Generation of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Abstract

Source: Tietz, S. M. et al. Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo. J. Vis. Exp. (2019)

This video demonstrates the method of inducing experimental autoimmune encephalomyelitis (EAE) by injecting myelin-derived peptides with an adjuvant to activate autoreactive T cells, which migrate to the brain. Pertussis toxin is administered to increase blood-brain barrier permeability, allowing these T cells and other immune cells to enter the brain. These immune cells cause demyelination, leading to impaired nerve transmission and development of EAE.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. EAE induction

1. Anesthetize C57BL/6 mice with isofluorane using a vaporizer system. To induce anesthesia in mice, expose the mouse to 4.5% isofluorane in oxygen in a small incubation chamber before transferring the mouse to a facemask with 2.1% isofluorane. Use an anesthesia unit equipped with a heating pad to prevent hypothermia of the mouse.
2. Fix the anesthetized mouse in one hand and first inject 30 µL of myelin oligodendrocyte glycoprotein (MOG)-peptide aa_35-55 (MOG_35-55)/ complete Freund's adjuvant (CFA)-emulsion subcutaneously into hind leg flanks (Figure 1: 1 + 2; in total 60 µL; left flank 30 µL and right flank 30 µL) in close proximity to the inguinal lymph nodes.
3. Place the mouse on its belly and inject 20 µL of MOGaa_35-55/CFA-emulsion into the left and right soft fatty tissue at the tail root (Figure 1: 3 + 4; in total 40 µL; left side tail root 20 µL and right side tail root 20 µL) and a little droplet of MOGaa_35-55/CFA-emulsion into the neck of the mouse (Figure 1: 5).
4. Inject 100 µL of Pertussis toxin (PTx) solution intraperitoneally into the mouse. Hold the mouse's head below the body center to avoid injection into the intestine.
5. Replace maintenance diet (extrudate major nutrients: crude protein 18.5%; crude fat 4.5%; crude fiber 4.5%; crude ash 6.5%; starch 35%; metabolic energy: 13.1 MJ/kg) to breeding diet (extrudate major nutrients: crude protein 23.5%; crude fat 5.5%; crude fiber 3%; crude ash 5.7%; starch 36%; metabolic energy: 14.3 MJ/kg) in order to provide the mice with food of higher energy content before and during the expected clinical disease.
6. Remove the face mask and transfer the mouse to its home cage with a warming pad. After 10 minutes, make sure the mouse is fully awake and motile.
7. Repeat the PTx injection (step 1.4) 48 hours after the first treatment.

2. Scoring of EAE mice

1. Check the health status of EAE mice every morning by taking a look inside the cages.
2. Score EAE mice every afternoon.
3. Take every individual mouse included in the EAE experiment out of the cage and check whether the tail has tonus by moving it upward with a finger. A healthy mouse will keep its tail up (the tail has a tonus). If clinical EAE has started, the tail tonus will be lower, visible by a gradual drop of the tail. Eventually, the mouse will not be able to lift its tail at all.
4. Place every individual mouse included in the EAE experiment on the clean bench and observe and document the walking behavior. See Table 1 for scoring criteria for the assessment of disease severity (the EAE score).
5. Assess and document the weight of every mouse included in the experiment.
6. To ensure adequate food and water uptake by mice displaying a clinical EAE score of 1 supply moistened food in a plastic dish on the bottom of the cage and refresh daily.

Results

Figure 1: Graphical representation of injection sites for EAE induction. 1) Injection site for 30 µL MOG-emulsion into the right flank, 2) injection site for 30 µL MOG-emulsion into the left flank, 3) injection site for 20 µL MOG-emulsion into the ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Breeding food	e.g. PROVIMI KLIBA SA	3336
Individually ventilated cages, Blue line Type II or III	e.g. Tecniplast	1145T, 1285L
Female C57BL/6J mice (8-12 weeks)	e.g. Janvier Labs		Females, 8-12 weeks
18G x 1½'' (1.2mm x 40mm) injection needle	e.g. BD, BD Microlance 3	304622
27G x ¾'' – Nr. 20 (0.4mm x 19mm) injection needle	e.g. BD, BD Microlance 3	302200
30G x ½'' (0.3 mm x 13 mm) injection needle	e.g. BD, BD Microlance 3	304000
Incomplete Freund's adjuvant (IFA)	e.g. Santa Cruz Biotechnology	sc-24648	Store at 4°C
Maintenance food	e.g. PROVIMI KLIBA SA	3436
MOGaa35-55 peptide	e.g. GenScript		Store at -80 °C
Mycobacterium tuberculosis H37RA	e.g. BD	231141	Store at 4 °C
NaCl 0.9 %	B. Braun	3535789
Omnican 50 30G x ½''	B. Braun	9151125S
Pertussis toxin	e.g. List biological laboratories, Inc.	180	Store at 4 °C
Stitch scissor	F.S.T	15012-12
syringe 1 ml	e.g. PRIMO	62.1002
syringe 10 ml	e.g. CODAN Medical ApS	2022-05