*4.4. Drug and Experimental Plan*

The TRPA1 antagonist ADM\_12, synthesized in the Laboratory of Prof. Cristina Nativi (University of Florence, Italy) and characterized by a high binding constant versus TRPA1 [23], was dissolved in saline and administered intraperitoneally (i.p.) at the dose of 30 mg/kg in a volume of 1 ml/kg [22–24].

The animals were randomly allocated in four groups of 12 animals each and assigned to different experimental sets, as shown in Table 2.

**Table 2.** Experimental groups and number (N) of animals per group that underwent the mechanical stimulation test (MST). The samples of the subsets were processed for the real time polymerase chain reaction (RT-PCR) or immunohistochemistry (IHC).


On day +27, sham and operated rats were treated with ADM\_12 or saline 1 h prior to the MST (Figure 8). The timing was chosen on the basis of previous studies reporting a significant effect of acute ADM\_12 treatment on behavioral responses [22–24]. At the end of the behavioral test, each rat was sacrificed with an i.p. overdose of pentobarbital (150 mg/kg). A subset of 6 rats per experimental group served for the detection of gene expression levels by means of real time polymerase chain reaction (RT-PCR); another subset of 6 animals per experimental group underwent the immunohistochemical evaluation of protein expression (Table 2).

**Figure 8.** Schematic representation of the experimental design.
