<i>P2RX7</i> ablation continues to reduce dystrophic pathology in 20-mo-old diaphragm and heart.

<p>(A) Representative immunofluorescence micrographs (left) and enumeration of CD11b- and CD68-expressing cells in 20-mo-old diaphragms showing reduced numbers of infiltrating leukocytes (CD11b <i>t-</i>test, <i>t</i> = 3.68, df = 6, <i>p</i> = 0.015) and macrophages (CD68⁺<i>t</i>-test, <i>t</i> = 4.73, df = 4, <i>p</i> = 0.009) in Pf-<i>mdx</i>/P2X7^−/− compared to <i>mdx</i> diaphragms. (B) Trichrome staining (left) and its average intensity in 20-mo-old diaphragms demonstrating no increase in fibrosis in Pf-<i>mdx</i>/P2X7^−/− over <i>mdx</i> mice (ANOVA, <i>F</i> = 60.32, df = 2, <i>n</i> = 5, 5, 3, <i>p <</i> 0.001; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.095; permutation analysis, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>F</i> = 4.47, <i>p</i> = 0.095). (C) Representative ImageJ output masks from morphometric analyses of diaphragm fibers (left) demonstrating the increased average minimum Feret diameter in <i>mdx</i>/P2X7^−/− compared to <i>mdx</i> diaphragms (ANOVA, <i>F</i> = 75.17, df = 2, <i>n</i> = 3, <i>p <</i> 0.001; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.006; permutation analysis, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>F</i> = 20.01, <i>p</i> = 0.099). (D) Graphs showing a lower total number of diaphragm fibers per unit area in Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i> mice (left; ANOVA, <i>F</i> = 52.77, df = 2, <i>n</i> = 3, <i>p <</i> 0.001; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.001; Pf-<i>mdx</i>/P2X7^−/− versus WT, <i>p</i> = 0.051) and the increased proportion of C/N fibers (right; <i>t-</i>test, <i>t</i> = 5, df = 4, <i>p</i> = 0.008). (E) Dystrophin immunofluorescence in representative transverse sections of 20-mo-old diaphragms showing the typical staining (green signal) in dystrophin-positive muscles and clusters of revertant dystrophin-positive fibers in dystrophic samples. The data show significantly fewer revertant fibers in 20-mo-old Pf-<i>mdx</i>/P2X7^−/− than in <i>mdx</i> diaphragms (<i>t-</i>test, <i>t</i> = 5.12, df = 4, <i>p</i> = 0.007). (F) Representative trichrome staining (left) of whole hearts from 20-mo-old mice showing a significant decrease in cardiac muscle damage (histological lesions) and average area of fibrosis (blue signal in trichrome staining) in Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i> mice (ANOVA, <i>F</i> = 166.29, df = 2, <i>n</i> = 4, 3, 3, <i>p <</i> 0.001; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p <</i> 0.001). (G) Representative examples of CD11b⁺ leukocyte marker staining (left; red immunofluorescence) and infiltrating cell counts demonstrating fewer infiltrations in Pf-<i>mdx</i>/P2X7^−/− compared to <i>mdx</i> hearts (ANOVA, <i>F</i> = 19.65, df = 2, <i>n</i> = 3, <i>p</i> = 0.002; Tukey’s test, Pf-<i>mdx</i>/P2X7^−/− versus <i>mdx</i>, <i>p</i> = 0.005). *<i>p <</i> 0.05, **<i>p <</i> 0.005, ***<i>p <</i> 0.001.