechanical hypersensitivity. Pharmacogenetic activation of spinal and sensory ganglionic glia was achieved by expressing Gq-coupled Designer Receptors Exclusively Activated by Designer Drugs (Gq-DREADD) in GFAP+ glia via Gfap-hM3Dq transgenic mice or targeted adeno-associated viral vector (AAV) delivery. Gq-DREADD agonist, clozapine N-oxide (CNO), was used to selectively activate Gq-GPCR signaling in GFAP+ glia and to decreases sensory neuronal excitability in vivo in awake animals. Afferent sensitivity and bladder function were evaluated by clinically-relevant visceral nociceptive assays and awake cystometry. RESULTS: Lumbosacral glial Gq-GPCR activation significantly attenuated VEGFA-induced visceral hypersensitivity, increased micturition efficiency, and shortened intermicturition intervals. The glia-mediated neuromodulatory effects were long-lasting and dose-dependent, suggesting broader implication of neuromodulation in treating pain and hypersensitivity in pelvic diseases. CONCLUSIONS: Our data demonstrated that sensory glial-mediated neuromodulation effectively alleviated visceral pain and regulated micturition in animal model of UCPPS by inhibiting afferent excitability in the lumbosacral spinal cord and respective sensory ganglia. These results suggest a strong therapeutic potential of targeting GFAP-expressing glia for the treatment of chronic pelvic pain and LUTS in UCPPS patients. Source of Funding: Colorado Pilot Program Mentored Award, CCTSI, PI: Alison Xiaoqiao XieR01 DK121506-01, PI: Anna Malykhina © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e35-e35 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alison Xie More articles by this author Randall Meacham More articles by this author Anna Malykhina More articles by this author Expand All Advertisement Loading ...

This Month in Anesthesiology

This Month in Anesthesiology

PA-Int5: An isatin-thiosemicarbazone derivative that exhibits anti-nociceptive and anti-inflammatory effects in Swiss mice.

Pain and inflammation are symptoms of various diseases, and they can be modulated by different pathways, thus highlighting the importance of investigating the therapeutic effects of novel compounds. Previous studies have shown that isatin-thiosemicarbazone exhibits antitumor, antifungal antibacterial and other biological properties. Based on the wide range of biological effects of these compounds, the aim of the present study was to investigate the central nervous system (CNS) performance, and the anti-nociceptive and anti-inflammatory activity of (Z)-2-(5-nitro-2-oxoindolin-3-ilidene)-N-hydroazinecarbothioamide (PA-Int5) in treated mice. Three doses of PA-Int5 were tested orally (1.0, 2.5 and 5.0 mg/kg) in the nociceptive and inflammatory animal models. Additionally, the potential sedative effects of PA-Int5 (5 mg/kg, oral gavage) were investigated using an open field and rotarod tests, to exclude any possible unspecific effects of the nociceptive assays. Anti-nociceptive activity was assessed using the acetic acid-induced abdominal contortion and formalin tests, whereas anti-inflammatory activity was assessed using a carrageenan-induced paw edema and zymosan-induced air-pouch models. PA-Int5 (5 mg/kg) induced anti-nociceptive activity in the abdominal contortion model. In the formalin test, PA-Int5 (at 2.5 and 5 mg/kg) reduced nociception in the second phase. At the higher dose tested, PA-Int5 did not affect spontaneous locomotion or motor coordination. The data revealed that at all doses tested, the compound significantly reduced paw edema following carrageenan administration. In the zymosan-induced air-pouch model, PA-Int5 potently inhibited leukocyte migration and protein levels at the site of inflammation. When combined, the results revealed, for the first time, that PA-Int5 exhibited anti-nociceptive and anti-inflammatory activities, and highlights its potential, as well that of other derivatives, as novel candidates for pain relief.

Kappa-opioid receptor-mediated thermal analgesia evoked by the intrathecal administration of the chemokine CCL1 in mice.

The chemokine CC motif ligand 1 (CCL1) participates in immune cell recruitment and, as other chemokines, is also involved in nociceptive processing. In contrast with previous reports indicating its participation in allodynia and cold hypernociception when spinally administered, its ability to evoke heat thermal analgesia, mediated by circulating leukocytes and endocannabinoids, after systemic administration has recently been reported. Aiming to explore the role played by CCL1 on spinal nociception, we study here the effect of its intrathecal administration on thermal nociception in mice. Behavioral nociceptive assays, immunohistochemical experiments, white cell blood depletion procedures and qRT-PCR experiments were performed. The intrathecal administration of CCL1 (0.3-30 ng) produced analgesia as measured by the unilateral hot plate test. This effect peaked 1 h after injection, was prevented by the CCR8 antagonist R243 and was accompanied by a reduction of c-Fos expression in spinal neurons. Whereas blood leukocyte depletion did not modify it, analgesia was abolished by the microglial inhibitor minocycline, but not the astroglial inhibitor aminoadipate. Furthermore, antinociception remained unmodified by the coadministration of cannabinoid type 1 or 2 receptors antagonists. However, it was reversed by naloxone but not by selective blockade of mu- or delta-opioid receptors. The inhibitory effect induced by the selective kappa-opioid receptor antagonist, nor-binaltorphimine, and by an anti-dynorphin A 1-17 antibody indicates that analgesia evoked by spinal CCL1 is mediated by endogenous dynorphins acting on kappa-opioid receptors. Endogenous dynorphin and microglia behave as key players in heat thermal analgesia evoked by spinal CCL1 in mice.

Peripheral signaling mechanisms underlying mu‐delta opioid synergy

In response to the ongoing opioid epidemic, and in an effort to develop new analgesics with improved safety profiles, researchers have invested significant energy into further understanding the various downstream signaling cascades initiated by opioid receptor activation. Recent studies have demonstrated that G protein-biased ligands demonstrate reduced side effects such as respiratory depression and tolerance. Further research has shown that bivalent or bifunctional opioid compounds can also result in differential signal transduction. We hypothesized that a combination of mu and delta opioid agonists would engage different signaling pathways than drugs that target only one opioid receptor. Therefore, we probed the signaling mechanisms underlying the behavioral analgesic synergy previously demonstrated by a 1:1 combination of loperamide, a peripherally-restricted mu-opioid agonist, and oxymorphindole, a delta-opioid agonist. Using the complete Freund's adjuvant model of inflammatory pain and the Hargreaves assay for thermal nociception, the anti-hyperalgesic effect of loperamide-oxymorphindole in male and female mice was challenged by co-administration of a recently developed antagonist against MOR-DOR heteromers, D24M. Then, mice were given an injection of loperamide, oxymorphindole, or their combination with and without pertussis toxin in order to assess the involvement of Gai/o signaling on the behavioral anti-hyperalgesia. Finally, in naïve mice, the role of G protein-coupled inwardly-rectifying potassium (GIRK) channels, a downstream target of G proteins, was investigated using the peptide inhibitor tertiapin-Q, as well as GIRK KO mice. The data demonstrate that D24M was significantly more potent in antagonizing the loperamide-oxymorphindole combination (p<0.01) than either drug alone, and that pertussis toxin significantly reduces the anti-hyperalgesic effect of the combination (p<0.05). Finally, both pharmacologic and genomic inhibition of GIRK2 function results in a significant reduction of the combination's anti-nociceptive effect (p<0.0001). From these data we conclude that the loperamide-oxymorphindole combination binds to heterodimeric opioid receptors, activates a G protein signaling pathway, and opens GIRK channels.

494 Mechanisms underlying MOR-DOR analgesic synergy in epidermal nerve fibers

We recently published electrophysiological data showing that the combination of the mu-opioid (MOR) agonist loperamide and the delta-opioid (DOR) agonist oxymorphindole (Lo-OMI), applied topically to mouse toe pads, prevents activation of nociceptive epidermal nerve fibers with high potency and efficacy (Uhelski et al., 2020). The present study tested the hypotheeis that i) heterodimeric opioid receptors mediate this analgesic action, and ii) inhibitory G proteins mediate this action via activation of G protein-coupled inwardly rectifying potassium (GIRK) channels. Recent studies have demonstrated that G protein-biased ligands demonstrate reduced side effects such as respiratory depression and tolerance. Using the complete Freund’s adjuvant model of inflammatory pain and the Hargreaves assay for thermal nociception, the anti-hyperalgesic effect of Lo-OMI in male and female mice was challenged by co-administration of a recently developed antagonist against MOR-DOR heteromers, D24M (Olson, Streicher et al., 2018). Then, mice were given an injection of Lo, OMI, or their combination with and without pertussis toxin in order to assess the involvement of Gai/o signaling on the behavioral anti-hyperalgesia. Finally, in naïve mice, the role of GIRK channels, a downstream target of G proteins, was investigated using the peptide inhibitor tertiapin-Q, as well as GIRK KO mice. The data demonstrate that D24M was significantly more potent in antagonizing the Lo-OMI combination (p<0.01) than either drug alone, supporting the heterodimeric opioid receptor hypothesis, and that pertussis toxin significantly reduces the anti-hyperalgesic effect of the combination (p<0.05), supporting the Gai/o signaling hypothesis. Finally, both pharmacologic and genomic inhibition of GIRK2 function results in a significant reduction of the combination’s anti-nociceptive effect (p<0.0001), supporting the requirement for GIRK channels.

Current Methods to Investigate Nociception and Pain in Zebrafish.

Pain is an unpleasant, negative emotion and its debilitating effects are complex to manage. Mammalian models have long dominated research on nociception and pain, but there is increasing evidence for comparable processes in fish. The need to improve existing pain models for drug research and the obligation for 3R refinement of fish procedures facilitated the development of numerous new assays of nociception and pain in fish. The zebrafish is already a well-established animal model in many other research areas like toxicity testing, as model for diseases or regeneration and has great potential in pain research, too. Methods of electrophysiology, molecular biology, analysis of reflexive or non-reflexive behavior and fluorescent imaging are routinely applied but it is the combination of these tools what makes the zebrafish model so powerful. Simultaneously, observing complex behavior in free-swimming larvae, as well as their neuronal activity at the cellular level, opens new avenues for pain research. This review aims to supply a toolbox for researchers by summarizing current methods to study nociception and pain in zebrafish. We identify treatments with the best algogenic potential, be it chemical, thermal or electric stimuli and discuss options of analgesia to counter effects of nociception and pain by opioids, non-steroidal anti-inflammatory drugs (NSAIDs) or local anesthetics. In addition, we critically evaluate these practices, identify gaps of knowledge and outline potential future developments.

Piroxicam reduces acute and chronic pain response in type 1 diabetic rats.

Pain associated with various underlying pathologies is a major cause of morbidity and diminished life quality in diabetic patients. Effective control of pain requires the use of analgesics with the best efficacy and with minimal side effects. Therefore, our aim in this study was to investigate the effects of non-steroidal anti-inflammatory drugs (NSAIDs) on pain in diabetic rats. In this study, we investigated the analgesic effects of drugs belonging to three different classes of NSAIDs in a rat model of diabetes. Four diabetic groups received normal saline, diclofenac, piroxicam and ketorolac, respectively, and four non-diabetic groups received normal saline, diclofenac, piroxicam and ketorolac. Type 1 diabetes was induced in rats by a single injection of streptozotocin (60mg/kgbw). Formalin (50µL of 2.5%) nociception assay was used to examine the effect of treatment with diclofenac, piroxicam and ketorolac on acute and chronic pain in healthy and diabetic rats. Piroxicam showed significant analgesic effects both in the acute phase of pain (5-10min after injection of formalin into the left hind paw), and in the chronic phase (20-60min after formalin injection) in healthy as well as diabetic rats. Diclofenac and ketorolac also reduced pain scores in healthy rats. However, these two drugs failed to diminish pain in diabetic rats. Our data point for better efficacy of piroxicam in controlling pain in diabetes.

Ultra-purified alginate gel implantation decreases inflammatory cytokine levels, prevents intervertebral disc degeneration, and reduces acute pain after discectomy

Lumbar intervertebral disc (IVD) herniation causes severe low back pain (LBP), which results in substantial financial and emotional strains. Despite the effectiveness of discectomy, there is no existing treatment for post-operative LBP induced by progressive IVD degeneration. Two key factors of LBP are intradiscal inflammation, indicated by tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), and sensory nerve ingrowth into the inner layer of the annulus fibrosus, triggered by nerve growth factor/high-affinity tyrosine kinase A (TrkA) signalling. In an animal models of discectomy, the bioresorbable ultra-purified alginate (UPAL) gel with an extremely low-toxicity has been effective in acellular tissue repair. We aimed to investigate whether UPAL gel can alleviate LBP using a rat nucleus pulposus (NP) punch model and a rabbit NP aspirate model. In both models, we assessed TNF-α and IL-6 production and TrkA expression within the IVD by immunohistochemistry. Further, histological analysis and behavioural nociception assay were conducted in the rat model. UPAL gel implantation suppressed TNF-α and IL-6 production, downregulated TrkA expression, inhibited IVD degeneration, and reduced nociceptive behaviour. Our results suggest the potential of UPAL gel implantation as an innovative treatment for IVD herniation by reducing LBP and preventing IVD degeneration after discectomy.

Manipulating Pharmacodynamic Efficacy with Agonist + Antagonist Mixtures: In Vitro and In Vivo Studies with Opioids and Cannabinoids

Pharmacodynamic efficacy of drugs to activate their receptors is a key determinant of drug effects, and intermediate-efficacy agonists are often useful clinically because they retain sufficient efficacy to produce therapeutically desirable effects while minimizing undesirable effects. Molecular mechanisms of efficacy are not well understood, so rational drug design to control efficacy is not yet possible; however, receptor theory predicts that fixed-proportion mixtures of an agonist and antagonist for a given receptor can be adjusted to precisely control net efficacy of the mixture in activating that receptor. Moreover, the agonist proportion required to produce different effects provides a quantitative scale for comparing efficacy requirements across those effects. To test this hypothesis, the present study evaluated effectiveness of fixed-proportion agonist/antagonist mixtures to produce in vitro and in vivo effects mediated by μ-opioid receptors (MOR) and cannabinoid type 1 receptors (CB1R). Mixtures of 1) the MOR agonist fentanyl and antagonist naltrexone and 2) the CB1R agonist CP55,940 and antagonist/inverse agonist rimonabant were evaluated in an in vitro assay of ligand-stimulated guanosine 5'-O-(3-[35S]thio)triphosphate binding and an in vivo assay of thermal nociception in mice. For both agonist/antagonist pairs in both assays, increasing agonist proportions produced graded increases in maximal mixture effects, and lower agonist proportions were sufficient to produce in vivo than in vitro effects. These findings support the utility of agonist-antagonist mixtures as a strategy to control net efficacy of receptor activation and to quantify and compare efficacy requirements across a range of in vitro and in vivo endpoints. SIGNIFICANCE STATEMENT: Manipulation of agonist proportion in agonist/antagonist mixtures governs net mixture efficacy at the target receptor. Parameters of agonist/antagonist mixture effects can provide a quantitative metric for comparison of efficacy requirements across a wide range of conditions.

Isolation and Pharmacological Characterization of Six Opioidergic Picralima nitida Alkaloids.

The seeds of the akuamma tree (Picralima nitida) have been used as a traditional treatment for pain and fever. Previous studies have attributed these effects to a series of indole alkaloids found within the seed extracts; however, these pharmacological studies were significantly limited in scope. Herein, an isolation protocol employing pH-zone-refining countercurrent chromatography was developed to provide six of the akuamma alkaloids in high purity and quantities sufficient for more extensive biological evaluation. Five of these alkaloids, akuammine (1), pseudo-akuammigine (3), akuammicine (4), akuammiline (5), and picraline (6), were evaluated against a panel of >40 central nervous system receptors to identify that their primary targets are the opioid receptors. Detailed in vitro investigations revealed 4 to be a potent kappa opioid receptor agonist, and three alkaloids (1-3) were shown to have micromolar activity at the mu opioid receptor. The mu opioid receptor agonists were further evaluated for analgesic properties but demonstrated limited efficacy in assays of thermal nociception. These findings contradict previous reports of the antinociceptive properties of the P. nitida alkaloids and the traditional use of akuamma seeds as analgesics. Nevertheless, their opioid-preferring activity does suggest the akuamma alkaloids provide distinct scaffolds from which novel opioids with unique pharmacologic properties and therapeutic utility can be developed.

Methylnaltrexone crosses the blood-brain barrier and attenuates centrally-mediated behavioral effects of morphine and oxycodone in mice

BackgroundAntagonism of peripheral opioid receptors by methylnaltrexone (MNTX) was recently proposed as a potential mechanism to attenuate the development of opioid analgesic tolerance based on experiments conducted in mice. However, reports indicate that MNTX is demethylated to naltrexone (NTX) in mice, and NTX may subsequently cross the blood-brain barrier to antagonize centrally-mediated opioid effects. The goal of this study was to determine whether MNTX alters centrally-mediated behaviors elicited by the opioid analgesics, morphine and oxycodone, and to quantify concentrations of MNTX and NTX in blood and brain following their administration in mice. MethodsCombinations of MNTX and morphine were tested under acute and chronic conditions in thermal nociceptive assays. Effects of MNTX and NTX pretreatment were assessed in an oxycodone discrimination operant procedure. Blood and brain concentrations of these antagonists were quantified after their administration using liquid chromatography-mass spectrometry. ResultsMNTX dose-dependently attenuated acute and chronic morphine antinociception. MNTX and NTX dose-dependently antagonized the discriminative stimulus effects of oxycodone. MNTX and NTX were detected in both blood and brain after administration of MNTX, confirming its demethylation and demonstrating that MNTX itself can cross the blood-brain barrier. ConclusionsThese results provide converging behavioral and analytical evidence that MNTX administration in mice attenuates centrally-mediated effects produced by opioid analgesics and results in functional concentrations of MNTX and NTX in blood and brain. Collectively, these findings indicate that MNTX cannot be administered systemically in mice for making inferences that its effects are peripherally restricted.

An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae.

Published assays for mechanical nociception in Drosophila have led to variable assessments of behavior. Here, we fabricated, for use with Drosophila larvae, customized metal nickel-titanium alloy (nitinol) filaments. These mechanical probes are similar to the von Frey filaments used in vertebrates to measure mechanical nociception. Here, we demonstrate how to make and calibrate these mechanical probes and how to generate a full behavioral dose-response from subthreshold (innocuous or non-noxious range) to suprathreshold (low to high noxious range) stimuli. To demonstrate the utility of the probes, we investigated tissue damage-induced hypersensitivity in Drosophila larvae. Mechanical allodynia (hypersensitivity to a normally innocuous mechanical stimulus) and hyperalgesia (exaggerated responsiveness to a noxious mechanical stimulus) have not yet been established in Drosophila larvae. Using mechanical probes that are normally innocuous or probes that typically elicit an aversive behavior, we found that Drosophila larvae develop mechanical hypersensitization (both allodynia and hyperalgesia) after tissue damage. Thus, the mechanical probes and assay that we illustrate here will likely be important tools to dissect the fundamental molecular/genetic mechanisms of mechanical hypersensitivity.