Complete Freund's Adjuvant Treatment Research Articles

Anti-arthritic and Antioxidant Effects of Trehalose in an Experimental Model of Arthritis.

The purpose of the present study was to study the potential anti-arthritic and antioxidant effects of trehalose in an experimental model of complete Freund's adjuvant (CFA)-induced arthritis. Arthritis was induced via subcutaneous injection of CFA (0.1) into the right footpad of each rat. Trehalose (10 mg/kg per day) and indomethacin (5 mg/kg) as a reference drug were intraperitoneally injected into CFA-induced arthritic rats from days 0 to 21. Changes in paw volume, pain responses, arthritic score, and oxidative/antioxidative parameters were determined. Trehalose administration could significantly decrease arthritis scores (p <0.01) and paw edema (p <0.001), and significantly increase the nociceptive threshold (p <0.05) in CFA-induced arthritic rats. Trehalose also significantly reduced the pro-oxidant-antioxidant balance values when compared to CFA treatment alone. In addition, no significant difference was found between the trehalose group and indomethacin as a positive control group. The current study suggests that trehalose has a protective effect against arthritis, which may be mediated by antioxidative effects of this disaccharide.

KATP Channel Prodrugs Reduce Inflammatory and Neuropathic Hypersensitivity, Morphine-Induced Hypersensitivity, and Precipitated Withdrawal in Mice.

Previous studies show ATP-sensitive potassium (KATP) channel openers can reduce hypersensitivity associated with chronic pain models in rodents, and reduce morphine tolerance. Many agonists of KATP channels are not soluble in physiologically relevant vehicles, requiring adaptation for clinical use. This study compared the antinociceptive activity of novel KATP channel targeting prodrugs, CKLP1, CKLP2, and CF3-CKLP. These prodrugs are activated by endogenous alkaline phosphatase enzymes present in the peripheral and central nervous systems. Analgesic capabilities of intrathecally injected prodrugs were tested in rodent models of spinal nerve ligation (SNL) and complete Freund's adjuvant (CFA) as models for neuropathic and inflammatory pain, respectively. CKLP1 and CKLP2 significantly increased mechanical paw withdrawal thresholds 1-2 hours after intrathecal administration in the SNL model, but all three prodrugs were able to attenuate hypersensitivity up to 7 days after CFA treatment. The reduction of opioid tolerance and opioid-induced hypersensitivity in mice treated chronically with morphine was significantly reduced in CKLP1 and CKLP2 treated animals. Prodrug cleavage was confirmed in mouse spinal cords using liquid chromatography. These studies may aid in the further development of KATP channel prodrugs for use in treatments of chronic pain, opioid tolerance, and withdrawal. SIGNIFICANCE STATEMENT: The cromakalim prodrugs, CKLP1, CKLP2, and CF3-CKLP1 reduced hypersensitivity in inflammatory and neuropathic pain models in male and female mice. CKLP1 and CKLP2 also reduced morphine-induced hypersensitivity in a mouse model of chronic morphine exposure. CKLP2 reduced jumping and rearing behaviors after naloxone-induced precipitated morphine withdrawal. Taken together, CKLP2 demonstrates the potential for development as a non-opioid analgesic drug.

A-80426 suppresses CFA-induced inflammatory pain by suppressing TRPV1 activity via NFκB and PI3K pathways in mice

ObjectivesPain is associated with many circumstances, including inflammatory reactions, which arise from modification of the features of signaling pathways. α2-adrenergic receptor antagonists are widely utilized in narcosis. Here, the authors focused on the narcotic effect of A-80426 (A8) on Complete Freund's Adjuvant (CFA) injections-triggered chronic inflammation pain in WT and TRPV1-/- mice and explored whether its antinociceptive impact was modulated via Transient Receptor Potential Vanilloid 1 (TRPV1). MethodCFA with or without A8 was co-administered to the mice, which were categorized randomly into four groups: CFA, A8, control, and vehicle. Pain behaviors underwent evaluation through mechanical withdrawal threshold, abdominal withdrawal reflex, and thermal withdrawal latency of WT animals. ResultsQuantitative polymerase chain reaction revealed that inflammation-promoting cytokines (IL-1β, IL-6, and TNF-α) were upregulated in Dorsal Root Ganglion (DRG) and Spinal Cord Dorsal Horn (SCDH) tissues of WT animals. A8 administration reduced the pain behaviors and production of pro-inflammatory cytokines; however, this effect was significantly reduced in TRPV1-/- mice. Further analysis showed that CFA treatment reduced the TRPV1 expression in WT mice and A8 administration increased its expression and activity. The co-administration of SB-705498, a TRPV1 blocker, did not influence the pain behaviors and inflammation cytokines in CFA WT mice; however, SB-705498 the effect of A8 in WT mice. In addition, the TRPV1 block decreased the NFκB and PI3K activation in the Dorsal Root Ganglia (DRG) and Spinal Cord Dorsal Horn (SCDH) tissues of WT mice. ConclusionsTogether, A8 exerted a narcotic impact on CFA-supplemented mice via the TRPV1-modulated NFκB and PI3K pathway.

Regulators impeding erythropoiesis following iron supplementation in a clinically relevant rat model of iron deficiency anemia with inflammation

AimsWhile elevated hepcidin levels with inflammation have been postulated as a putative mechanism hindering effective erythropoiesis after intravenous (IV) iron therapy in anemic patients undergoing surgery, little is known about the concomitant changes in other major regulators affecting erythropoiesis. This study investigated the activities of relevant regulators after iron replenishment in a rat model of iron deficiency anemia with inflammation. Main methodsInflammation was induced by administration of complete Freund's adjuvant (CFA) in male Sprague-Dawley rats. After 2 weeks of CFA treatment, the rats received IV iron (CFA‑iron) or saline (CFA-saline). The control group received saline instead of CFA and iron (saline-saline). At 1, 3, and 10 days after iron or saline treatment, inflammatory cytokines, oxidative markers, iron profiles, hepcidin, erythropoietin (EPO), erythroferrone (ERFE), fibroblast growth factor 23 (FGF 23), and expression of mRNA and proteins in the liver involved in hepcidin signaling pathways were measured. Key findingsCFA treatment and iron restriction decreased hemoglobin and serum iron levels, significantly increasing inflammatory and oxidative markers. Iron supplementation did not restore hemoglobin levels despite improved iron profiles. CFA injections increased hepcidin and FGF 23 levels and decreased EPO and ERFE levels, which further intensified after iron supplementation with concomitantly elevated levels of oxidative stress and inflammatory markers. SignificanceUnder inflammatory conditions, IV iron administration exacerbated inflammatory and oxidative stress and did not resolve anemia, even under iron deficiency conditions. Iron therapy exerted adverse influences on the changes in key regulators toward impeding erythropoiesis that was already impeded by inflammation.

Transcriptomic Analysis of Trigeminal Ganglion and Spinal Trigeminal Nucleus Caudalis in Mice with Inflammatory Temporomandibular Joint Pain

BackgroundPersistent facial pain heavily impacts the quality of life in patients with temporomandibular joint (TMJ) disorders. Previous studies have demonstrated that long non-coding ribonucleic acid (lncRNA) is an important regulator of pain. In this study, we aimed to analyze lncRNA expression in the whole transcriptome of trigeminal ganglia (TG) and spinal trigeminal nucleus caudalis (Sp5C) in a chronic inflammatory TMJ pain mouse model.MethodsChronic inflammatory TMJ pain was induced by intra-TMJ injection of complete Freund’s adjuvant (CFA). Mouse TG and Sp5C tissues were harvested on day 4 after CFA injection. The lncRNA expression patterns in the whole transcriptome of TG and Sp5C were profiled with RNA sequencing.ResultsWe observed that 38 lncRNAs and 849 mRNAs were differentially expressed after CFA treatment. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis further revealed relationships among those differentially expressed lncRNAs and mRNAs and their potential functions. Specific categories of biological process, cellular processes, and molecular function of the differentially expressed transcripts were ascertained.ConclusionOur results suggest that lncRNA expression in the whole transcriptome of trigeminal nociceptive system could contribute to the molecular mechanisms that underlie chronic inflammatory TMJ pain.

Pain-induced Impulsivity and Its Reduction with Amphetamine in Rats

Pain is known to produce various cognitive changes, including increased impulsivity in humans and animals. Increases in impulsivity may facilitate the development of psychiatric conditions such as substance use disorders and anxiety disorders. We have previously shown that Complete Freund's Adjuvant (CFA)-induced pain increases impulsivity in rats. Amphetamine is a central nervous system stimulant used to treat impulse control disorders by increasing monoaminergic activity in the brain. This study aimed to determine whether amphetamine can reduce pain-induced impulsivity and nociception in rats. Pain-induced impulsivity was assessed using a delay discounting task in male and female rats. Initial baseline tests were employed to screen for trait impulsivity and identify low and high impulsive rats. After intraplantar CFA injection, daily saline or amphetamine (0.25, 0.5, or 1.0 mg/kg) was given, and impulsivity was assessed over sixteen days. Animals were also examined for potential antinociceptive effects of amphetamine on CFA-induced tactile allodynia. CFA treatment increased impulsivity in high impulsive rats more than low impulsive rats. Repeated amphetamine administration blocked pain-induced impulsivity in a dose manner. Amphetamine began to block impulsivity after six daily injections. CFA-induced mechanical allodynia was not reduced by amphetamine. Our results show amphetamine's propensity to prevent pain-induced impulsivity, independent of treating nociception. While pain-induced cognitive deficits may proliferate from pain states, their treatment and continued expression may be independent of pain sensation.

Inhibition of Spinal Interleukin-33 Attenuates Peripheral Inflammation and Hyperalgesia in Experimental Arthritis.

Accumulating evidence indicates that the continuous and intense nociceptive from inflamed tissue may increase the excitability of spinal dorsal horn neurons, which can signal back and modulate peripheral inflammation. Previous studies have demonstrated that spinal interleukin (IL)-33 contributes to the hyperexcitability of spinal dorsal horn neurons. This study was undertaken to investigate whether spinal IL-33 can also influence a peripheral inflammatory response in a rat model of arthritis. Lentivirus-delivered short hairpin RNA targeting IL-33 (LV-shIL-33) was constructed for gene silencing. Rats with adjuvant-induced arthritis (AIA) were injected intrathecally with LV-shIL-33 3days before the complete Freund's adjuvant (CFA) injection. During an observation period of 21days, pain-related behavior and inflammation were assessed. In addition, the expression of spinal proinflammatory cytokines and the activation of spinal extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) pathways were evaluated on 9days after CFA treatment. The existence of tissue injury or inflammation in rats with AIA resulted in the upregulation of spinal IL-33, which is predominantly expressed in neurons, astrocytes, and oligodendrocytes. Intrathecal administration of LV-shIL-33 significantly alleviated hyperalgesia, paw swelling, and joint destruction, and attenuated the expression of proinflammatory cytokines [IL-6, IL-1β, and tumor necrosis factor-α (TNF-α)], as well as the activation of ERK and NF-κB/p65 in the spinal cord. Our data suggest that spinal IL-33 contributes to the development of both peripheral inflammation and hyperalgesia. Thus, interference with IL-33 at the spinal level might represent a novel therapeutic target for painful inflammatory disorders.

Changes in Hepcidin Levels in an Animal Model of Anemia of Chronic Inflammation: Mechanistic Insights Related to Iron Supplementation and Hepcidin Regulation.

We examined changes in hepcidin (closely associated with anemia of chronic inflammation (ACI)) and upstream regulatory pathways after intravenous (IV) iron supplementation in an ACI animal model. ACI was induced in male Sprague-Dawley rats by intraperitoneally administering complete Freund's adjuvant (CFA). Two weeks after starting CFA treatment, ACI rats received IV iron (CFA-iron) or vehicle (CFA-saline). Three days after IV iron treatment, iron profiles, hepcidin levels, and expression of proteins involved in the signaling pathways upstream of hepcidin transcription in the liver were measured. In CFA-treated rats, anemia with a concomitant increase in the levels of serum inflammatory cytokines and reactive oxygen species occurred. In CFA-iron rats, hemoglobin (Hb) concentration was still lower than that in control rats. In CFA-saline rats, hepatic hepcidin and ferritin levels increased compared with those in control rats and were further increased in CFA-iron rats. In CFA-saline rats, NADPH oxidase- (NOX-) 2, NOX-4, and superoxide dismutase levels in the liver were upregulated compared with those in control rats and their levels were further increased in CFA-iron rats. In CFA-saline rats, activities of the IL-6/STAT and BMP/SMAD pathways were enhanced in the liver compared with those in control rats and their levels were further increased in CFA-iron rats, whereas IL-6 expression remained unaffected after IV iron administration. In HepG2 cells, iron caused phosphorylation of STAT-3 and SMAD1/5 and knockdown of STAT-3 and SMAD1/5 using siRNAs reduced iron-induced hepcidin upregulation to levels similar to those in corresponding control cells. Renal erythropoietin expression and serum erythroferrone concentration were lower in CFA-iron rats than those in control rats. In ACI rats, IV iron supplementation did not recover Hb within three days despite an increase in hepatic ferritin levels, which might be attributable to an additional increase in hepcidin levels that was already upregulated under ACI conditions. Both STAT-3 phosphorylation and SMAD1/5 phosphorylation were associated with hepcidin upregulation after IV iron treatment, and this seems to be linked to iron-induced oxidative stress.

Complete Freund's adjuvant-induced protein dysregulation correlated with mirror image pain as assessed by quantitative proteomics of the mouse spinal cord

Inflammation or trauma occurring on one side of the body can cause pathological pain on the contralateral noninjured side in a phenomenon called mirror-image pain (MIP). Although some potential mechanisms involved in MIP have been reported, including those involving the immune system and glial cells as well as neural mechanisms, the molecular mechanisms are not well understood. In this study, we aimed to understand the molecular mechanisms in MIP using quantitative proteomics and whole-cell patch clamp recordings. Behavioral test results showed that complete Freund's adjuvant could induce MIP in the mice. The results of isobaric tags for relative and absolute quantification (iTRAQ) quantitative proteomics showed that 108 proteins were dysregulated, and these proteins may represent potential targets. Furthermore, bioinformatics analysis was applied to explore the potential molecular mechanisms during MIP after complete Freund's adjuvant (CFA) treatment. Parallel reaction monitoring (PRM) results showed that PKCδ and seven other dysregulated proteins were related to MIP after CFA treatment. Patch clamp recording results showed that CFA treatment could increase intrinsic excitability and spontaneous firing in spinal cord neurons during MIP. In summary, we found that CFA could induce MIP. The results of proteomic research on the spinal cord after CFA treatment could provide new insight into the molecular mechanisms of MIP. Moreover, the neuronal activity of spinal cord neurons was upregulated during MIP after CFA treatment. In summary, the results of the spinal cord proteomic profile provide a potential molecular mechanism for understanding MIP.

Pain-induced impulsivity is sexually dimorphic and mu-opioid receptor sensitive in rats.

Pain sensation can negatively impact cognitive function, including impulsivity. Pain-induced changes in impulsivity can contribute to development of psychiatric comorbidities found in those with chronic pain conditions. The goal of this study was to determine whether complete Freund's adjuvant (CFA)-induced pain manipulation enhances impulsivity in rats. Whether the pain-induced impulsivity is sexually dimorphic, and if mu-opioid receptors play a role in these processes. Male and female rats were screened for trait impulsivity and designated as high or low impulsive using a delay discounting task. Rats then received a hind paw injection of CFA, and their impulsivity was assessed for 16days. The effects of morphine on impulsivity were also examined. In a separate experiment, rats were pretreated with beta-funaltrexamine (β-FNA) to determine the role of mu-opioid receptors on impulsivity. CFA treatment increased impulsivity in males and females. The onset of CFA-induced impulsivity was faster in high impulsive females than males. Morphine blocked CFA-induced impulsivity in both sexes in a dose- and time-dependent manner. β-FNA prevented the actions of morphine on CFA-induced impulsivity in high impulsive males, but not high impulsive females. Moreover, β-FNA increased CFA-induced impulsivity in morphine naïve males, but not females. These findings demonstrate unique sex differences in CFA-induced impulsivity, response to morphine, and the impact of mu-opioid receptors. A better understanding of cognitive deficits and their mechanisms can provide insight into the development of substance abuse and psychiatric comorbidities that occur in people with chronic pain.

Lidocaine attenuates CFA-induced inflammatory pain in rats by regulating the MAPK/ERK/NF-κB signaling pathway.

Lidocaine is a commonly used local anesthetic that also confers analgesic effects, resistance to hyperalgesia and anti-inflammatory properties. The present study aimed to explore the effects of lidocaine on complete Freund's adjuvant (CFA)-induced inflammatory pain. In the present study, rats were subcutaneously injected with CFA to investigate the molecular mechanisms associated with lidocaine in an inflammation-induced pain model. Firstly, CFA was subcutaneously injected into the paws of Sprague-Dawley rats, following which lidocaine or saline and the ERK agonist recombinant human epidermal growth factor (rh-EGF) were injected via the tail vein. Rat behavior was then assessed at 0 and 4 h, 1, 4, 7 and 14 days after CFA treatment. Proinflammatory cytokine levels in the serum were measured using ELISA. Western blotting was performed to detect the protein levels of phosphorylated (p)-ERK1/2, ERK1/2 and NF-κB subunits p-p65 and p65. Reverse transcription-quantitative PCR was used to measure the mRNA expression of ERK1/2 and p65 in rat spinal cord tissues. The results showed that injection of CFA significantly reduced the mechanical withdrawal threshold, thermal withdrawal latency and the frequency of responses to cold stimulation in rats, whilst promoting tumor necrosis factor-α, interleukin (IL)-1β, IL-6 levels in addition to ERK1/2, p65 protein phosphorylation. These effects were alleviated by lidocaine treatment. Furthermore, treatment with rh-EGF reversed the protective effects of lidocaine on inflammatory pain caused by CFA. In conclusion, lidocaine inhibits the inflammatory response and pain through the MAPK/ERK/NF-κB pathway in a rat model of pain induced by CFA.

Tetrahydropalmatine exerts analgesic effects by promoting apoptosis and inhibiting the activation of glial cells in rats with inflammatory pain.

BackgroundPain is an unpleasant sensory experience that usually plays a protective role. Inflammatory pain is often severe and stubborn, which has a great impact on the quality of life of patients. However, there has been no breakthrough in the treatment strategy and mechanism of inflammatory pain.MethodsThis study investigated the analgesic effect of tetrahydropalmatine (THP) in rats injected with complete Freund's adjuvant (CFA)-induced inflammatory pain. Allodynia and gait analysis of rats were used to evaluate the analgesic effect at different time points before and after operation. THP (2.5, 5, and 10 mg/kg) was administered intraperitoneally once daily for 7 days post Day 3. The expression levels of TNF-α and IL-1β in the spinal cord were measured by enzyme-linked immunosorbent assay. The activation of astrocytes and microglial cells in the spinal cord was tested by western blot before and after THP treatment. The apoptosis of glial cells was tested by flow cytometry after treatment with THP in the primary cultured glial cell model.ResultsCFA treatment induced significant allodynia and caused abnormal gait in rats. Administration of THP at 10 mg/kg significantly alleviated CFA-induced inflammatory pain behaviors. Moreover, CFA-induced activation of glial cells and the increased levels of TNF-α and IL-1β were inhibited by THP administration. In addition, THP promotes apoptosis in primary cultured glial cells. This study suggests the possible clinical utility of THP in the treatment of inflammatory pain.ConclusionTHP plays an analgesic role by inhibiting the activation of glial cells and promoting apoptosis.

Fibroblast growth factor 13 stabilizes microtubules to promote Na+ channel function in nociceptive DRG neurons and modulates inflammatory pain

IntroductionFibroblast growth factor homologous factors (FHFs), among other fibroblast growth factors, are increasingly found to be important regulators of ion channel functions. Although FHFs have been link to several neuronal diseases and arrhythmia, its role in inflammatory pain still remains unclear. ObjectivesThis study aimed to investigate the role and mechanism of FGF13 in inflammatory pain. MethodsFgf13 conditional knockout mice were generated and CFA-induced chronic inflammatory pain model was established to measure the pain threshold. Immunostaining, western blot and quantitative real-time reverse transcription PCR (qRT-PCR) were performed to detect the expression of FGF13 in CFA-induced inflammatory pain. Whole-cell patch clamp recording was used to record the action potential firing properties and sodium currents of DRG neurons. ResultsConditional knockout of Fgf13 in dorsal root ganglion (DRG) neurons (Fgf13-/Y) led to attenuated pain responses induced by complete Freund's adjuvant (CFA). FGF13 was expressed predominantly in small-diameter DRG neurons. CFA treatment resulted in an increased expression of FGF13 proteins as well as an increased excitability in nociceptive DRG neurons which was inhibited when FGF13 was absent. The role of FGF13 in neuronal excitability of DRG was linked to its modulation of voltage-gated Na+ channels mediated by microtubules. Overexpression of FGF13, but not FGF13 mutant which lacks the ability to bind and stabilize microtubules, rescued the decreased neuronal excitability and Na+ current density in DRG neurons of Fgf13-/Y mice. ConclusionThis study revealed that FGF13 could stabilize microtubules to modulate sodium channel function in DRG neurons and modulate inflammatory pain. This study provides a novel mechanism for FGF13 modulation of sodium channel function and suggests that FGF13 might be a novel target for inflammatory pain treatment.

Endogenous transient receptor potential ankyrin 1 and vanilloid 1 activity potentiates glutamatergic input to spinal lamina I neurons in inflammatory pain.

Inflammatory pain is associated with peripheral and central sensitization, but the underlying synaptic plasticity at the spinal cord level is poorly understood. Transient receptor potential (TRP) channels expressed at peripheral nerve endings, including TRP subtypes ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), can detect nociceptive stimuli. In this study, we determined the contribution of presynaptic TRPA1 and TRPV1 at the spinal cord level to regulating nociceptive drive in chronic inflammatory pain induced by complete Freund's adjuvant (CFA) in rats. CFA treatment caused a large increase in the frequency of spontaneous excitatory postsynaptic currents (EPSCs) in lamina I, but not lamina II outer zone, dorsal horn neurons. However, blocking NMDA receptors had no effect on spontaneous EPSCs in lamina I neurons of CFA-treated rats. Application of a specific TRPA1 antagonist, AM-0902, or of a specific TRPV1 antagonist, 5'-iodoresiniferatoxin, significantly attenuated the elevated frequency of spontaneous EPSCs and miniature EPSCs, the amplitude of monosynaptic EPSCs evoked from the dorsal root in lamina I neurons of CFA-treated rats. AM-0902 and 5'-iodoresiniferatoxin had no effect on evoked or miniature EPSCs in lamina I neurons of vehicle-treated rats. In addition, intrathecal injection of AM-0902 or 5'-iodoresiniferatoxin significantly reduced pain hypersensitivity in CFA-treated rats but had no effect on acute nociception in vehicle-treated rats. Therefore, unlike neuropathic pain, chronic inflammatory pain is associated with NMDA receptor-independent potentiation in glutamatergic drive to spinal lamina I neurons. Endogenous presynaptic TRPA1 and TRPV1 activity at the spinal level contributes to increased nociceptive input from primary sensory nerves to dorsal horn neurons in inflammatory pain. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Peripheral Inflammation Accelerates the Onset of Mechanical Hypersensitivity after Spinal Cord Injury and Engages Tumor Necrosis Factor α Signaling Mechanisms.

Previously, we showed that noxious stimulation of the tail produces numerous detrimental effects after spinal cord injury (SCI), including an earlier onset and increased magnitude of mechanical hypersensitivity. Expanding on these observations, this study sought to determine whether localized peripheral inflammation similarly impacts the expression of mechanical hypersensitivity after SCI. Adult rats received a moderate contusion injury at the thoracic level (Tl0) or sham surgery, and were administered complete Freund's adjuvant (CFA) or vehicle in one hindpaw 24 hours later. Examination of locomotor recovery (Basso, Beattie, and Bresnahan [BBB] score) showed no adverse effect of CFA. Mechanical testing with von Frey hairs was done at time-points ranging from 1 h to 28 days after CFA or vehicle treatment, and rats were sacrificed at 1, 7, or 28 days for cellular assessment. Unlike vehicle-treated SCI rats where mechanical hypersensitivity emerged at 14 days, CFA-treated SCI rats showed mechanical hypersensitivity as early as 1 h after CFA administration, which lasted at least 28 days. CFA-treated sham subjects also showed an early onset of mechanical hypersensitivity, but this was maintained up to 7 days after treatment. Cellular assessments revealed congruent findings. Expression levels of c-fos, tumor necrosis factor α (TNFα), TNF receptors, and members of the TNFα signaling pathway such as caspase 8 and phosphorylated extracellular related kinase (pERK) were preferentially upregulated in the lumbar spinal cord of SCI-CFA rats. Meanwhile, c-jun was significantly increased in both CFA-treated groups. Overall, these results together with our previous reports, suggest that peripheral noxious input after SCI facilitates the development of pain by mechanisms that may require TNFα signaling.

Chronic inflammatory pain drives alcohol drinking in a sex-dependent manner for C57BL/6J mice.

Sex differences in chronic pain and alcohol abuse are not well understood. The development of rodent models is imperative for investigating the underlying changes behind these pathological states. In the present study, we investigated whether hind paw treatment with the inflammatory agent Complete Freund's Adjuvant (CFA) could generate hyperalgesia and alter alcohol consumption in male and female C57BL/6J mice. CFA treatment led to greater nociceptive sensitivity for both sexes in the Hargreaves test, and increased alcohol drinking for males in a continuous-access two-bottle choice (CA2BC) paradigm. Regardless of treatment, female mice exhibited greater alcohol drinking than males. Following a 2-h terminal drinking session, CFA treatment failed to produce changes in alcohol drinking, blood ethanol concentration (BEC), and plasma corticosterone (CORT) for both sexes. Two-hour alcohol consumption and CORT was higher in females than males, regardless of CFA treatment. Taken together, these findings have established that male mice are more susceptible to escalations in alcohol drinking when undergoing pain, despite higher levels of total alcohol drinking and CORT in females. Furthermore, the exposure of CFA-treated C57BL/6J mice to the CA2BC drinking paradigm has proven to be a useful model for studying the relationship between chronic pain and alcohol abuse. Future applications of the CFA/CA2BC model should incorporate manipulations of stress signaling and other related biological systems to improve our mechanistic understanding of pain and alcohol interactions.

Analgesic effect of mineral cream containing natural spa minerals for use on the skin.

Previous studies have shown that dissolved substances in some natural hot springs have analgesic/anti-nociceptive and anti-inflammatory actions. However, the mechanisms underlying how such dissolved substances exert these actions are not fully understood. In the present study on mice, we examined the analgesic/anti-nociceptive and anti-inflammatory properties of a mineral cream containing natural hot spring ingredients. The anti-nociceptive effects of the mineral cream were assessed by using the von Frey test. Application of the mineral cream to the hind paw of mice produced a significant anti-nociceptive effect compared to control. The anti-nociceptive effects of the mineral cream were also assessed following the injection of complete Freund's adjuvant (CFA) into the hind paws of mice after pre-treatment for one or four weeks with the mineral cream. Histological experiments with light microscopy showed that the mineral cream did not reduce inflammation caused by the CFA treatment. In addition, the mineral cream did not inhibit oxidative stress as evidenced by increased levels of oxidative metabolites (d-ROMs) and biological anti-oxidant potential (BAP). These results suggest that the mineral cream does not exert a protective effect against inflammation, and that the constituents of the mineral cream may produce their anti-nociceptive effects transdermally via different mechanisms including the nervous system.

Upregulation of CXCR4 through promoter demethylation contributes to inflammatory hyperalgesia in rats.

Chronic pain associated with inflammation is a common clinical problem, and the underlying mechanisms yet are incompletely defined. DNA methylation has been implicated in the pathogenesis of chronic pain. However, the specific genes regulated by DNA methylation under inflammatory pain condition remain largely unknown. Here, we investigated how chemokine receptor CXCR4 expression is regulated by DNA methylation and how it contributes to inflammatory pain induced by complete Freund's adjuvant (CFA) in rats. Intraplantar injection of CFA could not only induce significant hyperalgesia in rats, but also significantly increase the expression of CXCR4 mRNA and protein in the dorsal root ganglion (DRG). Intrathecal injection of CXCR4 antagonist AMD3100 significantly relieved hyperalgesia in inflammatory rats in a time- and dose-dependent manner. Bisulfite sequencing and methylation-specific PCR demonstrate that CFA injection led to a significant demethylation of CpG island at CXCR4 gene promoter. Consistently, the expression of DNMT3b was significantly downregulated after CFA injection. Online software prediction reveals three binding sites of p65 in the CpG island of CXCR4 gene promoter, which has confirmed by the chromatin immunoprecipitation assay, CFA treatment significantly increases the recruitment of p65 to CXCR4 gene promoter. Inhibition of NF-kB signaling using p65 inhibitor pyrrolidine dithiocarbamate significantly prevented the increases of the CXCR4 expression. Upregulation of CXCR4 expression due to promoter demethylation followed by increased recruitment of p65 to promoter of CXCR4 gene contributes to inflammatory hyperalgesia. These findings provide a theoretical basis for the treatment of chronic pain from an epigenetic perspective.

NLRP3 inflammasome signaling as an early molecular response is negatively controlled by miR-186 in CFA-induced prosopalgia mice

The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is the most frequently studied in the central nervous system and has been linked to neuropathic pain. In this study, a post-translational mechanism of microRNA (miR)-186 via regulating the expression of NLRP3 in the complete Freund's adjuvant (CFA)-treated mice was investigated. The injection of CFA was used to induce trigeminal neuropathic pain in mice. miRs microarray chip assay was performed in trigeminal ganglions (TGs). CFA treatment significantly increased the mRNA expression of NLRP3, interleukin (IL)-1β, and IL-18 in TGs compared to the control group. Moreover, 26 miRs were differentially expressed in TGs from trigeminal neuropathic pain mice, and the expression of miR-186 showed the lowest level of all the miRs. Further examination revealed that NLRP3 was a candidate target gene of miR-186. We delivered miR-186 mimics to CFA-treated mice. The head withdrawal thresholds of the CFA-treated mice were significantly increased by miR-186 mimics injection compared with CFA single treatment. The mRNA and protein expression of NLRP3, IL-1β, and IL-18 in TGs from trigeminal neuropathic pain mice were significantly inhibited by miR-186 mimics treatment compared to the CFA group. miR-186 was able to suppress the neuropathic pain via regulating the NLRP3 inflammasome signaling.

Protodioscin ameliorates oxidative stress, inflammation and histology outcome in Complete Freund's adjuvant induced arthritis rats.

Protective effect of protodioscin or methyl protodioscin against inflammation had been reported in various inflammation diseases. This study aimed to investigate the effect of protodioscin against Complete Freund's adjuvant (CFA)-induced arthritis rats. Rats randomly divided into model groups were injected with CFA, companied with different dose of protodioscin (50, 100, and 200mg/kg body weight). The histology, changes in biochemical parameters and inflammatory cytokines expression were detected for anti-inflammation effect evaluation of protodioscin. CFA treatment induced arthritic rats with swelling paw, ankle inflammation, and area of lymphocyte infiltration, upregulated inflammatory cytokines (IL-1β, TNF-α, cyclo-oxygenase 2, and IL-6 as well as prostaglandin E2), articular elastase, myeloperoxidase, lipid peroxidase and nitrite oxide levels, downregulated glutathione, catalase, and superoxide dismutase. In contrast, protodioscin ameliorated all the changes induced by CFA in rats, suggesting the anti-inflammatory effect of protodioscin. We concluded that protodioscin administration into CFA-induced arthritis rats protected against CFA-induced oxidative stress, neutrophil infiltration, and inflammation, suggesting the anti-inflammatory effect and the therapeutic potential of protodioscin for arthritis.