Quantification of Pain Thresholds by Von Frey Test

To quantify pain threshold, we utilized the von Frey test by assessing the escape behavior of the rats in response to stimuli. Prior to the test, the rats were acclimated to the experimental platform with a mesh-like floor surface for approximately 30 min. The von Frey filaments of different diameters (Aesthesio, DanMic Global, LLC, San Jose, CA, USA) were sequentially stimulated to the plantar region of the right hind paw. Each filament was pressed against the paw’s plantar surface for approximately 6 s until it curved. We observed whether escape behavior occurred and recorded the smallest filament stimulus that elicited such a response. This procedure was repeated five times, and the average value was determined as the paw withdrawal threshold (PWT) (Kim et al. 1997; Field et al. 1999; Dowdall et al. 2005).

All behavioral tests were conducted by researchers who maintained the confidentiality of the treatment group. The pain evaluation experiments were carried out at the animal testing facilities of Arcrise-Japan Ltd., in accordance with the Guidelines for the Appropriate Conduct of Animal Experiments set forth by the Science Council of Japan. The pain assessments were performed in a blinded manner, ensuring that the experimenters were unaware of the group assignments of the animals being evaluated.