For behavioural experiments, after habituation and baseline of pain sensitivity measurements, mice were randomized into treatment groups. In a first series of experiments, we explored whether the injection (20 μl per paw) of exemestane (1, 5, 10 nmol) or letrozole (10, 20 nmol), or their vehicle (5% DMSO) induced, in C57BL/6 or
Trpa1+/+ and
Trpa1−/− mice, an acute nociceptive behaviour and a delayed mechanical allodynia. In this set of experiments mechanical allodynia was measured just before (30 min) and 0.25, 0.5, 1, 2, 4 and 6 h post injection. Some C57BL/6 mice were pretreated with HC-030031 (100 mg kg
−1, i.p.) or capsazepine (10 mg kg
−1, i.p.) or their respective vehicles (4% DMSO and 4% Tween20 in isotonic solution), 60 min and 30 min, respectively, before exemestane (10 nmol) or letrozole (20 nmol) i.pl. injection. Mechanical allodynia was measured 60 min after AIs i.pl. injection.
In a second set of experiments, nociceptive behaviour and mechanical allodynia were assayed before and after systemic administration of exemestane (5 mg kg
−1, i.p. or 10 mg kg
−1, i.g.) and letrozole (0.5 mg kg
−1, i.p. or i.g.), or their vehicles (5% DMSO for i.p. or 0.5% carboxymethylcellulose, CMC, for i.g. administration), in C57BL/6 mice or
Trpa1+/+ and
Trpa1−/− mice. Mechanical allodynia was measured just before (30 min) and 1, 3, 6, 24, 48 h after injection. Some animals 2 h after AI administration received HC-030031 (100 mg kg
−1, i.p.) or its vehicle (4% DMSO and 4% Tween80 in isotonic solution), and mechanical allodynia and the forelimb grip strength were measured 1 and 3 h after vehicle or HC-030031. In a third series of experiments,
Trpa1+/+ and
Trpa1−/− mice were treated i.p. once a day for 15 consecutive days with exemestane or letrozole at the dose of 5 or 0.5 mg kg
−1, respectively, or with their vehicle (5% DMSO) and with i.g. exemestane or letrozole at the dose of 10 or 0.5 mg kg
−1, respectively, or with their vehicle (0.5% CMC). Mechanical allodynia and the forelimb grip strength were measured 10 min before and 1, 3, 6 and 24 h post administration at day 1, 5, 10 and 15.
To test whether PAR2 activation enhances the nocifensor behaviour evoked by exemestane and letrozole, in another experimental setting, the PAR2 activating peptide (PAR2-AP), SLIGRL-NH
2, (10 μg/10 μl i.pl.) or its reversed inactive form (PAR2-RP), LRGILS-NH
2, (10 μg per 10 μl i.pl.), were injected in the right hind paw. Ten minutes after i.pl. PAR2-AP or PAR2-RP injection, mice received exemestane (10 nmol per 10 μl i.pl.) or letrozole (20 nmol per 10 μl, i.pl.), or their vehicle (5% DMSO), in the plantar surface in the same paw injected with PAR2-AP or PAR2-RP, and the acute nociceptive behaviour was recorded. In another series of experiments H
2O
2 (0.5 μmol per 10 μl, i.pl.) or its vehicle was injected and the acute nocifensor behaviour to H
2O
2, which did not last longer than 5 min, was recorded for 10 min. Ten minutes after vehicle/H
2O
2, exemestane (10 nmol per 10 μl i.pl.) or letrozole (20 nmol per 10 μl, i.pl.) was injected in the same paw injected with H
2O
2 or vehicle and the acute nociceptive behaviour in response to AIs was recorded. Three hours after systemic administration of exemestane (5 mg kg
−1, i.p.) or letrozole (0.5 mg kg
−1, i.p.), mice were locally injected with H
2O
2 (0.5 μmol per 20 μl, i.pl.) or its vehicle and both acute nocifensor behaviour and mechanical allodynia were recorded.
Acute nocifensive response. AITC (10 nmol per paw), exemestane (10 nmol per paw), letrozole (20 nmol per paw) or their vehicles (5% DMSO), H
2O
2 (0.5 μmol per paw) or its vehicle (isotonic solution) and PAR2-AP or PAR2-RP (10 μg per paw) (10 or 20 μl) were injected into the paw of C57BL/6,
Trpa1+/+ and
Trpa1−/− mice, and immediately after injection animals were placed in a plexiglas chamber. The total time spent licking and lifting the injected hind paw was recorded for 5 min as previously described30 (
link).
Mechanical stimulation (von frey hair test). Mechanical threshold was measured in C57BL/6,
Trpa1+/+ and
Trpa1−/− mice after both local (i.pl.) administration of AITC (10 nmol per paw), exemestane (10 nmol per paw), letrozole (20 nmol per paw) or their vehicles (5% DMSO), H
2O
2 (0.5 μmol per paw) or its vehicle (isotonic solution), and systemic (i.p.) administration of exemestane (5 mg kg
−1, i.p.) or letrozole (0.5 mg kg
−1, i.p.) at different time points by using the up-and-down paradigm66 (
link). Mechanical nociceptive threshold was determined before (basal level threshold) and after different treatments. The 50% mechanical paw withdrawal threshold response (in g) was then calculated from these scores, as previously described66 (
link)67 (
link).
Forelimb grip strength test. The grip strength test was performed with a grip strength meter (Ugo Basile, Varese, Italy), as previously reported68 (
link). Mice were allowed to grasp a triangular ring attached to a force transducer and gently pulled away by the base of the tail until the grip was broken. The test was repeated four times and the mean peak force values (g) were calculated for each animal. The grip strength was measured in C57BL/6,
Trpa1+/+ and
Trpa1−/− mice 10 min before and 1, 3, 6 and 24 h post AI administration.
Fusi C., Materazzi S., Benemei S., Coppi E., Trevisan G., Marone I.M., Minocci D., De Logu F., Tuccinardi T., Di Tommaso M.R., Susini T., Moneti G., Pieraccini G., Geppetti P, & Nassini R. (2014). Steroidal and non-steroidal third-generation aromatase inhibitors induce pain-like symptoms via TRPA1. Nature Communications, 5, 5736.
