Adjuvant Induced Arthritis - Adjuvants

In 1956, Pearson found that rats immunized with Complete Freund’s Adjuvant (CFA), mineral oil containing heat-killed Mycobacterium tuberculosis (MT) particles, developed polyarthritis. This arthritis model, called Adjuvant-Induced Arthritis (AIA), has previously been used as a model for rheumatoid arthritis (RA). However, AIA is a more appropriate model for reactive arthritis rather than RA, due to the differences in histological and immunological features (1, 2)

Chondrex, Inc. provides CFA containing various concentrations of MT for inducing AIA. However, CFA containing 1 mg/ml MT is seldom effective for inducing AIA. For effective induction, CFA containing 10 mg/ml MT is recommended, although CFA containing 5 mg/ml of MT is also capable of inducing AIA. For more information, please see our Protocol for Induction of Adjuvant-Induced Arthritis in Rats or contact us.

*The Collagen-Induced Arthritis (CIA) Model for rats or mice and the Collagen Antibody-Induced Arthritis (CAIA) Model for mice are better suited for pre-clinical studies of RA. Please follow the links for more details.

Complete Freund's Adjuvants for AIA

Product Content of M. Tuberculosis H-37 RA Quantity Catalog # Price (USD)
Complete Freund's Adjuvant,  2 mg/ml Complete Freund's Adjuvant, 2 mg/ml 2 mg/ml 5 ml 7009 54.00
Complete Freund's Adjuvant,  3 mg/ml Complete Freund's Adjuvant, 3 mg/ml 3 mg/ml 5 ml 7015 65.00
Complete Freund's Adjuvant,  5 mg/ml Complete Freund's Adjuvant, 5 mg/ml 5 mg/ml 5 ml 7023 107.00
Complete Freund's Adjuvant, 10 mg/ml Complete Freund's Adjuvant, 10 mg/ml 10 mg/ml 5 ml 7027 154.00
Complete Freund's Adjuvant, 20 mg/ml Complete Freund's Adjuvant, 20 mg/ml 20 mg/ml 5 ml 7024 279.00

Muramyl Dipeptide, MDP

Product Catalog # Price (USD)
Muramyl Dipeptide, MDP, 2 mg Muramyl Dipeptide, MDP, 2 mg 9065 94.00

Table of Contents
    AIA Protocol and Timeline
    Evaluating AIA
    Factors to Consider 

AIA Protocol and Expected Results

AIA can easily be induced in susceptible strains of rats by a single subcutaneous injection of CFA (0.05-0.1 ml) containing 10 mg/ml MT (Catalog #7027) into a footpad or at the base of the tail. Before each injection, it is vital to re-suspend the adjuvant as the mycobacterial particles will settle. Failure to re-suspend the adjuvant could lead to inconsistent distribution of mycobacterial particles. 

Choose footpad or base of the tail injection depending on the purpose of the study. 

1) Footpad injection
Subcutaneously inject 0.05 ml of CFA containing 10 mg/ml MT into the footpad of a rear paw. Severe and acute inflammation will be observed within 30 minutes of the injection. The inflammation peaks within 3-4 days and lasts for 20-25 days. Secondary arthritis in the non-injected paws typically appears within 12-14 days of the injection. The arthritis peaks within 2-3 days of its onset and lasts for 20 to 25 days.

2) Base of the tail injection
Inject 0.1 ml of CFA containing 10 mg/ml MT subcutaneously at the base of the tail. Arthritis will appear in one or all paws between 12-14 days after the injection and last for 20 to 25 days.

Figure 1. Evaluating anti-inflammatory agents using the rat AIA model.
AIA was induced in SD rats by injecting 0.05 ml CFA (10 mg/ml MT) in the right hind paw footpad on Day 0. Severe inflammation in the paw developed immediately after injection. Secondary arthritis developed in the left hind paw footpad on day 12-14 after injection. The effect of the anti-inflammatory test compound was determined by comparing footpad volumes in treated and untreated rats. 

Note 1:
It is not necessary to use an emulsified adjuvant to induce AIA. However, to avoid inconsistent dosage of M. tuberculosis, CFA containing 20 mg/ml MT (Catalog # 7024) can be emulsified with an equal volume of saline or buffer solution and used for sensitization. This method has been reported for inducing AIA in F344 rats with mycobacterial peptidoglycans and muramyl dipeptides (3). 

Note 2:
Particle sizes of M. tuberculosis powder affects AIA development (4). Chondrex, Inc.'s MT powder is ground extensively before CFA preparation. This step increases incidence and consistency of severity of AIA in many rat strains.

For more information, please see our Protocol for Adjuvant-Induced Arthritis (AIA) in Rats.


Evaluating AIA

1. Clinical Arthritis Scores
The severity of arthritis can be scored by visual inspection. Chondrex, Inc. recommends scoring arthritis on a scale of 0 to 4 in each paw. For more information, please see our Arthritis Scoring System Guide

The tail base injection method scores arthritis in all four paws (maximum score 16), while the footpad injection method scores arthritis in three paws, excluding the adjuvant-injected paw (maximum score 12). 

2. Paw Thickness and Volume Assessment
Foot swelling associated with arthritis severity can be objectively determined by measuring paw thickness and volume. Paw thickness can be measured using a caliper such as the Mitsutoyo loop-handle dial thickness gauge, while paw volume can be measured using a plethysmograph. Figure 1 provides sample data of evaluating anti-inflammatory agents by measuring paw volume.

3. Local Hyperthermia Assessment
Local hyperthermia is a very accurate indicator of the severity of acute inflammatory reaction and is highly recommended because of its simplicity and sensitivity. In performing this measurement, the surface temperature of each paw can be measured by a contact-type thermometer (Thermomex TH-10, Natsume Seisakusho, Tokyo). 

4. Cytokine and Chemokine Analysis
In AIA rats, cytokine and chemokine levels in sera and paw extracts may correlate with inflammatory status and show different profiles in especially acute and chronic inflammatory phases. Furthermore, anti-inflammatory treatments would change these cytokine and chemokine levels (5,6). To evaluate these dynamics, samples must be prepared in appropriate periods depending on the target markers. 

Factors to Consider for the AIA Model

1. Protocols
Many research institutes require approval of animal study protocols from their Institutional Animal Care and Use Committee (IACUC). Please consult with the IACUC of your institution before purchasing products intended for use in animal studies. If you are planning on using the footpad injection route, IACUC approval is especially important as many institutions will restrict this procedure.    

Chondrex, Inc. provides a Protocol for Adjuvant-Induced Arthritis (AIA) in Rats. However, animal studies show often inconsistent results and involve many factors. We recommend performing a small pilot study to optimize the protocols for your purposes, before proceeding with a large-scale study.

If you need help with planning your pilot study or a large-scale study, please contact us.

2. Animal Vendors
Even within the same strain, there can be differences in the genetic background and bacterial flora among rats from different vendors. These differences affect how the rats will respond to various reagents, thus impacting experimental results. Chondrex, Inc. recommends performing a pilot study with a defined protocol to test animals from different vendors in order to choose an appropriate vendor for your studies.

3. Housing Conditions
Environmental factors are also important for inducing AIA. Housing conditions can affect the microbial composition of experimental animals, which in turn can affect disease phenotypes (7). To minimize this variation, Chondrex, Inc. recommends housing rats in SPF conditions whenever possible. However, if SPF housing conditions are not possible, we recommend thoroughly cleaning your housing facility before receiving the rats and during the experiment. 

4. Rat Strain and Age
Table 1 outlines the susceptibility of common rat strains to AIA. However, differences in the microbial composition of experimental animals will vary between vendors and can affect the disease phenotype(7). Ideally, 6-12 weeks old rats ought to be used for AIA studies because younger rats (1-7 days old) and older rats (>9 months old) are resistant to AIA.

Chondrex, Inc. recommends running a small pilot study with animals from your chosen vendor and desired age to ensure AIA susceptibility.

Table 1. Susceptibility of common rat strains to the Adjuvant Induced Arthritis Model.


1.    P. H. Wooley, Animal models of rheumatoid arthritis. Current Opinion in Rheumatology. 3, 407–420 (1991).
2.    L. Bevaart, M. J. Vervoordeldonk, P. P. Tak, Evaluation of therapeutic targets in animal models of arthritis: How does it relate to rheumatoid arthritis? Arthritis & Rheumatism. 62, 2192–2205 (2010).
3.    O. Kohashi et al., Susceptibility to adjuvant-induced arthritis among germfree, specific-pathogen-free, and conventional rats. Infect. Immun. 26, 791–794 (1979).
4.    R. Best, R. Christian, D. A. Lewis, Effect of particle size of dried mycobacteria on adjuvant induced arthritis in the rat. Agents and Actions. 14, 265–268 (1984).
5.    Z. Szekanecz et al., Temporal expression of inflammatory cytokines and chemokines in rat adjuvant-induced arthritis. - PubMed - NCBI. Arthritis & Rheumatism. 43, 1266–1277 (2000).
6.    X. Cai et al., Manipulation of the induction of adjuvant arthritis in Sprague-Dawley rats. Inflamm. Res. 55, 368–377 (2006).
7.    I. I. Ivanov et al., Induction of Intestinal Th17 Cells by Segmented Filamentous Bacteria. Cell. 139, 485–498 (2009).


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