males to be equally susceptible to EAE. More important is the source of the rats. LEW rats obtained from different colonies can differ significantly in their susceptibility to EAE (Gould et al., 1994). In our experience, DA rats are uniformly susceptible to EAE, although they also tend to develop adjuvant arthritis in response to the mycobacterial component of the CFA. Rats with arthritis occasionally have difficulty walking, and it is important not to misinterpret this as neurological impairment due to EAE. In contrast, arthritis is not a typical feature of MBP/CFA immunization in the LEW strain.
It should be reemphasized that commercial preparations of complete Freund's adjuvant (CFA) do not contain a sufficient quantity of mycobacteria to stimulate an optimal encephalitogenic response in rats. Thus, it is necessary to supplement the adjuvant with mycobacteria. Mycobacterium butyricum is as effective as M. tuberculosis with regard to eliciting EAE in rats. It is also important to ensure that the MBP/CFA emulsion is stable before immunizing the rats. A stable emulsion should not disperse when a drop is placed on an aqueous surface, e.g., in a beaker of water.
EAE can be transferred with MBP-activated spleen cells from syngeneic MBP/CFA-immu-nized donor rats (Richert et al., 1979). Alternatively, comparable activation can be achieved by culturing the spleen cells with concanavalin A (Panitch and McFarlin, 1977). Thus, an investigator without a supply of MBP can conduct adoptive transfer experiments using mito-gen instead of antigen.
Unfractionated rat spleen and lymph node cells respond poorly in proliferation assays, but optimal responses can be obtained with enriched populations of T cells to which irradiated thymocytes have been added as a source of antigen-presenting cells (Malotky et al., 1994). This method is highly satisfactory for evaluating in vitro rat T cell responses to MBP, as illustrated in Figure 15.2.1. Lymph node T cells perform consistently better than splenic T cells in this assay, perhaps because small numbers of macrophages contaminate the spleen T cell preparations. It is widely recognized that rat splenic macrophages inhibit rat T cell prolif-erative responses (Weiss and Fitch, 1977).
A single injection of myelin basic protein (MBP) in complete Freund's adjuvant (MBP/CFA) or of spinal cord homogenate/CFA
at the recommended dosage should induce paralytic experimental autoimmune encephalomyelitis (EAE) in Lewis (LEW) or Dark Agouti (DA) rats. Clinical signs usually become apparent by day 10 to 12. EAE is graded on a scale of 1+ to 3+, based on clinical severity. 1+ EAE is manifested by loss of tail tonicity, i.e., a "limp" tail when the rat is held in one's hand. 2+ EAE is defined as hind-limb weakness or paresis, which is characterized by ataxic gait, i.e., a tendency for the hind quarters to sway when the animal walks. However, it is important to ascertain that an unsteady gait is not the result of discomfort caused by the footpad immunization. 3+ EAE is characterized by complete hind limb paralysis, in which the rat drags itself using the front feet and is unable to move the hind legs. 3+ EAE often and 2+ EAE occasionally are accompanied by urinary incontinence, which manifests itself as soiling of the fur. The disease typically progresses from mild clinical signs to severe paralysis, occasionally within a matter of hours. The rats recover spontaneously, usually by day 18 to 20 postimmunization (Willenborg, 1979; Welch et al., 1980).
The onset of adoptive EAE with 2-5 x 107 MBP-activated spleen cells from MBP/CFA-immunized donors occurs in 4 to 5 days, maximal clinical signs are usually apparent 24 to 36 hr later, and spontaneous recovery occurs by day 7 to 9. Encephalitogenic T cell lines should induce disease at 2-5 x 106 cells/rat.
Induction of active experimental autoimmune encephalomyelitis (EAE) requires ~1 hr to prepare the emulsion and inject the rat. Daily weighing and evaluation require ~ xh hr. Preparation of guinea pig myelin basic protein (MBP) requires ~4 days. Adoptive transfer of EAE requires 3 days to prepare the cells, <1 hr to inject the rats, and 4 to 5 days for disease to develop.
Establishing an MBP-specific T cell line requires 3 days to prepare activated T cells, 7 to 10 days to expand lymphoblasts, and 3 days to restimulate with antigen. Each rest/stimulation cycle requires 10 days to 2 weeks.
The T cell proliferation assay requires <1 day to set up the plates, 3 days of culture, 18 hr for labeling, and ~0.5 day to harvest cells and measure incorporation.
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Animal Models for Autoimmune and
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