Autoimmune Encephalomyelitis in Mice

Mouse strain

Myelin peptide/protein

Sequence

Peptide/protein dose (μg)

Pertussis

Clinical course

toxin

BALB/c

Whole PLP (10)

From : bovine

200

Yes

Chronic

PLP_180–199 (10)

WTTCQSIAFPSKTSASIGSL

200

Yes

C57BL/6

MOG_35–55 (11)

MEVGWYRSPFSRVVHLYRNGK

200

Yes

Chronic

PLP_178–191 (12)

NTWTTCQSIAFPSK

200

Yes

PLP_180–199 ^a

WTTCQSIAFPSKTSASIGSL

200

Yes

SJL/J

Whole MBP (13)

From: mouse, rat, or bovine

200–400

Yes

Relapsing-remitting

MBP_84–104 (14)

VHFFKNIVTPRTPPPSQGKGR

200^b

Yes

MBP_89–101 (15)

VHFFKNIVTPRTP

200

Yes

MOG_92–106 (16, 17)

DEGGYTCFFRDHSYQ

200^b

Yes

Whole PLP (18)

From: rat or bovine

200

Yes

PLP_57–70 (19)

YEYLINVIHAFQYV

100

Yes

PLP_104–117 (20, 21)

KTTICGKGLSATVT

Yes

PLP_139-151 (22)

HSLGKWLGHPDKF

PLP_178-191 (23)

NTWTTCQSIAFPSK

200

PLP_180-199 ^a

WTTCQSIAFPSKTSASIGSL

200

Yes

ABH

Whole MOG (16)

From: rat

200^b

Yes

Chronic-relapsing

MOG_8-22 (16, 24)

PGYPIRALVGDEQED

200^b

Yes

PLP_56-70 (24)

DYEYLINVIHAFQYV

100^b

Yes

PL/J, B10.PL

Whole MBP (25)

From: rat or guinea pig

200

Yes

Chronic/acute monophasic

MBP_Ac1-11 (26, 25)

Ac-ASQKRPSQRSK

100

Yes

MBP_35-47 (27)

TGILDSIGRFFSG

200

Yes

MOG_35-55 (28)

MEVGWYRSPFSRVVHLYRNGK

200

Yes

PLP_43-64 (29)

EKLIETYFSKNYQDYEYLINVI

150

Yes

C3H/HeJ

Whole PLP (18)

From: rat or bovine

200

Yes

Chronic/atypical

PLP_190-209 (19, 30)

SKTSASIGSLCADARMYGVL

100

Yes

PLP_215-232 (31, 19)

PGKVCGSNLLSICKTAEFQ

100

Yes

^aTerry, Harp, and Miller, unpublished data, Fig. 2

^bMice require a second immunization on day 7

Fig. 1

Induction of EAE in the SJL/J mouse using PLP_139–151. Immunization of SJL/J mice with 50 μg of the PLP_139–151 peptide results in a relapsing-remitting course of EAE

Fig. 2

Induction of EAE in the C57BL/6 mouse using MOG_35–55. Immunization of C57BL/6 mice with 200 μg of the MOG_35–55 peptide results in a chronic course of EAE

Fig. 3

Induction of EAE in the BALB/c, C57BL/6, and SJL/J mouse using PLP_180–199. Immunization of BALB/c and C57BL/6 mice with 200 μg of the PLP_180–199 peptide results in a chronic course of EAE, but causes relapsing-remitting disease in SJL/J mice

Table 2

Transgenic mouse models of experimental autoimmune encephalomyelitis

Background	TCR specificity	Cell-inducing disease	% of spontaneous disease	Age of onset (in weeks)	Clinical course
B10.PL/TCR tg B10.PL/TCR tg × RAG-1^−/−	MBP_Ac1–11 (32, 33)	CD4	14–44 %	5–20	Chronic/AM
B10.PL/TCR tg B10.PL/TCR tg × RAG-1^−/−			100 %	6–20	Chronic
C57BL/6 HLA-DR2/TCR tg DR2/TCR tg × RAG-2^−/−	huMBP_84–102 (34)	CD4	4 %	ND	Variable
C57BL/6 HLA-DR2/TCR tg DR2/TCR tg × RAG-2^−/−			100 %	7–15
C57BL/6 HLA-DR15/TCR tg DR15/TCR tg × RAG-2^−/−	huMBP_85–99 (35)	CD4	60 %	16–24	Chronic
C57BL/6 HLA-DR15/TCR tg DR15/TCR tg × RAG-2^−/−			80–100 %	5–16
SJL/J 5B6	PLP_139–151 (36)	CD4	40–60 %	6 and older	Chronic
C57BL/6 2D2	MOG_35–55 (37)	CD4	4–15 %	10–20	Chronic
			30–40 %	10–52	Optic neuritis
C57BL/6 2D2 × IgH_MOG	MOG_35–55 (38, 39)	CD4	50–60 %	4–10	Chronic with lesions only in spinal cord and optic nerve
		B cells			Chronic with lesions only in spinal cord and optic nerve
SJL/J TCR1640	MOG_92–106 (40)	CD4	60–90 %	8–23	RR on female
		B cells			PP on male
C57BL/6 B7.2 expressed on microglia and T cells	ND (41, 42)	CD8	100 %	12–30	Chronic
C57BL/6 ODC-OVA × OT-I	OVA_257–264 (43)	CD8	90–100 %	1–3	Chronic/lethal
C57BL/6 HLA-A3/TCR tg	huPLP_45–53 (44)	CD8	4 %	ND	Motor deficit
NOD 1C6 × IgH_MOG	MOG_35–55 (45)	CD4	45–80 %	12–18	RR to chronic
		CD8
		B cells

AM acute monophasic, RR relapsing-remitting, PP primary progressive, tg transgenic, ODC oligodendrocytes, ND not defined

2 Materials

2.1 Active Induction of EAE

Female SJL/J, C57BL/6, or BALB/c mice at 6–8 weeks old (Jackson Laboratories).

Small animal clippers (e.g., model A5, blade size 50; Oster).

Incomplete Freund’s Adjuvant (IFA; Difco).

Mycobacterium Tuberculosis H37Ra, inactivated and desiccated (Difco).

Desired myelin protein or peptide (see Table 1).

Phosphate-buffered saline (PBS).

Pertussis Toxin, if required (see Table 1; List Biologicals).

15 mL polystyrene test tubes.

18 and 25 G needles.

10.

1 mL glass tuberculin syringes with Luer-Lok (VWR).

2.2 Adoptive Induction of EAE

Materials listed in Section 2.1.

Dissection forceps and scissors.

100 μm sieves (Becton Dickinson).

1 mL syringes.

50 mL conical polystyrene tubes.

Recombinant mouse IL-12, if required (R&D Systems).

175 cm² culture flasks.

37 °C, 6 % CO₂ tissue culture incubator.

30.5 G needles.

10.

Complete Roswell Park Memorial Institute (cRPMI): 440 mL of calcium-free, l-glutamine-free RPMI medium, 5 mL of 100× l-glutamine, 5 mL of 100× Penicillin-Streptomycin, 500 μL of 55 μM 2-mercaptoethanol, and 50 mL of Fetal Bovine Serum (FBS).

2.3 Isolation of CNS Infiltrating Leukocytes

Institution-approved anesthetic.

Dissection forceps and scissors.

30 mL syringes with Luer-Lok.

21.5 G needles.

PBS.

Non-treated plastic petri dishes, 60 mm.

5 mL syringes.

18 G needle.

Stainless steel wire mesh, 200–300 μm.

10.

15 mL polystyrene tubes.

11.

50 mL conical polystyrene tubes.

12.

Liberase, low Thermolysin concentration (Roche).

13.

DNase I (Sigma).

14.

Percoll (GE Healthcare).

15.

10× no calcium, no magnesium, no phenol red Hank’s Balanced Salt Solution (HBSS).

16.

10× no calcium, no magnesium HBSS.

17.

0.5 M EDTA, pH 8.

18.

FBS.

3 Methods

3.1 Active Induction of EAE

Shave the back of the mice using the small animal clippers (see Note 1 ).

Prepare complete Freund’s adjuvant (CFA) by combining 50 mL IFA and 200 mg M. tuberculosis H37Ra, resulting in a final concentration 4 mg/mL M. tuberculosis (see Note 2 ).

Prepare an emulsion of CFA and desired protein/peptide (Table 1) by mixing 1 mL of CFA with 1 mL of desired peptide/protein diluted in PBS. Repeatedly draw up and expel the liquid from a 1 mL glass syringe into a 15 mL polystyrene test tube, using an 18 G needle (see Notes 3 – 5 ).

Slowly draw up the emulsion into a new 1 mL glass syringe using an 18 G needle, taking care not to introduce air bubbles. Replace the 18 G needle with a 25 G needle.

Inject 100 μL of emulsion subcutaneously into the shaved backs of the mice, distributing evenly over three injection sites. One injection should be placed on the midline of the back just below the shoulders, and two on either side of the midline on the lower back.

Refer to Table 1 to determine if pertussis toxin is needed for the mouse strain and initiating protein/peptide you are using. If required, dilute pertussis toxin to 1 μg/mL in sterile PBS (see Note 6 ). Inject 200 μL of diluted pertussis toxin (200 ng per mouse) into the peritoneal cavity or intravenously on the day of disease induction, and again 48 h after induction (see Note 7 ).

Monitor mice every day to observe the development of clinical symptoms, which usually occur between day 10 and 28 post-induction (see Figs. 1, 2, and 3). Symptoms are measured using the scoring system shown in Table 3.

Table 3

Clinical scoring of mice with experimental autoimmune encephalomyelitis

Clinical score

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