Table 1.
Mouse MR Protocols.
Fig 1.
Sectioning scheme for histology results.
Fig 2.
Whole-body micro-dystrophin gene transfer to the musculature using rAAV6.
AAV6.CK8.H3μDys was administered by retro-orbital injection of 3-month-old mdx mice at a dose of 1013 vg, and tissues were analyzed 6 months later. All striated muscles were found to express widespread levels of the human micro-dystrophin. Shown are representative cross-sections of the TA, EDL, soleus, gastrocnemius, quadriceps, and diaphragm muscles immunostained with a rabbit polyclonal antibody against the N-terminal domain of dystrophin. Top row, muscles from wild-type mice; middle row, muscles from mdx mice; bottom row, muscles from mdx mice injected with rAAV6.CK8.H3μDys. Scale bar: 100 μm.
Fig 3.
Anti-dystrophin staining for TA, GA and SOL muscles.
Anti-dystrophin sAnti-dystrophin staining (green) shows clusters of transduced myofibers in representative sections of (A) TA, (B) GA, and (C) SOL muscles. Negative fibers (red) revealed morphological features of necrosis. (D) Between 50 to 65% of the total cross-sectional myofiber area of the selected muscles stained positive for dystrophin (n = 4). Scale bar: 500 μm.
Fig 4.
Systemic administration of rAAV6-μDys enhances the structural properties of EDL muscles in dystrophic mice.
(A) Treatment restored expression of dystrophin in the majority of EDL myofibers of treated mice, in contrast with no expression in the muscles of untreated mice and complete expression in the muscles of wild-type mice. (B) Dystrophin-positive myofibers of treated EDL muscles (mdx T Dys(+)) were larger in diameter (median myofiber diameter; wild-type, 54 μm; dystrophic, 43 μm; treated dystrophin-positive fibers, 52 μm). Shown is the mean and distribution (25th and 75th percentile are represented by the box, and the whiskers represent the farthest diameter of muscle fibers). (C) There was no change in central nucleation compared with the myofibers in the muscles of untreated mice. (D) EDL muscle mass was restored to wild-type values in treated dystrophic mice. n = 3 for WT; n = 3 for mdx; and n = 4 for mdx T. Error bars represent SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 5.
Systemic administration of rAAV6-μDys improves some functional properties of EDL muscles in dystrophic mice.
(A) The maximum peak isometric force-producing capacity of EDL muscles was unchanged among wild-type, untreated, and treated mice. (B) Treatment did not improve the peak isometric-force-producing capacity of EDL muscles as normalized for cross-sectional area (wild-type, 250 ± 10; mdx, 149 ± 18; treated mdx, 182 ± 12 kN/m2). (C) The muscles of treated mice show improved resistance to contraction-induced injury when subjected to consecutive eccentric contractions of 20% beyond optimal muscle length. Bars represent the mean ± SEM percentage of the initial optimal muscle contraction. Treated muscles were significantly (*P < 0.05; ***P < 0.001) protected from contraction-induced injury when compared to mdx mice. (D) A comparison of the fourth versus the first contraction demonstrates response to injury following treatment was significantly improved over untreated mdx. n = 7 for WT; n = 8 for mdx; and n = 4 for mdx T. Error bars represent SEM.
Fig 6.
Immunofluorescent staining of 9 month EDL muscle for laminin (red), dystrophin (green), and nuclei (blue).
(A) A composite cross-sectional image of an EDL muscle from a treated mdx mouse and a detail of the boxed area. (B) Control C57BL/6 EDL muscle sections demonstrate normal sarcolemmal localization of dystrophin and normal morphology. (C) Staining of EDL sections from mdx mice reveals the absence of dystrophin and demonstrates morphological characteristics of dystrophy, including variation in fiber size and abundant centrally located myonuclei. Scale bars: 100 μm (A-C); 1 mm (composite image).
Fig 7.
3D visualization of longitudinal T2w imaging.
T2w images for a normal mouse (A) and an mdx4cv mouse before (B) and 8 weeks (C), 16 weeks (D) and 24 weeks (E) after AAV vector mediated gene therapy. 3D muscle volume with necrotic regions segmented with high resolution in red. 3D T2w image acquired for the same normal mouse (F) and the four time points (G-J) for the mdx4cv mouse. Scale bar = 5 mm.
Table 2.
Values measured of MR parameters for each group, separated by muscle type and imaging time point.
Fig 8.
T2 values analyzed for TA, GA and SOL muscles.
Graphs for the quantified image values taken from the T2 modality for different muscle types of TA, GA and SOL muscles—all images were reviewed and measured. The graphs on the left column depict average T2% change values for both treated and untreated mdx mice at each time point for each muscle type while the graphs on the right display averaged T2 values for all mice including the normal (the normal mice had both pre and post time point data values averaged). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
Fig 9.
ADC values analyzed for TA, GA and SOL muscles.
Graphs for the quantified image values taken from the ADC modality for different muscle types of TA, GA and SOL muscles—all images were reviewed and measured. The graphs on the left column depict average ADC % change values for both treated and untreated mdx mice at each time point for each muscle type while the graphs on the right display averaged ADC values for all mice including the normal (the normal mice had both pre and post time point data values averaged).
Fig 10.
MTR values analyzed for TA, GA and SOL muscles.
Graphs for the quantified image values taken from the MTR modality for different muscle types of TA, GA and SOL muscles—all images were reviewed and measured. The graphs on the left column depict average MTR % change values for both treated and untreated mdx mice at each time point for each muscle type while the graphs on the right display averaged MTR values for all mice including the normal (the normal mice had both pre and post time point data values averaged). *P < 0.05.
Fig 11.
Histology was done on the left hindlimbs of all mice involved in the study. Representative T2 images are present (A) and (B). (C) Representative H&E images of an untreated mdx mouse at 1.6x and (E) is a 20x magnification of the gastrocnemius (GA) muscle (boxed area in (C)). (I) Representative Masson trichrome staining from an untreated mdx mouse at 1.6x and (F) is a 20x magnification of the GA muscle (boxed area in (C)). (D) Representative H&E images of a treated mdx mouse at 1.6x magnification and (G) is a 20x magnification of the tibialis anterior (TA) muscle (boxed area in (D)). (J) Representative Masson trichrome staining from a treated mdx mouse at 1.6x magnification and (H) is a 20x magnification of the TA muscle (boxed area in (J)).