Enhanced scratching evoked by PAR-2 agonist and 5-HT but not histamine in a mouse model of chronic dry skin itch

Abstract

Chronic itch is a symptom of many skin conditions and systemic disease, and it has been hypothesized that the chronic itch may result from sensitization of itch-signaling pathways. We induced experimental chronic dry skin on the rostral back of mice, and observed a significant increase in spontaneous hindlimb scratches directed to the dry skin. Spontaneous scratching was significantly attenuated by a PAR-2 antibody and 5-HT2A receptor antagonist, indicating activation of these receptors by endogenous mediators released under dry skin conditions. We also observed a significant increase in the number of scratch bouts evoked by acute intradermal injections of a protease-activated receptor (PAR)-2 agonist and serotonin (5-HT), but not histamine. We additionally investigated if pruritogen-evoked activity of dorsal root ganglion (DRG) neurons is enhanced in this model. DRG cells from dry skin mice exhibited significantly larger responses to the PAR-2 agonist and 5-HT, but not histamine. Spontaneous scratching may reflect ongoing itch, and enhanced pruritogen-evoked scratching may represent hyperknesis (enhanced itch), both potentially due to sensitization of itch-signaling neurons. The correspondence between enhanced behavioral scratching and DRG cell responses suggest that peripheral pruriceptors that respond to proteases and 5-HT, but not histamine, may be sensitized in dry skin itch.

Fig. 1

Spontaneous and pruritogen-evoked scratching behavior in AEW- and control mice. A: Bar graph plots mean number of scratch bouts recorded over a 30-min period without id injection (spontaneous) or following id injection of saline, histamine, the PAR-2 agonist SLIGRL-NH2, and 5-HT. Black bars: AEW dry skin treatment group; open bars: control water treatment; gray bars: naïve (untreated) group. Error bars: SEM. *: AEW group significantly different compared water and naïve groups for each stimulus condition (p<0.05 for all comparisons; repeated-measures ANOVA). #: significantly different compared to saline injection in water-treated group (p<0.05, unpaired t-test). B: Graph as in A, with vehicle-associated scratching subtracted. *: AEW group significantly different compared water group (p<0.05 for both; repeated-measures ANOVA). N.s.: after subtracting vehicle-associated scratching, there was no difference between AEW and W groups for histamine-evoked scratching behavior. (n =6–9/group).

Fig. 2

Reduced spontaneous scratching following treatment with PAR-2 antibody or 5-HT2A antagonist ketanserin. A: PAR-2 antibody. AEW-treated mice received an id injection of PAR-2 antibody, or vehicle (IgG antibody) within the AEW treatment area. Graph plots mean number of scratch bouts recorded over 80 min observation period, beginning 5 min following antibody pretreatment. *: significantly different from vehicle; p<0.05, paired t-test, n=6/group). B: Ketanserin. AEW-treated mice received systemic ketanserin (3 mg/kg, ip) or vehicle (5% tween 80 in saline). The graph plots the mean number of scratch bouts recorded over an 80 min period, beginning 30 min after ketanserin or vehicle pretreatment. *: significantly different from vehicle; p<0.05, paired t-test, n=6/group).

Fig. 3

Venn diagrams showing relative proportions of DRG cells responsive to histamine, PAR-2 agonist SLIGRL-NH2, and 5-HT, and various combinations, for each treatment group. A: Water-treated (W) controls (n=1,391 cells). 81%, 72% and 76% of histamine-, PAR-2 agonist- and 5-HTresponsive cells, respectively, were also activated by capsaicin. B: AEW (n=726). Proportions of histamine-, PAR-2 agonist-, and 5-HT-sensitive cells in AEW group were significantly greater compared to W group (p<0.05 for each, Fisher’s exact test). 66%, 58% and 85% of histamine-, PAR-2 agonist- and 5-HT-responsive cells, respectively, were also activated by capsaicin.

Fig. 4

Individual examples of pruritogen-evoked responses of DRG cells. A: Histamine. Graph plots normalized 340/380 nm ratio vs. time for DRG cells from AEW-and W-treated mice. Histamine was applied at time 0:30 (red bar). B: PAR-2 agonist. C: 5-HT. Note larger responses of cells from AEW group to PAR-2 agonist and 5-HT.

Fig. 5

DRG cells from AEW-treated mice show enhanced responses to PAR-2 agonist and 5-HT but not histamine. A: Histamine. Mean normalized ratiometric responses of DRG cells from each treatment group vs. time relative to histamine perfusion (red bar). Error bars: SEM. B: Mean peak response (% of baseline) of DRG cells to histamine for each treatment group. N.s.: no significant difference compared to W. C, E: as in A for PAR-2 agonist and 5-HT, respectively. D, F: as in B for PAR-2 agonist and 5-HT, respectively. *: significantly different compared to W (p<0.05, unpaired t-test). (n =14–23/group). Naïve data from cervical DRG cells (n=719) obtained from untreated mice. Dry skin treatment enhanced pruritogen-evoked scratching behavior in mice and responses of dorsal root ganglion cells, indicating peripheral sensitization of itch-signaling afferent fibers.