Histological Methods to Assess Skeletal Muscle Degeneration and Regeneration in Duchenne Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a progressive disease caused by the loss of function of the protein dystrophin. This protein contributes to the stabilisation of striated cells during contraction, as it anchors the cytoskeleton with components of the extracellular matrix through the dystrophin-associated protein complex (DAPC). Moreover, absence of the functional protein affects the expression and function of proteins within the DAPC, leading to molecular events responsible for myofibre damage, muscle weakening, disability and, eventually, premature death. Presently, there is no cure for DMD, but different treatments help manage some of the symptoms. Advances in genetic and exon-skipping therapies are the most promising intervention, the safety and efficiency of which are tested in animal models. In addition to in vivo functional tests, ex vivo molecular evaluation aids assess to what extent the therapy has contributed to the regenerative process. In this regard, the later advances in microscopy and image acquisition systems and the current expansion of antibodies for immunohistological evaluation together with the development of different spectrum fluorescent dyes have made histology a crucial tool. Nevertheless, the complexity of the molecular events that take place in dystrophic muscles, together with the rise of a multitude of markers for each of the phases of the process, makes the histological assessment a challenging task. Therefore, here, we summarise and explain the rationale behind different histological techniques used in the literature to assess degeneration and regeneration in the field of dystrophinopathies, focusing especially on those related to DMD.

Chronic Hypoxia Impairs Skeletal Muscle Repair via HIF-2α Stabilization

“…However, chronic hypoxia significantly reduced the number of proliferative MuSC (an average of 183 MuSC per TA cross-section with 18% Ki67 pos ; Figure 3A, 3B). In addition, we noticed frequent incidences of immunostaining artifacts that resembled degenerative myofibers, characteristic of an earlier regeneration stage under normoxia (Figure 3A, upper left panel of the "wildtype, hypoxia" group) [54]. Comparing the damaged TA muscles in wildtype mice to those in HIF-2α SCKO mice (with HIF-2α ablation in MuSC) exposed to identical chronic hypoxia, we found that the latter group had more proliferative MuSC (an average of 235 MuSC per TA cross-section with 25% Ki67 pos ; Figure 3A, 3B).…”

A deep learning-based automated image analysis for histological evaluation of broiler pectoral muscle

“…Automatic methods for fiber geometry analysis were shown to operate with similar accuracy and less user-introduced variability to manual quantification. Although there are several histological methods to assess skeletal muscle degeneration and regeneration ( Dubuisson et al, 2022 ), almost all were developed for mice and humans with myofibers that are considered relatively uniform compared to the highly variable terrain of broiler skeletal muscle. To our knowledge, we are the first to introduce an automated image analysis workflow designed for broiler skeletal muscle, providing a rapid and high-precision histological evaluation of tens of thousands of myofibers.…”

Extracellular Matrix Proteomics: The mdx-4cv Mouse Diaphragm as a Surrogate for Studying Myofibrosis in Dystrophinopathy

“…Dystrophin-lacking mdx-type mice exhibit varying degrees of fibrotic changes in the general musculature [46][47][48] but show severe reactive myofibrosis in the diaphragm muscle [49][50][51]. This histopathological hallmark in mdx-type diaphragm muscles is of considerable importance in the field of dystrophinopathy research [52,53], since endomysial fibrosis has been clearly established as a reliable marker that correlates well with motor loss in Duchenne patients [54]. The aged mdx-4cv mouse diaphragm, especially, shows high levels of extracellular matrix deposition [55], making it a suitable surrogate for studying reactive myofibrosis in dystrophinopaty, as discussed in this review.…”