D) Total sum behaviour over the 15?min observation period after intradermal injection of SLIRGL (50?g/mouse) and 1 hr pre-treatment FSLLRY (50?g/ mouse, intradermal); *P?0.05, **P?0.01 ***P?0.001, One Way ANOVA followed by Dunnetts test vehicle group. responded to hr-CatS decreased in Rabbit Polyclonal to NCAPG the presence of a PAR2 antagonist or in cultures of neurons from TRPV1?/? mice. Taken together, our results indicate CatS acts as a pruritogen via PAR2 activation in TRPV1-expressing sensory neurons. test and Mann-Whitney test. Calcium imaging data were analysed using GraphPad Prism 5 and are presented as the mean??SEM. n represents the number of experiments analysed. Statistical analyses were conducted using one-way ANOVA followed by Dunnetts multiple comparison test 20?min buffer incubation to determine the percentage of cells that responded Tripelennamine hydrochloride to application of substances. 3.?Results 3.1. Acute injection of cathepsin S induces scratching behaviour Intradermal injection of hr-CatS (1C20?g/mouse) at the nape of the neck induced significant scratching behaviour compared to saline injection, as indicated by the time spent scratching the neck area during a 15?min observation period (Fig. 1A), number of paw lifts towards the neck (itching bouts, measured as scratching-which it is assumed are in response to an itch) (Fig. 1B), composite scores obtained by combining time and itching bouts (Fig. 1C) and total behaviour scores (Fig. 1D). All itching time and itching bouts behaviour parameters reached statistical significance at 10?min after injection of 20?g of hr-CatS and ceased by 15?min after injection (Fig. 1ACD). The injection of heat inactivated hr-CatS (20?g/mouse) did not induce scratching behaviour (Fig. 1 A-D). As expected, the PAR2 receptor agonist SLIGRL-NH2 (10C100?g/mouse) also induced scratching behaviour following intradermal injection at the neck (Fig. 2ACD). At 5?min after 50 or 100?g/mouse, SLIGRL-NH2 injection was associated with significant time spent scratching (Fig. 2A) and significant paw lifts towards the neck (itching bouts) (Fig. 2B), which resulted in significant composite scores (Fig. 2C) and total behaviour scores (Fig. 2D). Total SLIGRL-NH2 itch-like behaviour values were nearly double those associated with CatS (102.2??30.5 vs. 55.4??9.6 respectively), which nevertheless produced significant pruritic effect at the highest deliverable dose according to solubility in saline (vehicle). Open in a separate window Fig. 1 Activated recombinant hr-CatS induces itch-like behaviour. A) Itching time (time spent scratching) over the 15?min observation period after intradermal injection in the nape of the neck. B) Number of paw lifts (Itching bouts) over the 15?min observation period. C) Composite scores over the 15?min observation period. D) Total sum behaviour over the 15?min observation period. Data are mean?+?SEM of 10 mice per group. *P?0.05, **P?0.01, Two-way ANOVA followed by Dunnetts test 1?g in panels A, B and C. One Way ANOVA followed by Dunnetts test 1?g dose in composite behaviour (panel D). Open in a separate window Tripelennamine hydrochloride Fig. 2 SLIGRL-NH2 induces itch-like behaviour. A) Itching time (time spent scratching) over the 15?min observation period after intradermal injection in the nape of the neck. B) Number of paw lifts (Itching bouts) over the 15?min observation period. C) Composite scores over the 15?min observation period. D) Total sum behaviour over the 15?min observation period. Data are mean?+?SEM of 7 mice per group *P?0.05, **P?0.01,***P?0.001, Two-way ANOVA followed by Dunnetts test 10?g in panels A,B and C. One-Way ANOVA followed by Dunnetts test 10?g dose in panel D. As local injection of hr-CatS in peripheral sites (intraplantar) is known to exert a pro-nociceptive effect (Barclay et al., 2007, Zhao et al., 2014), we Tripelennamine hydrochloride differentiated CatS-induced itch-like behaviour from pain-like behaviour using the cheek injection model in which wiping (pain-related) and scratching (itch-related) behaviour can be recorded. We observed that hr-CatS (20?g/mouse) induced wiping behaviour, which lasted for less than 10?s, but also scratching behaviour, which lasted for an average of 25?s over the.
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- Each swab was transported in a micro centrifuge tube with 200 L ice cold SPG and vigorously vortexed prior to dilution of the SPG with HBSS 1?:?2, 1?:?20, 1?:?200 and 1?:?2000