**4. Materials and Methods**

This study was conducted in accordance with the animal research guidelines of the Kyoto Prefectural University of Medicine, Kyoto, Japan (code no. M29-22, 1 April 2017).

#### *4.1. CIA Model*

Rat CIA models have many points of similarity with human RA and are widely used for RA studies in vivo. To create such models for this study, type II collagen (CII; Collagen Research Center, Tokyo, Japan) and Freund's incomplete adjuvant (FIA; Sigma-Aldrich, Saint Louis, MO, USA) were blended and emulsified on ice in a 1:1 ratio. Then, to create rat CIA models, 200 μL of the CII/FIA solution was intracutaneously injected into 32 8-week-old male Dark Agouti (DA) rats (Shimizu Laboratory Suppliers, Kyoto, Japan) at the base of the tail [30]. The rats weighed between 140 and 190 g.
