Characterization Studies of the Multiple Autoimmune Disease (MAD) Rat:

Rheumatoid Arthritis

S Champagne, B.S., RLAT, M Gardner, J Ross, ALAT, S Duclos, B.S., K Guberski, B.S., C Hogan, M.S.

Biomedical Research Models Inc., Worcester, MA

.

Thank you very much to Drs. John Mordes and Ellen Gravallese for histology work done for

this project.

Also, many thanks to the BRM technical staff.

Biomedical Research Models, Inc. has developed an inbred rat strain with susceptibility

to Multiple Autoimmune Diseases (MAD). The MAD rats were derived from congenic

LEW1.WR1 rats. During inbreeding (F55) selection was made for autoimmune features.

The goal was to develop a reliable model to test the efficacy of treatments designed to

prevent or reverse autoimmune disease and for screening for adverse effects of these

therapies.

In the present study we report on the characterization of the susceptibility of MAD rats to

Mycobacterium tuberculosis adjuvant induced arthritis (AIA). Rats were induced with

graded doses of adjuvant, and some treated with the reference compounds

methotrexate or dexamethasone. Arthritis symptoms were less severe, more transient

and variable in males versus females. Dexamethasone completely inhibited, while

methotrexate partially inhibited the appearance of clinical scores in both genders.

Primary endpoints were arthritis clinical scores, caliper measurements, and body

weights.

A novel physical finding that was observed was the appearance of skin nodules in

arthritic animals coincident with the progression of arthritis symptoms. Nodules appeared

as erythematous lesions, both raised and flat, on the tail, ears, and paws and persisted

throughout the study. Histologically the nodules were found to be characteristic of early

rheumatoid nodules found in human patients with rheumatoid arthritis (RA). This finding

is not found in other models of arthritis, mice or rats, and suggests that AIA in the MAD

rat represents a useful model of RA for testing potential therapeutic agents.

Biomedical Research Models, Inc.

508-459-7544

www.brmcro.com

The following summary is excerpted from Current Protocols in Immunology Unit 15.4

(Supplement 19, John Wiley and Sons, Inc):

Adjuvant induced arthritis (AIA) is an induced form of chronic arthritis to which some rat

strains are susceptible. AIA is typically induced with mycobacterium suspended in oil. The

disease is a T cell–mediated autoimmune arthritis that is frequently used to study

immunological aspects of rheumatoid arthritis (RA) and other arthritic or inflammatory

diseases in humans. It is also used as a model for developing and testing anti-inflammatory

drugs. There are no defined autoantigens in AIA; in this respect, the model resembles

spontaneous arthritic diseases in humans. In all susceptible rat strains, the inflammatory

process of AIA is self-remitting, although usually the disease is severe and leads to

permanent joint malformations, including ankylosis.

Compared to many other models of induced experimental autoimmunity, AIA is induced by

substances that do not contain a defined self-antigen. It can therefore be used to model how

environmental perturbants can lead to autoimmunity.

Score Description of Symptoms

0 No arthritis

1 Redness or swelling of one toe/finger joint

2

Mild redness and/or swelling of more than one

toe/finger joint +/or mild swelling of the metatarsal

(palm) area.

3 Moderate redness and swelling of the ankle or

wrist

4 Severe: entire paw redness and swelling; unable

to walk

MAD rats were group housed at up to 5 per cage in standard polycarbonate cages with

contact bedding. Rats were fed Harlan Teklad #7012 diet, and acidified, filtered tap water

(1 mL of 1N HCl/liter) ad libitum. Cages with any animals exhibiting an arthritis clinical

score of ≥ 3 will received Transgel , food was placed on the floor of cages, and affected

animals were monitored for dehydration. Cage side observations were performed daily.

Body weights were recorded 3 times per week for the duration of the 30 day study.

Adjuvant was administered in a volume of 0.03 mL per animal ID at the base of the tail in

one site. A 100 µL Hamilton syringe was used with a 25 G needle attached. Adjuvant was

administered on Day 1. Adjuvant consisted of dried, heat-killed M. tuberculosis mixed with

Incomplete Freund’s adjuvant (IFA) to a concentration of 10mg/mL.

Dexamethasone Administration for Group 3: Five times weekly (Monday through

Friday) by oral gavage beginning on Day 1. Dose per rat was 0.5 mg/kg.

Methotrexate Administration for Group 4: Two times weekly by oral gavage starting on

Day 3. Dose per rat was 1.0 mg/kg.

Injection Site/Skin Observations: The injection sites on the tails as well as the skin (to

note the presence of nodules) were observed once daily beginning on Day 6.

Arthritis Clinical Scoring: Clinical scoring for arthritis began on Day 3, and continue 3x

weekly until Day 29 - 30 of the study. Table 1 outlines the scoring system. All four paws are

evaluated for a possible maximum clinical score of 16 per rat per day.

Caliper Measurements: Caliper measurements were performed (hind paws only) on Day

3, and then three times weekly for the duration of the study until Day 29 - 30 of the study.

Rear paw widths and thickness (height) were measured.

We completed a characterization of the Mycobacterium tuberculosis AIA model in MAD

rats. Having determined the susceptibility of our rat strain to AIA, we proceeded to define the

optimum dose of the inducing agent to administer, the characteristics of the disease course in

each gender, and the effect of two reference compounds on disease course in this model.

Primary endpoints were arthritis clinical scores, caliper measurement of rear paws, and body

weights. Results from these studies are shown in Figures 1 and 2.

We found that these rats are very sensitive to the induction of inflammatory arthritis though

they did not show significant changes in body weight over the observation period as a result

of disease severity. The disease course differed from what we have reported in collagen

induced arthritis (CIA) in this strain rat (2); it more closely resembled the progression of

rheumatoid arthritis as seen in humans. Symptoms initially appeared distally in the digits,

progressed to the mid-paw area, and then moved into the ankle and wrist joints; in contrast

CIA progresses through the paw in the reverse direction. The clinical scoring system was

modified accordingly to reflect the disease progression. Symptoms were chronic for the 30

day testing period.

Figure 1 shows the mean arthritis clinical scores for male and female rats induced with

graded doses of adjuvant. The dose of 0.3 mg of adjuvant induced the most robust clinical

scores in both genders. Arthritis clinical symptoms in general were less severe, more

transient, and more variable in animals within a group in the males when compared to the

females. We have since therefore used females for subsequent studies.

Figure 2 shows the mean arthritis clinical scores and rear paw volumes calculated from

caliper measurements. Treatment with dexamethasone completely suppressed onset of

arthritis, while methotrexate dampened progression of both clinical scores and paw volume.

An unexpected novel physical finding that was observed in this model was the appearance

of skin nodules in arthritic animals coincident with the progression of arthritis symptoms

(Figure 3). Nodules were readily apparent by Day 15 - 20 in a large proportion of animals

depending on the dose of adjuvant. Nodules appeared as erythematous lesions, both raised

and flat, on the skin of the tail, ears, and paws. These nodules typically appear at onset of

arthritis clinical scores. At optimal doses of adjuvant, the nodules persisted throughout the 30

day course of the study. Histologically the nodules were unequivocally granulomatous, with

pallisading histiocytes and necrotizing centers. In a follow-up study, nodules presented

primarily in the ears, coinciding with the onset of joint inflammation. Histological analysis of

multiple nodules collected at necropsy during the peak of clinical arthritis scoring confirmed

that the nodules very closely resembled early rheumatoid nodules found on patients with RA

(Figure 4).

These nodules have not been observed in our studies with antigen-specific collagen

induced arthritis in this strain or in mice (data not shown). The clinical course of AIA in the

MAD rat (initially affecting small joints) displays more clinical fidelity as a model of human RA

than does the CIA models. We believe that future studies will be warranted to determine the

presence or absence of rheumatoid factor, anti-citrullinated protein antibodies (ACPA), or

anti-cyclic citrullinated peptide antibodies, which are frequently detected in the blood of RA

patients.

Rats were also tested weekly for the presence of elevated urinary glucose to screen for

possible co-presentation of T1D with AIA. No glycosuria was detected in any rats over the

course of the study (data not shown), indicating that the M. tuberculosis induction stimulus is

unable on its own to induce diabetes in the MAD rat strain.

We conclude that the MAD rat is now a useful system to model human autoimmune arthritis

which develops in the absence of exogenously administered antigens and in which rheumatoid

skin nodules are frequent.

Figure 1. Clinical scoring (A and B) and rear paw swelling (C and D) were assessed in male (A and C) and female (B and D) MAD rats after

induction of AIA with 1, 0.3, or 0.1 mg M. tuberculosis adjuvant. Results are presented as group mean ± SEM. N/group = 4.

Arthritis Clinical Scores

0 10 20 300

5

10

15No treatment

AIA

AIA dexamethasone

AIA methotrexate

Day

Art

hri

tis C

linic

al S

co

res

Rear Paw Volume

0 10 20 300

50

100

150No treatment

AIA

AIA Dexamethasone

AIA Methotrexate

Day

Paw

vo

lum

es (

mm

2)

Figure 2. Validation study for rat M. tuberculosis induced AIA in female

MAD rats. (A) Arthritis clinical scores. (B) Rear paw volumes. Rats were

either uninduced (No treatment), or induced with 0.3 mg M. tuberculosis

(designated AIA in the legend). AIA induced rats were either not treated

(designated AIA in the legend), or induced and treated with

dexamethasone or methotrexate. Results are means ± SEM for n=10

rats per group.

A B

Figure 4: Left hand photo shows a cross section of a normal rat ear. The right hand photo shows a

cross section of a rat ear with a rheumatoid nodule.

0 10 20 300

4

8

12

16

Study Day

Clin

ical S

co

re

0 10 20 300

4

8

12

16

Vehicle

0.3 mg M. tuberculosis

0.1 mg M. tuberculosis

1 mg M. tuberculosis

Study Day

Clin

ical S

co

re

3 6 8 10 13 15 17 20 22 24 29 30

0

50

100

150

200

Study Day

Paw

vo

lum

es (

mm

2)

3 6 8 10 13 15 17 20 22 24 29 30

0

50

100

150

200

Study Day

Paw

vo

lum

es (

mm

2)

A B C D

These studies are classified as USDA Pain Category E for the use of CFA and unrelieved

mild/moderate pain with humane end points for severe clinical scores. All studies were

performed after extensive review and approval of the BRM IACUC.

Figure 3. Skin nodules appear coincident

with onset of arthritis clinical symptoms in

male and female MAD rats induced for

AIA. Left panel: Appearance of red

nodules on the margin of the ear. Right

panel: Skin nodules can be seen on the

tail of a rat with AIA. Arthritis (erythema

and edema) is also apparent in the joints

of the tail as well as the paws (not shown).