Contextual Fear Conditioning Test

The contextual FC test was utilized to evaluate cognitive function (35). The contextual FC test consisted of a training phase at 24 h prior to ORIF surgery and an evaluation phase on days 1 and 7 after ORIF (36), when hippocampal-dependent memory was assessed (37).

During the training phase, rats were placed in a chamber (Ugo Basile, Italy) and allowed to adapt to the environment for 120 s. After adaption, a 20 s 70-dB tone (conditional stimulus) was delivered, followed by an interval of 25 s. After the interval, an 0.70 mA electrical foot shock was delivered to the rat for 2 s (unconditional stimulus). After six pairs of conditional-unconditional stimuli, the rats learned the association and had established long-term memory. The pairs of conditional-unconditional stimuli were separated by 60 s inter-training intervals. Each training chamber was cleaned with 75% ethyl alcohol before placement of the next rat and was illuminated only with a 10 W bulb in a dark experimental room.

During the evaluation phase, rats were placed again in the training chamber for 5 min without tone and foot shock (38). Each animal's freezing behavior (without any movements) was analyzed by using the ANY-Maze Video Tracking System (Stoelting, Illinois, USA). The percentage of time spent exhibiting freezing behavior was calculated using the formula of 100^*f/5 min, where f was the total of freezing time within 5 min. The results in this experiment were used to assess hippocampus-dependent memory (35, 37).