Evidence of induced muscle regeneration persists for years in the mouse

Abstract

Introduction: Efficient repositioning of centralized nuclei after injury has long been assumed, with centralized nuclei frequently cited as indicators of ongoing regeneration. However, reports of centralized nuclei that persist after full recovery of fiber area and muscle force production call into question the time course of nuclear repositioning.

Methods: We evaluated regeneration after cardiotoxin-induced damage in 10-week-old mice by quantifying intracellular and extracellular pathology at 2 and 94 weeks post-injection.

Results: Centrally nucleated fibers were still prevalent at 94 weeks post-injection, representing > 25% of muscle fibers. Areas with > 90% centrally nucleated fibers could still be identified. Extra-myocellular indicators of regeneration (e.g., fibrosis and fatty infiltration) also remained significantly elevated at the 94-week time-point.

Discussion: These findings indicate that not all nuclei are repositioned at the conclusion of induced muscle regeneration. Muscle Nerve 58:858-862, 2018.

Keywords: cardiotoxin; centralized nuclei; fatty infiltration; nuclear positioning; regeneration.

Figure 1.

Representative H&E stained sections from cardiotoxin (CTX) and saline (SAL) injected TA muscles at 2 weeks (2W) and 94 weeks (94W) post injection. Centralized nuclei are abundant in CTX injected muscle at both 2W (center left) and 94W (right column, each image represents the TA muscle from a different mouse in this group), but rare in SAL groups. Scale bar = 100 μm.

Figure 2.

A. The full TA muscle cross-section was reconstructed from individual H&E stained sections and centrally nucleated fibers (CNFs) marked (red dots). CNF marks are displayed within the TA perimeter outline (dotted white line) to create a visual map. Areas of high concentration of CNFs are evident in both CTX groups while CNFs are fewer and sporadically distributed in SAL groups. The left column is a representative map from the indicated group while the right column includes TA muscles from all 3 mice in the 94W CTX group for comparison. B. Quantification of CNFs as a percentage of total fibers. CTX groups had dramatically more CNFs than age-matched SAL groups, with significantly more CNFs at 2 weeks than at 94 weeks. Data are mean ± SEM.

Figure 3.

A. Picrosirius Red stained sections for visualization of extracellular matrix (red=collagen, yellow=muscle fibers). At 2 weeks, CTX muscles have uniformly increased collagen staining compared with SAL. At 94 weeks, focal areas of increased collagen staining can still be identified in the CTX group. Scale bar = 200 μm. B. Quantification of collagen area fraction from Picrosirius Red stained sections in areas of high CNF. Collagen area fraction is significantly elevated in both CTX groups compared with age-matched SAL, with significantly more area occupied by collagen at 2 weeks than at 94 weeks. C. Decellularized TA muscles stained with Oil Red O for visualization of the quantity and distribution of intramuscular adipocyte lipid (red). CTX muscles have qualitatively more lipid staining than SAL and lipid distribution appears aligned with the fiber direction while in SAL muscles, lipid staining tends to be concentrated around blood vessels. Scale bar = 1mm D. Optical density reading of extracted Oil Red O dye from constructs – a validated surrogate for lipid content. Both CTX groups have significantly elevated OD readings compared with age-matched SAL, with a further significant increase from 2 to 94 weeks. Data are mean ± SEM.