estimation of total lipid volume from decellularized muscles by lipid extraction

Decellularizasyon Yoluyla İskelet Kası Yağ İnfiltrasyonunun Miktar Tayini

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.

Yazar Spotlight: İskelet Kası Yağ İnfiltrasyonunun Decellularizasyona Dayalı Miktar Tayini  

İskelet Kası Yağ İnfiltrasyonunun Decellularizasyona Dayalı Miktar Tayini

Watch this Scientific Journal Video about Estimation of Total Lipid Volume from Decellularized Muscles by Lipid Extraction at JoVE.com

Estimation of Total Lipid Volume from Decellularized Muscles by Lipid Extraction  

Lipid Ekstraksiyonu ile Decellularized Kaslardan Toplam Lipid Hacminin Tahmini

ORO ile boyanmış hücrelerden arındırılmış iskelet kaslarını, 96 oyuklu bir plakanın ayrı kuyucuklarında 200 mikrolitre izopropanole aktararak başlayın. Plakaya vurarak çözeltiyi çalkalayın. Ve hücreden arındırılmış kas süspansiyonunda kırmızı küreler görülmeyene kadar kası mikroskop altında bir pipet ucuyla mekanik olarak ezin.Yine, 75 mikrolitreyi plakanın iki temiz kuyusuna aktarmadan önce her bir oyuktaki hücre süspansiyonunu yukarı ve aşağı pipetleyerek karıştırın. Plakayı örtün ve bir spektrofotometre veya bir plaka okuyucu kullanarak yinelenen 75 mikrolitrelik kuyucukların absorbansını okuyun.

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Research

JoVE Journal

Biology

Estimation of Total Lipid Volume from Decellularized Muscles by Lipid Extraction

275 Görüntüleme. ORO ile boyanmış hücrelerden arındırılmış iskelet kaslarını, 96 oyuklu bir plakanın ayrı kuyucuklarında 200 mikrolitre izopropanole aktararak başlayın. Plakaya vurarak çözeltiyi çalkalayın. Ve hücreden arındırılmış kas süspansiyonunda kırmızı küreler görülmeyene kadar kası mikroskop altında bir pipet ucuyla mekanik olarak ezin.Yine, 75 mikrolitreyi plakanın iki temiz kuyusuna aktarmadan önce her bir oyuktaki hücre süspansiyonunu yukarı ve aşağı...

Video: Estimation of Total Lipid Volume from Decellularized Muscles by Lipid Extraction  

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.