5.6. Complete Freund’s Adjuvant (CFA)-Provoked Arthritis Model

The chronic inflammatory CFA model was adopted to reveal the immunomodulatory potential of rosuvastatin in RA. The best antiedematogenic dose of rosuvastatin (40 mg/kg) estimated from formaldehyde-provoked arthritis was used in this model. Four different groups with six animals allocated to each group were formed, where group I was designated as healthy rats with no induction with CFA, group II served as the disease control group, group III was provided with piroxicam (10 mg/(kg day)), and group four received rosuvastatin (40 mg/(kg day)) for 28 days. CFA (0.1 mL) was injected in the left hind paw of rats on day 1 only for provoking arthritis.

Changes in the paw volume were assessed on a weekly basis by utilizing a digital plethysmometer for all treatment groups. The percentage protection from CFA-instigated arthritis was calculated according to the following formula:³⁷

Macroscopic scoring criteria were used to assign the arthritic score to all animals assigned to different treatment groups by examining the animals on a weekly basis. Score 1 was assigned for erythema evident in toes only, score 2 was given when there were swelling and erythema of paws, score 3 for swelling evident in ankles, and score 4 was given if there was swelling of the whole leg.³⁸

Changes in body weight were assessed for all animals in different treatment groups on a weekly basis.

Blood was collected via cardiac puncture after 28 days for the estimating hematological and biochemical parameters including WBCs, RBCs, platelets, ESR, Hb, C-reactive protein, RF, AST, ALP, ALT, urea, and creatinine.

After 28 days, ankle joints of all rats included in different treatment groups were dissected for radiographic analysis, and afterward, histological analysis was performed for these ankle joints.³⁹ Inflammatory cell infiltration, bone erosion, and pannus formation were analyzed by a blinded histopathologist. Scores from 0 to 4 were given according to the following criterion: 0 for normal, 1 for minimal, 2 for mild, 3 for moderate, and 4 for severe changes.⁴⁰

RNA was extracted according to the TRIzol method. cDNA was synthesized according to the instructions provided by the kit’s manufacturer (Thermo Scientific). The real-time polymerase chain reaction was executed using a Bio-Rad system to amplify and quantify the PCR product. Primer sequences for the above-mentioned genes and GAPDH were used from a previous study⁴¹ and were synthesized by a commercial manufacturer.