Magnetic resonance imaging at 7T reveals common events in age-related sarcopenia and in the homeostatic response to muscle sterile injury

Abstract

Skeletal muscle remodeling in response to various noxae physiologically includes structural changes and inflammatory events. The possibility to study those phenomena in-vivo has been hampered by the lack of validated imaging tools. In our study, we have relied on multiparametric magnetic resonance imaging for quantitative monitoring of muscle changes in mice experiencing age-related sarcopenia or active regeneration after sterile acute injury of tibialis anterior muscle induced by cardiotoxin (CTX) injection. The extent of myofibrils' necrosis, leukocyte infiltration, and regeneration have been evaluated and compared with parameters from magnetic resonance imaging: T2-mapping (T2 relaxation time; T2-rt), diffusion-tensor imaging (fractional anisotropy, F.A.) and diffusion weighted imaging (apparent diffusion coefficient, ADC). Inflammatory leukocytes within the perimysium and heterogeneous size of fibers characterized aged muscles. They displayed significantly increased T2-rt (P<0.05) and F.A. (P<0.05) compared with young muscles. After acute damage T2-rt increased in otherwise healthy young muscles with a peak at day 3, followed by a progressive decrease to basal values. F.A. dropped 24 hours after injury and afterward increased above the basal level in the regenerated muscle (from day 7 to day 15) returning to the basal value at the end of the follow up period. The ADC displayed opposite kinetics. T2-rt positively correlated with the number of infiltrating leucocytes retrieved by immunomagnetic bead sorting from the tissue (r = 0.92) and with the damage/infiltration score (r = 0.88) while F.A. correlated with the extent of tissue regeneration evaluated at various time points after injury (r = 0.88). Our results indicate that multiparametric MRI is a sensitive and informative tool for monitoring inflammatory and structural muscle changes in living experimental animals; particularly, it allows identifying the increase of T2-rt and F.A. as common events reflecting inflammatory infiltration and muscle regeneration in the transient response of the tissue to acute injury and in the persistent adaptation to aging.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Study design.

Schematic representation of the experimental design of the longitudinal study realized in acutely injured mice.

Figure 2. MRI and histological features of skeletal muscles in young and old mice.

Analysis of basal levels of T2-rt and F.A. in 8-weeks old C57Bl/6 wt mice (n = 8) and 18 months old C57Bl/6 wt mice (n = 6). A quantitative comparison of T2-rt, F.A. and histology in the two groups of mice is reported for tibialis anterior (TA) muscle (panel A, B and D). Quantitative analysis of T2-rt and F.A. for gastrocnemius (G) muscle is reported in panel A, B. *p

Figure 3. Dynamic changes of MRI parameters during muscle damage and repair.

The graphs show the dynamic trends of T2-rt (panel A), F.A. (panel B) and ADC (panel C) in a longitudinal study of C57Bl/6 mice damaged with CTX and followed by MRI before and at day1, 3, 5, 7, 10, 15 and 30 after damage. Red lines indicate tibialis anterior (TA) parameters while green lines represent gastrocnemius (G) parameters. Asterisks (*) label the tibialis anterior parameters that significantly differ from control gastrocnemius muscle parameters. p

Figure 4. Muscle T2 and FA mapping during damage and repair.

Panel A: qualitative comparison between T2-rt map (T2 Map), F.A. map (DTI) and histological findings (H&E) before and at distinct time points after damage. Panel B: The dynamic trends of T2-rt and F.A. in tibialis anterior (TA) muscle are reported. Three animals/time point were studied. Asterisks (*) label the tibialis anterior parameters that significantly differ from basal parameters. p

Figure 5. Correlations between MRI and histological changes.

The graph in panel A represents correlation analysis between T2-rt and the histological score computing both muscle damage and infiltration (panel A). n = 3 for each time point. The time points considered for the correlation analysis were day 0, 1, 3, 5, 7, 10, 15, 30. The same correlation analysis was performed for T2-rt with the number of infiltrating leukocytes in tibialis anterior (TA) muscle (panel B). Each dot represents the average number of infiltrating leukocytes and the average T2-rt at distinct time points. Time points considered for the correlation analysis were day 0, 1, 3, 5, 7, 10, and 15 after CTX injection. The graph in panel C represents the correlation analysis between F.A. values and the percentage of regenerating fibers in tibialis anterior muscle. Each dot represents the average number of regenerating fibers and F.A. at distinct time points. Time points considered for the correlation analysis were day 3, 5, 7, 10, 15, 30.