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. Author manuscript; available in PMC: 2019 Jan 9.
Published in final edited form as: Nat Med. 2018 Jul 9;24(8):1268–1276. doi: 10.1038/s41591-018-0083-x

Fig.6.

Fig.6

MrgprX1+ sensory afferents selectively innervate human conjunctiva and mediate itch in a humanized mouse model. (A-B) Representative images showing immunofluorescent staining of sensory neurons from MRGPRX1;Mrgpr-clusterΔ-/-and Mrgpr-clusterΔ-/- mice using a newly generated antibody for hMrgprX1. Arrows indicate sensory neurons labeled by hMrgprX1 antibody. (C-D) Representative images showing immunofluorescent staining of human conjunctiva and cornea using hMrgprX1 antibody. Arrows indicate sensory fibers labeled by hMrgprX1 antibody. The dashed lines indicate the boundary between the epithelium and stroma. Images shown are representative of three biologically independent samples. Scale bars: 100 μm. (E-F) Representative traces of action potentials elicited by CQ (1 mM) stimulation in hMrgprX1-expressing sensory neurons (as determined by calcium imaging using BAM8-22, n=5). The recorded neurons received pretreatments of vehicle control and 100 μM HC030031 (100 μM) (E) or in reverse order (F). (G) Scratching responses induced by conjunctiva challenge of allergen OVA in immunized WT,Mrgpr-clusterΔ-/-, and MRGPRX1;Mrgpr-clusterΔ-/- mice with or without pretreatments of 1% QX-314 with 4mM chloroquine (30min, n=6 and 3 (WT group), 5 and 6 (KO group), 6 and 7 (X1 group); 24hr, n=3 and 3 (WT group), 4 and 6 (KO group), 6 and 7 (X1 group)). Statistical analysis by two tailed Student’s t-test (WT vs. WT/QX314, 30min *P=0.011, 24hr *P=0.03; X1 vs. X1/QX314, 30 min ***P=0.0007; 24hr, **P=0.007). (H) Representative calcium transients of hMrgprX1-expressing heterologous cells in response to NPFF (6 μM) and BAM8-22 (2 μM). Arrows indicate BAM8-22 responsive hMrgprX1-expressing cells that were activated by NPFF. (I) Scratching responses induced by conjunctival application of NPFF (2.5 nmole in 2.5 μl) in WT (n=5), Mrgpr-clusterΔ-/- (n=6), and MRGPRX1;Mrgpr-clusterΔ-/- mice (n=6). Statistical analysis by one-way ANOVA (p=0.0082) followed by two tailed Student’s t-test (WT vs. KO, *p=0.016; KO vs. X1, ***p=0.0001). All data are expressed as mean±s.e.m.