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.

Keywords: Duchenne muscular dystrophy; animal model; degeneration; dystrophin; histology; immunofluorescence; immunohistology; myofibre; regeneration; skeletal muscle.

Figure 3

Morphological analysis of the muscle fibres of a wild type vs. an mdx mouse. (A) Representative image of the quadriceps of a wild type (WT) C57BL/10SCSNJ mouse. The periphery of the myofibres was labelled using an antibody against laminin (red) and nuclei were stained with DAPI (blue) as in [63]. (B) Representative image of the same muscle of a C57BL/10SCSN-Dmdmdx J mouse with the same staining. Below both images, the corresponding fibre cross sectional area (CSA) cartography automatically obtained after the analysis of the images shown in A and B with the program MuscleJ ([55], see Table S1). The green-scale colour code indicates the myofibre CSA in μm². This program also quantifies centrally nucleated fibres (CNFs) and the number of nuclei per fibre. At the bottom of the figure, the automatically obtained cartography shows a colour code depending on the number of central nuclei quantified in each fibre (white for 0, yellow for 1, orange for 2 and red for 3 or more nuclei). Note that mdx muscle samples are characterised by an irregular distribution of fibre CSA and high numbers of CNFs. Scales: 100 μm.

Figure 4

Molecular mechanism explaining the staining of degenerating fibres with IF of IgG. (A) Dystrophic muscles have damaged myofibres with sarcolemmal ruptures or pores due to the lack of functional dystrophin. The size of these pores is big enough for serum proteins such as albumin or immunoglobulins (Igs) to enter the leaky membrane and stay in the cytoplasm. (B) To recognise these degenerating fibres, incubation of dystrophic mouse muscle cross sections with a solution containing a fluorescently tagged antibody recognising mouse IgG will yield labelling of the myofibres that contain those Igs in their cytoplasm.

Figure 5

Regulation of connective tissue regeneration in healthy and dystrophic muscles. (A) Proliferation and differentiation of FAPs into myofibroblasts or adipocytes (markers in grey cursive letters) are regulated by pro- and antifibrotic signals released mainly by immune cells. While TNFα released by M1 macrophages induces FAP apoptosis [236], anti-inflammatory cytokines such as TGF-β and connective tissue growth factor (CTGF) promote their proliferation and differentiation into ECM-producing fibroblasts [237]. Moreover, eosinophil-produced interleukin 4 (IL-4) also contributes to FAP differentiation, and while activation of the IL-4 signalling cascade promotes mouse FAP differentiation into fibroblasts and contributes to normal connective tissue remodelling, its inhibition produces their differentiation into adipocytes and is associated with increased intramuscular fat, glucose dysregulation and muscle weakening [238,239]. (B) In healthy muscle, upon injury, tight regulation of the innate immune response with balanced release of profibrotic and antifibrotic factors produces the correct regeneration and maturation of damaged myofibres and parallel reconstruction of the extracellular matrix (ECM). However, in patients with DMD and animal models of the disease, continuous cycles of degeneration and regeneration contribute to chronic inflammation, constant release of profibrotic TGF-β and dysregulation of the ECM component deposition, leading to substitution of muscle fibres with fibrotic and adipose tissues.

Figure 1

Histopathological examination in dystrophic muscles. Main events and processes that can be assessed through histological tests in muscles lacking dystrophin. These muscles go through multiple rounds of degeneration/regeneration/maturation, ultimately leading to impaired regeneration, chronic inflammation and replacement of muscle fibres with connective tissue, thus producing fibrosis, fat deposition and muscle wasting. Each of these events will be explained in the sections indicated in the figure.

Figure 2

Pathological changes seen in the rectus femoris muscle of mdx mice stained with haemotoxylin and eosin. (A) General appearance of muscle fibres in a 3-month-old wild type (WT) animal. Fibres (pink) show similar sizes and little connective tissue within the perimysium (white arrows) and the endomysium (white arrowheads). Nuclei (dark purple) are located at the periphery of the muscle fibre (black arrows) and capillaries are evident (light pink) within the endomysium (black arrowheads). (B) Clusters of necrotic fibres with basophilic inflammatory cells inside (dark purple) and fragmented sarcoplasm (*) surrounded by regular fibres and regenerative fibres (+) in an age-matched mdx mouse. (C) Mild and (D) severe inflammatory cell infiltration, respectively, phagocytising necrotic fibres (black dashed line), surrounded by fibrotic tissue ($). (E) Clusters of small rounded centrally nucleated fibres in similar, early, stage of regeneration or immaturity (black dashed line) adjacent to a group of medium-sized regenerating fibres in later stages of maturation (+). (F) In some areas of the muscle, non-centrally nucleated hypertrophic fibres (x), larger than the fibres seen in WT muscles, can be observed. Scales: 100 μm.