EAE Induction by Active Immunization in SJL Mice

Recommended protocol for use with Hooke Kit™ PLP_139-151/CFA Emulsion (EK-0120) or PLP_139-151/CFA Emulsion PTX (EK-0121).

Hooke Kit™ choice and Mouse Substrains

Disease susceptibility varies according to mouse substrain.

Summary

SJL mice develop remitting-relapsing paralysis after immunization with PLP_139-151 peptide emulsified in Complete Freund’s Adjuvant. Mice develop EAE 11-14 days after immunization, but completely or partially recover within 7-10 days. Around 25-30 days after the immunization, most mice (60-100%) develop a second wave of paralysis (relapse).

Materials needed

Qty	Description
1	Hooke Kit™ PLP_139-151/CFA Emulsion (EK-0120) or Hooke Kit™ PLP_139-151/CFA Emulsion PTX (EK-0121)
10	SJL mice, females (Taconic Farms model SJL-F or Jackson Laboratory strain SJL/J)

Method

Mice should be 8-10 weeks old, acclimated at your facility for at least 4 days before immunization.

Inject the mice with the emulsion, subcutaneously, at two sites on the back, injecting 0.1 ml at each site, total of 0.2 ml of emulsion per mouse. Keep the needle inserted into the subcutaneous space for 10 to 15 seconds after each injection to avoid leakage of the emulsion. Alternatively, a light pull on the syringe plunger at the end of each injection will prevent leakage.
Only if using Hooke Kit™ PLP_139-151/CFA Emulsion PTX (EK-0121), inject the pertussis toxin (syringe with clear filling) intraperitoneally within 2 hours of injection of the emulsion. Inject 0.1 ml per mouse.
Check mice for signs of EAE daily (see EAE scoring), starting on day 7 after the immunization.
As soon as the first signs of paralysis occur, provide mice with food pellets and wet food on the floor of the cage, and easily accessible water. Transgel (Charles River Laboratories) may be used as a source of water and nutrients during the most severe paralysis.

EAE Scoring

Typically, EAE is scored on scale 0 to 5:

Score	Clinical Observations
0	No obvious changes in motor functions of the mouse in comparison to non-immunized mice. When picked up by the tail, the tail has tension and is erect. Hind legs are usually spread apart. When the mouse is walking, there is no gait or head tilting.
1	Limp tail. When the mouse is picked up by the tail, instead of being erect, the whole tail drapes over your finger.
2	Limp tail and weakness of hind legs. When mouse is picked up by tail, legs are not spread apart, but held closer together. When the mouse is observed when walking, it has a clearly apparent wobbly walk.
3	Limp tail and complete paralysis of hind legs (most common). OR Limp tail with paralysis of one front and one hind leg. OR ALL of: Severe head tilting, Walking only along the edges of the cage, Pushing against the cage wall, Spinning when picked up by the tail.
4	Limp tail, complete hind leg and partial front leg paralysis. Mouse is minimally moving around the cage but appears alert and feeding. Usually, euthanasia is recommended after the mouse scores level 4 for 2 days. When the mouse is euthanized because of severe paralysis, score of 5 is entered for that mouse for the rest of the experiment.
5	Complete hind and complete front leg paralysis, no movement around the cage. OR Mouse is spontaneously rolling in the cage. OR Mouse is found dead due to paralysis. If mouse is alive, euthanize the mouse immediately if it scores 5. Once mouse is scored 5, the same score is entered for all the days for the rest of the experiment.

Most researchers also give mice “in-between” scores (i.e. 0.5, 1.5, 2.5, 3.5) when the clinical picture lies between two defined scores.

Expected Results

EAE will be consistently induced in 90-100% of the mice, with onset of paralysis between 9 and 14 days after immunization, and mean maximum score of the first paralytic episode of 2 to 3. Incidence of relapses will be 60% to 100%, with mean maximum score of the second paralytic episode of 1.5 to 2.5.

Daily dosing of mice with a potential therapeutic often delays the onset of EAE and decreases severity of EAE due to stress of the procedures. Careful handling of the mice will minimize these effects. To reduce effects of stress, we recommend sham dosing, 7 and 4 days before of immunization of the mice.