2.3.2. The Acetic-Acid-Induced Writhing Method

Each group (n = 7) of mice received the same doses of recombinant crotamine (0.04, 0.13, 0.4, and 1.2 mg kg⁻¹) intraperitoneally (Table 2). Control animals received only saline (100 μL). After 20 min [14], 100 μL of a 0.6% (v/v) acetic-acid solution was intraperitoneally administered, and mice were immediately returned to the transparent acrylic observation cage (Supplementary Figure S1). The number of writhes was then measured for 20 min [15]. To evaluate antagonistic effects of naloxone, 100 μL of naloxone (4 mg kg⁻¹) was subcutaneously administered 10 min [8] before the intraperitoneal injection of the same recombinant crotamine doses or saline as above. Naloxone works to reverse opioid overdose in the body for only 30 to 90 min [16].

Experimental design for the acetic-acid-induced writhing method (n = 7 in each group).