Fig 1: Multiple doses of CV maintains physiological saliva secretion, acinar cell architecture, and functional innervation in the long term.(A) Timeline for radiation, treatments, and saliva collections. (B) Citrate-induced saliva output was measured once every 2 weeks from 35 to 91 days after radiation. (C) Maximal saliva secretion in response to systemic pilocarpine administration at 92 days. Data were normalized to non-IR controls. (D to G) Representative images and quantification of AQP5+ (D and E) and MUC10+ (F and G) acinar cells. Epithelial cells and nuclei were stained for E-cadherin (CDH1, red) and nuclei (Hoechst 33342, blue). (H to K) Representative images (H) and quantification of mitotic (pHH3+) acinar progenitor cells (I) (SOX2+) and transit-amplifying cells (J) (SOX2-). Quantification of SOX2+ acinar cells is shown in (K). (L to N) Representative images (L) and quantification of parasympathetic nerves marked by GFRa2 (M) [green, three-dimensional (3D) surface reconstructed with Imaris] and the neuropeptide VIP (N) (red, 3D surface reconstructed using Imaris). Hoechst 33342, nuclei (blue). GFRa2+ filaments with diameters of less than 1.5 µm (innervating the acini) were quantified. VIP was normalized to GFRa2+ filament volumes. (O to Q) Representative images (O) and quantification of pro-reparative (CD68+CD206+) M2-polarizing macrophages (P) and CD3+ T cells (Q). Scale bars, 40 µm (D, F, H, and O) and 10 µm (L). Dots in the bar graphs represent biological replicates. Data are expressed as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; a one-way ANOVA with Dunnett’s multiple comparison test for all graphs except in (B); a two-way ANOVA with Dunnett’s multiple comparison test was applied to (B).