Figure 3

PACAP1-38 increases migraine-like pain behavior in mice with mast cells expressing the human receptor MRGPRX2. (A) PACAP1-38 dose-dependent induced release of β-hexosaminidase from WT LAD2 human mast cells compared to LAD2 lacking MRGPRX2. N = 3, Error bar: S.E.M. *p < 0.05, ****p < 0.001. (B) Diagram showing the mating strategy to generate humanized (X2^+/+) mice. (C,D) Representative flow cytometric profile of MrgprB2 Cre MRGPRX2⁺ (X2⁺) mast cells. Cells were harvested from the meninges (C) or peritoneal cavity (D) and mast cells were identified as live c-kit⁺ FcεRI⁺ cells. An n = 3 of mice per group for both meningeal and peritoneal extracts. (E) Average change in intracellular calcium (red line) in peritoneal mast cells from MrgprB2 Cre⁺ MRGPRX2⁺ (X2⁺) or MrgprB2 Cre⁻ MRGPRX2⁺ (X2⁻) mice activated by PACAP1-38 (1 μM), with ± 95% confidence intervals shown (black points), n = 3 per group. (F) The percentage of MrgprB2 Cre⁺ MRGPRX2⁺ (X2⁺) or MrgprB2 Cre⁻ MRGPRX2⁺ (X2⁻) peritoneal mast cells responding to PACAP1-38. Two-tailed unpaired Student’s t-test, ***p < 0.001, n = 3 mice per group. (G,H) The non-invasive dural stimulation model was used to apply PACAP1-38 (1 μM) to the MrgprB2 Cre⁺ MRGPRX2⁺ (X2⁺) or MrgprB2 Cre⁻ X2⁺ (X2⁻) mouse meninges. (G) Mechanical hypersensitivity was measured in male mice prior to dural injection and then 1 h, 2 h, 4 h, and 24 h after dural stimulation with PACAP1-38; X2⁺ n = 7, X2⁻ n = 7. (H) Female mice were also tested for mechanical hypersensitivity at 1 h, 2 h, 4 h, and 24 h after dural stimulation with PACAP1-38; X2⁺ n = 8, X2⁻ n = 8. Error bar: S.E.M. *p < 0.05, ***p < 0.001 comparing X2⁺ to X2⁻ mice. Diagram created with BioRender.