Non-arthritic Rats Research Articles

Withaferin A alleviates inflammation in animal models of arthritis by inhibiting the NF-κB pathway and cytokine release

Withaferin A, a steroid lactone from Withania somnifera, exhibits anti-inflammatory, immunomodulatory, and antioxidant properties. This study investigated the effects of withaferin A on collagen-induced arthritis (CIA) rats, focusing on NF-κB p65 regulation and cytokine release. Withaferin A (50 mg/kg b.wt., orally) or methotrexate (0.25 mg/kg b.wt., i.p., as a reference drug) was given to CIA rats daily for 20 days postarthritis induction. Joints were removed from nonarthritic and arthritic rats to assess the levels of NO, MPO, interleukin (IL)-1β, IL-6, IL-10, TNF-α, COX-2, and NF-κB via ELISA. Furthermore, the mRNA expression of IL-1β, IL-10, TNF-α, COX-2, iNOS, and NF-κB was also assessed through qPCR. Treatment with withaferin A significantly inhibited the levels of inflammatory cytokines and the transcription factor NF-κB; suppressed the expression of IL-1β, IL-10, TNF-α, COX-2, iNOS, and NF-κB in the joint tissue of CIA rats; and reduced cartilage and bone destruction, as shown by H&E staining. To confirm the results obtained from biochemical and molecular studies and to determine the molecular target of withaferin A, we performed a molecular simulation of the potential targets of withaferin A, which identified the NF-κB pathway as its target. These results suggested that withaferin A effectively attenuated rheumatoid arthritis progression by inhibiting the activation of the NF-κB pathway and the downstream secretion of inflammatory cytokines.

Silibinin's Effects against Methotrexate-Induced Hepatotoxicity in Adjuvant-Induced Arthritis Rat Model.

Methotrexate (MTX) is the first drug of choice to treat several diseases, including rheumatoid arthritis. However, its administration is accompanied by severe side effects, most commonly hepatotoxicity. Hence, alternative therapies with a lower toxicity and fewer side effects are needed. This study aimed to investigate the antioxidant and hepatoprotective effects of silibinin (SIL, natural agent) against MTX-induced hepatotoxicity in an adjuvant-induced arthritis (AIA) rat model. Arthritic rats were treated with SIL (100 mg/kg) and/or methotrexate (2 mg/kg). Non-arthritic rats, arthritic untreated rats, and arthritic rats who received the vehicle were followed in parallel. SIL alleviated the systemic consequences of arthritis by restoring lost weight, decreasing the erythrocyte sedimentation rate, and ameliorating joint damage, which was evident both micro- and macroscopically. Additionally, SIL prevented the histopathological alterations in the liver and significantly reduced the liver damage caused by MTX and AIA, as shown by a decrease in the markers of liver damage (ALT and AST). Furthermore, SIL relieved the oxidative stress induced by AIA and MTX in liver tissue by decreasing the lipid peroxidation (MDA) levels and enhancing the antioxidant defense system (GSH levels; catalase and superoxide dismutase (SOD) activities). In conclusion, our results suggest that SIL is a potent antioxidant and hepatoprotective agent in arthritic rats. It markedly attenuated the progression and severity of the arthritic disease and eased the oxidative stress in liver tissue by improving the pro-oxidant/antioxidant balance.

Effects of tofacitinib in early arthritis-induced bone loss in an adjuvant-induced arthritis rat model.

Objectives The main goal of this work was to analyse how treatment intervention with tofacitinib prevents the early disturbances of bone structure and mechanics in the rat model of adjuvant-induced arthritis. This is the first study to access the impact of tofacitinib on the skeletal bone effects of inflammation. Methods Fifty Wistar rats with adjuvant-induced arthritis were randomly housed in experimental groups, as follows: non-arthritic healthy group (n = 20); arthritic non-treated group (n = 20); and 10 animals undergoing tofacitinib treatment. Rats were monitored during 22 days after disease induction for the inflammatory score, ankle perimeter and body weight. Healthy non-arthritic rats were used as controls for comparison. After 22 days of disease progression, rats were killed and bone samples collected for histology, micro-CT, three-point bending and nanoindentation analysis. Blood samples were also collected for quantification of bone turnover markers and systemic cytokines. Results At the tissue level, measured by nanoindentation, tofacitinib increased bone cortical and trabecular hardness. However, micro-CT and three-point bending tests revealed that tofacitinib did not reverse the effects of arthritis on the cortical and trabecular bone structure and on mechanical properties. Conclusion Possible reasons for these observations might be related to the mechanism of action of tofacitinib, which leads to direct interactions with bone metabolism, and/or to the kinetics of its bone effects, which might need longer exposure.

The Effects of a 5-Chromene-yl-thiazolidin-2,4-dione Derivative in Alleviating Oxidative Stress in Adjuvant-Induced Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease that reduces life quality and requires long-life therapy. Quercetin (Que) is a natural flavonoid with antioxidant and anti-inflammatory effects. (3-(2-(4-Chlorophenyl)-2-oxoethyl)-5-((6-methyl-4-oxo-4H-chromen-3-yl)methylene) thiazolidine-2,4-dione (TZD) is a thiazolidinedione derivative synthesized in our laboratory. This study was designed to investigate the antioxidant effects of the Que and TZD derivative administration in adjuvant-induced arthritic (AIA) rats. AIA was induced in Wistar rats by the intraplantar injection of Freund�s complete adjuvant (FCA), unilaterally in the right hind paw. The control non-arthritic rats and the arthritic rats were treated with Que (30 mg/kg/day) or TZD derivative (12 mg/kg/day) for 21 days. The antioxidant effects of 5-chromen-yl- thiazolidinedione were compared to Que. The serum levels of malondialdehyde (MDA) and protein carbonyl (PC) groups, the superoxide dismutase (SOD) and catalase (CAT) activity were assessed. AIA rats showed significatly increased oxidative stress parameter levels in the blood. The results indicated that the TZD derivative decreased the blood oxidative stress parameters in the treated arthritic rats, compared to Que. The antioxidant effects of 5-chromen-yl-thiazolidinedione in AIA suggest its therapeutic properties for the clinical treatment of RA.

Effects of tofacitinib in early arthritis-induced bone loss in an adjuvant-induced arthritis rat model.

The main goal of this work was to analyse how treatment intervention with tofacitinib prevents the early disturbances of bone structure and mechanics in the rat model of adjuvant-induced arthritis. This is the first study to access the impact of tofacitinib on the skeletal bone effects of inflammation. Fifty Wistar rats with adjuvant-induced arthritis were randomly housed in experimental groups, as follows: non-arthritic healthy group (n = 20); arthritic non-treated group (n = 20); and 10 animals undergoing tofacitinib treatment. Rats were monitored during 22 days after disease induction for the inflammatory score, ankle perimeter and body weight. Healthy non-arthritic rats were used as controls for comparison. After 22 days of disease progression, rats were killed and bone samples collected for histology, micro-CT, three-point bending and nanoindentation analysis. Blood samples were also collected for quantification of bone turnover markers and systemic cytokines. At the tissue level, measured by nanoindentation, tofacitinib increased bone cortical and trabecular hardness. However, micro-CT and three-point bending tests revealed that tofacitinib did not reverse the effects of arthritis on the cortical and trabecular bone structure and on mechanical properties. Possible reasons for these observations might be related to the mechanism of action of tofacitinib, which leads to direct interactions with bone metabolism, and/or to the kinetics of its bone effects, which might need longer exposure.

CXCL12/CXCR4 Axis Regulates Aggrecanase Activation and Cartilage Degradation in a Post-Traumatic Osteoarthritis Rat Model.

We evaluated the role of the CXCL12/CXCR4 (C-X-C motif chemokine ligand 12/C-X-C chemokine receptor type 4) axis in aggrecanase-mediated cartilage degradation, and explored the underlying mechanism in a post-traumatic osteoarthritis rat model. Expression of CXCL12/CXCR4 and ADAMTS-5 was analyzed in the knees of osteoarthritic and non-arthritic rats using Western blot, ELISA, immunohistochemistry and immunofluorescence. Rodent studies were performed using Sprague-Dawley rats, with animals divided into three groups: Destabilization of the medial meniscus/AMD3100-treated (DMM/AMD3100-treated), DMM/PBS-treated, and sham controls. Rats were sacrificed after eight weeks, and samples were collected for histology and immunohistochemistry analyses. IL-1-pretreated primary chondrocytes were cultured with untreated control, CXCL12a, siNC + CXCL12a, or siRNA CXCR4 + CXCL12a, and analyzed for expression of relevant markers and cellular pathways. Higher levels of CXCL12 were detected in the knee fluid of osteoarthritic subjects, with strong staining for CXCR4 in chondrocytes and CXCL12 in synoviocytes together with enhanced expression of ADAMTS-5. DMM/AMD3100-treated rats showed a significantly reduced immunological response, with minimal evidence of pathology in both histological and immunohistochemical analyses. Treatment with CXCL12a increased the expression of ACAN, RUNX-2, and ADAMTS-4/5 in IL-1-pretreated primary chondrocytes, together with a decrease in the expression of SOX-9. Molecular analyses revealed strong induction of NF-κB activation, along with phosphorylation of MAPKs, and activation of canonical Wnt/β-catenin signaling. In conclusion, inhibition of SDF-1α/CXCR4 signaling axis was able to inhibit aggrecanase expression and lessen cartilage degeneration in post-traumatic osteoarthritis rats.

Arthritis induces early bone high turnover, structural degradation and mechanical weakness.

BackgroundWe have previously found in the chronic SKG mouse model of arthritis that long standing (5 and 8 months) inflammation directly leads to high collagen bone turnover, disorganization of the collagen network, disturbed bone microstructure and degradation of bone biomechanical properties. The main goal of the present work was to study the effects of the first days of the inflammatory process on the microarchitecture and mechanical properties of bone.MethodsTwenty eight Wistar adjuvant-induced arthritis (AIA) rats were monitored during 22 days after disease induction for the inflammatory score, ankle perimeter and body weight. Healthy non-arthritic rats were used as controls for compar-ison. After 22 days of disease progression rats were sacrificed and bone samples were collected for histomorphometrical, energy dispersive X-ray spectroscopical analysis and 3-point bending. Blood samples were also collected for bone turnover markers.ResultsAIA rats had an increased bone turnover (as inferred from increased P1NP and CTX1, p = 0.0010 and p = 0.0002, respectively) and this was paralleled by a decreased mineral content (calcium p = 0.0046 and phos-phorus p = 0.0046). Histomorphometry showed a lower trabecular thickness (p = 0.0002) and bone volume (p = 0.0003) and higher trabecular sepa-ration (p = 0.0009) in the arthritic group as compared with controls. In addition, bone mechanical tests showed evidence of fragility as depicted by diminished values of yield stress and ultimate fracture point (p = 0.0061 and p = 0.0279, re-spectively) in the arthritic group.ConclusionsWe have shown in an AIA rat model that arthritis induc-es early bone high turnover, structural degradation, mineral loss and mechanical weak-ness.

AB0160 Effects of TOFACITINIB (CP-690,550) on lipid biomarkers in rat adjuvant-induced arthritis (AIA) model and in patients with active rheumatoid arthritis

BackgroundTofacitinib (CP-690,550) is a novel, oral Janus kinase (JAK) inhibitor being investigated as a targeted immunomodulator and disease-modifying therapy in rheumatoid arthritis (RA). Administration of tofacitinib to patients (pts) with...

Letter: Ulcerogenic potential of indomethacin in arthritic and non-arthritic rats.

Letter: Ulcerogenic potential of indomethacin in arthritic and non-arthritic rats.

Differences in the injury/sprouting response of splenic noradrenergic nerves in Lewis rats with adjuvant-induced arthritis compared with rats treated with 6-hydroxydopamine

Sympathetic nerves in the spleen undergo an injury and sprouting response with development of adjuvant-induced arthritis (AA), a model of rheumatoid arthritis (RA). The objective of the present study was to determine whether this injury and sprouting response is disease-specific or occurs in a non-specific manner similar to injury and sprouting responses following sympathectomy with specific neurotoxins. Changes in noradrenergic (NA) innervation in spleens from Lewis rats 28 days following adjuvant treatment to induce arthritis and/or local 6-hydroxydopamine (6-OHDA) treatment to destroy NA nerves were examined using immunocytochemistry for tyrosine hydroxylase (TH). We observed significant increases in sympathetic innervation of hilar regions, sites of nerve entry into the spleen, and a striking decline in innervation of splenic regions distant to the hilus in arthritic compared to non-arthritic rats. While increased hilar and decreased distal NA innervation in arthritic rats was strikingly similar to that of non-arthritic 6-OHDA-treated rats, there were differences in splenic compartments innervated by sympathetic nerves between these groups. In 6-OHDA-treated rats, NA nerves re-innervated splenic compartments normally innervated by sympathetic nerves. In arthritic rats, sympathetic nerves returned to normally innervated splenic compartments, but also abundantly innervated red pulp. These findings suggest that splenic sympathetic nerves undergo a disease-associated injury/sprouting response with disease development that alters the normal pattern and distribution of NA innervation. The altered sympathetic innervation pattern is likely to change NA signaling to immune cell targets, which could exert long-term regulatory influences on initiation, maintenance, and resolution of immune responses that impact disease pathology.

The CO 2-SFE crude lipid extract and the free fatty acid extract from Perna canaliculus have anti-inflammatory effects on adjuvant-induced arthritis in rats

The anti-inflammatory (AI) activity of a supercritical fluid extract (CO 2-SFE) of tartaric acid-stabilised Perna canaliculus mussel powder, and of the free fatty acid (FFA) class separated from the CO 2-SFE extract by column chromatography, was investigated in the rat adjuvant arthritis model. Administration of the CO 2-SFE extract (100 mg/kg BW/day s.c.) for 15 days post-adjuvant inoculation significantly reduced rear paw swelling by 34% and the deterioration in total body condition by 52% in arthritic rats, compared to vehicle controls. These observations were accompanied by a decreased serum ceruloplasmin oxidase activity, and reduced inflammatory response of the spleen. The mussel FFA extract given at one third of the dose (30 mg/kg BW/day s.c.) and for a shorter treatment period (5 days during the inflammatory phase) achieved an even greater AI activity, and was equipotent to piroxicam (2 mg/kg BW/day s.c.). Preliminary toxicology assessment using both arthritic and non-arthritic (healthy) rats revealed no significant differences between the mussel treatment groups and respective vehicle controls in either organ weights, tissue histology or selected biochemical parameters. These results indicate the CO 2-SFE crude lipid extract and its FFA components from stabilised P. canaliculus mussel powder contain biologically significant AI activity in vivo, with no apparent adverse side effects.

The role of gonadal hormones on opioid receptor protein density in arthritic rats

The purpose of this study was to evaluate the effects of the gonadal hormones on the opioid receptor protein levels of Freund's adjuvant-treated (arthritic) male and female Lewis rats. Following a paw pressure nociception assay, the midbrain and spinal cord tissues were collected for comparison of mu, delta, and kappa opioid receptor protein levels. The effects of Freund's adjuvant-induced hyperalgesia resulted in significantly decreased nociception thresholds in both males and females, compared to vehicle treated animals in the paw pressure test. It was hypothesized that the presence or lack thereof of gonadal hormones would alter nociception, an effect temporally correlated with a change in opioid receptor protein expression. Nociceptive thresholds were altered by arthritis in both sexes, but not further altered by gonadal changes in males. A small, but significant increase in threshold was shown in ovariectomized females. In spite of the small gonadal-induced changes in the nociceptive threshold sensitivity to pressure, significant changes in the plasticity of the opioid system were observed. There was a significant increase in kappa opioid receptor protein levels in the spinal cord of arthritic ovariectomized females. Mu opioid receptor and kappa opioid receptor protein levels in the spinal cord tissue of non-arthritic male rats were significantly higher than in arthritic rats, a difference eliminated by gonadectomy. Gonadectomy produced similar results in the mu opioid receptor protein level in the male midbrain tissue as well. Sex differences were observed in both the mu and kappa opioid receptor protein levels. The spinal cord tissue of male rats, regardless of the presence of gonads or arthritis displayed significantly greater levels of mu opioid receptor protein levels than females. The removal of gonadal hormones appears to have opposite effects in males and females in terms of opioid receptor proteins, but not nociception as quantified by the paw pressure test. The role of changes in the plasticity of the opioid systems in response to arthritis or gonadal hormones remains to be elucidated.

The antinociceptive effect of Δ9-tetrahydrocannabinol in the arthritic rat involves the CB 2 cannabinoid receptor

Cannabinoid CB 2 receptors have been implicated in antinociception in animal models of both acute and chronic pain. We evaluated the role both cannabinoid CB 1 and CB 2 receptors in mechanonociception in non-arthritic and arthritic rats. The antinociceptive effect of Δ 9-tetrahydrocannabinol (Δ 9THC) was determined in rats following administration of the cannabinoid CB 1 receptor-selective antagonist, SR141716A, the cannabinoid CB 2 receptor-selective antagonist, SR144528, or vehicle. Male Sprague–Dawley rats were rendered arthritic using Freund’s complete adjuvant and tested for mechanical hyperalgesia in the paw-pressure test. Arthritic rats had a baseline paw-pressure of 83 ± 3.6g versus a paw-pressure of 177 ± 6.42g in non-arthritic rats. SR144528 or SR141716A (various doses mg/kg; i.p.) or 1:1:18 (ethanol:emulphor:saline) vehicle were injected 1 h prior to Δ 9THC (4mg/kg; i.p) or 1:1:18 vehicle and antinociception determined 30min post Δ 9THC. AD 50's for both antagonists were calculated with 95% confidence limits. In addition, midbrain and spinal cord were removed for determination of cannabinoid CB 1 and CB 2 receptor protein density in the rats. SR144528 significantly attenuated the antinociceptive effect of Δ 9THC in the arthritic rats [AD 50 = 3.3 (2.7–4) mg/kg], but not in the non-arthritic rats at a dose of 10/mg/kg. SR141716A significantly attenuated Δ 9THC-induced antinociception in both the non-arthritic [AD 50 = 1.4 (0.8–2) mg/kg] and arthritic rat [AD 50 = 2.6 (1.8–3.1) mg/kg]. SR141716A or SR144528 alone did not result in a hyperalgesic effect as compared to vehicle. Our results indicate that the cannabinoid CB 2 receptor plays a critical role in cannabinoid-mediated antinociception, particularly in models of chronic inflammatory pain.

Arthritis-induced increase in cholecystokinin release in the rat anterior cingulate cortex is reversed by diclofenac

Given a hypothesised role for CCK in the anterior cingulate cortex (ACC) for the sensation of pain, the aim of the present study was to investigate whether the increased CCK release could be affected by two different analgesic drugs, morphine and the non-selective cyclooxygenase inhibitor diclofenac. Since opioids stimulate CCK release in other CNS regions we have also studied the effect of morphine by itself on the CCK-LI release in the ACC of non-arthritic rats. Three to seven hours after intraarticular carrageenan injection, at the time when the animals are known to show pain-related behaviour, in vivo microdialysis in awake rats revealed increased CCK-LI release in the ACC. The CCK-LI release was significantly attenuated by diclofenac (25 mg/kg i.m.), but not by morphine (10 mg/kg s.c.). Neither diclofenac (25 mg/kg i.m.) nor morphine (5 or 10 mg/kg s.c.) affected the CCK-LI release in the ACC in non-arthritic rats. The results obtained with diclofenac indicate that prostaglandins contribute to the increased CCK-LI release in the ACC during monoarthritis. However, the lack of effect of morphine suggests that the CCK release in the ACC is not directly related to the sensation of pain. Further on, the failure of morphine to affect the extracellular level of CCK-LI in the ACC in control animals as well as in animals with carrageenan-induced monoarthritis is in contrast to previous studies on the frontal cortex or the dorsal horn of the spinal cord, in which similar doses of morphine stimulate CCK release. Thus, compared to these regions, CCK release may be differently regulated in the ACC.

Changes in the density and distribution of sympathetic nerves in spleens from Lewis rats with adjuvant‐induced arthritis suggest that an injury and sprouting response occurs

Previously we demonstrated reduced norepinephrine concentrations in spleens from Lewis rats with adjuvant-induced arthritis (AA), an animal model of rheumatoid arthritis. This study extends these findings, examining the anatomical localization and density of sympathetic nerves in the spleen with disease development. Noradrenergic (NA) innervation in spleens of Lewis rats was examined 28 days following adjuvant treatment to induce arthritis or vehicle for the adjuvant by using fluorescence histochemistry for catecholamines, with morphometric analysis and immunocytochemistry for tyrosine hydroxylase. In AA rats, sympathetic nerve density in the hilar regions, where NA nerves enter the spleen, was increased twofold over that observed in vehicle-treated rats. In contrast, there was a striking twofold decline in the density of NA nerves in splenic regions distal to the hilus in arthritic rats compared with nonarthritic rats. In both treatment groups, NA nerves distributed to central arterioles, white pulp regions, trabeculae, and capsule. However, NA nerve density was reduced in the white pulp but was increased in the red pulp in AA rats compared with non-AA rats. These findings indicate an injury/sprouting response with disease development whereby NA nerves die back in distal regions and undergo a compensatory sprouting response in the hilus. The redistribution of NA nerves from white pulp to red pulp suggests that these nerves signal activated immune cells localized in the red pulp in AA. Although the mechanisms of this redistribution of NA nerves into the red pulp are not known, it may be due to migration from white pulp to red pulp of target immune cells that provide trophic support for these nerves. The redistribution of NA nerves into the red pulp may be critical in modulating immune functions that contribute to the chronic inflammatory stages of arthritis.

Nociceptive Sensitivity and Opioid Antinociception and Antihyperalgesia in Freund's Adjuvant-Induced Arthritic Male and Female Rats

The present study was designed to examine sex differences in complete Freund's adjuvant (CFA)-induced mechanical hyperalgesia and sex differences in opioid antinociception and anti-hyperalgesia. Female rats developed inflammation and hyperalgesia faster and exhibited greater peak hyperalgesia than male rats. In arthritic (CFA-treated) rats, lower thresholds were observed during estrus and proestrus, and in nonarthritic (vehicle-treated) rats, lower thresholds were observed during proestrus. Morphine and oxycodone were more potent in male than female arthritic rats, and butorphanol was more potent and effective in male than female arthritic rats. The potency of morphine was increased in arthritic rats, although to a greater magnitude in males. The potency of oxycodone was increased in male but not female arthritic rats. The potency of butorphanol was increased in arthritic male rats and the maximal antinociceptive effect of butorphanol was increased in arthritic female rats, but it did not result in greater than 20% antinociception. Morphine, oxycodone, and butorphanol all produced antihyperalgesic effects (returning thresholds of arthritic rats to the thresholds of nonarthritic rats) with greater potency in males than females. The peripherally acting opioid agonist loperamide produced intermediate levels of antinociception in male and female arthritic rats and no antinociception in nonarthritic rats. Loperamide was more potent in male than female arthritic rats at producing antihyperalgesia. These data demonstrate sex differences in arthritis-induced hyperalgesia and responsiveness to opioid analgesics. In arthritic rats, the antinociceptive effects of opioid agonists are most probably mediated by both central and peripheral opioid receptors, whereas their antihyperalgesic effects are mediated primarily by actions at peripheral opioid receptors.

The antinociceptive effect of Delta 9-tetrahydrocannabinol in the arthritic rat

Our study addressed the hypothesis that spinal release of endogenous opioids underlies Delta 9-tetrahydrocannabinol (Δ 9-THC)-induced antinociception in Freund's adjuvant-induced arthritic and nonarthritic rats. The paw-pressure test was used to assess the antinociceptive effects of Δ 9-THC versus those of morphine, and opioid and cannabinoid receptor-selective antagonists were used to characterize the involved receptors. Cerebrospinal fluid was collected after Δ 9-THC injection (i.p.) for the measurement of endogenous opioid peptides. Our results indicate that morphine or Δ 9-THC is equally potent and efficacious in both nonarthritic and arthritic rats. Δ 9-THC-induced antinociception is attenuated by the κ opioid receptor antagonist, nor-binaltorphimine, in arthritic rats only. Δ 9-THC induces increased immunoreactive dynorphin A ( idyn A) levels in nonarthritic rats while decreasing idyn A in arthritic rats. We hypothesize that the elevated idyn A level in arthritic rats contributes to hyperalgesia by interaction with N-methyl- d-aspartate receptors, and that Δ 9-THC induces antinociception by decreasing idyn A release.

Modulation of the gait deficit in arthritic rats by infusionsof muscimol and bicuculline

Gait analysis in the adjuvant-induced arthritic rat model of chronic pain was used to examine the role of GABAA receptors in the development of pain. Drug solutions were administered continuously at 5±0.75 μl/h for 14 days via Alzet® osmotic pumps (2ML2) placed under the skin of the back. The GABAA receptor agonist, muscimol, produces a dose-dependent reversal of the gait deficits seen in arthritic rats without reducing the tibiotarsal joints inflammatory edema or the histological picture of joint erosion and inflammation. The higher infusion rate for muscimol, 20 μg/h, caused the gait for the arthritic rats to be indistinguishable from that of normal non-arthritic rats. In normal, non-arthritic rats, muscimol did not show any effect on gait. The GABAA receptor antagonist bicuculline showed small but significant exacerbation of stride length (P<0.05), single and double stance time (P<0.05) and swing time deficits (P<0.05) in the arthritic rats, but no changes in measures of gait in the normal control rat. The results suggest that the development of arthritic pain is increased in the absence of GABAA receptor tone and that increasing GABAA receptor tone can reduce arthritic pain but does not affect the disease process.

Modulation of the gait deficit in arthritic rats by infusions of muscimol and bicuculline

Gait analysis in the adjuvant-induced arthritic rat model of chronic pain was used to examine the role of GABA A receptors in the development of pain. Drug solutions were administered continuously at 5±0.75 μl/h for 14 days via Alzet ® osmotic pumps (2ML2) placed under the skin of the back. The GABA A receptor agonist, muscimol, produces a dose-dependent reversal of the gait deficits seen in arthritic rats without reducing the tibiotarsal joints inflammatory edema or the histological picture of joint erosion and inflammation. The higher infusion rate for muscimol, 20 μg/h, caused the gait for the arthritic rats to be indistinguishable from that of normal non-arthritic rats. In normal, non-arthritic rats, muscimol did not show any effect on gait. The GABA A receptor antagonist bicuculline showed small but significant exacerbation of stride length ( P<0.05), single and double stance time ( P<0.05) and swing time deficits ( P<0.05) in the arthritic rats, but no changes in measures of gait in the normal control rat. The results suggest that the development of arthritic pain is increased in the absence of GABA A receptor tone and that increasing GABA A receptor tone can reduce arthritic pain but does not affect the disease process.

Chemokine receptor expression and in vivo signaling pathways in the joints of rats with adjuvant-induced arthritis.

This study was undertaken to characterize the role of CC chemokines and their receptors in rat adjuvant-induced arthritis (AIA), a model for rheumatoid arthritis (RA). Furthermore, we investigated the signaling pathways associated with CC receptors as well as the cell type distribution of the receptors. Using TaqMan real-time reverse transcription-polymerase chain reaction, Western blot analysis, and immunohistochemistry, we defined chemokine and chemokine receptor messenger RNA (mRNA) expression, CC chemokine receptor (CCR) protein activation during the disease course, CCR-associated signaling pathways, and immunopositive CCR5, phosphorylated signal transducer and activator of transcription 1 (p-STAT-1), and p-STAT-3 cells in rat AIA versus control joints. We showed significant up-regulation of CCR1, CCR2, CCR5, and macrophage inflammatory protein 1beta/CCL4 mRNA in AIA on post-adjuvant injection day 18, coincident with peak inflammation. Additionally, increases in tyrosine phosphorylation of CCR1 (days 14, 18, 21, and 24), CCR2 (days 14 and 18), and CCR5 (days 14, 18, and 21) were detected in AIA rats compared with control (nonarthritic) rats. JAK-1, STAT-1, and STAT-3 were associated with CCR1 and were highly tyrosine phosphorylated on days 14 and 18. Moreover, CCR2 was associated with JAK-2, STAT-1, and STAT-3 on day 18. The association of STAT-1 and STAT-3 with CCR5 on days 18 and 21 correlated with JAK-1 phosphorylation and binding on day 18. However, the activation of JNK was not associated with CCR5 activation in rat AIA. Immunohistochemical analysis demonstrated that the expression of CCR5, p-STAT-1, and p-STAT-3 was detected on synovial lining cells, macrophages, and endothelial cells in arthritic rat ankles on post-adjuvant injection day 18. While the majority of the CCR5 and p-STAT-1 immunostaining was on synovial lining cells and macrophages, p-STAT-3 was predominantly expressed on endothelial cells. CCR1, CCR2, and CCR5 mRNA expression and tyrosine phosphorylation increased with peak inflammation in the AIA model. CCR1, CCR2, and CCR5 tyrosine phosphorylation are associated with the JAK/STAT-1/STAT-3 pathway at different stages of rat AIA, as well as with macrophage and endothelial cell infiltration. However, their signaling activation overlaps with peak inflammation. Up-regulation and activation of CCRs may play a role in macrophage and endothelial cell infiltration in rat AIA joints in addition to activating the associated signaling pathways. The downstream intermediate signaling proteins associated with CC receptors may be used as potential tools to control inflammation in RA.