Rheumatoid Arthritis (RA) is a chronic inflammatory joint disease characterized by persistent synovitis, systemic inflammation, and erosion of bones and cartilage, which can ultimately lead to joint ankylosis and deformity. RA animal models are widely used for the study of pathogenesis and therapeutic methods. Various animal models have been established for the study of RA etiology, pathogenesis, influencing factors, and the research of new therapeutic targets and the evaluation of new therapies.

RA animal models can be broadly divided into two categories: induced and transgenic models. Common induced models include Collagen-Induced Arthritis (CIA) and Adjuvant-Induced Arthritis (AA), while transgenic mouse models include human TNF-α transgenic models, etc.

Freund's Adjuvant, invented by Jules Freund in the 1940s, is an oil-in-water antigen emulsion made by mixing an antigen aqueous solution with an oil agent and adding an emulsifier. It is the most commonly used adjuvant in animal experiments.

Freund's Adjuvant is divided into Complete Freund's Adjuvant (CFA) containing Mycobacterium tuberculosis and Incomplete Freund's Adjuvant (IFA) without Mycobacterium tuberculosis. They are mainly used to induce Collagen-Induced Arthritis (CIA) and Adjuvant-Induced Arthritis (AA) models in mice.

Figure 1. Human rheumatoid arthritis

Figure 2. Mouse rheumatoid arthritis

1 Collagen-Induced Arthritis (CIA) (for reference only)

1.1 Mouse Model

The CIA model is one of the most widely used RA mouse models. Immunizing animals with heterologous type II collagen can induce an autoimmune response against type II collagen in joint cartilage, with clinical manifestations of polyarticular peripheral arthritis.

Model Construction Method: Bovine type II collagen (CII) is dissolved in glacial acetic acid at 4°C overnight. Then, heat-killed Mycobacterium tuberculosis (BCG) is placed in liquid paraffin to prepare Complete Freund's Adjuvant (CFA). The two are mixed and emulsified to prepare a type II collagen emulsion. This emulsion is injected subcutaneously at the base of the mouse's tail at 0.1-0.2 mL to induce inflammation. On the 21st day after the initial immunization, a booster injection of 0.1-0.2 mL of type II collagen emulsion is administered intraperitoneally. After the first immunization, mice will have local inflammatory reactions that heal within about a week; after the adjuvant, mice will develop joint swelling starting from day 24, progressing from the hind feet to the front feet and then to the tail.

1.2 Rat Model

Model Construction Method: Bovine type II collagen (CII) is mixed with Incomplete Freund's Adjuvant (IFA) in equal volumes and emulsified. On the first day, rats are injected intradermally at multiple sites, and a week later, they receive an intraperitoneal boost. The model is obtained within 3-7 days after the second immunization. Seven days after sensitization, the ankle joints will be slightly swollen and red, and the symptoms will worsen after 3 weeks, with skin ulceration possible; after 5-6 weeks, pathological changes further worsen, with joint cartilage and subchondral bone being eroded and destroyed by pannus.

2 Adjuvant-Induced Arthritis (AA) (for reference only)

AA model is the most widely used rat RA model, with two types: Incomplete Freund's Adjuvant (IFA) and Complete Freund's Adjuvant (CFA).

Model Construction Method: Rats are injected intradermally to induce inflammation. The model generally shows continuous inflammatory symptoms 10-20 days after induction, peaking around 20 days. The inflammation is mainly in the ankle joint, which can affect the footpad and the whole foot. The joint swelling symptoms are similar to those of clinical RA patients, but immune dysfunction phenomena may occur.

3 Collagen-Induced Arthritis (CIA) Modeling Steps (for reference only)

• Bovine type II collagen (CII) is dissolved in glacial acetic acid solution at a concentration of 2 mg/mL and kept overnight at 4°C.
• Incomplete Freund's Adjuvant is supplemented with heat-killed Mycobacterium tuberculosis to a concentration of 2-5 mg/mL. The concentration of Mycobacterium tuberculosis in the 60718ES Complete Freund's Adjuvant provided by Yisheng Biology is less than 10 mg/mL.
• Bovine type II collagen acetic acid solution is mixed with Complete Freund's Adjuvant in equal volumes and emulsified.
• Each experimental mouse is given 4-6 subcutaneous injections on the back, totaling 0.1-0.2 mL.
• Three weeks later, Incomplete Freund's Adjuvant is mixed with bovine type II collagen acetic acid solution in equal volumes and emulsified, and each mouse is given 3-5 subcutaneous injections at the base of the tail, totaling 0.1-0.2 mL.

4 Adjuvant-Induced Arthritis (AA) Modeling Steps (for reference only)

• Prepare relevant reagents, noting the setting of experimental and control groups.
• Observation and recording: Generally, more than 80% of mice show symptoms of arthritis 7-12 days after the second immunization. Clinical symptoms are graded based on the redness and activity of the mouse's joint, and observations are recorded before the experiment and on the 3rd, 5th, 7th, and 12th days after the experiment, with grading criteria as follows:

Table 1 Scoring Criteria

• Mouse measurement: Use a mouse paw volume meter to measure the joint volume of the mouse's hind limb before and after the experiment. Measure the volume of about 5 mm below the knee joint of each mouse's hind leg three times, record the average value, and the volume is proportional to the degree of joint redness and swelling. Detection is performed every three days.
• Pathological examination: Take mice from the experimental and control groups, remove the skin, fix with 4% formaldehyde for more than 48 hours, decalcify with 5% nitric acid for 2 hours, immerse in xylene, and embed in paraffin. Make 6 mm sections, stain with HE, and observe with a conventional optical microscope to establish a scoring standard for pathological diagnosis.

5 Experimental case images (excerpted from literature)