Ten- to 12-week-old male mice were used for all procedures, were housed in groups of 10, and were maintained at 21°C ± 2°C on a 12-hour light/dark cycle with food and water ad libitum. All experimental procedures were approved by the local ethical review process committee and the UK Home Office. DBA/1 mice were bred at the Kennedy Institute of Rheumatology (London, UK) and B6 mice were purchased from Harlan UK (Bicester, Oxfordshire, UK). To reduce the risk of fighting amongst males, mice from different cages were not mixed beyond 6 weeks of age. All mice were immunised intradermally in two sites at the base of the tail with 200 μg of bovine, chicken, or mouse type II collagen in CFA as described previously [13 (link)]. To prepare the CFA, 100 mg of desiccated killed Mycobacterium tuberculosis H37Ra (BD Biosciences, Oxford, Oxfordshire, UK) was ground with a pestle and mortar to produce a fine powder and then suspended in 30 mL of incomplete Freund's adjuvant (BD Biosciences). It was observed that fighting amongst male mice reduced the incidence of arthritis. Hence, to reduce the risk of fighting, mice from different cages were not mixed beyond 6 weeks of age. Each experiment was performed on a minimum of two occasions.
For macroscopic assessment of arthritis, the thickness of each affected hind paw was measured daily with microcalipers (Kroeplin GmbH, Schlüchtern, Germany) and the diameter was expressed as an average for inflamed hind paws per mouse. Animals were also scored for clinical signs of arthritis [13 (link)] as follows: 0 = normal, 1 = slight swelling and/or erythema, 2 = pronounced oedematous swelling, and 3 = joint rigidity. Each limb was graded thus, allowing a maximum score of 12 per mouse. After completion of the experiment, mice were sacrificed and hind paws were immersion-fixed in 10% (vol/vol) buffered formalin and decalcified with 5.5% EDTA (ethylenediaminetetraacetic acid) in buffered formalin.
For histological assessment of arthritis, arthritic mice were killed up to 2 weeks after disease onset (early arthritis, n = 8) or 6 to 8 weeks following onset (late arthritis, n = 8). Joints were decalcified and paraffin-embedded, and sections (10 μm) were stained (haematoxylin and eosin) for conventional histology. Joints were classified according to the presence or absence of inflammatory cell infiltrates (defined as focal accumulations of leukocytes). Histological analysis was performed in a blinded fashion by a trained histopathologist (AS) (N = 8 per point).
For macroscopic assessment of arthritis, the thickness of each affected hind paw was measured daily with microcalipers (Kroeplin GmbH, Schlüchtern, Germany) and the diameter was expressed as an average for inflamed hind paws per mouse. Animals were also scored for clinical signs of arthritis [13 (link)] as follows: 0 = normal, 1 = slight swelling and/or erythema, 2 = pronounced oedematous swelling, and 3 = joint rigidity. Each limb was graded thus, allowing a maximum score of 12 per mouse. After completion of the experiment, mice were sacrificed and hind paws were immersion-fixed in 10% (vol/vol) buffered formalin and decalcified with 5.5% EDTA (ethylenediaminetetraacetic acid) in buffered formalin.
For histological assessment of arthritis, arthritic mice were killed up to 2 weeks after disease onset (early arthritis, n = 8) or 6 to 8 weeks following onset (late arthritis, n = 8). Joints were decalcified and paraffin-embedded, and sections (10 μm) were stained (haematoxylin and eosin) for conventional histology. Joints were classified according to the presence or absence of inflammatory cell infiltrates (defined as focal accumulations of leukocytes). Histological analysis was performed in a blinded fashion by a trained histopathologist (AS) (N = 8 per point).
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