2.3.2. Pain-Like Behavior Assessment

For rodents, pain cannot be directly measured but it can be assessed from “pain-like” behaviors, for example the withdrawal of the rodent’s tail or foot from a nociceptive stimulus. Such a method is analgesiometry or the Randall–Selitto test, which is the most commonly used method to quantify nociception in animal studies. The test was developed in 1957 as an evaluation method for pain levels in rodents, by measuring the animal’s resistance to mechanical pain and it is considered to be the most objective pain-like behavior assessment method by comparison with cold/heat sensibility evaluation [42]. The testing involves a mechanical force of increasing intensity applied on the animal’s foot or tail, which is maintained until the animal shows a retracement behavior (the animal retracts its foot or tail as a sign of pain) [43].

The pain-like behavior of the animals from the study was appraised on days 7, 14, and 21 using a bench-top Ugo Basile (Gemonio, Italy) analgesia-meter.

During testing, the animals were immobilized in a cotton towel in order to ensure uninterrupted access at the animal’s foot. Previously, before the peripheral nerve injury induction, the animals were accommodated to the testing procedure and the analgesia-meter for seven consecutive days. During pain evaluation, the mechanical pressure was directed on the dorsal part of both posterior feet (normal foot—N and experiment foot—E), in the same point for each animal (between the tip of the cone-shape and the plane surface of the foot). The mechanical pressure was constantly increased until the detection of a nociceptive response. The animals’ nociceptive behavior was observed in a subjective manner, by the researcher. The maximal force applied was 300 g to prevent tissue damage or other similar problems.