Non-invasive tracking of disease progression in young dystrophic muscles using multi-parametric MRI at 14T

Abstract

In this study, multi-parametric magnetic resonance imaging (MRI) was conducted to monitor skeletal muscle changes in dystrophic (mdx4cv) and age-matched control (C57BL/6J) mice starting at 3 weeks of age. The objective of this study was to evaluate and characterize changes in muscle tissue characteristics of hind limbs in young, dystrophic mice using MRI. Mdx4cv (n = 25) and age-matched C57BL/6J (n = 5) were imaged at 3, 5, 7, 9, and 11 weeks of age. Multiple MR measurements were taken from the tibialis anterior, gastrocnemius, and soleus muscles. There were significant differences between dystrophic and control groups for all three muscle types when comparing transverse relaxation times (T2) in lower hind limb muscles. Additionally, fractional anisotropy, radial diffusivity, and eigenvalue analysis of diffusion tensor imaging also demonstrated significant differences between groups. Longitudinal relaxation times (T1) displayed no significant differences between groups. The earliest time points in the magnetization transfer ratio measurements displayed a significant difference. Histological analysis revealed significant differences in the tibialis anterior and gastrocnemius muscles between groups with the mdx mice displaying greater variability in muscle fiber size in later time points. The multi-parametric MRI approach offers a promising alternative for future development of a noninvasive avenue for tracking both disease progression and treatment response.

Fig 1. Cross-sectional area for GA, SOL, and TA muscles.

Comparisons of muscle maximum cross-sectional area (CSA_max) between mdx^4cv and control groups for GA, SOL, and TA muscles at each time point.

Fig 2. T₂, T₁, and MTR values analyzed for TA, GA, and SOL muscles.

Graphs displaying the average longitudinal values of the mdx^4cv and control mice muscles in the T₂, T₁, and MTR measures. T₂ values for the mdx^4cv mice were significantly higher at all time points versus age-matched controls. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. Significant higher T₂ values were detected in ctrl mice at 3 weeks of age compared to other time-points. ‡p ≤ 0.05.

Fig 3. Representative T₂, T₁, and MTR color maps acquired from lower hind limbs of a mdx^4cv mouse followed longitudinally over 11 weeks.

The T₂, T₁ and MTR maps are overlaid onto T₂ weighted images to display the change in values for each time point.

Fig 4. Fractional Anisotropy (FA), Mean Diffusivity (MD) and Radial Diffusivity (RD) of TA, GA, and SOL muscles.

Graphs displaying average longitudinal FA, MD, and RD values for mdx^4cv and control muscles. FA values from GA muscles of mdx^4cv mice are significantly lower at all time points versus age-matched controls. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Fig 5. Eigenvalues analyzed for TA, GA, and SOL muscles.

Graphs displaying average longitudinal eigenvalues λ₁, λ₂, and λ₃ of mdx^4cv and control mouse muscles. λ₁ values for mdx^4cv mice were significantly lower at the 3- and 5-week time points versus age-matched controls in all muscles (top panels), while λ₃ values for mdx^4cv mice were significantly higher at 9 and 11 weeks (bottom panels). λ₂ values were not significantly different between groups until the 11-week time point in the TA muscle (middle panels). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Fig 6. Average muscle fiber area, and λ₃ versus average muscle fiber area for TA and GA muscles.

Individual muscle fiber areas were measured for the TA and GA muscles and then averaged for comparison between groups. Mdx^4cv mice exhibit significantly reduced average individual muscle fiber area for both TA and GA muscles. This data was then plotted against the λ₃ values for correlation of diffusivity across single muscle fibers and average fiber size. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Fig 7. Histogram Analysis for individual muscle fiber area of TA and GA muscle fibers in 11-week old mdx^4cv and control mice.

Muscle fiber areas measured for the TA and GA muscle differences between groups. Area values were binned into intervals of 500 μm² and overlaid for both frequency and distribution comparisons.

Fig 8. Longitudinal tissue analysis for mdx^4cv mice at each timing point (3, 5, 7, 9, and 11 weeks of age) at 10x and 20x magnification.

Representative H&E histology images of the TA muscle in mdx^4cv mice. Images are taken from the same area of muscle for both 10x and 20x images (scale bar represents 100 μm).

Fig 9. 11-week comparison of TA and GA muscles of control and mdx^4cv mice (both H&E and trichrome staining) at 20x magnification.

Comparisons of representative histology images for both groups of animals (scale bar represents 100 μm).