*4.2. Collagen-Induced Arthritis (CIA) Rat Model*

Animal procedures were performed in accordance with the ethical animal welfare and experimentation, and approved by the Portuguese official authority regulating laboratory animal sciences (DGAV). Fifteen female Wistar rats (7–8 weeks old) were purchased from Charles River Laboratories (Barcelona, Spain) and housed in a clean environment. Animals were randomly divided into CIA (*n* = 10) and healthy control (*n* = 5) groups. On day 0, CIA rats were immunized with 200 μL of CII-IFA emulsion (200 μg collagen/rat) with a 25-gauge needle via subcutaneous injection at the base of the tail. Seven days after, a booster injection of CII-IFA emulsion was administrated to ensure a high incidence and severity of arthritis. Animals were maintained under general isoflurane anesthesia during these interventions, and all the arthritis-inducing procedures were performed according to the guidelines of Chondrex and based in previous reports [9,15]. At day thirteen after CIA induction, one animal from CIA group was lost due to a problem of gastric obstruction.

#### *4.3. Monitoring of Clinical Arthritis Development*

The animal health condition and physical activity was monitored daily according to the recent guidelines of refinement in RA research [32]. Three times per week the animals were weighed, paw swelling was measured with digital caliper at the metacarpus level, and x-ray was performed. Each rat paw was scored individually according to signs of joints (interphalangeal, metacarpophalangeal, carpal and tarsal) inflammation, adapting the Chondrex arthritis scoring system: (0) normal, (1) mild redness and swelling of the ankle or digits, (2) moderate redness and swelling of ankle or wrist, (3) severe redness of the entire paw, (4) maximally inflamed limb with the involvement of multiple joints. The arthritis index was determined by the sum of the score of each front and hind paw with the total maximum of 16.
