Fig. 5.

Pharmacological silencing of MrgprA3⁺ neurons reduces both acute and allergic ocular itch. (A) Representative traces of action potentials elicited by repeated chloroquine (CQ) application were chosen from 10 recorded MrgprA3⁺ sensory neurons in the control group. (B) Representative traces of action potentials induced by application of QX-314 plus CQ and subsequent CQ alone were chosen from 8 recorded MrgprA3⁺ neurons in the QX-314 group. (C) Changes in the membrane potential (MP) in control (n=10) and QX-314 (n=8) groups.Statistical analysis by two tailed Student’s t-test (***P=0.00003). (D) The proportion of firing neurons in control and QX-314 groups. (E) Ocular scratching responses induced by conjunctival application of CQ (12 mM) after pretreatments of 4mM CQ and 1% QX-314 (QX-314 group, n=6), compared with the control group pretreated with 4 mM CQ alone (n=7). Statistical analysis by two tailed Student’s t-test (***P=0.0001). (F) Allergen ovalbumin (OVA)-induced ocular scratching responses at different time points after pretreatment of 4mM CQ and 1% QX-314, compared with controls pretreated with 4 mM CQ alone (n=14 (pre-treatment), 6 (30min), 5 (1h), 6 (3h), 10 and 9 (24h), 5 (48h) mice/group). Statistical analysis by two tailed Student’s t-test (30min, ***P=0.0006; 1h, **P=0.007; 3h, **P=0.006; 24h, **P=0.006). (G) Allergen ovalbumin (OVA)-induced ocular scratching responses at different time points after pretreatment of pheniramine (0.4%, 2 μl) or vehicle control (n=6 (pre-treatment), 6 and 5 (10min), 6 and 7 (1h), 9 (24h) mice/group). Statistical analysis by two tailed Student’s t-test (**P=0.007). (H) A diagram summarizing the predominant role of MrgprA3⁺ sensory neurons in ocular itch. (I) Representative images of vehicle-treated (control), QX-314-treated and pheniramine-treated conjunctivae under allergic conjunctivitis. The dashed lines indicate the boundary between the epithelium and stroma of the conjunctivae; white arrows indicate the mast cells. (J) Quantitative analysis of mast cell number after different treatments (each dot represents a conjunctiva explant; n=6 conjunctivae from three mice per group).Statistical analysis by one-way ANOVA followed by two tailed Student’s t-test (before vs. cont. ***P=0.0003; cont. vs. QX314 ***P=0.00004; QX314 vs. phenir ***P=0.000001) (K) Representative H&E staining showing inflammatory cell infiltration in vehicle, QX-314 and pheniramine-treated allergic conjunctiva after allergen (OVA) challenges. (L) Representative images showing the expression of neuropeptide CGRP (red) in MrgprA3⁺ sensory neurons (green), as indicated by arrows. (M) Representative images showing mast cell in the conjunctivae after treatments of CGRP (0.5 nmol in 2.5 μl) or saline. (N) Quantitative analysis of mast cell number after CGRP treatments (each dot represents a conjunctiva explant; n=4 conjunctivae from three mice per group). Statistical analysis by two tailed Student’s t-test (***P=0.0001). All data are expressed as mean±s.e.m. All images shown are representatives of three biologically independent mice.Scale bars: 50 μm