decellularization of skeletal muscle

Quantificazione dell'infiltrazione adiposa del muscolo scheletrico mediante decellularizzazione

The accumulation of adipocytes between myofibers within the skeletal muscle is a prominent feature of disparate conditions, from type 2 diabetes to sarcopenia to musculoskeletal injury1,2,3,4,5,6,7. Comprehensive assessment of this intramuscular adipose tissue (IMAT) is critical to understanding the pathogenesis of these conditions, as IMAT deposition is strongly correlated with insulin resistance3,8,9,10 and poor skeletal muscle function11,12,13,14,15. Although these associations have been noted for decades, the mechanisms associated with and the origin of IMAT still remain an area of intense investigation. This is partly because most studies assessing skeletal muscle fatty infiltration have been performed in humans, where mechanistic investigations are limited16,17. However, more recently, small animal models, including mice, have been utilized to help pinpoint cellular regulation of IMAT development and signaling18,19,20. This work aims to provide a new tool for use with small animal models for qualitatively visualizing and quantifying skeletal muscle fatty infiltration.
Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT)6,21, magnetic resonance imaging (MRI)16,17,22,23, and ultrasound (US)17,24. These imaging techniques typically identify a defined region of interest (ROI) in a muscle and acquire image slices within that region, although comprehensive approaches have also been employed25,26,27. These image slices are subjected to qualitative grading6 and quantified via pixel thresholding28. Similar approaches have been utilized in animals previously29,30; however, they are costly and require access to small animal imaging systems. Spatial resolution via CT and MRI use also presents a major issue, as they are unable to delineate IMAT adipocytes from skeletal muscle fibers within a voxel and instead rely on subjective separation of primarily muscle regions and primarily IMAT regions31,32. As such, the inability to accurately identify fat or muscle tissue also presents inaccurate quantification of representative amounts of these tissues.
Due to these limitations, current techniques to assess skeletal muscle fatty infiltration in small animal models most commonly rely on histology as an inexpensive and accessible alternative33,34. Standard staining procedures, including hematoxylin and eosin (H&#38;E), oil red O (ORO), and immunostaining for adipocyte markers such as perilipin, allow for simple detection and visualization of adipocytes comprising fatty infiltration within the muscle. However, histology approaches are rarely comprehensive, and typically, IMAT qualitative or quantitative assessment is limited to a single section34. Lipid extraction has also been used to quantify total muscle lipids35; however, this technique fails to distinguish between intramyocellular lipid (IMCL) and intramuscular adipose tissue (IMAT) stores36. In summary, current methodologies to visualize and quantify fat in muscle remain limited either by financial costs or the specific detection of IMAT.
Here, we describe a detailed method for assessing skeletal muscle fatty infiltration both by qualitative visualization and multi-scale quantification. This methodology employs a simple decellularization technique that removes myocellular structures, including IMCL, but keeps the larger IMAT adipocyte-derived lipid droplets intact. Validation of the specificity of this technique has been published37, including using lipid extraction to show the depletion of IMCL with decellularization, &#181;CT to show the retention of IMAT patterning with decellularization, and histology to show the similar size distribution of IMAT lipid droplets compared with those identified with decellularization. Once decellularized, muscles can be stained with lipid-soluble dyes for qualitative visualization of the pattern and the extent of fatty infiltration and/or quantitative imaging of individual IMAT lipid droplets. Dyes can subsequently be extracted with isopropanol, and the optical density (OD) of the resulting solution can be used to estimate the IMAT lipid volume. The stringent validation of this technique has been published elsewhere37. This article provides a detailed protocol to use this methodology with mouse muscles and provides troubleshooting tips to support the adoption of this method in other applications, such as muscles from other species or other tissues.

Autore in primo piano: Quantificazione basata sulla decellularizzazione dell'infiltrazione adiposa del muscolo scheletrico  

Quantificazione basata sulla decellularizzazione dell'infiltrazione grassa del muscolo scheletrico

Watch this Scientific Journal Video about Decellularization of Skeletal Muscle at JoVE.com

Decellularization of Skeletal Muscle   

Decellularizzazione del muscolo scheletrico 

Per iniziare, ispeziona i muscoli di interesse sezionati dal topo sacrificato per l'assenza di schegge ossee o bordi frastagliati e tagliali via con forbici affilate, se necessario. Pesare i muscoli puliti utilizzando una bilancia analitica e registrare il loro peso. Dopo aver posizionato il muscolo in almeno 0,1 millilitri di SDS all'1% per milligrammo di peso, posizionare la piastra del pozzetto su uno shaker a dondolo impostato a 50-80 Hertz.Ispezionare visivamente periodicamente la soluzione SDS. Quando la soluzione diventa torbida, rimuovere la soluzione con una pipetta senza aspirare i muscoli e sostituirla con un volume uguale di SDS fresca all'1%. Quando la soluzione rimane limpida per 24 ore, rimuovere la soluzione SDS finale dai muscoli decellularizzati e sostituirla con un volume uguale di PBS.Ispezionare i muscoli decellularizzati con uno stereomicroscopio e rimuovere con cura eventuali peli o detriti attaccati al muscolo utilizzando una pinza. Rimuovere con cautela il PBS e sostituirlo con un volume uguale di formaldeide al 3,7% prima di rimettere la piastra nello scuotitore a dondolo per 24 ore.

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Biology

Decellularization of Skeletal Muscle

376 Views. Per iniziare, ispeziona i muscoli di interesse sezionati dal topo sacrificato per l'assenza di schegge ossee o bordi frastagliati e tagliali via con forbici affilate, se necessario. Pesare i muscoli puliti utilizzando una bilancia analitica e registrare il loro peso. Dopo aver posizionato il muscolo in almeno 0,1 millilitri di SDS all'1% per milligrammo di peso, posizionare la piastra del pozzetto su uno shaker a dondolo impostato a 50-80 Hertz.Ispezionare visivamente periodicamente la...

Video: Decellularization of Skeletal Muscle   

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic ...

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Biologia

Intramuscular Adipose Tissue (IMAT) Visualization with Oil Red O Staining

Visualizzazione del tessuto adiposo intramuscolare (IMAT) con colorazione Oil Red O

Estimation of Total Lipid Volume from Decellularized Muscles by Lipid Extraction

Stima del volume lipidico totale da muscoli decellularizzati mediante estrazione lipidica

Quantification of Intramuscular Adipose Tissue (IMAT) Lipid Droplet Metrics Using Confocal Imaging

Quantificazione delle metriche delle goccioline lipidiche del tessuto adiposo intramuscolare (IMAT) utilizzando l'imaging confocale