Pain In Formalin Test Research Articles

Ganoderma tsuage promotes pain sensitivity in aging mice

Advances in modern medicine have extended human life expectancy, leading to a world with a gradually aging society. Aging refers to a natural decline in the physiological functions of a species over time, such as reduced pain sensitivity and reaction speed. Healthy-level physiological pain serves as a warning signal to the body, helping to avoid noxious stimuli. Physiological pain sensitivity gradually decreases in the elderly, increasing the risk of injury. Therefore, geriatric health care receives growing attention, potentially improving the health status and life quality of the elderly, further reducing medical burden. Health food is a geriatric healthcare choice for the elderly with Ganoderma tsuage (GT), a Reishi type, as the main product in the market. GT contains polysaccharides, triterpenoids, adenosine, immunoregulatory proteins, and other components, including anticancer, blood sugar regulating, antioxidation, antibacterial, antivirus, and liver and stomach damage protective agents. However, its pain perception-related effects remain elusive. This study thus aimed at addressing whether GT could prevent pain sensitivity reduction in the elderly. We used a galactose-induced animal model for aging to evaluate whether GT could maintain pain sensitivity in aging mice undergoing formalin pain test, hot water test, and tail flexes. Our results demonstrated that GT significantly improved the sensitivity and reaction speed to pain in the hot water, hot plate, and formalin tests compared with the control. Therefore, our animal study positions GT as a promising compound for pain sensitivity maintenance during aging.

Endomorphin-2 analogs with C-terminal esterification display potent antinociceptive effects in the formalin pain test in mice

Previously, we have investigated three C-terminal esterified endomorphin-2 (EM-2) analogs EM-2-Me, EM-2-Et and EM-2-Bu with methyl, ethyl and tert-butyl ester modifications, respectively. These analogs produced significant antinociception in acute pain at the spinal and supraspinal levels, with reduced tolerance and gastrointestinal side effects. The present study was undertaken to determine the analgesic effects and opioid mechanisms of these three analogs in the formalin pain test. Our results demonstrated that intracerebroventricular (i.c.v.) administration of 0.67–20 nmol EM-2 analogs EM-2-Me, EM-2-Et and EM-2-Bu produced dose-dependent antinociceptive effects in both phase Ⅰ and phase Ⅱ of formalin pain. EM-2-Me and EM-2-Bu displayed more potent antinociception than morphine. Especially, EM-2-Bu exhibited the highest antinociception in phase Ⅱ of formalin pain, with the ED50 value being 2.1 nmol. Naloxone (80 nmol, i.c.v.) completely antagonized the antinociceptive effects of EM-2-Me, EM-2-Et and EM-2-Bu (20 nmol, i.c.v.) in both phase I and phase Ⅱ of formalin pain, suggesting a central opioid mechanism. Nevertheless, the antinociception induced by EM-2-Me might be involved in the release of dynorphin A, which subsequently acted on κ- opioid receptor. EM-2-Bu produced the antinociception probably by the direct activation of both μ- and δ-opioid receptors. EM-2-Me, EM-2-Et and EM-2-Bu also produced significant analgesic effects after peripheral administration, and the central opioid receptors were involved. Furthermore, EM-2-Bu had no influence on the locomotor activity after i.c.v. injection. The present investigation demonstrated that C-terminal esterified modifications of EM-2 will be beneficial for developing novel therapeutics in formalin pain.

Contribution of the intra-hippocampal orexin system in the regulation of restraint stress response to pain-related behaviors in the formalin test.

Stress-induced antinociception (SIA) is due to the activation of several neural pathways and neurotransmitters that often suppress pain perception. Studies have shown that the orexin neuropeptide system is essential in pain modulation. Therefore, this study aimed to investigate the role of orexinergic receptors in the hippocampal CA1 region in modulating SIA response during the formalin test as an animal model of inflammatory pain. The orexin-1 receptor (OX1r) antagonist, SB334867, at 1, 3, 10, and 30 nmol or TCS OX2 29 as an orexin-2 receptor (OX2r) antagonist at the same doses were microinjected into the CA1 region in rats. Five minutes later, rats were exposed to restraint stress (RS) for 3 h, and pain-related behaviors were monitored in 5-min blocks for the 60-min test period in the formalin test. Results showed that applying RS for 3 h reduced pain responses in the early and late phases of the formalin test. The main findings showed that intra-CA1 injection of orexin receptor antagonists reduced the antinociception caused by stress in both phases of the formalin test. In addition, the contribution of OX2r in mediating the antinociceptive effect of stress was more prominent than that of OX1r in the early phase of the formalin test. However, in the late phase, both receptors worked similarly. Accordingly, the orexin system and its two receptors in the CA1 region of the hippocampus regulate SIA response to this animal model of pain in formalin test.

The integrative role of orexin-1 and orexin-2 receptors within the hippocampal dentate gyrus in the modulation of the stress-induced antinociception in the formalin pain test in the rat.

The stressful experiences, by triggering a cascade of hormonal and neural changes, can produce antinociception commonly referred to as stress-induced antinociception (SIA). Orexin neuropeptides have an essential role in stress responses and pain modulation. The dentate gyrus receives orexinergic projections and has been shown to be involved in pain processing. The current study investigated the possible role of orexin-1 and orexin-2 receptors (OX1r and OX2r, respectively) within the dentate gyrus in SIA in a rat model of formalin-induced pain behavior in one hind paw. Male Wistar rats weighing 230-250 g underwent stereotaxic surgery and a cannula was implanted in their brains, above the dentate gyrus region. Either SB334867 or TCS OX2 29 (OX1r and OX2r antagonists, respectively) was microinjected into the dentate gyrus region at a range of doses at 1, 3, 10, and 30 nmol (control group received DMSO 12% as vehicle), 5 min before the forced swim stress (FSS) exposure. The formalin test was performed to assess pain-related behaviors. The results indicated that FSS exposure relieves pain-related behavior in the early and late phases of the formalin test. Blockade of intra-dentate gyrus OX1 or OX2 receptors reduced the antinociceptive responses induced by FSS in the formalin test, with more impact during the late phase. Our findings support the potential role of intra-dentate gyrus orexin receptors as target sites of orexin neurons in painful and stressful situations. Therefore, understanding the exact mechanisms of SIA and the role of the orexinergic system in this phenomenon can lead to identifying the strategies to guide future research and offer a new approach to discovering new pain therapeutic agents.

Increased nociceptive behaviors and spinal c-Fos expression in the formalin test in a rat repeated cold stress model

Repeated cold stress (RCS) can trigger the development of fibromyalgia (FM)-like symptoms, including persistent deep-tissue pain, although nociceptive changes to the skin have not been fully characterized. Using a rat RCS model, we investigated nociceptive behaviors induced by noxious mechanical, thermal, and chemical stimuli applied to plantar skin. Neuronal activation in the spinal dorsal horn was examined using the formalin pain test. In rats exposed to RCS, nociceptive behavioral hypersensitivity was observed in all modalities of cutaneous noxious stimuli: the mechanical withdrawal threshold was decreased, and the heat withdrawal latency was shortened one day after the cessation of stress. The duration of nocifensive behaviors in the formalin test was prolonged in phase II but not in phase I. The number of c-Fos-positive neurons increased in the entire dorsal horn laminae I-VI, ipsilateral, but not contralateral, to formalin injection at the L3-L5 segments. The duration of nocifensive behavior in phase II was significantly and positively correlated with the number of c-Fos-positive neurons in laminae I-II. These results demonstrate that cutaneous nociception is facilitated in rats exposed to RCS for a short time and that the spinal dorsal horn neurons are hyperactivated by cutaneous formalin in the RCS model.

Investigation of the Antinociceptive Activity of the Hydroethanolic Extract of Junglas nigra Leaf by the Tail-Immersion and Formalin Pain Tests in Rats

BackgroundJuglans (J.) nigra leaf is obtained from a plant that is used in traditional medicine in some countries to alleviate inflammatory diseases.AimThe aim of this study was to compare the effects of J. nigra extract on acute nociceptive and inflammatory pain in rats.MethodsAntinociceptive activity was examined in Wistar rats by the tail-immersion and formalin tests. Motor function was assessed using the rotarod test. Plant extract was administered intraperitoneally.ResultsIn the tail-immersion test, the maximal antinociceptive effect of the plant extract (100–330 mg/kg) was about 24–30% and is the result of the effect of a high concentration of ethanol. In the formalin test, the plant extract (41.3–330 mg/kg) significantly and dose-dependently inhibited nociception in both phases of the test with similar maximal effects of about 76% and 85%. Only the plant extract at the dose of 330 mg/kg caused a significant time-dependent reduction in time spent on the rotarod.ConclusionsIn rats, the preventive systemic administration of the hydroethanolic extract of J. nigra leaf reduced chemically but not thermally induced pain. Higher efficacy was obtained in pain associated with inflammation and tissue injury. The antinociceptive effect is dose-dependent and may be limited by motor impairment.

Antinociceptive Effects and Interaction Mechanisms of Intrathecal Pentazocine and Neostigmine in Two Different Pain Models in Rats

Background Pentazocine produces a wide variety of actions in the treatment of perioperative analgesia. Neostigmine is a cholinesterase inhibitor used to antagonize the residual effects of muscle relaxants and also produces an analgesic effect. Objectives To investigate the analgesic effects of intrathecally injected pentazocine and neostigmine and their interaction. Methods Sprague–Dawley rats were used to test the analgesic effect of pentazocine and neostigmine using the paw formalin pain model and the incision mechanical allodynia model. Pentazocine (3, 10, 30, and 100 μg), neostigmine (0.3, 1, 3, and 10 μg) or a pentazocine-neostigmine mixture were separately injected to evaluate their antinociceptive effects alone on the treatment groups. The corresponding control group received an intrathecal injection containing the same volume of saline. The formalin pain test, or the plantar incision pain behavior test were performed 30 minutes later. Isobolographic analysis was used to evaluate the interaction between pentazocine and neostigmine. Intrathecally administered selective mu-opioid receptor antagonist CTAP, selective kappa-opioid receptor antagonist nor-Binaltorphimine (nor-BNI), nonselective opioid receptor antagonist naloxone, and muscarinic acetylcholine receptor antagonist atropine were also used to test the possible interaction mechanism. These antagonists were used 30 minutes before the pentazocine and neostigmine mixtures which were intrathecally injected. Results Intrathecally administered pentazocine (3, 10, 30, and 100 μg) and neostigmine (0.3, 1, 3, and 10 μg) alone had a marked dose-related impact on suppressing the biphasic responses in the formalin test. Pentazocine (3, 10, 30, and 100 μg) and neostigmine (0.3, 1, 3, and 10 μg) alone attenuated the mechanical allodynia in a plantar incision model in a dose-dependent manner. Isobolographic analysis revealed that the mixture of intrathecal pentazocine and neostigmine synergistically decreased both phase I and II activity in the formalin test and mechanical allodynia in the plantar incision model. Pretreatment of intrathecally administered nor-BNI, naloxone, atropine, but not CTAP, antagonized the analgesic effect of the pentazocine-neostigmine mixture. Conclusions All of these results suggest that the combined application of pentazocine and neostigmine is an effective way to relieve pain from formalin and acute incision mechanical allodynia. The synergistic effect between pentazocine and neostigmine is mostly attributed to the kappa-opioid receptor and the cholinergic receptor in the spinal cord.

Nociceptive chemical hypersensitivity in the spinal cord of a rat reserpine-induced fibromyalgia model

The pathological mechanisms of fibromyalgia (FM) are largely unknown. Recently, a rat reserpine-induced pain model showing exaggerated pain-related behaviors to mechanical and thermal stimuli has been used in FM research. However, the model has not been fully characterized. Here, we investigated nociceptive hypersensitivity to chemical stimuli and its spinal mechanisms to further characterize the model. The rat model was induced by administering reserpine to the nervous system. Nociceptive behaviors to chemical stimuli were quantified using the formalin pain test, and neuronal activation of the stimuli was examined using spinal c-Fos immunohistochemistry and electrophysiological recordings of superficial dorsal horn (SDH) neurons. The duration of pain-related behaviors was prolonged in both phases I (0−5 min) and II (10−60 min) and the interphase; and the number of c-Fos-immunoreactive nuclei increased in laminae I−II, III−IV, and V−VI at the spinal segments L3−L5 on the side ipsilateral to the formalin injection, and these factors were significantly and positively correlated. The action potentials of SDH neurons induced by formalin injection were markedly increased in rats treated with reserpine. These results demonstrate that pain-related behaviors are facilitated by noxious chemical stimuli in a rat reserpine-induced FM model, and that the behavioral hypersensitivity is associated with hyperactivation of SDH neurons.

Protopanaxadiol alleviates neuropathic pain by spinal microglial dynorphin A expression following glucocorticoid receptor activation.

New remedies are required for the treatment of neuropathic pain due to insufficient efficacy of available therapies. This study provides a novel approach to develop painkillers for chronic pain treatment. The rat formalin pain test and spinal nerve ligation model of neuropathic pain were used to evaluate antinociception of protopanaxadiol. Primary cell cultures, immunofluorescence staining, and gene and protein expression were also performed for mechanism studies. Gavage protopanaxadiol remarkably produces pain antihypersensitive effects in neuropathic pain, bone cancer pain and inflammatory pain, with efficacy comparable with gabapentin. Long-term PPD administration does not induce antihypersensitive tolerance, but prevents and reverses the development and expression of morphine analgesic tolerance. Oral protopanaxadiol specifically stimulates spinal expression of dynorphin A in microglia but not in astrocytes or neurons. Protopanaxadiol gavage-related pain antihypersensitivity is abolished by the intrathecal pretreatment with the microglial metabolic inhibitor minocycline, dynorphin antiserum or specific κ-opioid receptor antagonist GNTI. Intrathecal pretreatment with glucocorticoid receptor)antagonists RU486 and dexamethasone-21-mesylate, but not GPR-30 antagonist G15 or mineralocorticoid receptor antagonist eplerenone, completely attenuates protopanaxadiol-induced spinal dynorphin A expression and pain antihypersensitivity in neuropathic pain. Treatment with protopanaxadiol, the glucocorticoid receptor agonist dexamethasone and membrane-impermeable glucocorticoid receptor agonist dexamethasone-BSA in cultured microglia induces remarkable dynorphin A expression, which is totally blocked by pretreatment with dexamthasone-21-mesylate. All the results, for the first time, indicate that protopanaxadiol produces pain antihypersensitivity in neuropathic pain probably through spinal microglial dynorphin A expression after glucocorticoid receptor activation and hypothesize that microglial membrane glucocorticoid receptor/dynorphin A pathway is a potential target to discover and develop novel painkillers in chronic pain.

The anti-inflammatory and analgesic activities of the ethyl acetate extract of Viburnum taitoense Hayata

Ethnopharmacological relevanceViburnum taitoense Hayata has been used as folk medicine by the minority people in Southwestern China for a long history, especially in Guangxi Zhuang Autonomous Region. The minority in Guangxi including Zhuang, Miao and Yao people use the ethanol extract of V. taitoense Hayata to treat the fracture, kill the pain of rheumatism because of its definite therapeutic effects. Aim of the studySo far, the scientific investigation of V. taitoense Hayata is done very little. Here, we first prepared the ethyl acetate extract of V. taitoense (EEVt), secondly measured the contents of phenols, flavonoids, and terpenoids in EEVt, and thirdly, the anti-inflammatory and analgesic activities of EEVt were investigated by invitro model of RAW 264.7 cells and invivo models of inflammation and pain in rats and mice. Materials and methodsThe contents of phenols, flavonoids, and terpenoids in EEVt were determined by UV spectrophotometry, respectively. The anti-inflammatory effect of EEVt (5, 25, 50, 100, and 200 μg/mL) in vitro was tested by determining its inhibitory effect on the nitric oxide production of RAW264.7 cells activated by lipopolysaccharide (LPS). The anti-inflammatory and analgesic effects of EEVt in vivo were investigated in the following experimental rats and mice models: carrageenan-induced paw edema, corton-oil-induced ear edema, acetic acid writhing test, and formalin pain test. ResultsThe contents of total phenolic, total flavonoids, and total triterpenoids in V. taitoense were measured to be 3.46 ± 0.04%, 2.38 ± 0.04%, and 14.96 ± 0.17%, respectively. In vitro test showed that EEVt at different tested dosages (5, 25, 50, 100, and 200 μg/mL) had no significant toxicity to RAW264.7 macrophages. At dosages of 37.5 and 75 μg/mL of EEVt significant inhibitory (p < 0.001) on the productions of nitric oxide (NO). High dosage (200 μg/mL) of EEVt displayed highly significant inhibitory (p < 0.001) on the productions of proinflammatory cytokines IL-6, IL-1β, and TNF-α from the LPS-induced RAW264.7 macrophages. EEVt showed obvious anti-inflammatory activity at different time points after carrageenan injection (p < 0.05) in vivo test, and its anti-inflammatory activity reached the strongest 4 h. Similarly, through the ear swelling test, EEVt (200 mg/kg) showed significant (p < 0.05) anti-inflammatory activity. Besides, formalin and acetic acid writhing experiments also showed that EEVt has significant (p < 0.05) analgesic activity. ConclusionEEVt was confirmed to be definite anti-inflammatory and analgesic effects, and the phytochemicals of EEVt was disclosed to be rich in triterpenoids, which was worthy to be further investigated.

Azadirone from Toona sinensis possesses anti-inflammatory and analgesic activities via suppressing MAPK signal pathway

This study was designed to investigate the anti-inflammatory, analgesic activity of azadirone from Toona sinensis (Juss.) M. Roem., Meliaceae, and explore its potential mechanisms. Anti-inflammatory activities of azadirone were evaluated using dimethylbenzene-induced ear edema, acetic acid–induced vascular permeability, and cotton ball granuloma in mice; in addition, its analgesic activities were investigated using acetic acid writhing, hot plate pain, and formalin pain tests. The results showed that azadirone had obvious anti-inflammatory and analgesic effects in animals. Further studies revealed that azadirone can reduce levels of TNF-α, IL-6, and IL-1 in lipopolysaccharide-stimulated RAW 264.7 cells. In addition, azadirone can downregulate COX-2 and substance P in spinal cord tissues of formalin-stimulated mice. Also, expressions of ERK, P38, and JNK in RAW 264.7 cells were suppressed by azadirone. Collectively, azadirone has promising anti-inflammatory and analgesic potentials, and its mechanism is closely related to the reduction of pro-inflammatory cytokines and the blocking of MAPK signal pathway.

Interaction between hyperbaric oxygen therapy and prostaglandin synthesis inhibition in the formalin pain test

BackgroundPain is the main symptom of inflammatory diseases, such as diabetes mellitus, hypertension, osteoarthritis among others. Lipopolysacaccharide from E. coli (LPS) has been used to induce an inflammatory process. Prostaglandins has a role in inflammation so that NSAIDs are useful pharmacological tools to treat it. Hyperbaric oxygen therapy (HBOT) has also been used in some of these diseases. However, information about pharmacological interaction with HBOT and its mechanism is scarce. So, the purpose of this work was to study the interaction between LPS, HBOT and NSAIDs using a formalin pain test.Methods96 adults male Wistar rats (150 to 200 g) were used and 2 groups treated with LPS or its vehicle were formed and each of them was divided in the next subgroups (n=8 each): Control, indomethacin, rofecoxib, HBOT, HBOT/indomethacin and HBOT/rofecoxib. 20 μL of 2% formalin was injected into the plantar surface of the right hind paw. Then nociceptive behavior was evaluated as the number of flinches in 1 min every 5 for 60 min. LPS and HBOT were applied 4 and 2.5 h prior to formalin respectively. Additionally, PGE2 and PGI2 was quantified using RP‐HPLC‐UV.ResultsFormalin produces a nociceptive behavior that decreased in the first 10 min (phase I) and in the next 50 increased to a plateau and then become to reduce (phase II, inflammatory). LPS increased and HBOT and drugs treatment reduced the phase II, while PGI2 levels were increased with LPS and reduced with HBOT and drugs. The HBOT seems to increase the indomethacin antinociceptive effect but HBOT effect was lesser than control and levels of prostaglandins were similar than in absence of HBOT. LPS treatment reduced the rofecoxib and HBOT effect increasing PGE2 and PGI2 levels respectively. Finally, when HBOT is associated with LPS and indomethacin, an increase on AUC of formalin and PGI2 levels was observed.ConclusionsThe HBOT and prostaglandin synthesis inhibition were effective antinociceptive interventions. However, indomethacin but not rofecoxib increased the HBOT antinoceptive effect diminishing PGI2 levels. In presence of an inflammatory process, the effect of HBOT and rofecoxib but no indomethacin, the antinociception was lost due to an increase in prostaglandin synthesis. Therefore, there are an interaction between HBOT and PG synthesis inhibition and the presence of inflammation reduced the beneficial effects of physical or pharmacological interventions.Support or Funding InformationThe authors would like to acknowledge CONACYT (557096) for their financial support

Opioid-sparing effects of cannabinoids on morphine analgesia: participation of CB1 and CB2 receptors.

Much of the opioid epidemic arose from abuse of prescription opioid drugs. This study sought to determine if the combination of a cannabinoid with an opioid could produce additive or synergistic effects on pain, allowing reduction in the opioid dose needed for maximal analgesia. Pain was assayed using the formalin test in mice and the carrageenan assay in rats. Morphine and two synthetic cannabinoids were tested: WIN55,212-2 (WIN), which binds to both CB1 and CB2 receptors, and possibly TRPV1 channels; and GP1a, which has activity at CB2 receptors and is reported to inhibit fatty acid amide hydrolase, thus raising levels of endogenous cannabinoids. Morphine in combination with WIN in the formalin test gave synergistic analgesia. Studies with selective antagonists showed that WIN was acting through CB1 receptors. Morphine in combination with GP1a in the formalin test was sub-additive. In the carrageenan test, WIN had no added effect when combined with morphine, but GP1a with morphine showed enhanced analgesia. Both WIN and Gp1a used alone had analgesic activity in the formalin pain test, but not in the carrageenan pain test. The ability of a cannabinoid to produce an additive or synergistic effect on analgesia when combined with morphine varies with the pain assay and may be mediated by CB1 or CB2 receptors. These results hold the promise of using cannabinoids to reduce the dose of opioids for analgesia in certain pain conditions.

Effects of Salmon Calcitonin on the Concentrations of Monoamines in Periaqueductal Gray in Formalin Test

Background:The receptors of salmon calcitonin, located on certain areas of the brain such as the periaqueductal gray matter, are responsible for pain modulation.Aims:The effects of intracerebroventricular injection of salmon calcitonin on the behavioral response to pain and on the levels of monoamines in the periaqueductal gray were explored using a biphasic animal model of pain.Study Design:Animal experiment.Methods:A total of 45 male rats were divided into four groups (n=6). Salmon calcitonin was injected into the lateral ventricle of the brain (1.5 nmol, with a volume of 5 μL). After 20 min, 2.5% formalin was subcutaneously injected into the right leg claw, and pain behavior was recorded on a numerical basis. At the time of the formalin test, the periaqueductal gray area was microdialized. High-performance liquid chromatography method was used to gauge the levels of monoamines and their metabolites.Results:Intracerebroventricular injections of salmon calcitonin resulted in pain reduction in the formalin test (p<0.05). The dialysate concentrations of serotonin, dopamine, norepinephrine, 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic, and 4-hydroxy-3-methoxyphenylglycol increased in the periaqueductal gray area in different phases of the formalin pain test (p<0.05).Conclusion:Salmon calcitonin reduced pain by increasing the concentrations of monoamines and the metabolites derived from them in the periaqueductal gray area.

Arachidonoyl‐2‐chloroethylamide alleviates inflammatory pain in a sex‐dependent manner using the formalin test

Previous studies have shown that certain cannabinoids have analgesic effects. Specifically, activation of cannabinoid receptor (CB) 1 has been demonstrated to produce analgesia in multiple animal models. Arachidonyl‐2‐chloroethylamide (ACEA) is a synthetic agonist for CB1 receptors. The activation of CB1 with ACEA could enable chronic inflammatory pain patients to manage this discomfort. ACEA has not been extensively studied in animal models; therefore, not much is known about the dose effectiveness, development of tolerance, or sex‐specific effects.To study the analgesic effects of ACEA, we treated male and female C57BL6J mice prior to the formalin pain test. Administration of formalin (2.5% intraplantar) produces a biphasic pain response, modeling both acute and inflammatory pain. We believe that administration of ACEA at a dose of 0.5 mg per kg intraperitoneal will reduce inflammatory pain in this model. Also, we suggest that daily pretreatment with ACEA prior to the formalin test will cause desensitization to this compound; therefore, the initial analgesic effect will not be seen. To examine sex‐specific effects, we also tested and compared male and female mice. Moreover, vaginal lavage was performed daily on female mice to track the estrous cycle.Preliminary data suggests that females experience higher pain sensitivity compared with males when ACEA 0.5 mg per kg was used. Furthermore, our preliminary data shows that chronic administration in females results in estrous cycle changes. We concluded that there is a sex‐dependent difference in analgesic response to ACEA, and that it may be hormonally‐mediated.Support or Funding InformationFunded by NIDA DA044999‐01A1 (JG) and Texas Tech University Health Sciences Center School of Medicine #121035 (JG).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanistic assessment of the analgesic, anti-inflammatory and antipyretic actions of Dalbergia saxatilis in animal models

Context: Aqueous root extract of Dalbergia saxatilis, Hook, f., (Leguminosae) (DS) is reported useful for toothache, pains, and fever, but not scientifically proven. Objective: This study determined its effectiveness in pain, inflammation, and fever, applying scientific models. Materials and methods: Swiss mice or Sprague–Dawley rats (n = 5) were pretreated with distilled water, DS (100 or 200 mg/kg), or standard drug for 30 min. The analgesic activity was measured by acetic acid writhing, tail flick, tail immersion, tail clip, hot plate, and formalin pain tests; anti-inflammatory effects were determined via carrageenan and dextran rat paw oedema tests; antipyretic activity was measured by Escherichia coli lipopolysaccharide (ECL) and turpentine in rabbits, and d-amphetamine sulphate (d-AS) pyrexia test in rats. Results: Writhing frequency inhibition was produced by 200 mg/kg DS (33.10%), aspirin (38.19%) and morphine (93.68%). Unlike morphine, DS did not produce significant prolongation of the reaction times in the hot-plate, tail immersion, tail flick, and tail clip tests. In the first and second phases of formalin test, respectively, % inhibition was: 200 mg/kg DS (25.70% and 0%), aspirin (4.76% and 67.33%), morphine (81.42% and 66.11%); for carrageenan and dextran tests, significant difference was recorded between 200 mg/kg DS and control up to 6 h. Significant reduction in ECL, turpentine and d-AS pyrexia was recorded at 100 and 200 mg/kg DS. Conclusion: DS produces mild non-steroidal analgesic and anti-inflammatory, as well as significant antipyretic actions involving cyclooxygenase, α2 adrenoceptor and interleukin-1 β1 due to any of glycosides, saponins or phenolic tannins.

Restraint training for awake functional brain scanning of rodents can cause long-lasting changes in pain and stress responses.

With the increased interest in longitudinal brain imaging of awake rodents, it is important to understand both the short-term and long-term effects of restraint on sensory and emotional processing in the brain. To understand the effects of repeated restraint on pain behaviors and stress responses, we modeled a restraint protocol similar to those used to habituate rodents for magnetic resonance imaging scanning, and studied sensory sensitivity and stress hormone responses over 5 days. To uncover lasting effects of training, we also looked at responses to the formalin pain test 2 weeks later. We found that while restraint causes acute increases in the stress hormone corticosterone, it can also cause lasting reductions in nociceptive behavior in the formalin test, coupled with heightened corticosterone levels and increased activation of the "nociceptive" central nucleus of the amygdala, as seen by Fos protein expression. These results suggest that short-term repeated restraint, similar to that used to habituate rats for awake functional brain scanning, could potentially cause long-lasting changes in physiological and brain responses to pain stimuli that are stress-related, and therefore could potentially confound the functional activation patterns seen in awake rodents in response to pain stimuli.

Intrathecal RGS4 inhibitor, CCG50014, reduces nociceptive responses and enhances opioid-mediated analgesic effects in the mouse formalin test.

The regulator of G-protein signaling protein type 4 (RGS4) accelerates the guanosine triphosphatase activity of G(αi) and G(αo), resulting in the inactivation of G-protein-coupled receptor signaling. An opioid receptor (OR), a G(αi)-coupled receptor, plays an important role in pain modulation in the central nervous system. In this study, we examined whether (1) spinal RGS4 affected nociceptive responses in the formalin pain test, (2) this RGS4-mediated effect was involved in OR activation, and (3) the µ-OR agonist-induced antinociceptive effect was modified by RGS4 modulation. Formalin (1%, 20 µL) was injected subcutaneously into the right hindpaws of male 129S4/SvJae×C57BL/6J (RGS4(+/+) or RGS4(-/-)) mice, and the licking responses were counted for 40 minutes. The time periods (seconds) spent licking the injected paw during 0 to 10 minutes (early phase) and 10 to 40 minutes (late phase) were measured as indicators of acute nociception and inflammatory pain response, respectively. An RGS4 inhibitor, CCG50014, and/or a µ-OR agonist, [D-Ala², N-MePhe⁴, Gly-ol]-enkephalin (DAMGO), were intrathecally injected 5 minutes before the formalin injection. A nonselective OR antagonist, naloxone, was intraperitoneally injected 30 minutes before the CCG50014 injection. Mice that received the formalin injection exhibited typical biphasic nociceptive behaviors. The nociceptive responses in RGS4-knockout mice were significantly decreased during the late phase but not during the early phase. Similarly, intrathecally administered CCG50014 (10, 30, or 100 nmol) attenuated the nociceptive responses during the late phase in a dose-dependent manner. The antinociceptive effect of the RGS4 inhibitor was totally blocked by naloxone (5 mg/kg). In contrast, intrathecal injection of DAMGO achieved a dose-dependent reduction of the nociceptive responses at the early and late phases. This analgesic effect of DAMGO was significantly enhanced by the genetic depletion of RGS4 or by coadministration of CCG50014 (10 nmol). These findings demonstrated that spinal RGS4 inhibited the endogenous or exogenous OR-mediated antinociceptive effect in the formalin pain test. Thus, the inhibition of RGS4 activity can enhance OR agonist-induced analgesia. The enhancement of OR agonist-induced analgesia by coadministration of the RGS4 inhibitor suggests a new therapeutic strategy for the management of inflammatory pain.

Suppression of nociception by Solanum incanum (Lin.) Diclomethane root extract is associated antiinflammatory activity

Solanum incanum is an herb that is an important African folklore remedy for several ailments such as inflammation, pain, fever, microbial diseases, and neoplastic disorders. The herb possesses several pharmacological activities that include antinociceptive effect to thermal pain test models, antipyretic, antimicrobial and anticancer activity. However, there are no reported studies on its anti-inflammatory activity and effects on chemical pain test models. The aim of this study was to evaluate the effect of the root extract on inflammation and formalin pain test model. In the anti-inflammatory assay, white Wistar rats were injected intraperitoneally with doses of the herb diclofenac and the vehicle. Thirty minutes later the animals were injected with 50µg of 5% formalin in the sub-plantar region of the left hind paw to induce inflammation and the diameter of the paw measured using a digital caliper. The difference between the initial paw diameter and subsequent readings was quantified as the edema developed in the paw. To assess the effect of the herb on leukocyte migration, carrageenan was injected intraperitoneally into the white albino mice after thirty minutes following subcutaneous administration of the herb extracts and controls. Four hours later, normal saline was injected into the peritoneum and a peritoneal lavage performed and the total number of leukocytes in the fluid determined using a Neubauer chamber. In the antinociceptive assay, white Wistar rats were injected intraperitoneally with doses of the herb extracts. Thirty minutes later the animals were injected with 50µg of 5% formalin in the sub-plantar region of the left hind paw the total time spent in flinching, lifting, biting and licking the hind paw was quantified as the latency of nociception. The dichloromethane extract exhibited significant (p &lt; 0.05) anti-inflammatory and 2rd phase antinociceptive effect, with 50 mg doses inhibiting highly significant (p &lt; 0.001) effect in the early phase. Since the second phase of nociception is both inflammatory and neurogenic these results suggest that the anti-inflammatory activity of S. incanum plays an important role in its antinociception.

Antinociceptive activity of Syzygium cumini leaves ethanol extract on orofacial nociception protocols in rodents

Context: Syzygium cumini (L.) Skeels (Myrtaceae) is a tree with dark purple fruits, popularly known as “jambolão” or “jambolan”. In folk medicine, this plant is used for the treatment of diabetes and inflammatory conditions.Objective: We investigated the antinociceptive effect of ethanol extract (EE) from S. cumini leaves on orofacial nociception.Material and methods: The antinociceptive effects of the EE obtained from the leaves of S. cumini were evaluated in mice using formalin- and glutamate-induced orofacial nociception.Results: ESI-MS/MS analyses demonstrated that major constituents in the analyzed samples coincided with the mass of the phenolic acids and flavonoids. In pharmacological approach, pre-treatment with EE (100, 200, or 400 mg/kg, p.o.) significantly reduced (p < 0.05 or p < 0.01) the percentage of paw licks time during phase 2 (43.2, 47.1, and 57.4%, respectively) of a formalin pain test when compared to control group animals. This effect was prevented by pretreatment with glibenclamide and NG-nitro-l-arginine (l-NOARG). The extract, all doses, also caused a marked inhibition (p < 0.01 or p < 0.001) of glutamate-induced orofacial nociception (38.8, 51.7, and 54.7%) when compared with the control group. No effect was observed with the rota-rod model.Conclusions: We can suggest that the antinociceptive effect of the EE is mediated by peripheral mechanisms, possibly involving KATP channels and the nitric oxide pathways. These effects appear to be related to the presence of flavonoids compounds, such as quercetin.