post-CFA Injection Research Articles

Onosma bracteatum Wall Aqueous-Ethanolic Extract Suppresses Complete Freund's Adjuvant-Induced Arthritis in Rats via Regulation of TNF-α, IL-6, and C-Reactive Protein.

Onosma bracteatum Wall (O. bracteatum) has been used traditionally for the management of arthritis; however, its therapeutic potential warrants further investigation. This study aimed to evaluate the anti-arthritic effects of the aqueous-ethanolic extract of O. bracteatum leaves (AeOB) in a rat model of complete Freund's adjuvant (CFA)-induced arthritis. Rats were treated with AeOB (250, 500, and 750 mg/kg), indomethacin (10 mg/kg), or a vehicle control from days 8 to 28 post-CFA injection. Arthritic score, paw diameter, and body weight were monitored at regular intervals. X-ray radiographs and histopathological analysis were performed to assess arthritic severity. Inflammatory cytokines tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and C-reactive protein (CRP) were quantified by qPCR and icromatography. Phytochemical analysis of AeOB revealed alkaloids, flavonoids, phenols, tannins, Saponins, and glycosides. AeOB also exhibited antioxidant potential with an IC50 of 73.22 µg/mL in a DPPH assay. AeOB and diclofenac exhibited anti-inflammatory and anti-arthritic activities. Rats treated with AeOB at 750 mg/kg and indomethacin showed significantly reduced arthritic symptoms and joint inflammation versus the CFA control. The AeOB treatment downregulated TNF-α and IL-6 and decreased CRP levels compared with arthritic rats. Radiography and histopathology also showed improved prognosis. These findings demonstrate the anti-arthritic potential of AeOB leaves.

Orofacial anti-hypernociceptive effect of citral in acute and persistent inflammatory models in rats

Orofacial pain has significant psychological and physiological effects. Citral (3,7-dimethyl-2,6-octadienal) is the main component of Cymbopogon citratus (DC) Stapf, an herb with analgesic properties. Although citral has been considered a potent analgesic, its putative effects on orofacial pain are still unknown. ObjectiveThe objective of this study is to test the hypothesis that citral modulates orofacial pain using two experimental models: formalin-induced hyperalgesia in the vibrissae area and during persistent temporomandibular hypernociception using Complete Freund's Adjuvant - CFA test. MethodsFor the formalin test, citral (100 and 300 mg/kg, oral gavage) or its vehicle (Tween 80, 1 %) were given 1 h before the formalin injection subcutaneously (sc) into the vibrissae area. For the CFA model, we analyzed the prophylactic (100 mg/kg of citral by oral gavage, 1 h before CFA injection) and the chronic therapeutic (citral treatment 1-hour post-CFA injection and daily post-CFA injection) effect of citral or its vehicle in animals treated with CFA for 8 days. ResultsCitral caused a decrease in formalin-induced local inflammation and the time spent performing nociceptive behavior in a dose-dependent fashion. Similarly, prophylactic and therapeutic citral treatment decreased the CFA-induced persistent mechanical hypernociception in the temporomandibular area. ConclusionOur data strengthen the notion that citral plays a powerful antinociceptive role by decreasing orofacial hypernociception in formalin and CFA models.

Emerging role of microglia and astrocyte in the affective-motivational response induced by a rat model of persistent orofacial pain

Few studies are approaching the neural basis underlying the aggregation of emotional disorders in orofacial pain despite the stress, depression, and anxiety are some of the most commonly reported risk factors. Using a persistent orofacial pain rat model induced by complete Freund's adjuvant (CFA) injection into the temporomandibular joint, we have investigated the plasticity astrocytes and microglia key brain regions for the affective-emotional component of pain. We measured the expression and morphologic pattern of reactivation of glial fibrillary acidic protein (GFAP, astrocyte marker) and Iba-1 (microglial marker) by western blotting and immunohistochemistry analysis. The results showed no alterations on motor activity during inflammatory pain, indicating an exclusive effect of nociceptive behavior on the plasticity of limbic regions. CFA-induced temporomandibular inflammation changed GFAP and Iba-1 expression in distinct regions related to emotional behavior in a time-dependent manner. A significant increase in GFAP and Iba-1 expression was observed in the central nucleus of the amygdala, hippocampus and periaqueductal grey matter from day 3 to day 10 post-CFA injection. Moreover, a positive correlation between GFAP and Iba-1 upregulation and an increased mechanical hypersensitivity was observed. Conversely, no change on GFAP and Iba-1 expression was observed in the hypothalamus and colliculus during orofacial inflammatory pain. Our data suggest an important role for glial cells in the affective-motivational dimension of orofacial pain beyond their well-explored role in the traditional nociceptive transmission circuits.

Effects of buprenorphine on acute pain and inflammation in the adjuvant-induced monoarthritis rat model

Background and aimAnimal modelling of arthritis is often associated with pain and suffering. Severity may be reduced with the use of analgesia which is, however, often withheld due to concerns of introducing a confounding variable. It is therefore important to design and validate pain relief protocols that reduce pain without compromising the scientific objectives. The present study evaluated the effect of buprenorphine analgesia in the immediate post-induction period of an adjuvant-induced monoarthritic rat model. The aim of this study was to extend previous work on refinement of the model by alleviating unnecessary pain. MethodsMale and female Sprague Dawley rats were injected with 20 μl of complete Freund's adjuvant (CFA) into the left ankle. Rats were treated with buprenorphine, either injected subcutaneously or ingested voluntarily, and were compared to rats given subcutaneous injections with vehicle (saline or pure nut paste) or carprofen the first three days post CFA-injection. Measurements of welfare, clinical model-specific parameters and pain-related behaviour were assessed. ResultsBuprenorphine, administered either subcutaneously (0.10 or 0.15 mg/kg, twice daily) or by voluntary ingestion in nut paste (1.0 or 3.0 mg/kg, twice daily), improved mobility, stance, rearing and lameness scores significantly 7 h post CFA-injection. Mechanical hyperalgesia peaked at 7 h and was significantly lower in buprenorphine-treated animals, compared to vehicle-treated animals. Joint circumference was highest 24–72 h after CFA injection. Animals treated with buprenorphine did not decrease in joint circumference, opposite carprofen treated animals. ConclusionBuprenorphine, administered either subcutaneously or by voluntary ingestion, provides adequate analgesia for both sexes within the first 24 h post CFA-injection. Buprenorphine treatment improved clinical scores and appeared not to suppress the inflammatory response. The present study supports previous findings that voluntarily ingested buprenorphine is an effective alternative to repeated injections.

Fragile X mental retardation protein-regulated proinflammatory cytokine expression in the spinal cord contributes to the pathogenesis of inflammatory pain induced by complete Freund's adjuvant.

Studies have verified that Fragile X mental retardation protein (FMRP), an RNA-binding protein, plays a potential role in the pathogenesis of formalin- and (RS)-3,5-dihydroxyphenylglycine-induced abnormal pain sensations. However, the role of FMRP in inflammatory pain has not been reported. Here, we showed an increase in FMRP expression in the spinal dorsal horn (SDH) in a rat model of inflammatory pain induced by complete Freund's adjuvant (CFA). Double immunofluorescence staining revealed that FMRP was mainly expressed in spinal neurons and colocalized with proinflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)]. After consecutive intrathecal injection of fragile X mental retardation 1 small interfering RNA for 3days post-CFA injection, FMRP expression in the SDH was reduced, and CFA-induced hyperalgesia was decreased. In addition, the CFA-induced increase in spinal TNF-α and IL-6 production was significantly suppressed by intrathecal administration of fragile X mental retardation 1 small interfering RNA. Together, these results suggest that FMRP regulates TNF-α and IL-6 levels in the SDH and plays an important role in inflammatory pain.

Recurring Real-Time Monitoring of Inflammations in Living Mice with a Chemiluminescent Probe for Hypochlorous Acid

Recurring Real-Time Monitoring of Inflammations in Living Mice with a Chemiluminescent Probe for Hypochlorous Acid

Celastrol ameliorates inflammatory pain and modulates HMGB1/NF-κB signaling pathway in dorsal root ganglion

Evidences reported that high mobility group box 1 (HMGB1) played a pivotal role in the modulation of chronic inflammatory pain. Celastrol, a bioactive component extracted from Tripterygium wilfordii Hook, possesses anti-inflammatory activity, but the underlying mechanism remains to be fully clarified. We aim to investigate whether HMGB1 in dorsal root ganglion (DRG) participates in the effect of celastrol on inflammatory pain. Complete Freund’s adjuvant (CFA)-induced inflammatory pain rat model was used. Paw withdrawal latency (PWL) was detected to evaluate the effects of celastrol on CFA-evoked inflammatory pain. After application of celastrol (1mg/kg, i.p.) on day 1, 3, 7 and 14 post-CFA injection, the expression levels of HMGB1, NF-κB, some proinflammatory markers, GFAP and CD11b in DRG were determined by qRT-PCR and western blot analysis. These results showed that celastrol significantly suppressed HMGB1, NF-κB and IL-1β mRNA and protein expression in DRG and alleviated CFA-evoked thermal hyperalgesia. Furthermore, celastrol obviously inhibited COX-2 protein expression and down-regulated IL-6, IL-17, TNF-α, MCP-1, GFAP and CD11b mRNA levels in DRG of CFA rats. Collectively, the present study firstly provide evidences of the anti-inflammatory effect of celastrol via suppressing CFA-induced the activation of HMGB1/NF-κB signaling pathway in DRG, which maybe a potential therapeutic target for celastrol alleviating inflammatory pain.

A non-reflexive method based on the variability of temperature and bioimpedance in measuring inflammatory hyperalgesia and analgesia in mice

BackgroundPreclinical studies measure withdrawal responses to evoking thermal and mechanical stimuli instead of the more clinically important spontaneous pain. New methodTherefore, we studied the effect of peripheral inflammation induced by intraplantar administration of complete Freund’s adjuvant (CFA) in mice on the variability of temperature and bioimpedance as an index of pain produced by inflammation. To this end, we initially determined mathematical scores based on changes in temperature and bioimpedance (STB) for animals with an inflamed paw and compared these scores with commonly used measures of inflammatory pain. We then pharmacologically validated the tool using dexamethasone. ResultsThe STB analysis resembled the response found in the von Frey Hair (vFH) test. The CFA-induced increase in STB and vFH tests were reversed by intraperitoneal administration of dexamethasone. The correlation between the STB and vFH measurements showed a high correlation coefficient (R2 = 0.911, p < 0.001). Comparison with existing methodOur results also demonstrated that CFA paw injection induced mechanical hyperalgesia in mice and remained virtually unaltered during all time-points tested for 5 days, as measured with vFHs. The administration of CFA into the paw induced a large increase in paw volume that was apparent 1 and 5 days after the injection. The CFA injection resulted in a significant (p < 0.05) decrease in the response latency to the heat stimulus, as evaluated on day 4 post-CFA injection. ConclusionsThe data presented here suggest that STB may provide a novel non-invasive approach for inflammatory pain detection.

Evaluation of the effect of andrographolide and methotrexate combined therapy in complete Freundʼs adjuvant induced arthritis with reduced hepatotoxicity

ObjectiveMethotrexate is one of the most widely used disease-modifying anti-rheumatic drugs. The hepatotoxicity of methotrexate resulted in poor compliance with therapy. The current study was designed to analyse the combined therapy of andrographolide (AD) and methotrexate (MTX) for complete Freund's adjuvant (CFA)-induced arthritis, focusing on hepatoprotective effects, oxidative stress and arthritic-related cytokines. MethodWistar rats were injected with CFA into the right hind paw. Ten days post-CFA injection, the Wistar rats were administered with 1% CMC-Na solution, methotrexate (2 mg/kg/week), AD (50 mg/kg/d) and combined therapy for 35 days. The anti-arthritic effect was assessed by paw volume, X-ray and serum tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β levels. Serum samples were also analysed for glutamic oxaloacetic transaminases (GOT), serum glutamic pyruvic transaminase (GPT), alkaline phosphatase (AKP) and lactate dehydrogenase (LDH). Liver tissue samples were used to examine the cellular antioxidant defence activities using catalase activity (CAT) and GSH as well as GSH-Px and MDA. Histopathology analysis was conducted to evaluate liver damage. ResultsAD treatment strengthened the anti-arthritic capacity of MTX. AD and MTX-combined therapy additively reduced the inflammatory symptoms in CFA rats. The combined therapy of AD and MTX showed hepatoprotective effect indicated by an improvement in the serum marker, possibly due to antioxidant action and confirmed by liver histopathological changes. Furthermore, the combined therapy significantly reduced serum TNF-α, IL-6 and IL-1β levels. ConclusionsA combined therapy of AD and methotrexate significantly alleviated MTX-induced hepatocellular injury and strengthened the anti-arthritic effect. Further clinical studies should be done to further verify the possibility of combined its clinical usage.

Abstract # 1773 Cytotoxic T cells may induce pain hypersensitivity in female but not male mice after nerve injury in an interferon-g-receptor-dependent manner

In our previous study, we observed that female mice, unlike males, do not require microglia to produce pain hypersensitivity after neuropathic or inflammatory injury. Using T-cell deficient mice, we found that female mutant mice “switch” to the “male” microglial system. Important questions include how microglial or T cell systems are recruited, how and which T cells are infiltrating into the spinal cord, and what T cells are releasing that can sensitize pain-relevant neurons. Immune cells were extracted from lumbar spinal cord of WT mice and IFNgRKO mice post SNI and CFA injection, and analyzed using flow cytometry. Additionally, mechanical allodynia was assessed. Analyzing the spinal cord microenvironment after SNI revealed that female mice displayed: (1) a 2-fold increase in CD8+ cytotoxic T cells, (2) upregulation of the anti-inflammatory microglia population, and (3) downregulation of anti-inflammatory T regulatory cells and pro-inflammatory microglia. In contrast, males showed: (1) a 2-fold decrease in CD8+ cytotoxic T cells, and (2) an increase in pro-inflammatory microglia. IFNgRKO female mice showed the same pain behavioural response as WT males wherein minocycline reversed allodynia. Consistent with this, IFNgRKO female, like WT male, mice did not show upregulation of CD8+ cytotoxic T cells. Our current data further suggest that female mice, unlike males, are using cytotoxic CD8+ T cells to induce neuropathic pain, and that this mechanism appears to be dependent on IFNgRs.

Light-emitting diode therapy reduces persistent inflammatory pain: Role of interleukin 10 and antioxidant enzymes

Background: During the last decades, the use of light-emitting diode therapy (LEDT) has increased significantly for the treatment of wound healing, analgesia and inflammatory processes. Nevertheless, scientific data on the mechanisms responsible for the therapeutic effect of LEDT are still insufficient. Thus, this study investigated the analgesic, anti-inflammatory and anti-oxidative effect of LEDT in the model of chronic inflammatory hyperalgesia. Experimental procedures: Mice injected with Complete Freund’s Adjuvant (CFA) underwent behavioral, i.e. mechanical and hot hyperalgesia; determination of cytokine levels (tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL-10), oxidative stress markers (protein carbonyls and thiobarbituric acid reactive species (TBARS)) and antioxidant enzymes (catalase (CAT) and superoxide dismutase (SOD)). Additionally, mice were pretreated with either naloxone or fucoidin and mechanical hyperalgesia was assessed. Results: LEDT inhibited mechanical and thermal hyperalgesia induced by CFA injection. LEDT did not reduce paw edema, neither influenced the levels of TNF-α and IL1-β; although it increased the levels of IL-10. LEDT significantly prevented TBARS increase in both acute and chronic phases post-CFA injection; whereas protein carbonyl levels were reduced only in the acute phase. LEDT induced an increase in both SOD and CAT activity, with effects observable in the acute but not in the chronic. And finally, pre-administration of naloxone or fucoidin prevented LEDT analgesic effect. Conclusions: These data contribute to the understanding of the neurobiological mechanisms involved in the therapeutic effect of LEDT as well as provides additional support for its use in the treatment of painful conditions of inflammatory etiology.

Cdk5 contributes to inflammation-induced thermal hyperalgesia mediated by the p38 MAPK pathway in microglia

Background: The mechanisms underlying cyclin-dependent kinase 5 (Cdk5)-mediated thermal hyperalgesia induced by inflammation remain poorly understood. In the present study, we examined thermal hyperalgesia provoked by peripheral injection of complete Freund׳s adjuvant (CFA) to test for Cdk5 signaling in the spinal dorsal horns of rats through the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway, which is known to function in mediating inflammatory pain.Methods: We induced the inflammatory pain model by plantar injection of CFA and compared the inhibitory effects of roscovitine and SB203580 on thermal hyperalgesia. We measured localization of Cdk5, p35, OX-42, and glial fibrillary acidic protein (GFAP) in the dorsal horn at 1 and 3 days after CFA injection using immunohistochemistry, and we measured protein levels of OX-42 and phosphorylated-p38 (p–p38) using Western blot analysis. Tumor necrosis factor-a (TNF-a) was measured by ELISA.Results: The maximum thermal hyperalgesia induced by CFA occurred at 1d following injection and decreased until 5 d. We found colocalization of the Cdk5 activator p35, the microglial marker OX-42 and p–p38 in the same microglial cells and neurons of the spinal cord at day 1 after CFA injection; however, we saw no colocalization of p35 and GFAP, a marker of activated astrocytes. The thermal hyperalgesia induced by CFA was inhibited by intrathecal administration of the Cdk5 inhibitor roscovitine and by the p38 inhibitor SB203580. Furthermore, the expression of OX-42, p–p38, and TNF-a was remarkably increased from days 1 to 5 post-CFA injection and were significantly reversed by roscovitine between 1 and 3 days.Conclusions: Cdk5, an upstream regulator of p38 and TNF-a, mediates CFA-induced thermal hyperalgesia. As such, pharmacological blocking of the generation of p–p38 mediated by Cdk5 may present a novel approach for diminishing inflammatory pain.This article is part of a Special Issue entitled SI: Spinal cord injury.

Development of an Effective Topical Liposomal Formulation for Localized Analgesia and Antiinflammatory Actions in the Complete Freund’s Adjuvant Rodent Model of Acute Inflammatory Pain

Background: Peripheral opioid receptor targeting has been well established as a novel target in clinical pain management for acute and chronic peripheral inflammatory pain. The physiochemical properties of the peripheral mu-opioid receptor agonist, loperamide HCl, limit the use of the free drug as an analgesic or anti-inflammatory agent, particularly for dermal delivery across intact skin. Objective: Our objective was to manufacture an effective topical formulation containing loperamide using liposomal delivery that would allow loperamide to produce analgesia and anti-inflammatory effects, by penetrating the epidermis to reach peripheral opioid receptors within the dermis of intact skin. Study Design: A randomized, double blind, controlled animal trial. Methods: Thirty-five adult male Wistar rats (200 – 250 g) were randomly divided into 5 groups: loperamide HCl-encapsulated liposomal gel, naloxone methiodide + loperamide HCl-encapsulated liposomal gel, free loperamide gel, empty liposomal gel, and 1% diclofenac gel (Voltaren®). Diclofenac gel was used as a positive control as it is clinically used as a topical analgesic and anti-inflammatory drug. Animals received an intraplantar injection of 150 µl Complete Freund’s Adjuvant (CFA) into the right hindpaw and experiments were performed 5 days post-CFA injection, which corresponded to the peak inflammatory response. All manufactured formulations were applied topically on both hind paws twice daily, whereas Voltaren gel was applied 3 times a day in accordance with the manufacturer’s instructions. The dose administered was 50 µl, which equated to 0.4 mg of loperamide HCl for the loperamide HCl treatment groups (low dose). Naloxone methiodide (1 mg/kg) was administered via intraplantar injection, 15 minutes prior to application of loperamide HCl-encapsulated liposomal gel to determine opioid receptor dependent activity. An investigator blinded to the treatment administered assessed time course of the antinociceptive and antiinflammatory effects using a paw pressure analgesiometer and plethysmometer, respectively. Results: Application of loperamide HCl in a liposomal gel formulation exerted analgesic and antiinflammatory effects exclusively in peripheral painful inflamed tissue. This formulation produced highly significant analgesic and anti-inflammatory effects over the 48-hour time course studied following topical administration in rats with CFA-induced inflammation of the paw. As expected, the diclofenac gel group showed significant antinociception over the duration of the study; however, this effect was lower in comparison to the loperamide HCl liposomal gel formulation. All other control groups showed no significant antinociceptive effects. In addition, all control groups (1% diclofenac gel, free loperamide gel, and empty liposomal gel) did not demonstrate a significant change in paw volume over 48 hours. Limitations: In vivo studies were performed in the well-established rodent model of acute inflammatory pain. We are currently studying this approach in chronic pain models known to have clinical activation of the peripheral immune-derived opioid response. Conclusions: The study demonstrates that topically applied loperamide encapsulated within liposomal systems has improved therapeutic efficacy over conventional formulations for the local treatment of acute peripheral inflammatory pain conditions where the skin has remained intact. Once in the inflamed peripheral tissue, loperamide provides analgesic and anti-inflammatory effects in a similar manner to peripheral endogenous opioids. This preparation optimises the retention of drug at the site where action is required. Key words: Pain, inflammation, opioids, loperamide, liposomes, topical drug delivery, peripheral opioid receptors

Fyn kinase‐regulated NMDA receptor‐ and AMPA receptor‐dependent pain sensitization in spinal dorsal horn of mice

Inhibition of Src-family protein tyrosine kinases (SFKs) in spinal dorsal horn has been established as an effective strategy for the alleviation of chronic pathological pain. As one of the important SFKs members, Fyn kinase is critical for synaptic plasticity and many pathophysiological processes. However, whether Fyn is involved in spinal sensitization is far from being elucidated. We manipulated Fyn activity by expressing a constitutively active Fyn mutant [Fyn(Y528F) ] or a catalytically null mutant [Fyn(K296M) ] in the spinal dorsal horn of mice, and performed behavioural and biochemical experiments to investigate the role of Fyn in regulating the nociceptive responses and the synaptic expression of ionotropic glutamate receptors. Spinal expression of Fyn(Y528F) alone in intact mice was sufficient to elicit persistent mechanical allodynia and thermal hyperalgesia, which lasted for at least 12 days. Fyn(Y528F) simultaneously enhanced the concentrations of N-methyl-D-aspartic acid (NMDA)-subtype and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype glutamate receptors at synaptosomal membrane fraction. Pharmacological inhibition of NMDA receptors or AMPA receptors greatly alleviated Fyn(Y528F)-induced pain hypersensitivity. To evaluate the contribution of Fyn to inflammatory pain, we expressed Fyn(K296M) before intradermal injection of complete Freund's adjuvant (CFA), finding that Fyn(K296M) had no effect on the induction of inflammatory pain within 3 h post-CFA injection, which, however, repressed the synaptic accumulation of NMDA receptors and AMPA receptors to attenuate the maintenance of chronic pain states. Fyn played a key role in the sustained sensitization of nociceptive behaviours by up-regulating the functions of ionotropic glutamate receptors in spinal dorsal horn.

Targeted Nanoparticles that Mimic Immune Cells in Pain Control Inducing Analgesic and Anti-inflammatory Actions: A Potential Novel Treatment of Acute and Chronic Pain Conditions

Background: The peripheral immune-derived opioid analgesic pathway has been well established as a novel target in the clinical pain management of a number of painful pathologies, including acute inflammatory pain, neuropathic pain, and rheumatoid arthritis. Objective: Our objective was to engineer targeted nanoparticles that mimic immune cells in peripheral pain control to deliver opioids, in particular loperamide HCl, specifically to peripheral opioid receptors to induce analgesic and anti-inflammatory actions for use in painful inflammatory conditions. This peripheral analgesic system is devoid of central opioid mediated side effects (e.g., respiratory depression, sedation, dependence, tolerance). Study Design: A randomized, double blind, controlled animal trial. Methods: Thirty-six adult male Wistar rats (200 - 250 g) were randomly divided into 6 groups: loperamide HCl-encapsulated anti-ICAM-1 immunoliposomes, naloxone methiodide + loperamide HCl-encapsulated antiICAM-1 immunoliposomes, loperamide HCl-encapsulated liposomes, empty anti-ICAM-1 immunoliposomes, empty liposomes, and loperamide solution. Animals received an intraplantar injection of 150 µL Complete Freund’s Adjuvant (CFA) into the right hindpaw and experiments were performed 5 days post-CFA injection, which corresponded to the peak inflammatory response. All formulations were administered intravenously via tail vein injection. The dose administered was 200 µL, which equated to 0.8 mg of loperamide HCl for the loperamide HCl treatment groups (sub-therapeutic dose). Naloxone methiodide (1 mg/kg) was administered via intraplantar injection, 15 minutes prior to loperamide-encapsulated anti-ICAM-1 immunoliposomes. An investigator blinded to the treatment administered assessed the time course of the antinociceptive and antiinflammatory effects using a paw pressure analgesiometer and plethysmometer, respectively. Biodistribution studies were performed 5 days post-CFA injection with anti-ICAM-1 immunoliposomes and control liposomes via tail vein injection using liquid scintillation counting (LSC). Results: Administration of liposomes loaded with loperamide HCl, and conjugated with antibody to intercellular adhesion molecule-1 (anti-ICAM-1), exerted analgesic and anti-inflammatory effects exclusively in peripheral painful inflamed tissue. These targeted nanoparticles produced highly significant analgesic and anti-inflammatory effects over the 48 hour time course studied following intravenous administration in rats with Complete Freund’s Adjuvant-induced inflammation of the paw. All control groups showed no significant antinociceptive or anti-inflammatory effects. Our biodistribution study demonstrated specific localization of the targeted nanoparticles to peripheral inflammatory tissue and no significant uptake into the brain. Limitations: In vivo studies were performed in the well-established rodent model of acute inflammatory pain. We are currently studying this approach in chronic pain models known to have clinical activation of the peripheral immune-derived opioid response. Conclusions: The study presents a novel approach of opioid delivery specifically to injured tissues for pain control. The study also highlights a novel anti-inflammatory role for peripheral opioid targeting, which is of clinical relevance. The potential also exists for the modification of these targeted nanoparticles with other therapeutic compounds for use in other painful conditions. Key words: Pain, inflammation; immune cells, opioids, ICAM-1, loperamide, targeted drug delivery, immunoliposomes

MTOR and its downstream pathway are activated in the dorsal root ganglion and spinal cord after peripheral inflammation, but not after nerve injury

Protein translation controlled through activation of mammalian target of rapamycin (mTOR) participates in many physiological and pathological processes. However, whether such activation is required for chronic pain is still unknown. Here, we examined activation of the mTOR signaling pathway during complete Freund's adjuvant (CFA)-induced chronic inflammatory pain and L5 spinal nerve ligation (SNL)-induced neuropathic pain in rats. Western blot analysis showed significantly increased levels of phosphorylated mTOR (p-mTOR) and phosphorylated p70S6 kinase 1 (p-S6K1, a downstream effector of mTOR) in the ipsilateral L4/5 spinal cord 2h, 1 day, 3 days, and 7 days after intraplantar CFA injection and in the ipsilateral L4/5 dorsal root ganglions (DRGs) 1 and 3 days after CFA injection. Immunohistochemistry also demonstrated increases in number of p-mTOR-labeled neurons in the ipsilateral L4/5 DRGs and in density of p-mTOR-labeled immunoreactivity in the ipsilateral L4/5 superficial dorsal horn 1 day after CFA injection. Moreover, intrathecal administration of rapamycin, a selective inhibitor of mTOR, significantly blocked CFA-induced mechanical allodynia and thermal hyperalgesia 1 day post-CFA injection. Interestingly, expression of neither p-mTOR nor p-S6K1 was markedly altered on days 3, 7, or 14 after L5 SNL in L5 spinal cord or DRG. These findings indicate that in DRG and spinal cord, mTOR and S6K1 are activated during chronic inflammatory pain, but not during neuropathic pain. Our results strongly suggest that mTOR and its downstream pathway contribute to the development of chronic inflammatory pain.

Spinal Cord Brain-Derived Neurotrophic Factor Levels Increase after Dexamethasone Treatment in Male Rats with Chronic Inflammation

Dexamethasone is widely used in the therapy of chronic inflammatory diseases for its pain-modulating effects. The objective of this study was to evaluate the effect of dexamethasone on nociception and local inflammation, and the levels of brain-derived neurotrophic factor (BDNF) in the spinal cord in male rats with chronic inflammation induced by complete Freund’s adjuvant (CFA). Rats were randomly divided into a control group (not manipulated) and 2 CFA-induced chronic inflammation groups (in the 15th post-CFA injection): 1 injected with vehicle (saline solution) and 1 received dexamethasone (0.25 mg/kg) for 8 days. The hot-plate and electronic von Frey tests were performed 24 h after the end of treatment. BDNF spinal cord levels were determined by enzyme-linked immunosorbent assay (ELISA). The level of inflammation in the tibiotarsal joint (the ankle region) was evaluated histologically at the end of treatment. Dexamethasone produced significantly increased latency in the hot-plate test (one-way ANOVA, p < 0.05) and withdrawal threshold in the electronic von Frey test (p < 0.005). The dexamethasone group showed increased spinal cord BDNF levels compared to the other groups (one-way ANOVA p, < 0.05). Histological analysis showed a local inflammatory response only in animals treated with vehicle, which demonstrated that the dexamethasone treatment decreased the inflammatory process. Our findings corroborate the antinociceptive and anti-inflammatory properties of dexamethasone. In addition, we showed that the dexamethasone treatment increased BDNF levels in the spinal cord; its pain- modulating effects can be attributed to this effect.

Anti-arthritic activities of Annona muricata L. leaves extract on complete Freund's adjuvant (CFA) – induced arthritis in rats

Annona muricata L. (Annonaceae) (AML) has been used as folkloric herbal medicine in many regions throughout the world in treating fever, cough, diarrhea, sedative, rash, ring worm and lactation for women afterbirth (1). The present study was carried out to investigate the anti-arthritic effects of AML ethanolic extract in complete Freund's adjuvant (CFA)-induced arthritis in rats. Edema in the left hind paw of rat was induced by the subcutaneous injection of 0.1ml of heat-killed Mycobacterium tuberculosis in mineral oil (5mg/ml). AML (3, 10, 30 and 100mg/kg) or indomethacin (10mg/kg) as positive control was administered orally once a day between days 1 and 14 post-CFA injection. Edema volume in each paw was measured by using a plethysmometer for 14 days. Levels of tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) in local tissue were measured using enzyme-linked immunosorbent assay (ELISA) on day 14 post-CFA injection. The result exhibited that AML at all doses (3, 10, 30 and 100mg/kg) were significantly (p&lt;0.05) reduced the edema by 48.39, 66.67, 79.57 and 72.04% respectively. AML at higher doses 30 and 100mg/kg significantly suppressed the local tissue TNF-α level by 42.81 and 51.82% and IL-1β level by 35.57 and 39.79% respectively on day 14 post-CFA injection. For TNF-α, the efficacies of the extract at doses 30 and 100mg/kg were higher compared to the effect of indomethacin (51.82%). In conclusion, the findings suggest that AML present notable anti-arthritic activities that may be mediated by suppressing pro-inflammatory cytokines.

Measuring persistent temporomandibular joint nociception in rats and two mice strains

Temporomandibular joint (TMJ) pain has been reported to last for prolonged periods in humans. In rodents a variety of methods have been used to measure TMJ nociception, but for most of these methods the period of measurement has been minutes to a couple of hours. In addition, most measurement protocols required restraint or training of the animal. Previous studies from our laboratory demonstrated that feeding behavior, particularly meal duration, was an indicator of TMJ nociception in unrestrained and untrained male and female Sprague–Dawley rats for up to two days. In this study, we first found that injection of complete Freund's adjuvant (CFA) into the TMJ of rats significantly lengthened meal duration for 19 days and also decreased meal frequency for 42 days. Interestingly, the meal duration varied significantly from day to day within the 19 day period. TMJ interleukin-1β (IL-1β) and calcitonin gene-related peptide (CGRP) were significantly elevated in the TMJ tissues of CFA-injected animals and the level of these markers was attenuated as the meal duration decreased with time. Control animals injected with saline into the TMJ or CFA into the knee did not show a significant lengthening in meal duration but did show a decrease in meal frequency. In a second study, DBA/1LacJ mice given TMJ CFA injections showed a significantly lengthened meal duration on four of the seven days measured using end-of-the meal definition of 5 or 10 min. No other meal pattern changed significantly. Two days post-CFA injection, the DBA/1LacJ mice showed significantly elevated interleukin-6 (IL-6), but not elevated IL-1β. Seven days post-injection, both IL-6 and IL-1β were significantly elevated. No change in CGRP was detected. In this study C57Bl/6 mice also received TMJ CFA injections, but they did not show a lengthening in any meal pattern or significant increases in IL-1β, IL-6 or CGRP. Our data show, for the first time, that meal duration can be used to measure CFA-induced nociception in the TMJ over the course of several weeks in unrestrained rats and for up to seven days in the DBA/1LacJ mouse strain. In addition, C57Bl/6 mice are resistant to CFA-induced TMJ nociception at the same dose used in the DBA/1LacJ mice.

153 CAN PATIENTS WITH CENTRAL POST-STROKE PAIN BE IDENTIFIED USING A QUESTIONNAIRE?

Methods: The monoarthritis (MA) model induced by injection of CFA (Complete Freud’s Adjuvant) in the tibiotarsal joint was used. Immunohistochemistry against ATF-3 at timepoints 2, 4, 7 and 14 days post-CFA injection was performed in ipsiand contralateral L3, L4 and L5 Dorsal Root Ganglions (DRGs). Control rats were injected with saline or vehicle and sacrificed after 2 days. Results: Expression of ATF-3 was significantly increased at 2 and 4 days of MA, but it started diminishing after that period although in 7 and 14 days MA was still higher than controls. Rises were more considerable in L5. Control DRGs showed no significant ATF-3 expression. Conclusion: Data suggest ATF-3 expression, at early timepoints, is involved in the inflammatory pain process, at the periphery. Further studies are necessary to clarify the ATF-3 signaling pathways associated to this condition and the underlying mechanisms that contribute to this particular expression pattern.