Figure 5. CGRP and SP trigger tracheal neurogenic inflammation in response to denatonium.

(A and B) CGRP and SP measurements in excised tracheae with and without denatonium stimulation in WT (Trpm5^+/+) and Trpm5^–/– mice (n = 5–7 samples from 6–10 mice). (C and D) Staining of CGRP⁺ (C) and SP⁺ (D) nerve fibers (arrowheads) in tracheal slices of control (vehicle) and denatonium-treated WT mice. L, lumen; E, epithelium; LP, lamina propria. Scale bars: 50 μm (C and D). (E and F) Quantification of contacts between BCs and CGRP⁺ (E) and SP⁺ (F) nerve endings in whole-mount preparations of tracheae treated with vehicle or 1 mM denatonium (n = 15–29 image stacks containing 16–77 contacts/sample from 8 mice). (G and H) Treatment of tracheae with 1 mM denatonium significantly reduced the CGRP⁺ (G) and the SP⁺ (H) nerve fiber volume (normalized to the total stack volume) in Trpm5^+/+ but not in Trpm5^–/– mice (n = 12–23 volumes from 4 mice). (I) 3D reconstruction of a whole-mount tracheal preparation immunofluorescence with Imaris software (see Supplemental Methods). BCs (GFP, green) are approached by CGRP⁺ nerves. (J) Nerve endings approaching BCs are CGRP⁺ and SP⁺. Scale bars: 20 μm (left images in I and J) and 10 μm (right images). In A, B, and E–H, data are shown as single values ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA followed by Bonferroni’s multiple-comparison correction (A and B) or 2-tailed, unpaired Student’s t test (E–H).