*2.2. ADM\_12 Effect on Gene Expression*

The expression of *Trpa1*, calcitonin-related polypeptide alpha (*Calca*), and preprotachykinin-A, (*PPT-A*) was evaluated in the TG and cervical spinal cord (CSC) ipsilateral (ipsi) and contralateral (contra) to the IoN ligation, and in the medulla in toto. Because of the strong relationship between TRPA1 and TRPV1 channels [19–21], we also investigated the *Trpv1* mRNA expression levels in the same areas.

#### 2.2.1. *Trpa1* mRNA Expression

In the ipsilateral TG and CSC, and in medulla region, *Trpa1* mRNA expression levels were significantly increased in the IoN-CCI1 group compared with Sham1 and Sham2 groups (Figure 2). The increased mRNA levels were significantly reduced after treatment with ADM\_12 in IoN-CCI rats (IoN-CCI2 group) in the same regions (Figure 2). ADM\_12 administration did not provoke any changes in sham-operated rats.

A significant difference in mRNA levels, both in TG and CSC, was detected between sides in the IoN-CCI1 group; whereas, there was no difference between groups when comparing *Trpa1* mRNA levels on the contralateral side of TG and CSC (Figure 2A,B).

**Figure 2.** *Trpa1* mRNA expression in trigeminal ganglia (TGs) (**A**), cervical spinal cord (CSC) (**B**), and medulla (**C**). Data is expressed as mean + SEM. One way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison Test or Two-way ANOVA followed by Bonferroni post-hoc test, \*\*\* *p* < 0.001 vs. Sham1 and Sham2 (ipsi), ◦◦◦ *p* < 0.001 vs. IoN-CCI1 (ipsi), ˆˆˆ *p* < 0.001 vs. IoN-CCI1 (contra).

### 2.2.2. *Trpv1* mRNA Expression

In the ipsilateral TG and CSC, and in the medulla region, *Trpv1* mRNA expression levels were significantly increased in the IoN-CCI1 group compared with Sham1 and Sham2 groups (Figure 3). The mRNA levels of *Trpv1* were also significantly higher in the IoN-CCI1 group in the contralateral CSC when compared to Sham groups (Figure 3B), though this increase was less marked than the increase observed on the ipsilateral side. The increased mRNA levels in these areas were significantly reduced by ADM\_12 treatment in CCI rats (IoN-CCI2 group) in ipsilateral TG and CSC, and in medulla in toto (Figure 3). ADM\_12 administration did not provoke any changes in sham-operated rats.

**Figure 3.** *Trpv1* mRNA expression in TGs (**A**), CSC (**B**), and medulla (**C**). Data is expressed as mean + SEM. One way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison Test or Two-way ANOVA followed by Bonferroni post-hoc test, \*\*\* *p* < 0.001 vs. Sham1 and Sham 2 (ipsi), ◦◦◦ *p* < 0.001 vs. IoN-CCI1 (ipsi), ˆˆˆ *p* < 0.001 vs. IoN-CCI1 (contra), # *p* < 0.05 vs. Sham1 and Sham2 (contra).

A significant difference was seen between the ipsi- and contralateral side (both in TG and CSC) in the IoN-CCI1 (Figure 3A,B).
