Complete Freund's Adjuvant Model Research Articles

Toxicological analysis and anti-inflammatory and antioxidant evaluations of extract, fractions and secoxyloganin obtained from Guettarda viburnoides Cham. & Schltdl. in mice

Ethnopharmacological relevanceGuettarda viburnoides, "veludinho do campo," is traditionally used for the treatment of pain and inflammatory conditions in humans; however, only one scientific study has reported this effect in an ear inflammatory model. Therefore, it is necessary to explore other in vivo models and the chemical composition of this medicinal plant. Aim of the studyA chemical investigation of methanolic extract of G. viburnoides (MEGV) (leaves) led to the isolation of secoxyloganin (GV-1). In addition, the preclinical safety of MEGV (in acute and subacute toxicological models, gavage = p.o.), antioxidants of MEGV, ethyl acetate (EAFGV) and hydromethanolic (HMFGV) fractions were tested using free radical scavenging and lipid peroxidation methodologies, and the anti-inflammatory effects of MEGV, HMFGV and GV-1 (p.o.) were evaluated on carrageenan and complete Freund's adjuvant (CFA) models of inflammation in mice. Materials and MethodsMEGV was obtained from air-dried leaves by maceration with methanol at room temperature. MEGV was then purified by liquid-liquid partitioning, to obtain the EAFGV and HMFGV fractions. Purification of HMFGV afforded GV-1. The quantification of total phenols, flavonoids, flavonols, and condensed tannins was subsequently performed for MEGV. The antioxidant activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and oxidation of β-carotene were evaluated in MEGV and its fractions. The anti-inflammatory activity of MEGV (3, 30, and 100 mg/kg, p.o.) was assayed in carrageenan-induced models, followed by assessments of MEGV (100 mg/kg, p.o.), HMFGV (3 and 30 mg/kg, p.o.) and GV-1 (3 mg/kg, p.o.) in CFA- induced models in mice (including paw oedema, mechanical allodynia and cold sensitivity). Acute (14 days of MEGV, 2000 mg/kg, p.o.) and subacute (28 days, MEGV 30, 100, and 300 mg/kg, p.o.) toxicity was assessed in female Swiss mice. ResultsThe major compound was secoxyloganin (GV-1). The oral acute toxicity test of MEGV revealed no evidence of toxicity, indicating low toxicity according to the Organization for Economic Cooperation and Development (OECD) guidelines. In the subacute toxicity group, no clinical signs of toxicity or changes in body weight, water consumption, food consumption, or organ weight or morphology were observed after 28 days of gavage with MEGV (30, 100, and 300 mg/kg) compared with those in the control group. MEGV, EAFGV, and HMFGV showed significant free-radical scavenging and lipid peroxidation activities, with IC50 values ≤ 26.38 ± 4.56 μg/mL. In in vivo anti-inflammatory assays, MEGV (3, 30 and 100 mg/kg) reduced carrageenan-induced oedema (2 and 4 h) and hyperalgesia (3 and 4 h). In the CFA model, MEGV (100 mg/kg), HMFGV (30 mg/kg) and GV-1 (3 mg/kg) reduced inflammation (at 3, 4 and 24 h) in all parameters (oedema, mechanical allodynia and cold sensitivity). ConclusionThis study revealed that G. viburnoides has antioxidant and anti-inflammatory properties, and no toxicity was detected after acute or subacute gavage with MEGV, validating its traditional use in the treatment of inflammatory conditions.

Peripheral CaV2.2 channels in skin regulate prolonged heat hypersensitivity during neuroinflammation.

Neuroinflammation can lead to chronic maladaptive pain affecting millions of people worldwide. Neurotransmitters, cytokines, and ion channels are implicated in neuro-immune cell signaling but their roles in specific behavioral responses are not fully elucidated. Voltage-gated CaV2.2 channel activity in skin controls rapid and transient heat hypersensitivity induced by intradermal (id) capsaicin via IL-1α cytokine signaling. CaV2.2 channels are not, however, involved in mechanical hypersensitivity that developed in the id capsaicin animal model. Here, we show that CaV2.2 channels are also critical for heat hypersensitivity induced by id Complete Freund's Adjuvant (CFA). Id CFA, a model of chronic neuroinflammation, involves ongoing cytokine signaling for days leading to pronounced edema and hypersensitivity to sensory stimuli. Peripheral CaV2.2 channel activity in skin was required for the full development and week-long time course of heat hypersensitivity induced by id CFA but, paw edema and mechanical hypersensitivity were independent of CaV2.2 channel activity. CFA induced increases in several cytokines in hind paw fluid including IL-6 which was also dependent on CaV2.2 channel activity. Using IL-6 specific neutralizing antibodies in vivo we show that IL-6 contributes to heat hypersensitivity and, neutralizing both IL-1α and IL-6 was even more effective at reducing the magnitude and duration of CFA-induced heat hypersensitivity. Our findings demonstrate a functional link between CaV2.2 channel activity and the release of IL-6 in skin and show that CaV2.2 channels have a privileged role in the induction and maintenance of heat hypersensitivity during chronic forms of neuroinflammation in skin.Significance statement Neuroinflammation can lead to chronic maladaptive pain. Neurotransmitters, ion channels, cytokines, and cytokine receptors are implicated in neuron-immune signaling, but their importance in mediating specific behavioral responses are not fully elucidated. We show that the activity of peripheral CaV2.2 calcium ion channels in skin play a unique role in the induction and maintenance of heat hypersensitivity in the CFA model of prolonged neuroinflammation, but they are not involved in the development of edema and mechanical hypersensitivity. Blocking peripheral CaV2.2 channel activity reduces local cytokine levels in hind paws injected with CFA including IL-6, and neutralizing IL-6 reduces CFA-induced heat hypersensitivity. Our studies define key signaling molecules that act locally in skin to trigger and maintain heat hypersensitivity during chronic neuroinflammation.

Pyrazolo-Pyrimidinones with Improved Solubility and Selective Inhibition of Adenylyl Cyclase Type 1 Activity for Treatment of Inflammatory Pain.

Adenylyl cyclase isoform 1 (AC1) is considered a promising target for treating inflammatory pain. Our group identified the pyrazolyl-pyrimidinone scaffold as potent and selective inhibitors of Ca2+/CaM-mediated AC1 activity; however, the molecules suffered from poor aqueous solubility. The current study presents a strategy to improve aqueous solubility of the scaffold by reduction of crystal packing energy and increasing rotational degrees of freedom within the molecule. Structure-activity and property relationship studies identified the second generation lead 7-47A (AC10142A) that demonstrated and AC1 IC50 value of 0.26 μM and aqueous solubility of 74 ± 7 μM. After in vitro ADME characterization, the scaffold advanced to in vivo pharmacokinetic evaluation, demonstrating adequate levels of exposure. Finally, 7-47A exhibited antiallodynic efficacy in a rat complete Freund's adjuvant model for inflammatory pain showing improvement over previous iterations of this scaffold. These results further validate AC1 inhibition as a viable therapeutic strategy for treating chronic and inflammatory pain.

Peripheral Inflammation is accompanied by Cerebral Hypoperfusion in Mice.

Chronic pain is a disabling condition that is accompanied by neuropsychiatric comorbidities such as anxiety, depression, and cognitive decline. While the peripheral alterations are well-studied, we lack an understanding of how these peripheral changes can result in long-lasting brain alterations and the ensuing behavioral phenotypes. This study aims to quantify changes in cerebral blood perfusion using laser speckle contrast imaging (LSCI) in the murine Complete Freund's adjuvant (CFA) model of unilateral peripheral inflammation. Twenty female and 24 male adult C57BL/6 mice were randomly assigned to control (0.05ml saline) or 1 of 3 experimental groups receiving CFA (0.01ml, 0.05ml, and 0.1ml) on the right hindpaw. Three days after the intraplantar injections, animals were assessed for signs of pain (von Frey), working memory (y-maze), and anxiety (zero maze and open field), and subjected to craniotomy and in vivo LSCI of the parietal-temporal lobes. Unilateral administration of CFA resulted in signs of local inflammation, decreased mechanical thresholds on the affected hindpaw, signs of anxiety in the zero maze, as well as cerebral hypoperfusion in dose-dependent manner. To our knowledge, this is the first study using laser speckle contrast imaging to examine the effects of CFA-induced peripheral inflammation on cerebral blood perfusion. It serves as a first step in delineating the path by which insult to peripheral tissues can cause long-lasting brain plasticity via vascular mechanisms.

Peripheral CaV2.2 channels in skin regulate prolonged heat hypersensitivity during neuroinflammation.

Neuroinflammation can lead to chronic maladaptive pain affecting millions of people worldwide. Neurotransmitters, cytokines, and ion channels are implicated in neuro-immune cell signaling but their roles in specific behavioral responses are not fully elucidated. Voltage-gated CaV2.2 channel activity in skin controls rapid and transient heat hypersensitivity induced by intradermal capsaicin via IL-1α cytokine signaling. CaV2.2 channels are not, however, involved in mechanical hypersensitivity that developed in the same animal model. Here, we show that CaV2.2 channels are also critical for heat hypersensitivity induced by the intradermal (id) Complete Freund's Adjuvant (CFA) model of chronic neuroinflammation that involves ongoing cytokine signaling for days. Ongoing CFA-induced cytokine signaling cascades in skin lead to pronounced edema, and hypersensitivity to sensory stimuli. Peripheral CaV2.2 channel activity in skin is required for the full development and week-long time course of heat hypersensitivity induced by id CFA. CaV2.2 channels, by contrast, are not involved in paw edema and mechanical hypersensitivity. CFA induced increases in cytokines in hind paws including IL-6 which was dependent on CaV2.2 channel activity. Using IL-6 specific neutralizing antibodies, we show that IL-6 contributes to heat hypersensitivity and, neutralizing both IL-1α and IL-6 was even more effective at reducing the magnitude and duration of CFA-induced heat hypersensitivity. Our findings demonstrate a functional link between CaV2.2 channel activity and the release of IL-6 in skin and show that CaV2.2 channels have a privileged role in the induction and maintenance of heat hypersensitivity during chronic forms of neuroinflammation in skin.

S1P/S1PR1 signaling is involved in the development of nociceptive pain.

Pain is a complex perception involving unpleasant somatosensory and emotional experiences. However, the underlying mechanisms that mediate its different components remain unclear. Sphingosine-1-phosphate (S1P), a metabolite of sphingomyelin and a potent lipid mediator, initiates signaling via G protein-coupled receptors (S1PRs) on cell surfaces. It serves as a second messenger in cellular processes such as proliferation and apoptosis. Nevertheless, the neuropharmacology of sphingolipid signaling in pain conditions within the central nervous system remains largely unexplored and controversial. Chronic nociceptive pain models were induced in vivo by intraplantar injection of 20μL complete Freund's adjuvant (CFA) into the left hind paws. We assessed S1P and S1PR1 expression in the spinal cords of CFA model mice. Functional antagonists of S1PR1 or S1PR1-specific siRNA were administered daily following CFA model establishment. Paw withdrawal response frequency (PWF) and paw withdrawal latency (PWL) were measured to evaluate mechanical allodynia and thermal hyperalgesia, respectively. RT-PCR assessed interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α levels. Western blotting and immunofluorescence were used to analyze glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule (Iba1), STAT3, ERK, and p38 MAPK protein expression. In the chronic nociceptive pain model induced by CFA, S1P and S1PR1 expression levels were significantly elevated, leading to activation of spinal cord glial cells. S1PR1 activation also promoted MMP2-mediated cleavage of mature IL-1β. Additionally, S1PR1 activation upregulated phosphorylation of STAT3, ERK, and p38 MAPK in glial cells, profoundly impacting downstream signaling pathways and contributing to chronic nociceptive pain. The S1P/S1PR1 axis plays a pivotal role in the cellular and molecular mechanisms underlying nociceptive pain. This signaling pathway modulates glial cell activation and the expression of pain-related genes (STAT3, ERK, p38 MAPK) and inflammatory factors in the spinal dorsal horn. These findings underscore the potential of targeting the S1P system for developing novel analgesic therapies.

The impact of the migraine treatment onabotulinumtoxinA on inflammatory and pain responses: Insights from an animal model.

Migraine, a prevalent and debilitating disease, involves complex pathophysiology possibly including inflammation and heightened pain sensitivity. The current study utilized the complete Freund's adjuvant (CFA) model of inflammation, with onabotulinumtoxinA (BoNT/A) as a treatment of interest due to its use in clinical migraine management. Using an animal model, the study sought to investigate the role of BoNT/A in modulating CFA-induced inflammation, alterations in pain sensitivity, and the regulation of calcitonin gene-related peptide (CGRP) release. Further, we aimed to assess the changes in SNAP-25 through western blot analysis to gain insights into the mechanistic action of BoNT/A. BoNT/A or control was administered subcutaneously at the periorbital region of rats 3 days before the induction of inflammation using CFA. Periorbital mechanical sensitivity was assessed post-inflammation, and alterations in CGRP release were evaluated. Changes in SNAP-25 levels were determined using western blot analysis. Upon CFA-induced inflammation, there was a marked increase in periorbital mechanical sensitivity, with the inflammation side showing increased sensitivity compared to other periorbital areas. BoNT/A did decrease the withdrawal thresholds in the electronic von Frey test. Despite not being able to observe differences in pain thresholds or CGRP release, BoNT/A reduced baseline release under CFA inflamed conditions. Analysis of SNAP-25 levels in the trigeminal ganglion revealed both intact and cleaved forms that were notably elevated in BoNT/A-treated animals. These findings, derived from western blot analysis, suggest an effect on neurotransmitter release. Our investigation highlights the role of BoNT/A in reducing baseline CGRP in the context of inflammation and its involvement in SNAP-25 cleavage. In contrast, BoNT/A did not appear to alter facial pain sensitivity induced by inflammation, suggesting that mechanisms other than baseline CGRP could be implicated in the elevated thresholds in the CFA model.

Identification of 1,3,8-Triazaspiro[4.5]Decane-2,4-Dione Derivatives as a Novel δ Opioid Receptor-Selective Agonist Chemotype.

δ opioid receptors (DORs) hold potential as a target for neurologic and psychiatric disorders, yet no DOR agonist has proven efficacious in critical phase II clinical trials. The exact reasons for the failure to produce quality drug candidates for the DOR are unclear. However, it is known that certain DOR agonists can induce seizures and exhibit tachyphylaxis. Several studies have suggested that those adverse effects are more prevalent in delta agonists that share the (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)/4-[(αR*)-α-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl]-N,N-diethylbenzamide chemotype. There is a need to find novel lead candidates for drug development that have improved pharmacological properties to differentiate them from the current failed delta agonists. Our objective in this study was to identify novel DOR agonists. We used a β-arrestin assay to screen a small G-protein coupled receptors (GPCR)-focused chemical library. We identified a novel chemotype of DOR agonists that appears to bind to the orthosteric site based of docking and molecular dynamic simulation. The most potent agonist hit compound is selective for the DOR over a panel of 167 other GPCRs, is slightly biased toward G-protein signaling and has anti-allodynic efficacy in a complete Freund's adjuvant model of inflammatory pain in C57BL/6 male and female mice. The newly discovered chemotype contrasts with molecules like SNC80 that are highly efficacious β-arrestin recruiters and may suggest this novel class of DOR agonists could be expanded on to develop a clinical candidate drug. SIGNIFICANCE STATEMENT: δ opioid receptors are a clinical target for various neurological disorders, including migraine and chronic pain. Many of the clinically tested delta opioid agonists share a single chemotype, which carries risks during drug development. Through a small-scale high-throughput screening assay, this study identified a novel δ opioid receptor agonist chemotype, which may serve as alternative for the current analgesic clinical candidates.

A mouse model of chronic primary pain that integrates clinically relevant genetic vulnerability, stress, and minor injury.

Chronic primary pain conditions (CPPCs) affect over 100 million Americans, predominantly women. They remain ineffectively treated, in large part because of a lack of valid animal models with translational relevance. Here, we characterized a CPPC mouse model that integrated clinically relevant genetic (catechol-O-methyltransferase; COMT knockdown) and environmental (stress and injury) factors. Compared with wild-type mice, Comt+/- mice undergoing repeated swim stress and molar extraction surgery intervention exhibited pronounced multisite body pain and depressive-like behavior lasting >3 months. Comt+/- mice undergoing the intervention also exhibited enhanced activity of primary afferent nociceptors innervating hindpaw and low back sites and increased plasma concentrations of norepinephrine and pro-inflammatory cytokines interleukin-6 (IL-6) and IL-17A. The pain and depressive-like behavior were of greater magnitude and longer duration (≥12 months) in females versus males. Furthermore, increases in anxiety-like behavior and IL-6 were female-specific. The effect of COMT genotype × stress interactions on pain, IL-6, and IL-17A was validated in a cohort of 549 patients with CPPCs, demonstrating clinical relevance. Last, we assessed the predictive validity of the model for analgesic screening and found that it successfully predicted the lack of efficacy of minocycline and the CB2 agonist GW842166X, which were effective in spared nerve injury and complete Freund's adjuvant models, respectively, but failed in clinical trials. Yet, pain in the CPPC model was alleviated by the beta-3 adrenergic antagonist SR59230A. Thus, the CPPC mouse model reliably recapitulates clinically and biologically relevant features of CPPCs and may be implemented to test underlying mechanisms and find new therapeutics.

Triterpenes G-A and G-E from Galphimia glauca with anti-inflammatory and anti-arthritic activity in mice

Ethnopharmacological relevanceGalphimia glauca is a medicinal plant that treats inflammatory and anti-rheumatic problems. Its anti-inflammatory capacity has been reported pharmacologically, attributed to the triterpenes G-A and G-E. AimThe objective of the present work was to measure the anti-inflammatory and immunomodulatory effect of the methanolic extract (GgMeOH) of Galphimia glauca and the isolated galphimines G-A and G-E, first in an acute test of plantar edema with carrageenan, and later in the model of experimental-induced arthritis with CFA. The effect was measured by quantifying joint inflammation, the concentration of pro- (TNF-α, IL-6, IL-17) and anti-inflammatory (IL-10, and IL-4) cytokines, and the ADA enzyme in joints, kidneys, and spleen from mice with experimental arthritis. MethodThe extract and the active triterpenes were obtained according to established methods using different chromatographic techniques. Female ICR strain mice were subjected to intraplantar administration with carrageenan and treated with different doses of GgMeOH, G-A, and G-E; edema was monitored at different times. Subsequently, the concentration of TNF-a and IL-10 in the spleen and swollen paw was quantified. Meloxicam (MEL) was used as an anti-inflammatory control drug. The most effective doses of each treatment were analyzed using a complete Freunds adjuvant (CFA)-induced experimental arthritis model. Joint inflammation was followed throughout the experiment. Ultimately, the concentration of inflammation markers, oxidant stress, and ADA activity was quantified. In this experimental stage, methotrexate (MTX) was used as an antiarthritic drug. ResultsTreatments derived from G. glauca, GgMeOH (DE50 = 158 mg/kg), G-A (DE50 = 2 mg/kg), and G-E (DE50 = 1.5 mg/kg) caused an anti-inflammatory effect in the plantar edema test with carrageenan. In the CFA model, joint inflammation decreased with all natural treatments; GgMeOH and G-A inhibited the ADA enzyme in all organs analyzed (joints, serum, spleen, left and right kidneys), while G-E inhibited the enzyme in joints, serum, and left kidney. CFA caused an increase in the weight index of the organs, an effect that was counteracted by the administration of G. glauca treatments, which also modulate the response to the cytokines analyzed in the different organs (IL-4, IL-10, IL-17, IL-6, and TNF- α). ConclusionIt is shown, for the first time, that the GgMeOH extract and the triterpenes G-A and G-E of Galphimia glauca have an anti-arthritic effect (anti-inflammatory, immunomodulatory, antioxidant, and ADA inhibitor), using an experimental arthritis model with CFA. Therefore, knowledge of the plant as a possible therapeutic agent for this rheumatic condition is expanding.

Insights into the molecular mechanisms of Spirulina platensis against rheumatoid arthritis through integrative serum pharmacochemistry and network pharmacology analysis

Spirulina platensis traditionally and increasingly consumed as a premium food and complementary medicine in many Asian countries on account of its nutritive and pharmacological attributes. So far, Spirulina holds promise as a potential therapeutic agent for RA alleviation. However, the mechanistic basis of S. platensis against RA remains poorly clarified.The research strategy featuring serum pharmacochemistry and network pharmacology was constructed to pinpoint the potential bioactive components and efficiency mechanisms of S. platensis against RA. Firstly, a rat model of RA was established using Complete Freund's Adjuvant (CFA). Secondly, serum migrating components following Spirulina administration were profiled and relatively quantified as monitoring indexes for Spirulina bioavailability. Using network pharmacology strategy, potential target genes of transitional components and associated pathways were predicted. Lastly, the control mechanisms of S. platensis in CFA model were verified through a series of serum and synovial biochemical markers along with master RA-related pathways by western blotting. Nineteen circulating compounds, including four prototypes and fifteen metabolites were profiled and selected as candidate efficacy compounds successfully absorbed and maintained considerable concentrations in Spirulina-treated sera creating a great opportunity to exert the anti-RA potential. Among which, fatty acids and their conjugated metabolites represented the most relevant and abundant circulating metabolites. Regarding network pharmacology findings, the top predictive targets highly correlated with RA were NFKB, NLRP3, TNF, MMP-9, TLR4 and VEGFA. Relatedly, KEGG analysis dissected PI3K-Akt signaling pathway, MAPK signaling pathway, osteoclasts (OCs) differentiation and VEGF signaling pathway as the top-ranked RA pathways. Our efficacy experimental findings emphasized that Spirulina markedly downregulated the inflammatory mediators (IL-1, IL-6, IL-17, TNF-α and MMP-9) levels. Following Spirulina treatment, western blotting detected significant alterations in RA master proteins as TLR4, NLRP3, VEGF, Pro caspases-1, Caspase-1, and NF-κB p65. This integrated work illuminates the molecular mechanisms and efficacy constituents of S. platensis as referenced templates against RA. In the light of the above, systematized information on the metabolic fate of the absorbable Spirulina metabolites should be expanded through in-depth pharmacokinetics research for highly targeted drugs. In a broader sense, future investigations and adequate evidence should be explored to broaden the therapeutic strategies scenario in the era of personalized medicine to achieve a comprehensive “treat-to-target” approach which, undoubtedly, is of a great significance in the clinical settings.

Higenamine inhibits acute and chronic inflammatory pain through modulation of TRPV4 channels

Pain is the cardinal symptom of many debilitating diseases and results in heavy health and economic burdens worldwide. Asarum (Asarum sieboldii Miq.) is a commonly used analgesic in Chinese medicine. However, the analgesic components and mechanisms of asarum in acute and chronic pain mice model remain unknown. In this study, we first generated asarum water extract and confirmed strong analgesic properties in mice in both the acute thermal and mechanical pain models, as well as in the complete Freund's adjuvant (CFA) induced chronic inflammatory pain model. Second, we identified higenamine as a major component of asarum and found that higenamine significantly inhibited thermal and mechanical induced acute pain and CFA induced chronic inflammatory pain. Then, using Trpv4−/− mice, we found that TRPV4 is necessary for CFA induced thermal and mechanical allodynia, and demonstrated that higenamine analgesia in the CFA model is partly through TRPV4 channel inhibition. Finally, we found that GSK1016790A, a TRPV4 agonist, induced calcium response was significantly inhibited by higenamine in both cultured DRG neurons and TRPV4 transfected HEK293 cells. Consistent with calcium imaging results, higenamine pretreatment also dose-dependently inhibited GSK1016790A induced acute pain. Taken together, our behavior and calcium imaging results demonstrate that the asarum component higenamine inhibits acute and chronic inflammatory pain by modulation of TRPV4 channels.

Investigating antiarthritic potential of polyherbal emulgel

BackgroundRheumatoid arthritis (RA) is an inflammation of joints with increased cellularity of synovial tissue. Allopathic drugs possess several adverse effects, which have led to increase in the utilization of herbal medicines. Polyherbal emulgel resolves the bioavailability issue associated with hydrophobic drugs and can be used effectively in the treatment of RA. ObjectivesThe present study aimed at the formulation of polyherbal emulgel, and evaluation of in vitro anti-inflammatory activity and in vivo antiarthritic activity. MethodsSeven emulgels F-1 to F-7 were optimally formulated. In vitro anti-inflammatory activity was determined using protein denaturation method employing Diclofenac sodium as the standard. In antiarthritic study Complete Freund's Adjuvant (CFA) model was used. The various parameters were assessed, like paw volume, body weight, hematological parameters, antioxidant parameters, Rheumatic factor (RF), and histopathological study of ankle joint. ResultsF-4 and F-7 were found to be optimized formulations as compared to other formulations. The in vitro anti-inflammatory activity was found to be highest in F-4 with IC50 7.74 and F-7 with IC50 8.87 in comparison with Diclofenac sodium having IC50 57.0. Both formulations F-7 and F-4 showed a significant reduction in paw volume and normalization of body weights. The formulation F-7 even showed more potent antiarthritic activity than F-4 by decreasing white blood cells (WBC), lymphocytes, increasing packed cell volume (PCV), neutrophils, superoxide dismutase (SOD), catalase and decreasing malondialdehyde (MDA) levels in serum. This was further confirmed by histopathological study. ConclusionAs an anti-inflammatory agent, this newly developed emulgel was found to possess more therapeutic efficacy than commercially available diclofenac sodium.

Attenuation of CFA-induced arthritis through regulation of inflammatory cytokines and antioxidant mechanisms by Solanum nigrum L. leaves extracts.

Solanum nigrum L. is a popular traditional medicine for various inflammatory conditions including rheumatism and joint pain. The current study aimed to evaluate the anti-arthritic mechanism of Solanum nigrum L. Four extracts were prepared using n-hexane, methanol, chloroform, and water. The anti-nociceptive and anti-inflammatory activity was carried out with 100, 200, and 300mg/kg body wt. PO of each extract by the hot plate and carrageenan-induced paw oedema methods, respectively. The anti-arthritic study was performed with chloroform and aqueous extracts (300mg/kg) in complete Freund's adjuvant (CFA)-induced arthritis. Paw size (mm), ankle joint diameter (mm), and latency time (sec) were recorded on day 0 and every 4th day till 28days. The hematological, inflammatory, and oxidative biomarkers were estimated. Results showed that significant analgesia (p < 0.05) and reduction in paw inflammation were achieved with all extracts. The highest percent inhibition in Carrageenan-induced inflammation was achieved with 300mg/kg of chloroform (72.19%) and aqueous (71.30%) extracts, respectively. In the CFA model, both extracts showed a significant reduction in paw size and ankle joint diameter (p < 0.05). The RT-qPCR analysis revealed the upregulation of interleukin-4 and interleukin-10, and down-expression of interleukin-1β, interleukin-6, tumor necrosis factor-α, cycloxygenase-2, nuclear factor-κB, prostaglandin E synthase 2, and interferon-γ. A significant increase in superoxide dismutase, catalase, and glutathione levels was observed. Hence, it is concluded that Solanum nigrum L. leaf extracts regulate the expression of inflammatory markers and improve oxidative stress resulting in the attenuation of CFA-induced arthritis.

Activation of G-protein-coupled receptor 183 initiates inflammatory pain via macrophage CCL22 secretion

Chronic pain is a major public health problem with limited effective therapeutic options. G-protein-coupled receptors play a significant role in pain modulation; however, whether and how G-protein-coupled receptor 183 participates in pain regulation remain unclear. In the present study, we found that G-protein-coupled receptor 183 expression was specifically upregulated in the hind paws of mice in various inflammatory pain models. Activation of G-protein-coupled receptor 183 induced acute pain, whereas inhibition or silencing of this receptor alleviated mechanical allodynia and thermal hyperalgesia in complete Freund's adjuvant (CFA) model. Mechanistically, activating G-protein-coupled receptor 183 triggers pain responses via the upregulation of C-C motif chemokine 22(CCL22) in macrophages while blocking the CCL22 receptor C-C motif chemokine receptor 4 (CCR4) attenuates pain hypersensitivity. Taken together, our findings indicate that the G-protein-coupled receptor 183-CCL22 axis has a critical role in the development and maintenance of inflammatory pain.

Sex-specific biobehavioral regulation of persistent inflammatory pain by alcohol.

Although a large percentage of chronic pain patients consume alcohol to manage their pain, there is a significant gap in knowledge regarding the mechanisms underlying the antinociceptive effects of alcohol. To determine the longitudinal analgesic effects of alcohol, we utilized the complete Freund's adjuvant (CFA) model of inflammatory pain in adult female and male Wistar rats. Both somatic and negative motivational aspects of pain were measured using the electronic von Frey (mechanical nociception) system, thermal probe test (thermal nociception), and mechanical conflict avoidance task (pain avoidance-like behavior). Tests were conducted at baseline and 1 and 3 weeks following intraplantar CFA or saline administration. At both time points post-CFA, animals were treated with each of three doses of alcohol (intraperitoneal; 0, 0.5, and 1.0 g/kg) over separate days in a Latin square design. Alcohol produced dose-dependent mechanical analgesia and antihyperalgesia in females but only antihyperalgesia in males. Although alcohol continued to attenuate CFA-induced decreases in both thermal and mechanical nociceptive thresholds between 1 and 3 weeks post-CFA, it appeared less effective at increasing thresholds 3 weeks after CFA induction. These data suggest that individuals may develop tolerance to alcohol's ability to alleviate both somatic and negative motivational symptoms of chronic pain over time. We also discovered sex-specific neuroadaptations in protein kinase A-dependent phosphorylation of GluR1 subunits and extracellular signal-regulated kinase (ERK 1/2) phosphorylation in nociceptive brain centers of animals receiving an alcohol challenge 1 week post-CFA. Together, these findings illustrate a sex-specific regulation of behavioral and neurobiological indices of persistent pain by alcohol.

Characterization of pain-, anxiety-, and cognition-related behaviors in the complete Freund's adjuvant model of chronic inflammatory pain in Wistar-Kyoto rats.

Chronic pain is often associated with comorbid anxiety and cognitive dysfunction, negatively affecting therapeutic outcomes. The influence of genetic background on such interactions is poorly understood. The stress-hyperresponsive Wistar-Kyoto (WKY) rat strain, which models aspects of anxiety and depression, displays enhanced sensitivity to noxious stimuli and impaired cognitive function, compared with Sprague-Dawley (SD) counterparts. However, pain- and anxiety-related behaviors and cognitive impairment following induction of a persistent inflammatory state have not been investigated simultaneously in the WKY rats. Here we compared the effects of complete Freund's adjuvant (CFA)-induced persistent inflammation on pain-, negative affect- and cognition-related behaviors in WKY vs. SD rats. Male WKY and SD rats received intra-plantar injection of CFA or needle insertion (control) and, over the subsequent 4 weeks, underwent behavioral tests to assess mechanical and heat hypersensitivity, the aversive component of pain, and anxiety- and cognition-related behaviors. The CFA-injected WKY rats exhibited greater mechanical but similar heat hypersensitivity compared to SD counterparts. Neither strain displayed CFA-induced pain avoidance or anxiety-related behavior. No CFA-induced impairment was observed in social interaction or spatial memory in WKY or SD rats in the three-chamber sociability and T-maze tests, respectively, although strain differences were apparent. Reduced novel object exploration time was observed in CFA-injected SD, but not WKY, rats. However, CFA injection did not affect object recognition memory in either strain. These data indicate exacerbated baseline and CFA-induced mechanical hypersensitivity, and impairments in novel object exploration, and social and spatial memory in WKY vs. SD rats.

In vitro and in vivo Anti-arthritic and Antioxidant potential of Glycosmis pentaphylla in Complete Freund’s Adjuvant model

Rheumatoid arthritis (RA) is a systemic, chronic inflammatory and auto immune disorder. RA is provoked by genetical predisposition and environmental factors. Oxidative stress makes the human liable to diseases like diabetes, RA, cancer, atherosclerosis etc. Hence the study investigated the anti-arthritic and oxidative activity of ethanolic extract of Glycosmis pentaphylla (EEGP) as it has not been pharmacologically tested. Anti-arthritic potential was evaluated in vitro using protein denaturation by bovine serum albumin and in vivo by Complete Freund Adjuvant (CFA) models at 200 and 400mg/kg doses. Also, in vitro and in vivo antioxidant ability was appraised by DPPH scavenging and assessment of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and lipid peroxidation (LPO). The results exhibited concentration dependent inhibition of albumin denaturation with maximum results obtained at 800μg/ml. The results of CFA model depicted better protection against arthritic lesions and body weight alterations. Also G. pentaphylla remarkably ameliorated histopathological changes. Additionally, plant exhibited remarkable anti-oxidant activity.

Evidence for Endogenous Opioid Dependence Related to Latent Sensitization in a Rat Model of Chronic Inflammatory Pain.

Studies performed in a mouse model of chronic inflammatory pain induced by intraplantar injection of complete Freund's adjuvant (CFA) have shown that constitutive activation of the endogenous opioid signaling, besides serving as a mechanism of endogenous analgesia that tonically represses pain sensitization, also generates a state of endogenous opioid dependence. Since species-related differences concerning pain biology and addictive behaviors occur between mice and rats, the present study explored whether the coexistence of endogenous opioid analgesia and endogenous opioid dependence also characterizes a homologous rat model. To this aim, CFA-injured Wistar rats were treated with either 3 mg/kg or 10 mg/kg of the opioid receptor inverse agonist naltrexone (NTX) during the pain remission phase and monitored for 60 min for possible withdrawal behaviors. At 3 mg/kg, NTX, besides inducing the reinstatement of mechanical allodynia, also caused a distinct appearance of ptosis, with slight but nonsignificant changes to the occurrence of teeth chatters and rearing. On the other hand, 10 mg/kg of NTX failed to unmask pain sensitization and induced significantly lower levels of ptosis than 3 mg/kg. Such an NTX-related response pattern observed in the rat CFA model seems to differ substantially from the pattern previously described in the mouse CFA model. This supports the knowledge that mice and rats are not identical in terms of pharmacological response and stresses the importance of choosing the appropriate species for preclinical pain research purposes depending on the scientific question being asked.

Topical Calendula officinalis L. inhibits inflammatory pain through antioxidant, anti-inflammatory and peripheral opioid mechanisms

ObjectiveThe present study evaluated the antinociceptive activity of Calendula officinalis L. (Ca) cream on inflammatory hypernociception. MethodsCreams with different Ca concentrations were tested for their ability to ameliorate pain-related behavior and edema in rats using formalin test, carrageenan (Cg)-induced acute inflammation model, bradykinin (BK)-induced acute inflammation model, and complete Freund’s adjuvant (CFA)-induced chronic inflammation model. Using the formalin test, we also examined the implication of peripheral opioid receptors in the antinociceptive mechanisms of Ca cream, by means of Q-naloxone, a peripherally acting nonselective opioid antagonist. Furthermore, the effects of Ca cream compared with diclofenac on BK-induced edema were examined when the kininase II in tissue was preserved or inhibited by captopril. The local production of redox biomarkers in formalin model, tumor necrosis factor-α (TNF-α) in Cg model and histopathological changes in CFA model were also evaluated. ResultsA single application of Ca cream at a dose of 10% or 30% (w/w) decreased the formalin-induced licking/biting behavior during both phases of this test in a Q-naloxone-sensitive manner. This effect was associated with the reduction of oxidative stress in the injured paw and the edema associated with the later phase of formalin-induced pain. A single application of Ca cream (10% or 30%) reduced paw edema and thermal hypernociception in Cg-induced acute inflammation, corresponding with a local decrease in TNF-α. Ca cream also inhibited BK-induced spontaneous nociceptive behavior and paw inflammation in both paradigms studied. Repeated applications of Ca cream also decreased CFA-induced chronic inflammation, mechanical hypernociception and histopathological changes in the paw. ConclusionThese results reveal the topical antinociceptive and antiedematogenic effects of Ca cream. A modulatory action on peripheral opioid receptors associated with its antioxidant mechanism may be involved, at least in part, in its analgesic effects. These findings may have an impact on the clinical management of painful inflammatory diseases.