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
 
PLP180–199 (10)

WTTCQSIAFPSKTSASIGSL

200

Yes
 
C57BL/6

MOG35–55 (11)

MEVGWYRSPFSRVVHLYRNGK

200

Yes

Chronic
 
PLP178–191 (12)

NTWTTCQSIAFPSK

200

Yes
  
PLP180–199 a

WTTCQSIAFPSKTSASIGSL

200

Yes
 
SJL/J

Whole MBP (13)

From: mouse, rat, or bovine

200–400

Yes

Relapsing-remitting
 
MBP84–104 (14)

VHFFKNIVTPRTPPPSQGKGR

200b

Yes
  
MBP89–101 (15)

VHFFKNIVTPRTP

200

Yes
  
MOG92–106 (16, 17)

DEGGYTCFFRDHSYQ

200b

Yes
  
Whole PLP (18)

From: rat or bovine

200

Yes
  
PLP57–70 (19)

YEYLINVIHAFQYV

100

Yes
  
PLP104–117 (20, 21)

KTTICGKGLSATVT

50

Yes
  
PLP139-151 (22)

HSLGKWLGHPDKF

50

No
  
PLP178-191 (23)

NTWTTCQSIAFPSK

200

No
  
PLP180-199 a

WTTCQSIAFPSKTSASIGSL

200

Yes
 
ABH

Whole MOG (16)

From: rat

200b

Yes

Chronic-relapsing
 
MOG8-22 (16, 24)

PGYPIRALVGDEQED

200b

Yes
  
PLP56-70 (24)

DYEYLINVIHAFQYV

100b

Yes
 
PL/J, B10.PL

Whole MBP (25)

From: rat or guinea pig

200

Yes

Chronic/acute monophasic
 
MBPAc1-11 (26, 25)

Ac-ASQKRPSQRSK

100

Yes
  
MBP35-47 (27)

TGILDSIGRFFSG

200

Yes
  
MOG35-55 (28)

MEVGWYRSPFSRVVHLYRNGK

200

Yes
  
PLP43-64 (29)

EKLIETYFSKNYQDYEYLINVI

150

Yes
 
C3H/HeJ

Whole PLP (18)

From: rat or bovine

200

Yes

Chronic/atypical
 
PLP190-209 (19, 30)

SKTSASIGSLCADARMYGVL

100

Yes
  
PLP215-232 (31, 19)

PGKVCGSNLLSICKTAEFQ

100

Yes
 

aTerry, Harp, and Miller, unpublished data, Fig. 2

bMice require a second immunization on day 7



A319089_1_En_88_Fig1_HTML.gif


Fig. 1
Induction of EAE in the SJL/J mouse using PLP139–151. Immunization of SJL/J mice with 50 μg of the PLP139–151 peptide results in a relapsing-remitting course of EAE


A319089_1_En_88_Fig2_HTML.gif


Fig. 2
Induction of EAE in the C57BL/6 mouse using MOG35–55. Immunization of C57BL/6 mice with 200 μg of the MOG35–55 peptide results in a chronic course of EAE


A319089_1_En_88_Fig3_HTML.gif


Fig. 3
Induction of EAE in the BALB/c, C57BL/6, and SJL/J mouse using PLP180–199. Immunization of BALB/c and C57BL/6 mice with 200 μg of the PLP180–199 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−/−

MBPAc1–11 (32, 33)

CD4

14–44 %

5–20

Chronic/AM
   
100 %

6–20

Chronic

C57BL/6 HLA-DR2/TCR tg

DR2/TCR tg × RAG-2−/−

huMBP84–102 (34)

CD4

4 %

ND

Variable
   
100 %

7–15
 

C57BL/6 HLA-DR15/TCR tg

DR15/TCR tg × RAG-2−/−

huMBP85–99 (35)

CD4

60 %

16–24

Chronic
   
80–100 %

5–16
 

SJL/J 5B6

PLP139–151 (36)

CD4

40–60 %

6 and older

Chronic

C57BL/6 2D2

MOG35–55 (37)

CD4

4–15 %

10–20

Chronic
     
30–40 %

10–52

Optic neuritis

C57BL/6 2D2 × IgHMOG

MOG35–55 (38, 39)

CD4

50–60 %

4–10

Chronic with lesions only in spinal cord and optic nerve
   
B cells
   

SJL/J TCR1640

MOG92–106 (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

OVA257–264 (43)

CD8

90–100 %

1–3

Chronic/lethal

C57BL/6 HLA-A3/TCR tg

huPLP45–53 (44)

CD8

4 %

ND

Motor deficit

NOD 1C6 × IgHMOG

MOG35–55 (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




1.

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

 

2.

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

 

3.

Incomplete Freund’s Adjuvant (IFA; Difco).

 

4.

Mycobacterium Tuberculosis H37Ra, inactivated and desiccated (Difco).

 

5.

Desired myelin protein or peptide (see Table 1).

 

6.

Phosphate-buffered saline (PBS).

 

7.

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

 

8.

15 mL polystyrene test tubes.

 

9.

18 and 25 G needles.

 

10.

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

 


2.2 Adoptive Induction of EAE




1.

Materials listed in Section 2.1.

 

2.

Dissection forceps and scissors.

 

3.

100 μm sieves (Becton Dickinson).

 

4.

1 mL syringes.

 

5.

50 mL conical polystyrene tubes.

 

6.

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

 

7.

175 cm2 culture flasks.

 

8.

37 °C, 6 % CO2 tissue culture incubator.

 

9.

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




1.

Institution-approved anesthetic.

 

2.

Dissection forceps and scissors.

 

3.

30 mL syringes with Luer-Lok.

 

4.

21.5 G needles.

 

5.

PBS.

 

6.

Non-treated plastic petri dishes, 60 mm.

 

7.

5 mL syringes.

 

8.

18 G needle.

 

9.

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




1.

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

 

2.

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 ).

 

3.

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 ).

 

4.

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.

 

5.

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.

 

6.

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 ).

 

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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Jul 12, 2017 | Posted by in NEUROLOGY | Comments Off on Autoimmune Encephalomyelitis in Mice
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