EAE Induction by Active Immunization in C57BL/6 Mice

Recommended protocol for use with Hooke Kit™ MOG_35-55/CFA Emulsion PTX (EK-0110, EK-0112, or EK-0113).

Summary

C57BL/6 mice develop chronic paralysis after immunization with MOG_35-55 peptide emulsified in Complete Freund’s Adjuvant (CFA). Mice develop EAE 8-14 days after immunization and stay chronically paralyzed for the duration of the experiment (typically, mice are observed for 30-40 days).

Around 25% of the mice show exacerbation of EAE symptoms after initial partial recovery. This usually occurs 20-27 days after immunization.

Materials needed

Qty	Description
1	Hooke Kit™ MOG_35-55/CFA Emulsion PTX (EK-0110, EK-0112, or EK-0113)
10	C57BL/6 mice, females (Taconic Farms model B6-F or Jackson Laboratory strain C57BL/6J)

Disease susceptibility varies according to mouse age and substrain; we recommend use of the Hooke Kit™ variant optimized to match your mice.

Even using optimized reagents, our testing shows that Taconic mice develop somewhat more consistent EAE than Jackson mice. Mice of Taconic origin will have a more uniform time of onset and maximum severity score.

With a given Hooke Kit™, older mice generally develop more severe disease and more uniform disease onset.

Method

To induce EAE in C57BL/6 mice, use Hooke Kit™ MOG_35-55/CFA Emulsion PTX (EK-0110, EK-0112, or EK-0113).

Mice should be acclimated at your facility for at least 4 days before immunization.

Inject the mice with the emulsion (syringe with white content) 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 will prevent leakage.
Within 2 hours of injection of the emulsion, inject the first dose of pertussis toxin (syringe with clear filling) intraperitoneally. Inject 0.1 ml per mouse.
22-26 hours after injection of the emulsion, inject the second dose of pertussis toxin into the mice intraperitoneally; inject 0.1 ml of pertussis toxin (syringe with clear filling) into each 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 8 and 14 days after immunization, and mean maximum score of 2-3.

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 immunization of the mice.

The following illustrates typical results:

Results graph