1. Shake the suspension of heat-killed M. tuberculosis in IFA (to ensure even distribution of bacterial particles), then draw suspension into a 1-ml glass syringe attached to a 20-G needle.
Commercial sources of complete Freund's adjuvant are not suitable for inducing AA.
2. Weigh 6- to 12-week old pathogen-free Lewis rat or measure ankle and wrist joint thickness on both sides three times and use the average as a preimmunization (baseline) value.
3. Place rat in restraining device.
Alternatively, the rat may be transiently anesthestized using ether or other inhalent (unit 1.4).
4. Wet the skin in the central dorsal area at the base of the tail to increase visibility of the skin under the hair.
5. Replace the needle on the glass syringe with a 25-G needle.
6. Resuspend material in glass syringe by rolling between hands. Repeat two to five times for every rat to be immunized.
7. Inject 0.1 ml M. tuberculosis suspension intracutaneously (unit 1.6).
A local circumscribed elevation of the skin surface should remain after inoculation.
Alternatively, animals can be injected in the footpad, but then the injected limb cannot be scored.
8. Daily, on days 10 to 25, evaluate disease development by macroscopic inspection. Assess walking ability, and screen for skin redness and swelling at the site of ankle and wrist joints and small interphalangeal joints.
Intensity of arthritis can be scored by grading each paw from 0 to 4 based on erythema, swelling, and deformity of the joint: 0 = no erythema or swelling; 1 = slight erythema or swelling of one of the toes or fingers; 2 = erythema and swelling of more than one toe or finger; 3 = erythema and swelling of the ankle or wrist; 4 = complete erythema and swelling of toes or fingers and ankle or wrist, and inability to bend the ankle or wrist. All four legs are scored, so the highest possible arthritic index is 16. (For alternative scoring methods, see Commentary.)
9. Daily, on days 10 to 25, weigh each rat to follow body weight changes.
10. After day 25, evaluate and weigh each rat once every 2 to 3 days for an additional 2 weeks.
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