2.2. Neuropathic Pain Rat Model and Behavioral Assessment

We developed a neuropathic pain rat model by using unconsolidated constrictive ligatures surrounding the sciatic nerve [15]. Hyperalgesic responses were detected on the third day after surgery and lasted up to two months using this model. Behavioral experiments were performed between 9 and 11 am. During the blinded experiments, the animals were identified by earmarks assigning a number to each mouse, which were announced to the investigator only after completing the experiments and analysis.

Under anesthesia with 2-3% isoflurane in 100% oxygen applied for approximately 1-2 min, maintained through a nose cone to minimize animal suffering during the procedure as described previously, male SD rats were placed in the prone position. The 7 mm sciatic nerve was exposed, and then 4 ligatures (using 4.0 chromic gut) were tied unconsolidated with a spacing of approximately 1 mm near the sciatic trifurcation in the CCI group [15]. Some rats received sham procedures, that is, sciatic exposure without ligation, representing the control group. This kind of control method is beneficial to reduce the influence of extraneous model pain behavior due to receptor expression on the contralateral DRG [16, 17]. Rats were immediately placed on a heating pad (37°C) and administered analgesia (4 mg/kg carprofen subcutaneously) to minimize postprocedural suffering.

On day 3 postsurgery, the spontaneous pain behavior score [18] was rated as in our previous study [14]: 0 score, no obvious behavioral change; 1 score, claw slightly flexed; 2 score, obvious claw buckling and outward rollover; 3 score, lateral, medial margin of claw parts in contact with the glass plate but not bearing; 4 score, animal foot lift, not in contact with the glass plate; and 5 score, in addition to foot lift, licking and biting toe. Thermal hyperalgesia was indicated by testing the paw withdrawal latency (PWL) [19], and the paw withdrawal mechanical threshold (PWMT) was measured to determine the presence of mechanical hyperalgesia [20]. The nociceptive responses were determined using thermal and mechanical hyperalgesia plantar test instruments (Cat: 37370 and Cat: 37450, Ugo Basile, Italy). Concretely, the rats were acclimatized to the apparatus consisting of three individual Perspex boxes on a glass table. A mobile radiant heat source was located under the table and focused on the desired paw, the heat was increased gradually until a withdrawal response was evoked, and the latency of heat needed to cause the withdrawal response was recorded. To prevent tissue damage, an automatic cut-off at 30 sec was set. Rats were placed on a wire mesh floor in clear cylindrical plastic enclosures. Following 20 min of acclimation, a von Frey filament was placed on the plantar surface of the right hind paw, and the force was increased gradually until a withdrawal response was evoked. The amount of force needed to cause the withdrawal response was recorded. A maximum cut-off value of 50 g was used. Each trial was repeated 3 times at ~5 min intervals, and the mean force producing the withdrawal response was determined. These two behavioral tests were performed before ligation surgery and once every other day during the first 14 postoperative days. We only performed behavioral tests and separated the tissue samples on the ipsilateral side. A timeline diagram of the whole experiment is shown in Figure 1.

Timeline diagram of the experiment. Twelve rats were randomized into two groups and received CCI or sham surgery procedures on day 0. The pain behavioral tests were performed from day 3 to day 15. FG retrograde trace labeling was performed at day 15. Three days later, the DRG was carefully harvested for further experiments.