Nociceptive Assays Research Articles

Paeoniflorin regulates microglia-astrocyte crosstalk, inhibits inflammatory response, and alleviates neuropathic pain through HSP90AA1/HMGB1 signaling pathway

Given the unclear, complex pathogenesis of neuropathic pain and the potential of paeoniflorin in relieving neuropathic pain, this study aimed to further clarify the therapeutic effect of paeoniflorin on neuropathic pain and to preliminarily explore the possible protective mechanisms of paeoniflorin. Chronic constrictive injury-induced Sprague Dawley rats and lipopolysaccharide-induced BV-2 cells were used for in vivo and in vitro experiments, respectively. The exosome uptake assay of mouse astrocytes (PKH-67 fluorescent labeling) and the mechanical nociceptive assay (the von Frey fibrous filaments) were performed. The effects of paeoniflorin and its downstream mechanisms on microglial and astrocyte activation, inflammation-associated proteins and exosome marker were determined. Paeoniflorin alleviated mechanical abnormal pain, decreased levels of ionized calcium binding adapter molecule-1 (Iba-1), glial fibrillary acidic protein, Heat Shock Protein 90 Alpha Family Class A Member 1 (HSP90AA1, inflammatory factor) and High Mobility Group Box 1 (HMGB1, inflammation-related protein), and inhibited neuronal apoptosis in chronic constrictive injury rats or lipopolysaccharide-induced BV-2 cells. However, these effects were offset by HSP90AA1 overexpression in lipopolysaccharide-induced BV-2 cells. Exosomes of BV-2 cells could be absorbed by mouse astrocytes. In addition, HSP90AA1 overexpression reversed the effects of paeoniflorin on HMGB1 expression and inflammatory factors and proteins in mouse astrocytes co-cultured with exosome. Collectively, paeoniflorin alleviates neuropathic pain and inhibits inflammatory responses in chronic constrictive injury by modulating microglia-astrocyte crosstalk through HSP90AA1/HMGB1 pathways, which further evidences the potential of paeoniflorin in the treatment of neuropathic pain.

Mechanical Nociception Assay in Drosophila Larvae.

The nervous system of animals can sense and respond to noxious stimuli, which include noxious thermal, chemical, or mechanical stimuli, through a process called nociception. Here, we describe a simple behavioral assay to measure mechanically induced nociceptive responses in Drosophila larvae. This assay tests larval mechanosensitivity to noxious force with calibrated von Frey filaments. First, we explain how to construct and calibrate the customizable von Frey filaments that can be used to deliver reproducible stimuli of a defined force or pressure. Next, we describe how to perform the mechanical nociception assay on third-instar larvae. Through comparison of the responses of genotypes of interest, this assay can be useful for investigation of molecular, cellular, and circuit mechanisms of mechanical nociception. At the molecular level, prior studies have identified the importance of sensory ion channels such as Pickpocket/Balboa, Piezo, dTRPA1, and Painless. At the cellular level, the class IV multidendritic arborizing (md-da) neurons are the main mechanical nociceptor neurons of the peripheral system, but class III and class II md-da have been found to also play a role. At the circuit level, studies have shown that mechanical nociception relies on interneurons of the abdominal ganglia that integrate inputs from these various md-da neuron classes.

Optogenetic Stimulation of Nociceptive Escape Behaviors in Drosophila Larvae.

In animals, noxious stimuli activate a neural process called nociception. Drosophila larvae perform a rolling escape locomotion behavior in response to nociceptive sensory stimuli. Noxious mechanical, thermal, and chemical stimuli each trigger this same escape response in larvae. The polymodal sensory neurons that initiate the rolling response have been identified based on the expression patterns of genes that are known to be required for nociception responses. The synaptic output of these neurons, known as class IV multidendritic sensory neurons, is required for behavioral responses to thermal, mechanical, and chemical triggers of the rolling escape locomotion. Importantly, optogenetic stimulation of the class IV multidendritic neurons has also shown that the activation of those cells is sufficient to trigger nociceptive rolling. Optogenetics uses light-activated ion channels expressed in neurons of interest to bypass the normal physiological transduction machinery so that the cell may be activated in response to light that is applied by the investigator. This protocol describes an optogenetic technique that uses channelrhodopsin-2 (ChR2) to activate larval nociceptors and trigger nociceptive rolling. First, we explain how to set up the necessary genetic crosses and culture the larval progeny. Next, we describe how to perform the optogenetic nociception assay on third-instar larvae.

Assaying Nociception Behaviors in Drosophila Larvae During Parasitoid Wasp Attacks.

Nociception in fruit fly (Drosophila melanogaster) larvae is characterized by a stereotyped escape behavior. When a larva encounters a noxious (potentially harmful) stimulus, it responds by curving its body into a c-shape and rolling in a corkscrew-like manner around its long-body axis. This rolling behavior may serve to quickly remove the larva from the source of the noxious stimulus, and is particularly adaptive to escape from a common natural predator of fruit fly larvae: parasitoid wasps (Leptopilina boulardi). L. boulardi completes its life cycle by using fruit fly larvae as hosts for its offspring. Female wasps sting fly larvae with an ovipositor and lay an egg within the larva. The wasp offspring hatches inside the fly larva, consumes the fly tissues during pupation, and eventually emerges from the pupal case as an adult wasp. Fruit fly larvae respond to oviposition attacks by rolling, which causes the long flexible ovipositor to be wound around the larval body like a spool. This dislodges the wasp and allows the larva to attempt to escape by crawling. Rolling behavior is triggered by the activation of sensory neurons (nociceptors) whose function can inform our understanding of the mechanisms of nociception. In this protocol, we describe a simple behavioral assay to test and measure nociceptive responses in Drosophila larvae during oviposition attacks by female parasitoid wasps. First, we discuss parasitoid wasp husbandry and culturing methods in the laboratory. We then describe how to perform the wasp nociception assay on third-instar fruit fly larvae.

A novel marine-derived mitophagy inducer ameliorates mitochondrial dysfunction and thermal hypersensitivity in paclitaxel-induced peripheral neuropathy.

Mitochondrial dysfunction contributes to the pathogenesis and maintenance of chemotherapy-induced peripheral neuropathy (CIPN), a significant limitation of cancer chemotherapy. Recently, the stimulation of mitophagy, a pivotal process for mitochondrial homeostasis, has emerged as a promising treatment strategy for neurodegenerative diseases, but its therapeutic effect on CIPN has not been explored. Here, we assessed the mitophagy-inducing activity of 3,5-dibromo-2-(2',4'-dibromophenoxy)-phenol (PDE701), a diphenyl ether derivative isolated from the marine sponge Dysidea sp., and investigated its therapeutic effect on a CIPN model. Mitophagy activity was determined by a previously established mitophagy assay using mitochondrial Keima (mt-Keima). Mitophagy induction was further verified by western blotting, immunofluorescence, and electron microscopy. Mitochondrial dysfunction was analysed by measuring mitochondrial superoxide levels in SH-SY5Y cells and Drosophila larvae. A thermal nociception assay was used to evaluate the therapeutic effect of PDE701 on the paclitaxel-induced thermal hyperalgesia phenotype in Drosophila larvae. PDE701 specifically induced mitophagy but was not toxic to mitochondria. PDE701 ameliorated paclitaxel-induced mitochondrial dysfunction in both SH-SY5Y cells and Drosophila larvae. Importantly, PDE701 also significantly ameliorated paclitaxel-induced thermal hyperalgesia in Drosophila larvae. Knockdown of ATG5 or ATG7 abolished the effect of PDE701 on thermal hyperalgesia, suggesting that PDE701 exerts its therapeutic effect through mitophagy induction. This study identified PDE701 as a novel mitophagy inducer and a potential therapeutic compound for CIPN. Our results suggest that mitophagy stimulation is a promising strategy for the treatment of CIPN and that marine organisms are a potential source of mitophagy-inducing compounds.

Disrupted stress-induced analgesia in a neuropathic pain state is rescued by the endocannabinoid degradation inhibitor JZL195.

Acute stress normally engages descending brain pathways to produce an antinociceptive response, known as stress-induced analgesia. Paradoxically, these descending pain modulatory pathways are also involved in the maintenance of the abnormal pain associated with chronic neuropathic pain. It remains unclear how stress-induced analgesia is affected by neuropathic pain states. We therefore examined the impact of a chronic constriction nerve-injury (CCI) model of neuropathic pain on restraint stress-induced analgesia in C57BL/6 mice. Thirty minutes of restraint stress produced analgesia in the hotplate thermal nociceptive assay that was less in CCI compared to control mice who underwent a sham-surgery. In sham but not CCI mice, stress-induced analgesia was reduced by the opioid receptor antagonist naltrexone. The cannabinoid CB1 receptor antagonist AM281 did not affect stress-induced analgesia in either sham or CCI mice. Low-dose pre-treatment with the dual fatty acid amide hydrolase and monoacylglycerol lipase inhibitor JZL195 increased stress-induced analgesia in CCI but not sham mice. The JZL195 enhancement of stress-induced analgesia in CCI mice was abolished by AM281 but was unaffected by naltrexone. These findings indicate that the acute opioid-mediated analgesic response to a psychological stressor is disrupted in a nerve-injury model of neuropathic pain. Importantly, this impairment of stress-induced analgesia was rescued by blockade of endocannabinoid breakdown via a cannabinoid CB1 receptor dependent mechanism. These findings suggest that subthreshold treatment with endocannabinoid degradation blockers could be used to alleviate the disruption of endogenous pain control systems in a neuropathic pain state.

Digging deeper into pain: an ethological behavior assay correlating well-being in mice with human pain experience.

The pressing need for safer, more efficacious analgesics is felt worldwide. Preclinical tests in animal models of painful conditions represent one of the earliest checkpoints novel therapeutics must negotiate before consideration for human use. Traditionally, the pain status of laboratory animals has been inferred from evoked nociceptive assays that measure their responses to noxious stimuli. The disconnect between how pain is tested in laboratory animals and how it is experienced by humans may in part explain the shortcomings of current pain medications and highlights a need for refinement. Here, we survey human patients with chronic pain who assert that everyday aspects of life, such as cleaning and leaving the house, are affected by their ongoing level of pain. Accordingly, we test the impact of painful conditions on an ethological behavior of mice, digging. Stable digging behavior was observed over time in naive mice of both sexes. By contrast, deficits in digging were seen after acute knee inflammation. The analgesia conferred by meloxicam and gabapentin was compared in the monosodium iodoacetate knee osteoarthritis model, with meloxicam more effectively ameliorating digging deficits, in line with human patients finding meloxicam more effective. Finally, in a visceral pain model, the decrease in digging behavior correlated with the extent of disease. Ultimately, we make a case for adopting ethological assays, such as digging, in studies of pain in laboratory animals, which we believe to be more representative of the human experience of pain and thus valuable in assessing clinical potential of novel analgesics in animals.

Role of induced nitric oxide synthases in orofacial nociception/discomfort after dental tooth bleaching with hydrogen peroxide

ObjectiveTo evaluate the role of induced nitric oxide synthase (iNOS) in nociception/orofacial discomfort in rats submitted to tooth whitening with hydrogen peroxide (H₂O₂). Design:Wistar rats were divided into three groups (n = 24/group): a sham group not submitted to whitening treatment, a saline group submitted to whitening treatment, and a test group submitted to whitening treatment and blockade of iNOS with aminoguanidine 50 mg/kg/day. After 24 and 48 h, and 7 days, the animals were euthanized to collect trigeminal ganglia and maxillae to histomorphometric analysis (size of neuronal bodies and percentage of pulp area filled by vessels) and behavior/nociception (Grimace scales, scratching and biting counting, weight loss and nociception assay). ANOVA-1- or − 2-way tests were used (p < 0.05, GraphPadPrism 5.0). Results:The aminoguanidine-treated group showed a reduction in nociceptive threshold in the masseteric region (p < 0.001), Grimace scale scores (p < 0.001), number of scratching (p = 0.011) and body mass loss (p = 0.007). After 24 and 48 h of tooth bleaching, the saline group showed a significant increase in the mean area of the blood vessels (p = 0.020) and iNOS immunostaining in odontoblasts (p = 0.002) and non-odontoblasts cells (p = 0.025). Aminoguanidine reversed both increases. Tooth bleaching reduced the mean area of neuronal bodies, and aminoguanidine significantly reversed it (p = 0.019), but an increase in GFAP immunostaining in neuronal bodies did not reduce after seven-days or after aminoguanidine treatment (p = 0.003). Conclusion:iNOS blockage by aminoguanidine plays an important role in nociception and orofacial discomfort by control of inflammation in dental pulp after tooth bleaching with hydrogen peroxide (H₂O₂) 35%.

Unveiling the antinociceptive mechanisms of Methyl-2-(4-chloro-phenyl)-5-benzoxazoleacetate: insights from nociceptive assays in mice.

Methyl-2-(4-chloro- phenyl)-5-benzoxazoleacetate (MCBA), a synthetic benzoxazole derivative with established antipsoriatic efficacy, was investigated for potential antinociceptive effects. This study employs various nociceptive assays in mice to elucidate MCBA's antinociceptive mechanisms. MCBA's antinociceptive potential was tested against various nociception models induced by formalin, glutamate, capsaicin, a transient receptor potential vanilloid 1 (TRPV1) receptor agonist, and phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator. It was then assessed using the hot plate test and examined within the acetic acid-induced writhing test. During the acetic acid-induced writhing test, MCBA was pre-challenged against selective receptor antagonists such as naloxone, caffeine, atropine, yohimbine, ondansetron, and haloperidol. It was also pre-challenged with ATP-sensitive potassium channel inhibitor (glibenclamide) to further elucidate its antinociceptive mechanism. The results showed that oral administration of MCBA led to a dose-dependent and significant inhibition (p < 0.05) of nociceptive effects across all evaluated models at doses of 60, 120, and 240 mg/kg. Moreover, the efficacy of MCBA's antinociceptive potential was significantly counteracted (p < 0.0001) by specific antagonists: (i) directed at adenosinergic, alpha-2 adrenergic, and cholinergic receptors using caffeine, yohimbine, and atropine, respectively; and (ii) targeting ATP-sensitive potassium channels, employing glibenclamide. Antagonists aimed at opioidergic and serotoninergic receptors (naloxone and ondansetron, respectively) had poor utility in inhibiting antinociceptive activity. Conversely, the dopaminergic receptor antagonist haloperidol potentiated locomotor abnormalities associated with MCBA treatment. MCBA-induced antinociception involves modulation of glutamatergic-, TRVP1 receptors- and PKC-signaling pathways. It impacts adenosinergic, alpha-2 adrenergic, and cholinergic receptors and opens ATP-sensitive potassium channels.

Cold nociception as a measure of hyperalgesia during spontaneous heroin withdrawal in mice

Opioids are powerful analgesic drugs that are used clinically to treat pain. However, chronic opioid use causes compensatory neuroadaptations that result in greater pain sensitivity during withdrawal, known as opioid withdrawal-induced hyperalgesia (OWIH). Cold nociception tests are commonly used in humans, but preclinical studies often use mechanical and heat stimuli to measure OWIH. Thus, further characterization of cold nociception stimuli is needed in preclinical models. We assessed three cold nociception tests—thermal gradient ring (5–30 °C, 5–50 °C, 15–40 °C, and 25–50 °C), dynamic cold plate (4 °C to −1 °C at −1 °C/min, −1 °C to 4 °C at +1 °C/min), and stable cold plate (10 °C, 6 °C, and 2 °C)—to measure hyperalgesia in a mouse protocol of heroin dependence. On the thermal gradient ring, mice in the heroin withdrawal group preferred warmer temperatures, and the results depended on the ring's temperature range. On the dynamic cold plate, heroin withdrawal increased the number of nociceptive responses, with a temperature ramp from 4 °C to −1 °C yielding the largest response. On the stable cold plate, heroin withdrawal increased the number of nociceptive responses, and a plate temperature of 2 °C yielded the most significant increase in responses. Among the three tests, the stable cold plate elicited the most robust change in behavior between heroin-dependent and nondependent mice and had the highest throughput. To pharmacologically characterize the stable cold plate test, we used μ-opioid and non-opioid receptor-targeting drugs that have been previously shown to reverse OWIH in mechanical and heat nociception assays. The full μ-opioid receptor agonist methadone and μ-opioid receptor partial agonist buprenorphine decreased OWIH, whereas the preferential μ-opioid receptor antagonist naltrexone increased OWIH. Two N-methyl-d-aspartate receptor antagonists (ketamine, MK-801), a corticotropin-releasing factor 1 receptor antagonist (R121919), a β2-adrenergic receptor antagonist (butoxamine), an α2-adrenergic receptor agonist (lofexidine), and a 5-hydroxytryptamine-3 receptor antagonist (ondansetron) had no effect on OWIH. These data demonstrate that the stable cold plate at 2 °C yields a robust, reliable, and concise measure of OWIH that is sensitive to opioid agonists.

Rats chasing the dragon: A new heroin inhalation method

BackgroundDespite extensive human use of inhalation for ingesting opioids, models in rodents have mostly been limited to parenteral injection and oral dosing. Methods using electronic drug delivery systems (EDDS; “e-cigarettes”) have shown efficacy in rodent models but these do not faithfully mimic the most popular human inhalation method of heating heroin to the point of vaporization. New methodMiddle aged rats were exposed to vapor created by direct heating of heroin HCl powder in a ceramic e-cigarette type atomizer. Efficacy was determined with a warm water tail withdrawal nociception assay, rectal temperature and self-administration. ResultsTen minutes of inhalation of vaporized heroin slowed response latency in a warm water tail withdrawal assay and increased rectal temperature in male rats, in a dose-dependent manner. Similar antinociceptive effects in female rats were attenuated by the opioid antagonist naloxone (1.0 mg/kg, s.c.). Female rats made operant responses for heroin vapor in 15-minute sessions, increased their response rate when the reinforcement ratio increased from FR1 to FR5, and further increased their responding when vapor delivery was omitted. Anti-nociceptive effects of self-administered volatilized heroin were of a similar magnitude as those produced by the 10-minute non-contingent exposure. Comparison with existing methodsInhalation of directly volatilized heroin successfully produces heroin-typical effects, comparable to EDDS inhalation delivery. ConclusionsThis study shows that “chasing the dragon” methods of inhalation of heroin can be modeled successfully in the rat. Inhalation techniques may be particularly useful for longer term studies deep into the middle age of rats.

Standardized seed extract of Cajanus cajan (L) Millsp. produced antinociceptive and anti-inflammatory actions through inhibition of inflammatory mediators and activation of opioidergic signaling

Background and PurposePeripheral tissue damage and diseases could lead to persistent pain beyond possible resolution suggestive of disease-promoting condition which gives rise to chronic pain. The inability of current medications to manage some chronic inflammatory and painful conditions necessitate the need for better treatment options. Natural products have been a veritable source for the discovery of more efficacious and safer therapeutics. Cajanus cajan (L) Millsp, (Fabaceae) is used in traditional African medicines for the treatment of painful, gut disturbance and inflammatory conditions. Hence, the current study aims to investigate the anti-inflammatory and antinociceptive actions of the ethanol seed extract of Cajanus cajan (CC) in rodents and possible mechanisms of action. MethodsHigh performance liquid chromatography coupled with diode-array detection (HPLC-DAD) was used to identify 8 major flavonoids in CC. Antinociceptive activity was investigated using the acetic acid-induced writhing reflex, biphasic formalin-induced flinching behaviour and supraspinal hot plate-evoked nociception in mice. Carrageenan-induced rat paw oedema test was used to evaluate the anti-inflammatory action of CC in rats. Possible mechanism of antinociception and anti-inflammatory actions were examined using both in vivo model and molecular docking procedures against µ-opioid receptor (MOR), cyclooxygenase-2 (COX-2) and phospholipase A2 (PLA2) proteins, respectively. ResultsThe chromatographic procedure qualitatively and quantitatively confirmed the presence of rutin, quercetin, gallic acid, luteolin, pinostrobin, BiochaninA, formononetin and apigenin in CC. The in vivo nociceptive assay, showed that CC (50, 100, or 200 mg/kg, p.o.) significantly decreased mean number of writhing reflex with peak effect at 50 mg/kg (72% inhibition) when compared with vehicle control in mouse writhing test. Similarly, the biphasic formalin-induced nociception was significantly reduced by CC (50 mg/kg) with 24.61 and 66.73% inhibition of nociceptive reactions in both the early and late phases of formalin-induced inflammatory pain, respectively. In addition, CC significantly increased supraspinal thermally mediated pain threshold with maximum possible effects of 42.25 and 52.16% at CC 50 and 100 mg/kg, respectively, in hot-plate test. Similarly, CC100mg/kg caused time course inhibition of carrageenan-induced paw oedema in rat with peak effect (65.78%, CC 100 mg/kg; 6 h). Interestingly, the antinociceptive action of CC was blocked by the pre-treatment of mice with l-NG-nitro arginine methyl ester (L-NAME) or naloxone. The molecular docking analysis revealed prominent interactions of pinostrobin, physcion and lupeol with MOR, COX-2, and PLA2, respectively. ConclusionFindings from this study showed that Cajanus cajan seeds extract possesses anti-nociceptive and anti-inflammatory through inhibition of inflammatory mediators (PLA2/COX-2) and activation of opioidergic signalling.

Oral Oxycodone-Induced Respiratory Depression During Normocapnia and Hypercapnia: A Pharmacokinetic-Pharmacodynamic Modeling Study.

The widely prescribed opioid oxycodone may cause lethal respiratory depression. We compared the effects of oxycodone on breathing and antinociception in healthy young volunteers. After pharmacokinetic/pharmacodynamic (PK/PD) modeling, we constructed utility functions to combine the wanted and unwanted end points into a single function. We hypothesized that the function would be predominantly negative over the tested oxycodone concentration range. Twenty-four male and female volunteers received 20 (n=12) or 40 (n=12) mg oral oxycodone immediate-release tablets. Hypercapnic ventilatory responses (visit 1) or responses to 3 nociceptive assays (pain pressure, electrical, and thermal tests; visit 2) were measured at regular intervals for 7 hours. the PK/PD analyses, that included carbon dioxide kinetics, stood at the basis of the utility function: probability of antinociception minus probability of respiratory depression. Oxycodone had rapid onset/offset times (30-40 minutes) with potency values (effect-site concentration causing 50% of effect) ranging from 0.05 to 0.13 ng/mL for respiratory variables obtained at hypercapnia and antinociceptive responses. Ventilation at an extrapolated end-tidal carbon dioxide partial pressure of 55 mmHg, was used for creation of 3 utility functions, one for each of the nociceptive tests. Contrary to expectation, the utility functions were close to zero or positive over the clinical oxycodone concentration range. The similar or better likelihood for antinociception relative to respiratory depression may be related to oxycodone's receptor activation profile or to is high likeability that possibly alters the modulation of nociceptive input. Oxycodone differs from other μ-opioids, such as fentanyl, that have a consistent negative utility.

Influence of early-life adversity on responses to acute and chronic ethanol in female mice.

Stressful early-life experiences increase the risk of developing an alcohol use disorder. We previously found that male C57BL/6J mice reared under limited bedding and nesting (LBN) conditions, a model of early-life adversity, escalate their ethanol intake in limited-access two-bottle choice (2BC) sessions faster than control (CTL)-reared counterparts when exposed to chronic intermittent ethanol (CIE) vapor inhalation. However, the alcohol consumption of female littermates was not affected by LBN or CIE. In the present study, we sought to determine whether this phenotype reflected a general insensitivity of female mice to the influence of early-life stress on alcohol responses. In a first experiment, CTL and LBN females with a history of 2BC combined or not with CIE were tested in affective and nociceptive assays during withdrawal. In a second group of CTL and LBN females, we examined ethanol-induced antinociception, sedation, plasma clearance, and c-Fos induction. In females withdrawn from chronic 2BC, CIE increased digging, reduced grooming, and increased immobility in the tail suspension test regardless of early-life history. In contrast, LBN rearing lowered mechanical nociceptive thresholds regardless of CIE exposure. In females acutely treated with ethanol, LBN rearing facilitated antinociception and delayed the onset of sedation without influencing ethanol clearance rate or c-Fos induction in the paraventricular nucleus of the hypothalamus, paraventricular nucleus of the thalamus, central nucleus of the amygdala, or auditory cortex. CIE withdrawal produced multiple indices of negative affect in C57BL/6J females, suggesting that their motivation to consume alcohol may differ from air-exposed counterparts despite equivalent intake. Contrasted with our previous findings in males, LBN-induced mechanical hyperalgesia in chronic alcohol drinkers was specific to females. Lower nociceptive thresholds combined with increased sensitivity to the acute antinociceptive effect of ethanol may contribute to reinforcing ethanol consumption in LBN females but are not sufficient to increase their intake.

Generalized resistance to pruritogen-induced scratching in the C3H/HeJ strain.

Previously the effect of the pruritogens, such as histamine and chloroquine, was tested in 11 inbred mouse strains, and this study aimed to identify resistant and sensitive strains, consistent with the observation that underlies the large variability in human populations. In the present study, we used the low responder C3H/HeJ (C3H) and the more sensitive C57BL/6J (C57) strain to find out if resistance and sensitivity to develop pruritus is restricted to only histamine and chloroquine or extends to other known pruritogens as well. We tested five additional commonly known pruritogens. We established dose-response relationships by injecting four concentrations of the pruritogens in the range of 0.3, 1, 3, and ten-fold in the nuchal fold. Then we assessed the scratching behavior for 30 min after injection with an automated custom-designed device based on the bilateral implantation of mini-magnets in the hind paws and on single cages placed within a magnetic coil. We found that the resistance to pruritogens is a general phenotype of the C3H strain and extends to all pruritogens tested, including not only histamine and chloroquine, but also endothelin, trypsin, 5-HT (serotonin), the short peptide SLIGRL, and Lysophosphatidic acid (LPA). C57 was more sensitive to all pruritogens and, in contrast to C3H, dose-response relationships were evident for some of the pruritogens. In general, comparable peak scratch responses were observed for the 0.3-fold concentrations of the pruritogens in C57 whereas C3H required at least the ten-fold concentration and still displayed only between 5 and 33% of the scratch responses observed in C57 for the respective pruritogen. The general resistance to pruritogens and the low level of scratching behavior found in the C3H strain is an interesting trait and represents a model for the study of the heritability of itch. It is accompanied in C3H with a higher sensitivity in assays of nociception.

Opioids alter paw placement during walking, confounding assessment of analgesic efficacy in a postsurgical pain model in mice.

Introduction:Hind paw–directed assays are commonly used to study the analgesic effects of opioids in mice. However, opioid-induced hyperlocomotion can obscure results of such assays.Objectives:We aimed to overcome this potential confound by using gait analysis to observe hind paw usage during walking in mice.Methods:We measured changes in the paw print area after induction of postsurgical pain (using the paw incision model) and treatment with oxycodone.Results:Paw incision surgery reduced the paw print area of the injured hind paw as mice avoided placing the incised section of the paw on the floor. Surprisingly, oxycodone caused a tiptoe-like gait in mice, reducing the paw print area of both hind paws. Further investigation of this opioid-induced phenotype revealed that analgesic doses of oxycodone or morphine dose-dependently reduced the hind paw print area in uninjured mice. The gait changes were not dependent on opioid-induced increases in the locomotor activity; speed and paw print area had no correlation in opioid-treated mice, and other analgesic compounds that alter locomotor activity did not affect the paw print area.Conclusion:Unfortunately, the opioid-induced “tiptoe” gait phenotype prevented gait analysis from being a viable metric for demonstrating opioid analgesia in injured mice. However, this work reveals an important, previously uncharacterized effect of treatment with analgesic doses of opioids on paw placement. Our characterization of how opioids affect gait has important implications for the use of mice to study opioid pharmacology and suggests that scientists should use caution when using hind paw–directed nociceptive assays to test opioid analgesia in mice.

S-Ketamine oral thin film-Part 2: Population pharmacodynamics of S-ketamine, S-norketamine and S-hydroxynorketamine.

Ketamine is a versatile drug used for many indications and is administered via various routes. Here, we report on the pharmacodynamics of sublingual and buccal fast-dissolving oral-thin-films that contain 50 mg of S-ketamine in a population of healthy male and female volunteers. Twenty volunteers received one or two 50 mg S-ketamine oral thin films in a crossover design, placed for 10 min sublingually (n = 15) or buccally (n = 5). The following measurements were made for 6 h following the film placement: antinociception using three distinct pain assay; electrical, pressure, and heat pain, and drug high on an 11-point visual analog scale. Blood samples were obtained for the measurement of plasma S-ketamine, S-norketamine, and S-hydroxynorketamine concentrations. A population pharmacodynamic analysis was performed in NONMEM to construct a pharmacodynamic model of S-ketamine and its metabolites. P-values < 0.01 were considered significant. The sublingual and buccal 50 and 100 mg S-ketamine oral thin films were antinociceptive and produced drug high with effects lasting 2–6 h, although a clear dose-response relationship for antinociception could not be established. The effects were solely related to the parent compound with no contribution from S-norketamine or S-hydroxynorketamine. S-ketamine potency was lower for antinociception (C50 ranging from 1.2 to 1.7 nmol/mL) than for drug high (C50 0.3 nmol/ml). The onset/offset of effect as defined by the blood-effect-site equilibration half-life did not differ among endpoints and ranged from 0 to 5 min. In conclusion, the 50-mg S-ketamine oral thin film was safe and produced long-term antinociception in all three nociceptive assays with side effects inherent to the use of ketamine. The study was registered at the trial register of the Dutch Cochrane Center (www.trialregister.nl) under identifier NL9267 and the European Union Drug Regulating Authorities Clinical Trials (EudraCT) database under number 2020-005185-33.

Sex differences in choice-based thermal nociceptive tests in adult rats

Interest in the role of sex as a biological variable has increased, including a mandate for the study of both sexes in NIH-funded research. As sex differences exist in both human chronic pain conditions and rodent models of nociception, it is critical to understand the impact of sex in nociceptive assays. Choice-based thermal nociceptive tests permit the study of avoidance responses to thermal stimuli compared to traditional nociceptive assays, which measure nocifensive reactions. However, to date no comparison of male and female responses to choice-based tests has been published. Herein, we examined the effect of sex on two choice-based thermal nociceptive tests, the thermal gradient test and the temperature place preference test, in adult rats. The activation of a 10 °C-to-47 °C thermal gradient results in an increase in time spent in the 10 °C zone in females, compared to a reduction in males. Additionally, in a temperature place preference test pairing a surface temperature of 22 °C with either 5 °C, 10 °C, 47 °C, or 50 °C, females appeared to have overall greater tolerance for non-ambient temperatures. Males spent less than 50% of their time in every non-22 °C zone, whereas in females this was only observed when testing 5 °C and 50 °C. Together, these results suggest that male rats show more avoidance behavior than females to both hot and cold non-ambient temperatures when given free access to multiple zones, including at milder temperatures than those typically used to evoke a nociceptive response in traditional hot and cold plate tests.

Thermosensitive injectable graphene oxide/chitosan-based nanocomposite hydrogels for controlling the in vivo release of bupivacaine hydrochloride

Local anesthetics are commonly used for the management of intraoperative and postoperative acute and chronic pain caused by small invasive procedures. However, their short half-life and duration of action limit their clinical benefits. In this study, we proposed the incorporation of graphene oxide (GO) nanosheets to chitosan (CS)/β-glycerophosphate (GP) thermosensitive hydrogel system to form an injectable nanocomposite hydrogel (NCH) with improved mechanical properties and better control over the release of bupivacaine hydrochloride (BH). The prepared NCHs were characterized for their gelation time, porosity, swelling ratio, injectability, mechanical strength and in vitro drug release. In vivo, the efficacy of the prepared NCH containing 0.5 % w/v BH was evaluated using a thermal nociceptive assay in a rat model. The incorporation of GO significantly enhanced the physicochemical and mechanical properties of the hydrogel scaffolds in a concentration-dependent manner. Inclusion of 0.1% w/v GO resulted in 84% reduction in gelation time and 16% and 40% decrease in the porosity and swelling ratio of the NCHs, respectively. The mechanical strength of the CS/GP hydrogel scaffolds was also significantly improved in presence of GO. BH was slowly released from the NCHs containing 0.1% w/v GO and resulted in a 55% and 86.43% drug release after 6 and 24 h, respectively. In vivo studies showed that BH-loaded NCH significantly prolonged the local anesthetic effect and resulted in a 6.5-fold increase in blocking the pain sensory reflex compared to BH solution. These results indicate that the incorporation of GO significantly improved the physical and mechanical properties of CS/GP thermosensitive hydrogels and successfully sustained the effect of local anesthesia for more effective pain management.

Translating Outcomes from the Clinical Setting to Preclinical Models: Chronic Pain and Functionality in Chronic Musculoskeletal Pain.

Fibromyalgia (FM) is a chronic pain disorder characterized by chronic widespread musculoskeletal pain (CWP), resting pain, movement-evoked pain (MEP), and other somatic symptoms that interfere with daily functioning and quality of life. In clinical studies, this symptomology is assessed, while preclinical models of CWP are limited to nociceptive assays. The aim of the study was to investigate the human-to-model translatability of clinical behavioral assessments for spontaneous (or resting) pain and MEP in a preclinical model of CWP. For preclinical measures, the acidic saline model of FM was used to induce widespread muscle pain in adult female mice. Two intramuscular injections of acidic or neutral pH saline were administered following baseline measures, 5 days apart. An array of adapted evoked and spontaneous pain measures and functional assays were assessed for 3 weeks. A novel paradigm for MEP assessment showed increased spontaneous pain following activity. For clinical measures, resting and movement-evoked pain and function were assessed in adult women with FM. Moreover, we assessed correlations between the preclinical model of CWP and in women with fibromyalgia to examine whether similar relationships between pain assays that comprise resting and MEP existed in both settings. For both preclinical and clinical outcomes, MEP was significantly associated with mechanical pain sensitivity. Preclinically, it is imperative to expand how the field assesses spontaneous pain and MEP when studying multi-symptom disorders like FM. Targeted pain assessments to match those performed clinically is an important aspect of improving preclinical to clinical translatability of animal models.