Intra-articular Injection Of Carrageenan Research Articles

A CCL2/MCP-1 antagonist attenuates fibrosis of the infrapatellar fat pad in a rat model of arthritis

BackgroundFibrosis of the infrapatellar fat pad (IFP) is a feature of osteoarthritis and contributes substantially to the pain and dysfunction in patients’ joints. However, the underlying mechanisms remain unclear. C-C motif chemokine ligand-2 (CCL2) plays a central role in tissue fibrosis. Thus, we aimed to investigate the role of CCL2 in the development of IFP fibrosis in a rat model of arthritis, hypothesizing that a CCL2 antagonist could mitigate fibrotic progression.MethodsWe induced arthritis in male Wistar rats using intra-articular injections of carrageenan. Furthermore, to evaluate the effects of a CCL2 antagonist on protein expression and collagen deposition in the IFP of the rats, we transferred an N-terminal-truncated CCL2 gene into a rat model via electroporation-mediated intramuscular injection. Macrophage infiltration and collagen deposition in the IFP were analyzed in vivo. Groups were compared using the Mann–Whitney U test and Student’s t-test.ResultsWe identified infiltrating macrophages as well as increases in CCL2 and TGF-β levels as collagen deposition progressed. Gene transfer of the CCL2-antagonist before arthritis induction attenuated collagen deposition remarkably.ConclusionsWe provide initial evidence that anti-CCL2 gene therapy can effectively suppress the development of IFP fibrosis in a rat model. Thus, targeting CCL2 holds promise as a therapeutic strategy for managing tissue fibrosis in osteoarthritis patients.

Inhibitory effect of low‑intensity pulsed ultrasound on the fibrosis of the infrapatellar fat pad through the regulation of HIF‑1α in a carrageenan‑induced knee osteoarthritis rat model.

Fibrotic changes in the infrapatellar fat pad (IFP) are involved in the pathogenesis of knee osteoarthritis (KOA). HIF-1α is a transcription factor that is activated during hypoxia and is suggested to play a role in fibrosis in various organs. However, its participation in the fibrotic changes in IFP remains unclear. Therefore, we investigated the role of HIF-1α in IFP fibrosis using a carrageenan-induced KOA rat model and evaluated the potential of low-intensity pulsed ultrasound (LIPUS) as a novel treatment for KOA. A rat model was prepared by intra-articular injection of 0.5% carrageenan (50 µl) using 8-week-old male Wistar rats. Fibrosis of the IFP was evaluated histologically by hematoxylin and eosin and Sirius Red staining at 1 and 2 weeks after intra-articular injection. The mRNA expression levels of HIF-1α and fibrosis-related molecules, CTGF and VEGF, were analyzed using reverse transcription-quantitative PCR, and the DNA binding activity of HIF-1α was assessed using a binding assay. In addition, the effect of irradiation with LIPUS on the fibrosis of IFP was verified. Histological studies demonstrated a significant increase in the fibrosis of IFP 1 and 2 weeks after intra-articular injection of carrageenan, accompanied by overexpression of CTGF and VEGF, which was followed by upregulation of transcriptional activation of HIF-1α. Moreover, intervention with LIPUS for 2 weeks after injection of carrageenan attenuated fibrosis of IFP, accompanied by a significant reduction in the transcriptional activation of HIF-1α and decreased the gene expression levels of HIF-1α, CTGF, and VEGF. The present study demonstrated that activation of HIF-1α promoted fibrosis of IFP in carrageenan-induced arthritis in rats and that intervention with LIPUS decreased the activity of HIF-1α and inhibited fibrosis. These results suggest that LIPUS may serve as a novel approach for the treatment of KOA, through its modulation of HIF-1α.

Involvement of the tuberomammillary nucleus of the hypothalamus in the modulation of nociception and joint edema in a model of monoarthritis

AimsInvestigate the involvement of the histaminergic projections from tuberomammillary nucleus (TMN) to the spinal cord in the modulation of nociception and peripheral edema in a model of monoarthritis. Main methodsSubacute monoarthritis was induced by an intraarticular injection of carrageenan followed by LPS 72 h later. Disability and joint edema were assessed at the 3rd hour after LPS and at every hour up to 6 h. Key findingsIntrathecal administration of histamine potentiated joint incapacitation and edema, while the H1R antagonist cetirizine decreased both. The H3R agonist immepip decreased both incapacitation and edema, while the H3R antagonist thioperamide had the opposite effect. The microinjection of glutamate into the ventral TMN (vTMN) caused an increase of incapacitation and articular edema, whereas the blockade of this nucleus by cobalt chloride inhibited both parameters. Intrathecal administration of cetirizine prevented the increase of incapacitation and joint edema caused by glutamate microinjection into the vTMN. Similarly, an intrathecal injection of the NKCC1 cotransporter inhibitor bumetanide prevented the effects of glutamate microinjection into the vTMN, whereas coadministration of histamine with bumetanide only inhibited the potentiation of joint edema. A microinjection of orexin B into the vTMN potentiated incapacitation and joint edema, while coadministration of the OX1/2 receptor antagonist almorexant with orexin B did not. SignificanceThese data support the notion that TMN participates in the modulation of a peripheral inflammatory process by means of histaminergic projections to the spinal cord, and the hypothalamus may trigger TMN activation by means of glutamate and orexin.

Acute Mono-Arthritis Activates the Neurohypophysial System and Hypothalamo-Pituitary Adrenal Axis in Rats.

Various types of acute/chronic nociceptive stimuli cause neuroendocrine responses such as activation of the hypothalamo-neurohypophysial [oxytocin (OXT) and arginine vasopressin (AVP)] system and hypothalamo-pituitary adrenal (HPA) axis. Chronic multiple-arthritis activates the OXT/AVP system, but the effects of acute mono-arthritis on the OXT/AVP system in the same animals has not been simultaneously evaluated. Further, AVP, not corticotropin-releasing hormone (CRH), predominantly activates the HPA axis in chronic multiple-arthritis, but the participation of AVP in HPA axis activation in acute mono-arthritis remains unknown. Therefore, we aimed to simultaneously evaluate the effects of acute mono-arthritis on the activity of the OXT/AVP system and the HPA axis. In the present study, we used an acute mono-arthritic model induced by intra-articular injection of carrageenan in a single knee joint of adult male Wistar rats. Acute mono-arthritis was confirmed by a significant increase in knee diameter in the carrageenan-injected knee and a significant decrease in the mechanical nociceptive threshold in the ipsilateral hind paw. Immunohistochemical analysis revealed that the number of Fos-immunoreactive (ir) cells in the ipsilateral lamina I–II of the dorsal horn was significantly increased, and the percentage of OXT-ir and AVP-ir neurons expressing Fos-ir in both sides of the supraoptic (SON) and paraventricular nuclei (PVN) was increased in acute mono-arthritic rats. in situ hybridization histochemistry revealed that levels of OXT mRNA and AVP hnRNA in the SON and PVN, CRH mRNA in the PVN, and proopiomelanocortin mRNA in the anterior pituitary were also significantly increased in acute mono-arthritic rats. Further, plasma OXT, AVP, and corticosterone levels were significantly increased in acute mono-arthritic rats. These results suggest that acute mono-arthritis activates ipsilateral nociceptive afferent pathways at the spinal level and causes simultaneous and integrative activation of the OXT/AVP system. In addition, the HPA axis is activated by both AVP and CRH in acute mono-arthritis with a distinct pattern compared to that in chronic multiple-arthritis.

Mouse synovial mesenchymal stem cells increase in yield with knee inflammation.

Even though mouse studies have various advantages, harvesting an adequate number of synovial mesenchymal stem cells (MSCs) is difficult in mice. We investigated whether the total yield of MSCs increased in synovium with inflammation in mice. Infrapatellar fat pads (IFPs) were harvested from 10 knees of 5 mice 3, 7, and 14 days after intraarticular injection of carrageenan. Ten IFPs were also harvested from untreated knees as a control. Seven days after initial plating, the total yield of cells was compared among the 4 groups (n = 4–6). The harvested cells were analyzed for multipotentiality and surface epitopes. Furthermore, knee synovitis was compared among the 4 groups in histology. The number of cells in the 3 and 7 days treated group was significantly higher than the other groups. The harvested cells had characteristics of MSCs. Synovitis in the 3 and 7 days treated groups was significantly severer than the other groups. There seemed to be a relationship between the synovitis score and the total yield of cells derived from IFPs. In mice, it became possible to increase the yield 50-fold by inducing inflammation. This method makes it possible to analyze the molecular mechanisms of cartilage regeneration of synovial MSCs in mice models. © 2014 The Authors. © 2014 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 33:246–253, 2015.

Follistatin alleviates synovitis and articular cartilage degeneration induced by carrageenan.

Activins are proinflammatory cytokines which belong to the TGFβ superfamily. Follistatin is an extracellular decoy receptor for activins. Since both activins and follistatin are expressed in articular cartilage, we hypothesized that activin-follistatin signaling participates in the process of joint inflammation and cartilage degeneration. To test this hypothesis, we examined the effects of follistatin in a carrageenan-induced mouse arthritis model. Synovitis induced by intra-articular injection of carrageenan was significantly alleviated by preinjection with follistatin. Macrophage infiltration into the synovial membrane was significantly reduced in the presence of follistatin. In addition, follistatin inhibited proteoglycan erosion induced by carrageenan in articular cartilage. These data indicate that activin-follistatin signaling is involved in joint inflammation and cartilage homeostasis. Our data suggest that follistatin can be a new therapeutic target for inflammation-induced articular cartilage degeneration.

P34 Mechanisms underlying the protective effect of H2S donors against carrageenan-induced knee joint synovitis and nociception

Controversy exists as to whether H 2 S has a direct protective or deleterious effect on arthritis. Although we have evidence from our previous study that exogenous H 2 S delivered to the knee joint can produce a significant anti-inflammatory and anti-nociceptive effect against carrageenan-induced joint synovitis [1] , the mechanisms involved are unknown. To investigate this, synovitis was induced in Wistar rats (previously treated with Lawesson reagent [LR], a classical H 2 S donor) by intra-articular injection of carrageenan into the knee joint in the presence or absence of treatment with glibenclamide (antagonist at the sulfonylurea receptor 1 (SUR1) regulatory sub-unit of ATP-sensitive K + (K ATP ) channels), capsazepine (transient receptor potential vanilloid 1 antagonist TRPV1), and verapamil (L-type Ca 2+ channel blocker). Both nociceptive and inflammatory parameters were assessed 4 h later. Animal procedures were approved by the local Ethics Committee and injections were performed under isoflurane anaesthesia. As expected, LR significantly attenuated the carrageenan (CGN)-induced nociceptive response of the ipsilateral hindpaw (impaired gait and secondary tactile allodynia) and related inflammatory process. Suppression of CGN-induced synovitis formation by H 2 S donor was not reversed by the blockade of K + (K ATP ), TRPV1 or L-type Ca 2+ channels. However, CGN-induced knee oedema and associated nociception was suppressed by glibenclamide, capsazepine or verapamil alone to the same extent as by LR. Compared with control group, determinants of oxidative stress (e.g. glutathione S-transferase (GST) and glutathione reductase (GR)) in the synovial membrane of rats with synovitis showed no differences among groups. These findings demonstrate that exogenous administration of H 2 S donor Lawesson reagent-induced protective effect against CGN-mediated synovitis and referred allodynia does not seem to require activation of both K ATP channels and L-type Ca 2+ or TRPV1 receptors in rats. Thus, the full details of the mechanisms of action of H 2 S donors in CGN-induced synovitis remain to be elucidated. Financial support : CAPES, CNPq, FAPESP.

Blockade of nociceptive sensory afferent activity of the rat knee joint by the bradykinin B2 receptor antagonist fasitibant

The aim of this study was to determine in intact and inflamed knee joints of the rat, the effect of the bradykinin (BK) B2 receptor antagonist fasitibant (MEN16132) on nociceptor mechanosensitivity and hyperalgesia. Joint afferent sensory fibers of the medial articular nerve of anesthetized animals were electrophysiologically recorded, measuring nerve impulse activity evoked by passive innocuous and noxious movements of the joint, in intact and kaolin and carrageenan-injected joints. Knee joints of rats were also acutely inflamed by intra-articular injection of carrageenan alone. Long term duration of fasitibant antinociceptive effects were behaviorally evaluated using the incapacitance test. BK (100μM) injected into the saphenous artery, induced excitation and sensitization of multi- and single unit recordings. Fasitibant (300μM) injected prior to BK, reduced its excitatory effects as well as the overall increase of movement-evoked activity resulting from repeated injections of BK. Fasitibant did not affect movement-evoked activity of sensory fibers of intact, non-inflamed knee joints. Intra-articular fasitibant (100μg/knee) significantly reduced the carrageenan-induced inflammatory hyperalgesia measured with the incapacitance test up to four days after treatment. This antinociceptive effect was not obtained with systemic endovenous injection of the drug. Fasitibant prevents B2 receptor-mediated activation and sensitization of peripheral joint afferents and the ensuing inflammatory hyperalgesia, and may be a useful, novel drug for arthritis pain treatment.

ETB receptor activation as a mechanism of modulation of inflammatory pain and neurogenic inflammation in the temporomandibular joint of capsaicin-treated rats

Background: Endothelin (ET), a peptide best known for its vascular effects, also evokes pain and hyperalgesia, independently of its vascular actions. Data suggest that ET can have nociceptive effects, acting directly on receptors expressed in sensory neurons. As such, the aim this study was to investigate the direct effect of ET on hyperalgesia and edema, induced by carrageenan, on the temporomandibular joint (TMJ) of capsaicin-treated rats. Methods: Capsaicin was administered by subcutaneous injection to newborn, male Wistar rats. Inflammation was induced 60 days later by a single intra-articular injection of carrageenan into the left TMJ (control group received sterile saline). Inflammatory parameters, such as plasma extravasation, leukocyte influx and mechanical allodynia (measured as the head-withdrawal force threshold) were evaluated 4 h after edematogenic stimulus. ET-1 and ET-3, and the ET-B receptor (ETBR) antagonist were administered 3 min before edematogenic stimulus. ET and transient receptor potential vanilloid (TRPV1) mRNA expression was assessed by reverse-transcription polymerase chain reaction (RT-PCR). Edema formation was evaluated by measurement of the extravascular accumulation of injected 125I-human serum albumin into the TMJ soft tissues of anesthetized rats. Results: Capsaicin neonatal treatment significantly reduced edema formation, leukocyte influx and mechanical allodynia in TMJ, when compared to the control group, while the ETBR antagonist increased plasma extravasation and hyperalgesia in the capsaicin-treated group. ET-1 treatment reduced both plasma extravasation and myeloperoxidase activity. Capsular mRNA for ET-1 was significantly augmented in the TMJ of capsaicin-treated rats, when compared to controls. Conclusions: Our results suggest, for the first time, that ET-1, via ETBR activation, reduces plasma extravasation, leukocyte influx and inflammatory pain in the temporomandibular joint of capsaicin-treated rats.

Role of transient receptor potential vanilloid 4 in rat joint inflammation

To determine whether activation of transient receptor potential vanilloid 4 (TRPV-4) induces inflammation in the rat temporomandibular joint (TMJ), and to assess the effects of TRPV-4 agonists and proinflammatory mediators, such as a protease-activated receptor 2 (PAR-2) agonist, on TRPV-4 responses. Four hours after intraarticular injection of carrageenan into the rat joints, expression of TRPV-4 and PAR-2 in trigeminal ganglion (TG) neurons and in the TMJs were evaluated by real-time reverse transcription-polymerase chain reaction and immunofluorescence, followed by confocal microscopy. The functionality of TRPV-4 and its sensitization by a PAR-2-activating peptide (PAR-2-AP) were analyzed by measuring the intracellular Ca(2+) concentration in TMJ fibroblast-like synovial cells or TG neurons. Plasma extravasation, myeloperoxidase activity, and the head-withdrawal threshold (index of mechanical allodynia) were evaluated after intraarticular injection of selective TRPV-4 agonists, either injected alone or coinjected with PAR-2-AP. In the rat TMJs, TRPV-4 and PAR-2 expression levels were up-regulated after the induction of inflammation. Two TRPV-4 agonists specifically activated calcium influx in TMJ fibroblast-like synovial cells or TG neurons. In vivo, the agonists triggered dose-dependent increases in plasma extravasation, myeloperoxidase activity, and mechanical allodynia. In synovial cells or TG neurons, pretreatment with PAR-2-AP potentiated a TRPV-4 agonist-induced increase in [Ca(2+) ](i) . In addition, TRPV-4 agonist-induced inflammation was potentiated by PAR-2-AP in vivo. In this rat model, TRPV-4 is expressed and functional in TG neurons and synovial cells, and activation of TRPV-4 in vivo causes inflammation in the TMJ. Proinflammatory mediators, such as PAR-2 agonists, sensitize the activity of TRPV-4. These results identify TRPV-4 as an important signal of inflammation in the joint.

Follistatin alleviates synovitis and articular cartilage degradation induced by carrageenan in mice

Purpose: Osteoarthritis (OA), a chronic degenerative joint disorder characterized by articular cartilage destruction and osteophyte formation, is prevalent in our society as a major cause of disability. Molecular pathogenesis of OA is not fully understood, it is considered that acute joint inflammation plays significant roles in the onset of OA. This idea is supported by various animal experiments, since articular cartilage degradation is induced by the intra-articular injection of carrageenan or zymosan. Previously we showed that intra-articular injection of recombinant human BMP7 (rhBMP7) inhibited cartilage degradation induced by zymosan partly through the inhibition of inflammatory cytokine expression such as IL1-beta in the joint. From these data we hypothesized that TGF-βeta/BMP signal maintains joint homeostasis by controlling inflammatory status. Here we report that follistatin, an endogenous inhibitor for Activin which belongs to TGF-beta/BMP family and works as a proinflammatory cytokine, effectively alleviates synovitis and articular cartilage degradation induced by the intra-articular injection of carrageenan in mice. Methods: This study was approved and conducted in accordance with the guideline of the animal committee of Tokyo Medical and Dental University. Male C57Bl/6J mice (12weeks old) were purchased from ORIENTAL YEAST co.,Ltd (Tokyo, Japan). They were housed under a 12-h light-dark cycle and allowed food and water ad libitum. Twelve mice were randomly divided into two groups (n=6/group). Mice were anesthetized by the inhalation of 5% isoflurane in oxygen. Under deep anesthesia, a solution of 30μg lamda-carrageenan (Sigma-Aldrich) in 5μL saline was injected into the left knee joint through the lateral margin of the patella tendon. Recombinant mouse follistatin (25ng in 5μl in physiological saline, Sigma-Aldrich) was injected into the left knee at 30 minutes before carrageenan challenge. Mice were maintained in cage ad libitum for 3 days after the challenge. Knee joints were dissected, fixed in 4% paraformaldehyde, decalcified, embedded, and 5μm sagittal sections were prepared for histology. Integrity of articular cartilage and synovium was assessed by Hematoxylin and Eosin staining. To assess articular cartilage damage, three sections (apart from 150μm respectively) were stained by Safranin O and 400 μm in width of articular cartilage between anterior and posterior edge of medial meniscus was contoured into 3 areas according to the dyeability: Grade I; intact cartilage, Grade II; mildly denatured cartilage with reduced safranin O staining, and Grade III; severely denatured cartilage with no Safranin O staining. Each area was measured using Zeiss Axio Vision Image Analysis system. Kruskal-Wallis test followed by Tukey-Kramer methods was used for statistical analysis. Results: Image analyses indicated that dyeability of articular cartilage by Safranin O was significantly reduced by the single intra-articular injection of carrageenan at 3 days (Grade I: 21.4%, Grade II: 33.8%, Grade III: 44.8%), although we did not observe any obvious alteration in the articular surface structure at this stage. In contrast, the loss of dyeability after carrageenan injection was significantly improved by the pre-injection of follistatin (Grade I: 26.4%, Grade II: 73.6%, Grade III: 0%, p<0.05). Hematoxylin and Eosin staining showed that the cellularity of synovial tissue is greatly increased in carrageenan-injected mice. In contrast, these inflammatory responses were greatly alleviated by the pre-injection of follistatin. Conclusions: Carrageenan-induced arthritis is a well-established experimental model to investigate inflammation-mediated articular cartilage degradation in rodents. Here we report that follistatin effectively inhibits the loss of Safranin O dyeability of articular cartilage induced by carrageenan. Our data strongly suggest that the Activin signal pathway is involved in the process of joint inflammation and proteoglycan loss in the cartilage matrix. We believe that our experimental system will be of great use for analyzing the molecular events undergoing the onset of OA.

The assessment of bee venom responses in an experimental model of mono-arthritis using Tc-99m DPD bone scintigraphy

Several recent studies have shown that bee venom (BV) has an anti-nociceptive and anti-inflammatory effect on arthritis. However, objective methods for evaluation of the therapeutic effect of BV is insufficient in animal studies and clinical trials. Our purpose was to determine the usefulness of bone scintigraphy using Tc-99m DPD (3,3-diphosphono-1,2-propan-dicarbonacid) about effects of BV applied to carrageenan-induced mono-arthritis (CIA) model. Mono-arthritis was induced by an intra-articular injection of carrageenan in Sprague-Dawley rats. Administration of BV (0.8 mg/kg) was performed at 30 min before and at 4 h after the induction of mono-arthritis. We assigned rats to BV-before, BV-after, control-before and control-after groups and compared the results of each group by the weight-loading test and bone scintigraphy. The rats received an intravenous injection of 37 MBq of Tc-99m DPD by the tail vein and then scanning was performed at 4 and 24 h after the injection. Visual assessment and quantitative analysis were performed for both knees. The BV-before and BV-after groups were more improved than the control groups on the weight load test (p < 0.05). Bone scintigraphy showed lower activity in the BV-before group than in the control-before group (p < 0.05) on the 4 h imaging. However, a significant difference in the BV-before and BV-after groups was not observed on the 24 h imaging. BV had therapeutic effects by anti-nociceptive and anti-inflammatory activity in the CIA and bone scintigraphy performed on 4 h imaging provided visual and quantitative information for the assessment of the therapeutic response to BV as an objective method in mono-arthritis model.

Differing effects of exogenous and endogenous hydrogen sulphide in carrageenan-induced knee joint synovitis in the rat.

Recent findings suggest that the noxious gas H(2)S is produced endogenously, and that physiological concentrations of H(2)S are able to modulate pain and inflammation in rodents. This study was undertaken to evaluate the ability of endogenous and exogenous H(2)S to modulate carrageenan-induced synovitis in the rat knee. Synovitis was induced in Wistar rats by intra-articular injection of carrageenan into the knee joint. Sixty minutes prior to carrageenan injection, the rats were pretreated with indomethacin, an inhibitor of H(2)S formation (DL-propargylglycine) or an H(2)S donor [Lawesson's reagent (LR)]. Injection of carrageenan evoked knee inflammation, pain as characterized by impaired gait, secondary tactile allodynia of the ipsilateral hindpaw, joint swelling, histological changes, inflammatory cell infiltration, increased synovial myeloperoxidase, protein nitrotyrosine residues, inducible NOS (iNOS) activity and NO production. Pretreatment with LR or indomethacin significantly attenuated the pain responses, and all the inflammatory and biochemical changes, except for the increased iNOS activity, NO production and 3-NT. Propargylglycine pretreatment potentiated synovial iNOS activity (and NO production), and enhanced macrophage infiltration, but had no effect on other inflammatory parameters. Whereas exogenous H(2)S delivered to the knee joint can produce a significant anti-inflammatory and anti-nociceptive effect, locally produced H(2)S exerts little immunomodulatory effect. These data further support the development and use of H(2)S donors as potential alternatives (or complementary therapies) to the available anti-inflammatory compounds used for treatment of joint inflammation or relief of its symptoms.

Pain Behavior Measures to Quantitate Joint Pain and Response to Neurotoxin Treatment in Murine Models of Arthritis

To evaluate the validity of newly developed pain behavior measures in two murine models of inflammatory arthritis and to determine the ability of these measures to evaluate the analgesic effectiveness of intra-articular (IA) botulinum toxin type A (BoNT/A) for treatment of arthritis pain. Acute inflammatory arthritis was produced in adult female mice by IA injection of carrageenan and chronic inflammatory arthritis by IA injection of CFA. The presence of arthritis was confirmed by the presence of swelling and erythema. A menu of pain behavior measures was devised for quantitating pain in these models including tenderness, and spontaneous nocturnal wheel running. Toxicity due to neurotoxin was measured as gross limb weakness and impaired functional ability during wheel running. Tenderness measures and spontaneous nocturnal wheel-running are valid measures of arthritis pain and are sensitive to the effects of analgesia. Narcotic analgesics are effective, but in fully analgesic doses impair wheel-running. IA BoNT/A is an effective analgesic for chronic arthritis pain, but not for acute arthritis pain. High doses can produce local limb muscle weakness, which impairs wheel-running function. Doses of botulinum toxin that are not toxic retain their analgesic function. Tenderness and spontaneous pain behavior measures are valid and sensitive for the measurement of pain and analgesia in murine models of inflammatory arthritis. Effective narcotic analgesia produces a decline in function in mice similar to that seen in humans. IA neurotoxin is a promising therapy for chronic inflammatory arthritis but may not be effective for acute arthritis pain.

Pathophysiological basis of acute inflammatory hyperaemia in the rat knee: roles of cyclo-oxygenase-1 and -2.

The role of different isoforms of cyclo-oxygenase (COX) in mediating the acute (0-6 h) and late (24 h) phases of inflammation was investigated in the rat knee joint following intra-articular injection of carrageenan. The hyperaemic response was assessed transcutaneously using laser Doppler imaging (LDI). Samples were taken at corresponding time points for detection of synovial COX-1, COX-2 and inducible nitric oxide synthase (iNOS) mRNA, and measurement of urinary prostaglandin (PG) and nitric oxide metabolites (NO(x)). A non-selective COX inhibitor (indomethacin, 15 mg kg(-1) I.P.), a selective COX-2 inhibitor (SC-236, 16.8 mg kg(-1) I.P.) or vehicle were administered 1 h prior to carrageenan in the acute phase study. LDI scans were taken hourly for 4 h post-induction. Inflammatory hyperaemia in the vehicle group was attenuated in the indomethacin- (P < 0.001, two-way ANOVA) and SC-236-treated groups (P < 0.0001), with no difference between these treatments. At 24 h, I.V. infusion of indomethacin (0.1 mg min(-1)), increased vascular resistance (24 +/- 7.1 %; P < 0.05) compared to vehicle infusion, whereas SC-236 (0.11 mg min(-1)) did not. Resistance changes to indomethacin also differed from SC-236 (P < 0.05). Knee joint diameter progressively increased over 24 h (P < 0.0001, one-way ANOVA). Urinary PG levels increased by 6 h (P < 0.05), but returned to baseline by 24 h. COX-1 mRNA was detectable at all time points; COX-2 mRNA only at 3 h. Urinary NO(x) levels increased progressively over 24 h (P < 0.05), paralleled by induction of iNOS in the 3 and 24 h samples. Prostaglandin production via COX-2 appears to mediate the development of acute inflammatory hyperaemia, but nitrergic mechanisms may supervene subsequently. COX-1 but not COX-2 contributes to the maintenance of basal blood flow in the hyperaemic joint at 24 h.

Endogenous glucocorticoids modulate neutrophil migration and synovial P-selectin but not neutrophil phagocytic or oxidative function in experimental arthritis.

Pharmacologic glucocorticoids are powerful inhibitors of the inflammatory response at many levels, including leucocyte trafficking and function. The adhesion molecule P-selectin is a key participant in polymorphonuclear neutrophil (PMN) migration to sites of inflammation. The extent to which endogenous glucocorticoids influence PMN migration and activation is not clear. We used the glucocorticoid receptor antagonist RU486 to examine the effect of endogenous glucocorticoid blockade on PMN migration and function in carrageenan monoarthritis in the rat. Arthritis was induced by intraarticular injection of carrageenan and disease severity measured by PMN count in synovial lavage fluid. Decalcified frozen sections of injected joints were analysed for expression of P-selectin by immunohistochemistry. Adrenal glucocorticoid action was blocked in vivo with RU486 20 mg/kg. PMN phagocytosis and reactive oxygen species synthesis were measured by flow cytometry. Carrageenan injection was associated with severe arthritis (synovial lavage PMN 5.9 +/- 0.7 x 10(6), P < 0.01 versus control) which was dose-dependent. P-selectin was not detected in normal joints but was abundant in joints injected with 500 microg carrageenan. RU486 resulted in exacerbation of carrageenan arthritis (9.7 +/- 0.8 x 10(6), P < 0.05). RU486 also altered the threshold for disease induction, in that most RU486-treated animals were susceptible to arthritis at a dose of carrageenan (2.5 microg) which did not induce arthritis in most control-treated animals (P < 0.05), denoting an altered threshold for arthritis induction. RU486 treatment was associated with increased synovial P-selectin expression. Activation status as measured by PMN phagocytic and oxidative function were not influenced by endogenous glucocorticoid blockade. These findings suggest that endogenous glucocorticoids selectively influence PMN migration to inflamed joints via P-selectin expression, but have no effect on PMN activation status.

MR imaging of the arthritic rabbit knee joint using albumin-(Gd-DTPA) 30 with correlation to histopathology

The purpose of this study was to demonstrate a technique, in a pilot study, for measuring abnormal capillary permeability in synovial tissue of rabbit arthritic knees using dynamic MRI with a gadolinium-based blood pool agent. Arthritis, simulating rheumatoid arthritis, was induced in knees of 8 rabbits by intra-articular injection of carrageenan ( n = 4) or ovalbumin ( n = 4). Sequential fat presaturated T 1 -weighted Spoiled Grass images were obtained before and up to 30 min after intravenous administration of albumin-(Gd-DTPA) 30. Estimates of synovial tissue plasma-volume (PV), fractional-leak-rate (FLR), and permeability-surface-area-product (PS) were computed. Histologic correlation was obtained in the corresponding regions. Dynamic MRI showed extravasation of albumin-(Gd-DTPA) 30 into hypertrophic synovium in six of the eight arthritic knees. Histologic examination of these six knees showed markedly inflamed synovium. The two knees that did not show abnormal vascular permeability contained non-hypertrophic synovium. None of the rabbits showed abnormal permeability in muscle. MRI derived microvascular characteristics (PV, FLR and PS) correlated positively ( r 2 = 0.51, 0.97 and 0.86) with the histology. Factors involving the structural and functional microvascular characteristics of synovial tissue can be estimated non-invasively using albumin-(Gd-DTPA) 30. This technique may be useful for monitoring disease progression and treatment response in rheumatoid arthritis.

Morphological and mechanical study on the effects of experimentally induced inflammatory knee arthritis in rabbit long bones.

Inflammatory knee arthritis was induced by intraarticular injection of carrageenan twice a week for a total of 6 weeks in New Zealand White rabbits and the effects of the arthritis on the morphological and mechanical properties of the adjacent femur and tibia were evaluated 8 weeks after the first injection. Carrageenan-induced knee arthritis resulted in severe osteopenic changes and a dramatic decrease in bone strength of the entire ipsilateral femur and tibia, including the femoral head and distal tibia, but not the contralateral femur and tibia and the remote humerus. The osteoporotic changes of the adjacent bones of the inflammatory arthritic knee are the basis for the reduced mechanical strength of these bones. These findings may have clinical significance with regard to the mechanisms and consequences of osteoporotic changes in patients with rheumatoid arthritis.

Antiinflammatory effect of lipocortin 1 in experimental arthritis.

The glucocorticoid-induced antiinflammatory protein lipocortin 1 is present in arthritic synovium but its ability to regulate joint inflammation has not previously been studied. We investigated the role of lipocortin 1 in the antiinflammatory activity of glucocorticoids in an acute arthritis model induced by intraarticular injection of carrageenan. Compared to control joints (0.09 +/- 0.08 x 10(6) synovial fluid cell count), carrageenan injected joints exhibited marked infiltration of PMN (10.2 +/- 0.7 x 10(6), p < 0.001). Both intraperitoneal (1.0 mg/kg) and intraarticular administration (5 micrograms) of dexamethasone (DEX) significantly suppressed arthritis severity (p < 0.001 and 0.005, respectively), and the effects of DEX were significantly prevented by intra-articular injection of antilipocortin 1 mAb (p < 0.05). Carrageenan arthritis was also significantly inhibited by intraarticular administration of the N-terminal lipocortin 1 peptide Ac2-26 at doses of 1 or 2 mg/kg (p < 0.01). Intraarticular injection antilipocortin 1 mAb in the absence of DEX also significantly exacerbated arthritis severity (p < 0.005). In vitro treatment of PMN with DEX was associated with significant inhibition of phagocytosis (p < 0.005) and reactive oxygen species (ROS) generation (p < 0.001). Antilipocortin 1 mAb significantly reduced the inhibitory effects of DEX (p < 0.01 and 0.005, respectively). These results demonstrate that lipocortin 1 mediates the effects of exogenous glucocorticoids on neutrophil migration in carrageenan-induced acute arthritis, exerts an endogenous antiinflammatory influence, and mediates glucocorticoid inhibition of neutrophil activation.

Bisphosphonate (pamidronate/APD) prevents arthritis‐induced loss of fracture toughness in the rabbit femoral diaphysis

Patients with rheumatoid and other inflammatory arthritis have an increased risk for fracture. This study was designed to determine the effect of experimental inflammatory arthritis on the material properties (fracture toughness and shear modulus) and structural properties (torque, angular deflection, and absorbed energy) of femoral diaphyseal bone tested in torsion to fracture, as well as the effect on these properties of APD (3-amino-1-hydroxypropylidene-1,1-bisphosphonate), a drug known to block osteoclast activity. Two dose levels were investigated. Experimental inflammatory arthritis was induced by intra-articular injection of carrageenan into the right tibiofemoral joint, given over 7 weeks, in three groups of animals. Simultaneously, daily subcutaneous injections of APD were given to three groups of rabbits. Five groups (12 animals each) were established: normal, arthritis, normal/high dose APD, arthritis/high dose APD, and arthritis/low dose APD. The diaphyses of each excised right femur were loaded to fracture in torsion at an angular deflection rate of 8 degrees/sec. In the arthritis group, the fracture toughness was 39% lower than in the normal group, and the structural properties all were reduced significantly. By contrast, the shear modulus was unaffected by arthritis. In this study, the higher dose level (0.3 mg/kg of body weight) of APD prevented loss of fracture toughness and maintained the structural properties in experimental inflammatory arthritis; the low dose was not effective.