serious sex restrictions on susceptibility. Young rats (1 to 7 days old) are not susceptible, and old animals (>9 months) are relatively resistant. Wide variations in the frequency and severity of lesions are observed in different rat strains (see Table 15.4.1; Glenn and Gray, 1965; Swingle et al., 1969; Rosenthale, 1970; Battisto et al., 1982; Griffiths, 1988; Bersani-Amado et al., 1990).
AA is a rather aggressive, monophasic, and self-remitting form of arthritis. This means that the time period that can be used for measuring effects of drugs or other agents to modulate disease is limited. Therefore, in some cases it may be desirable to start the modulation before the disease becomes clinically apparent, e.g, between days 7 and 10 after immunization.
Monitoring of disease development is done most easily by macroscopic examination, and this method appears to be more sensitive than mechanical measurement of joint thickness. An arthritic index can be used to score the degree of periarticular erythema and edema as well as deformity of the joints (see Basic Protocol, step 8, and Trentham et al., 1977). Using this index, the severity of involvement of each paw is graded from 0 to 4, with a maximum possible score of 16 per rat. Other scoring methods in which each joint can be evaluated separately for swelling and erythema have been described (Taurog et al., 1985). The knee is excluded because neither swelling nor erythema of the knee could be reliably quantitated by physical examination. Nodules on ears or tails can be scored as well.
Additional monitoring of disease development can be performed by weight examination (see Fig. 15.4.2), histological examination of joint tissues, and radiographical examination or magnetic resonance imaging (MRI) of joints (Terrier et al., 1985). Mercury or water plethys-mography can be used to measure increase in hind paw volume (i.e., the volume of mercury or water displaced by the paw and the leg; Harris and Spencer, 1962; Parrott and Lewis, 1977). Swelling of the paw, for instance the ankle from the medial to the lateral malleolus, can also be quantitated by measuring thickness with a thickness-gauge caliper (Trentham et al., 1977; Larsson et al., 1985). Macroscopic scoring or histology is recommended for monitoring active inflammatory arthritis. Plethysmog-raphy or mechanical thickness measurements may be useful in the initial phases of arthritis development, where tissue swelling (edema of skin and soft tissues) goes together with the
Figure 15.4.2 Mean body weights of rats during the active phase of adjuvant arthritis (AA). Body weights of five 8-week-old Lewis rats immunized at day 0 for induction of adjuvant arthritis with 6 mg/ml Mycobacterium tuberculosis H37Ra in mineral oil (arthritis rats) are shown. Clinical arthritis was seen to develop from day 13 onwards. Mean body weights of nonimmunized animals of the same age (normal rats) are shown as a control. An immediate decline in weight development is seen at the time of arthritis development. At the time of AA remission (day 30) weight is increasing again.
inflammatory response. Later in the course of disease, increased joint thickness is caused by bony malformations, which no longer reflect the process of active inflammation.
The success rate of AA induction is largely dependent on the technical aspects of the immunization procedure. Optimal results are obtained when a local circumscribed elevation of the skin remains immediately after immunization. The development of skin ulceration at the site of immunization prior to disease development is a good predictor of success. In the absence of such ulceration, it is likely that the inoculation was not optimal.
Wide variation in success rates may occur depending on the batch of mycobacteria used, the source (supplier) of the animals, and housing and food conditions (Yiagou and Hadji-petrou-Kourounakis, 1983). As commercial CFA preparations may not have the optimal mycobacterial particle size, these authors advise grinding the dried bacteria with a mortar and pestle (Best et al., 1984). As an alternative for Mycobacterium tuberculosis H37Ra, M. butyricum or a mixture of three strains of M. tuberculosis (C, DT, and PN) may be used. Besides whole mycobacteria, cell walls of M. bovis BCG (Whitehouse et al., 1974) or Wax D
obtained from cell walls of M. tuberculosis H37Rv, H37Ra, C, DT, or PN may be used (Koga and Pearson, 1973; Kayashima et al., 1976; Koga et al., 1976). Mineral oil can be replaced by squalene, pristane, hexadecane, and 1-octadecane (Whitehouse et al., 1974).
To obtain good results, it may be advisable to start off by testing small groups of rats from different suppliers. The AA model is particularly sensitive to changes in exogenous factors, especially prior infection. In this regard, some rat strains have been shown to develop AA resistance under the influence of exposure to normal environmental bacterial flora (Kohashi et al., 1979).
The concentration of mycobacteria in the suspension can be reduced to 5 mg/ml or even lower; as a consequence, the intensity or incidence of disease may decrease. This may be of interest when novel antiinflammatory approaches are being tested, because it appears to make the model more sensitive to such manipulations.
In studies measuring resistance parameters, for instance following immunological manipulation, it is essential to expose control animals to the same experimental manipulations (e.g., sham inoculations or administration of the same oily adjuvants) as the experimental group. When such protocols include determinations of
Animal Models for Autoimmune and
Adjuvant Arthritis in the Rat
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