- The MAD rat strain was developed to be susceptible to multiple autoimmune diseases. This study characterized the susceptibility of MAD rats to adjuvant-induced arthritis (AIA), a model of rheumatoid arthritis. - MAD rats were found to be highly sensitive to developing inflammatory arthritis after AIA induction. Symptoms progressed from small to large joints similarly to human rheumatoid arthritis. - A novel finding was the appearance of skin nodules coinciding with arthritis, resembling early rheumatoid nodules in humans. Treatment with dexamethasone inhibited arthritis while methotrexate partially inhibited it.

1. 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 30
0
5
10
15
No treatment
AIA
AIA dexamethasone
AIA methotrexate
Day
ArthritisClinicalScores
Rear Paw Volume
0 10 20 30
0
50
100
150
No treatment
AIA
AIA Dexamethasone
AIA Methotrexate
Day
Pawvolumes(mm2
)
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 30
0
4
8
12
16
Study Day
ClinicalScore
0 10 20 30
0
4
8
12
16
Vehicle
0.3 mg M. tuberculosis
0.1 mg M. tuberculosis
1 mg M. tuberculosis
Study Day
ClinicalScore
3
6
8
10
13
15
17
20
22
24
29
30
0
50
100
150
200
Study Day
Pawvolumes(mm2
)
3
6
8
10
13
15
17
20
22
24
29
30
0
50
100
150
200
Study Day
Pawvolumes(mm2
)
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).