Hello, Amirreza, CFA is a well-established and classic agent for inducing arthritis. Below is the protocol for your reference.

CFA-induced Arthritis Model Protocol (Complete Freund’s Adjuvant-induced Arthritis Model)

1. Animals

Species and strain: Male Sprague-Dawley rats

Weight range: 180–220 g

Housing conditions: Temperature 22±2 °C, humidity 50%±10%, 12-hour light/dark cycle

Feeding and drinking: Free access to food and water

Acclimation: Animals were acclimated to the environment and handled for 3–4 days prior to the experiment

2. Arthritis Induction

Induction method: Single intra-articular injection of Complete Freund’s Adjuvant (CFA)

CFA dose: 100 μL CFA

Injection site: Knee joint cavity

Control group: Injected with the same volume of saline

Anesthesia: Sodium pentobarbital (50 mg/kg, ip) was administered before injection

3. Joint Swelling Assessment

Measurement time points: At various time points after CFA injection

Measurement method: Using a flexible tape measure to determine the knee joint circumference

4. Pain Sensitivity Tests

4.1 Mechanical Hyperalgesia Test

Apparatus: Transparent plastic cage placed on a perforated plastic mesh floor (mesh size approximately 1 cm²)

Acclimation: Rats were allowed to acclimate for 15 minutes before testing

Stimulation tool: Von Frey filaments

Stimulation site: Plantar surface of the hind paw

Response threshold assessment: Up-down method was used to record the force (g) required to elicit a 50% withdrawal response

4.2 Cold Stimulus Hyperalgesia Test

Apparatus: Homemade cold plate (temperature maintained at 2 °C)

Response latency: Time from placement on the cold plate to the first paw shake

5. Serum Cytokine Detection

Blood collection time point: Day 19 after CFA injection

Anesthesia: Sodium pentobarbital (50 mg/kg, ip)

Serum preparation: Blood was stored at 4 °C for 30 minutes, centrifuged at 3000×g for 10 minutes, and the supernatant was collected and stored at -20 °C

Detection targets: TNF-α, IL-1, IL-2, IL-10

Detection method: ELISA

6. Histopathological Examination of Synovial Tissue

Sampling time point: Day 19 after CFA injection

Tissue fixation: Fixed in 4% glutaraldehyde for 6 hours

Decalcification: Standard decalcification procedure

Embedding and sectioning: Paraffin embedding, routine sectioning

Staining: HE staining and Van Gieson’s staining (VG staining)

7. Statistical Analysis

Data presentation: Mean±SD

Statistical methods: ANOVA, repeated measures analysis of variance, and Student’s Newman-Keuls test for post hoc comparisons when appropriate

Software: SPSS 10.0

Significance level: P

Lucian Miles Thanks for your help

If I do my injection in the tail or paws, would it be the same as this protocol?

Hello, Amirreza,

RA also can be induced by subcutaneous injection of 0.1 mL Complete Freund’s Adjuvant (CFA) at the tail base and footpad.

You can refer to this article for tail injection in rats: https://pubmed.ncbi.nlm.nih.gov/26345515/.

This article discusses footpad injection: https://www.sciencedirect.com/science/article/pii/S0378874109005285.

I also found a review article that you may find useful: https://journals.iium.edu.my/kom/index.php/imjm/article/view/2026.