BackgroundTo facilitate the development of therapies for rheumatoid arthritis (RA), the Innovative Medicines Initiative BTCure has combined the experience from several laboratories worldwide to establish a series of protocols for different animal models of arthritis that reflect the pathogenesis of RA. Here, we describe chronic pristane-induced arthritis (PIA) model in DA rats, and provide detailed instructions to set up and evaluate the model and for reporting data.MethodsWe optimized dose of pristane and immunization procedures and determined the effect of age, gender, and housing conditions. We further assessed cage-effects, reproducibility, and frequency of chronic arthritis, disease markers, and efficacy of standard and novel therapies.ResultsOut of 271 rats, 99.6% developed arthritis after pristane-administration. Mean values for day of onset, day of maximum arthritis severity and maximum clinical scores were 11.8±2.0 days, 20.3±5.1 days and 34.2±11 points on a 60-point scale, respectively. The mean frequency of chronic arthritis was 86% but approached 100% in long-term experiments over 110 days. Pristane was arthritogenic even at 5 microliters dose but needed to be administrated intradermally to induce robust disease with minimal variation. The development of arthritis was age-dependent but independent of gender and whether the rats were housed in conventional or barrier facilities. PIA correlated well with weight loss and acute phase reactants, and was ameliorated by etanercept, dexamethasone, cyclosporine A and fingolimod treatment.ConclusionsPIA has high incidence and excellent reproducibility. The chronic relapsing-remitting disease and limited systemic manifestations make it more suitable than adjuvant arthritis for long-term studies of joint-inflammation and screening and validation of new therapeutics.

ATP is an important neuronal and astroglial signaling molecule in the brain. In the present study, brain slices were prepared from the prefrontal cortex (PFC) of Wistar rats and 2 lines of mice. P2X₇ receptor immunoreactivity was colocalized with astro- and microglial but not neuronal markers. Whole-cell patch-clamp recordings showed that, in astroglial cells, dibenzoyl-ATP (BzATP) and ATP caused inward currents, near the resting membrane potential. The inactivity of α,β-methylene ATP, as well as the potency increases of BzATP and ATP in a low divalent cation (X²(+))-containing superfusion medium suggested the involvement of P2X₇ receptors. This idea was corroborated by the inhibition of these current responses by PPADS, Brilliant Blue G, A 438079, and calmidazolium. Ivermectin, trinitrophenyl-adenosine-5'-triphosphate, and cyclopentyl-dipropylxanthine did not alter the agonist effects. The reversal potential of BzATP was near 0 mV, indicating the opening of cationic receptor channels. In a low X²(+) superfusion medium, ATP-induced current responses in PFC astroglial cells of wild-type mice but not of the P2X₇ knockouts. Hence, cortical astroglia of rats and mice possess functional P2X₇ receptors. These receptors may participate in necrotic/apoptotic or proliferative reactions after stimulation by large quantities of ATP released by central nervous system injury.

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Rheumatoid arthritis (RA)—a widespread chronic inflammatory disease in industrialized countries—is characterized by a persistent and progressive joint destruction. The chronic pro-inflammatory state results from a mutual activation of the innate and the adaptive immune system, while the exact pathogenesis mechanism is still under discussion. New data suggest a role of the innate immune system and especially polymorphonuclear granulocytes (PMNs, neutrophils) not only during onset and the destructive phase of RA but also at the chronification of the disease. Thereby the enzymatic activity of myeloperoxidase (MPO), a peroxidase strongly abundant in neutrophils, may be important: While its peroxidase activity is known to contribute to cartilage destruction at later stages of RA the almost MPO-specific oxidant hypochlorous acid (HOCl) is also discussed for certain anti-inflammatory effects. In this study we used pristane-induced arthritis (PIA) in Dark Agouti rats as a model for the chronic course of RA in man. We were able to shown that a specific detection of the HOCl-producing MPO activity provides a sensitive new marker to evaluate the actual systemic inflammatory status which is only partially detectable by the evaluation of clinical symptoms (joint swelling and redness measurements). Moreover, we evaluated the long-term pharmacological effect of the well-known anti-inflammatory flavonoid epigallocatechin gallate (EGCG). Thereby only upon early and continuous oral application of this polyphenol the arthritic symptoms were considerably diminished both in the acute and in the chronic phase of the disease. The obtained results were comparable to the treatment control (application of methotrexate, MTX). As revealed by stopped-flow kinetic measurements, EGCG may regenerate the HOCl-production of MPO which is known to be impaired at chronic inflammatory diseases like RA. It can be speculated that this MPO activity-promoting effect of EGCG may contribute to the pharmacological mode of action of this polyphenol.

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