*4.5. Real Time-PCR*

The trigeminal ganglia (TG), cervical spinal cord (CSC, C1-C2 level), and medulla (bregma, −13.30 to −14.60 mm; Paxinos and Watson 4th edition), containing the Sp5C, of each animal were quickly removed after completing the MST on day +27 and frozen at –80 ◦C. Samples were then processed to evaluate the expression levels of the genes encoding for TRPA1 (*Trpa1*), TRPV1 (*Trpv1*), CGRP (*Calca*), SP (*PPT-A*), IL-1beta (*IL-1beta*), IL-6 (*IL-6*), and TNF-alpha (*TNF-alpha*). mRNA levels were analyzed by RT-PCR, as previously described [24,114,115]. After tissue homogenization by means of ceramic beads (PRECELLYS, Berthin Pharma, Montigny-le-Bretonneux, France), total RNA was extracted with TRIzol®reagent (Invitrogen, Carlsbad, California, USA) and quantified by measuring the absorbance at 260/280 nm using a nanodrop spectrophotometer (Euroclone, Pero (MI), Italy). Following cDNA generation with the iScript cDNA Synthesis kit (BIO-RAD, Hercules, California, USA), gene expression was analyzed using the Fast Eva Green supermix (BIO-RAD). Primer sequences were obtained from the AutoPrime software (http://www.autoprime.de/AutoPrimeWeb) (Table 3). The amplification was performed through two-step cycling (95–60◦C) for 45 cycles with a Light Cycler 480 Instrument RT-PCR Detection System (Roche, Milan, Italy). The expression of the housekeeping

gene, glyceraldehyde 3-phosphate dehydrogenase (*GAPDH*), remained constant in all the experimental groups considered. All samples were assayed in triplicate.


**Table 3.** Primer sequences.
