*4.2. Experimental Design*

This study was divided into three parts.

In part one, the effects of short-term or long-term 100 Hz EA stimulation on CFA induced pain were monitored. For short-term EA stimulation experiments, rats were randomly assigned into four groups (*n* = 9/group): (1) control group, (2) CFA group, (3) CFA + EA group, and (4) CFA + sham EA group. Rats were treated by acupuncture with (EA) or without (sham EA) 100 Hz electric current stimulation for 3 days after CFA injection. Pain behavioral tests were conducted according to the schedule (Figure 1A), i.e., on days -3, -2, -1, and zero before EA stimulation, and on days 1 and 3 after EA stimulation, respectively. After 3 days post EA stimulation, rats were sacrificed for tissue collection. The L4–6 DRG and lumbar spinal cord were removed for immunofluorescence staining or western blotting. For long-term EA stimulation experiments, rats were assigned to four groups as above, and administered with EA or sham EA for 14 days after CFA injection. Pain behavioral tests were conducted according to the schedule (Figure 1B), i.e., on days -3, -2, -1 and zero before EA stimulation, and on days 1, 3, 7 and 14 after EA stimulation. After fourteen days post EA stimulation, tissues were collected and applied as above.

In part two, the involvement of P2X3 in EA-mediated pain modulation was explored through the administration of a P2X3 antagonist (A317491) or agonist (α β-me ATP) via intraplantar injection (i.pl.) or intrathecal injection (i.t.). Rats were randomly assigned to eight groups (*n* = 5/group), i.e., (1) control + i.pl. vehicle group, (2) control + i.pl. α β-me ATP group, (3) CFA + i.pl. A317491 group, (4) CFA + i.pl. vehicle group, (5) control + i.t. vehicle group, (6) control + i.t. α β-me ATP group, (7) CFA + i.t. A317491 group, and (8) CFA + i.t. vehicle group. For i.pl., antagonists or agonists were administered ipsilaterally on day three after CFA injection, and paw withdrawal thresholds (PWT) were recorded according to the schedule (Figure 7A). For i.t., antagonists or agonists were delivered via implanted PE tubes. PWT was recorded according to the schedule (Figure 7C).

In part three, to explore the role of P2X3 in DRG and SCDH on the analgesic effect of 100 Hz EA stimulation on CFA rats, we investigated whether peripheral subcutaneous injection or central intrathecal injection of P2X3 inhibitors could simulate the analgesic effect of 100 Hz EA (Figure 8). A group of 42 adult male rats (*n* = 6/group) were divided into a (1) CFA + i.pl. vehicle group, (2) CFA

+ i.pl. A317491 group, (3) CFA + 100 Hz + i.pl. vehicle group, (4) CFA + i.t. vehicle group, (5) CFA + i.t. A317491 group, (6) CFA + 100 Hz + i.t. vehicle group, and a (7) CFA + 100 Hz + i.t. A317491 group. Then, we observed whether peripheral subcutaneous injection or central intrathecal injection of a P2X3 agonist (α β-me ATP) could reverse the analgesic effect of 100 Hz EA (Figure 9). We randomly assigned 36 adult male rats to six groups, including a (1) CFA + i.pl. vehicle group, (2) CFA + 100 Hz + i.pl. α β-me ATP group, (3) CFA + 100 Hz + i.pl. vehicle group, (4) CFA + i.t. vehicle group, (5) CFA + 100 Hz + i.t. α β-me ATP group, and a (6) CFA + 100 Hz + i.t. vehicle group.
