Fig 1.
Experimental partitioning of the diaphragm to quantify regional variability in muscle microstructure.
Top: Schematic representing the defined regions of the diaphragm. The diaphragm was split into two halves, bisecting the ventral region. Left: Histological cross-sections of the diaphragm were used to quantify CSA, regenerating fibers, and macrophages. Right: Whole mounted diaphragm was used to quantify sarcomere length, interstitial space, and branched fibers. Representative confocal images of the left (red = laminin, blue = CD68+ macrophages, green = nuclei) and right (red = alpha-actinin, blue = collagen I) halves of the immunostained diaphragms obtained from healthy and mdx mice (60x objective). Bar = 10 μm.
Fig 2.
Mdx diaphragms exhibit a clear progression of muscular dystrophy as mice age.
(A, C, E): Sarcomere length, the interstitial space ratio, and the percent of branched fibers are spatially heterogeneous in both the healthy and dystrophic diaphragm. Dystrophic diaphragms have shorter sarcomeres and more interstitial space and branched fibers than control mice. * = P<0.05 compared to dorsal, φ = P<0.05 compared to midcostal. (B, D, F): Regional differences in diaphragm microstructure are altered with age. Interstitial space varies with age, whereas the percent of branched fibers decreases with age in healthy and dystrophic diaphragms. * = P<0.05 compared to three months, φ = P<0.05 compared to seven months. D, M, V: dorsal, midcostal, and ventral regions. Values are means ± SD.
Fig 3.
Mdx diaphragms have greater interstitial space and more branched fibers than healthy diaphragms.
Representative confocal images (60x objective) of sarcomere length, interstitial space, and branched fibers in diaphragms from 3, 7, and 10 month old mdx and healthy mice in the dorsal, midcostal, and ventral regions (red = alpha-actinin). Bar = 25 μm.
Fig 4.
Healthy and dystrophic diaphragms are regionally diverse.
(A, C, E): CSA, the percentage of regenerating fibers and macrophages exhibit spatial variations. Dystrophic diaphragms have a greater percentage of regenerating fibers and macrophages and smaller CSAs than controls. * = P<0.05 compared to dorsal, φ = P<0.05 compared to midcostal. (B, D, F): Regional differences in microstructure are altered with age. In three and ten month old mdx mice, the number of macrophages is increased in the midcostal region compared with the ventral and dorsal region. The number of regenerating fibers increases with age in the ventral region in mdx diaphragms. * = P<0.05 compared to three months, φ = P<0.05 compared to seven months. D, M, V: dorsal, midcostal, and ventral regions. Values are means ± SD.
Fig 5.
Mdx diaphragms have more regenerating fibers and macrophages than healthy diaphragms.
Representative confocal images (60x objective) of CSA, regenerating fibers, and macrophages in diaphragms from 3, 7, and 10 month old mdx and healthy mice in the dorsal, midcostal, and ventral regions (red = laminin, blue = CD68+ macrophages, green = nuclei). Bar = 25 μm.
Fig 6.
The microstructure of dystrophic diaphragms is dependent on region and age.
(A): As the percentage of regenerating fibers increases, sarcomere length decreases. (B): In mdx diaphragms, the CSA and the percentage of branching fibers decreased with age. (C): When CSA is compared to interstitial space ratio, the age groups cluster together. D, M, V: dorsal, midcostal, and ventral regions. For each age group of healthy and mdx mice, the mean of each region is plotted for two metrics.