Pig models for Duchenne muscular dystrophy - from disease mechanisms to validation of new diagnostic and therapeutic concepts.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal muscles and heart. Animal models are essential for preclinical evaluation of novel diagnostic procedures and treatment strategies. Gene targeting/editing offers the possibility of developing tailored pig models for monogenic diseases. The first porcine DMD model was generated by deletion of DMD exon 52 (DMDΔ52) in cultured kidney cells, which were used for somatic cell nuclear transfer to produce DMDΔ52 offspring. The animals resembled clinical, biochemical, and pathological hallmarks of DMD, but died before sexual maturity, thus preventing their propagation by breeding. This limitation was overcome by the generation of female heterozygous DMDΔ52 carrier pigs, which allowed the establishment of a large breeding colony. In this overview, we summarize how porcine DMD models have been used for dissecting disease mechanisms, for validating multispectral optoacoustic tomography as an imaging modality for monitoring fibrosis, and for preclinical testing of a CRISPR/Cas9 based approach to restore an intact DMD reading frame. Particular advantages of porcine DMD models include their targeted design and the rapid disease progression with early cardiac involvement, facilitating translational studies in reasonable time frames.

A scalable, clinically severe pig model for Duchenne muscular dystrophy.

Large-animal models for Duchenne muscular dystrophy (DMD) are crucial for the evaluation of diagnostic procedures and treatment strategies. Pigs cloned from male cells lacking DMD exon 52 (DMDΔ52) exhibit molecular, clinical and pathological hallmarks of DMD, but die before sexual maturity and cannot be propagated by breeding. Therefore, we generated female DMD+/- carriers. A single founder animal had 11 litters with 29 DMDY/-, 34 DMD+/- as well as 36 male and 29 female wild-type offspring. Breeding with F1 and F2 DMD+/- carriers resulted in an additional 114 DMDY/- piglets. With intensive neonatal management, the majority survived for 3-4 months, providing statistically relevant cohorts for experimental studies. Pathological investigations and proteome studies of skeletal muscles and myocardium confirmed the resemblance to human disease mechanisms. Importantly, DMDY/- pigs displayed progressive myocardial fibrosis and increased expression of connexin-43, associated with significantly reduced left ventricular ejection fraction, at 3 months. Furthermore, behavioral tests provided evidence for impaired cognitive ability. Our breeding cohort of DMDΔ52 pigs and standardized tissue repositories provide important resources for studying DMD disease mechanisms and for testing novel treatment strategies.

A new method for physical disector analyses of numbers and mean volumes of immunohistochemically labeled cells in paraffin sections.

BACKGROUND: In the neurosciences, the physical disector method represents an established quantitative stereological method for unbiased sampling and counting of cells in histological tissue sections of known thickness. Physical disector analyses are conventionally performed using plastic-embedded tissue samples, because plastic-embedding causes a comparably low and definable shrinkage of the embedded tissue, and the thickness of thin plastic sections can be determined adequately. However, immunohistochemistry protocols often don't work satisfactorily in sections of plastic-embedded tissue. NEW METHOD: Here, a new methodological approach is presented, allowing for physical disector analyses of immunohistochemically labeled cells in paraffin sections. The embedding-related tissue shrinkage is standardized by using defined tissue sample volumes and paraffin volumes, and the extent of tissue shrinkage can be determined accurately from the sample volumes prior to and after embedding. Co-embedding of polyethylene section thickness standards together with the tissue samples allows the precise determination of individual paraffin section thicknesses by spectral reflectance measurements. RESULTS AND COMPARISON WITH EXISTING METHOD(S): The applicability of the new method is demonstrated by physical disector analysis of immunohistochemically identified somatotroph cells in paraffin sections of porcine pituitary gland tissue. With consideration of individual shrinkage factors and section thicknesses, the cell numbers and mean volumes estimated in paraffin disector sections do not significantly differ from the results obtained by analyses of plastic-embedded pituitary tissue samples of the identical animals (2.4% average difference). CONCLUSIONS: The featured method enables combination of paraffin section immunohistochemistry and physical disector analyses for unbiased quantitative stereological analyses of different cell types.

Detection of collagens by multispectral optoacoustic tomography as an imaging biomarker for Duchenne muscular dystrophy.

Biomarkers for monitoring of disease progression and response to therapy are lacking for muscle diseases such as Duchenne muscular dystrophy. Noninvasive in vivo molecular imaging with multispectral optoacoustic tomography (MSOT) uses pulsed laser light to induce acoustic pressure waves, enabling the visualization of endogenous chromophores. Here we describe an application of MSOT, in which illumination in the near- and extended near-infrared ranges from 680-1,100 nm enables the visualization and quantification of collagen content. We first demonstrated the feasibility of this approach to noninvasive quantification of tissue fibrosis in longitudinal studies in a large-animal Duchenne muscular dystrophy model in pigs, and then applied this approach to pediatric patients. MSOT-derived collagen content measurements in skeletal muscle were highly correlated to the functional status of the patients and provided additional information on molecular features as compared to magnetic resonance imaging. This study highlights the potential of MSOT imaging as a noninvasive, age-independent biomarker for the implementation and monitoring of newly developed therapies in muscular diseases.