estimation of total lipid volume from decellularized muscles by lipid extraction

탈세포화(decellularization)를 통한 골격근 지방 침투의 정량화

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.

저자 스포트라이트: 골격근 지방 침투의 탈세포화 기반 정량화  

골격근 지방 침윤의 탈세포화 기반 정량화

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  

지질 추출에 의한 탈세포화된 근육의 총 지질 부피 추정

ORO로 염색된 탈세포화된 골격근을 96웰 플레이트의 개별 웰에서 200마이크로리터의 이소프로판올로 옮기는 것으로 시작합니다. 플레이트를 두드려 용액을 저어줍니다. 그리고 세포가 제거된 근육 현탁액에 빨간색 구체가 보이지 않을 때까지 현미경으로 피펫 팁으로 근육을 기계적으로 분쇄합니다.다시 말하지만, 75마이크로리터를 플레이트의 두 개의 깨끗한 웰로 옮기기 전에 위아래로 피펫팅하여 각 웰의 셀 현탁액을 혼합합니다. 플레이트를 덮고 분광 광도계 또는 플레이트 리더를 사용하여 복제된 75마이크로리터 웰의 흡광도를 읽습니다.

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JoVE Journal

Biology

Estimation of Total Lipid Volume from Decellularized Muscles by Lipid Extraction

275 조회수. ORO로 염색된 탈세포화된 골격근을 96웰 플레이트의 개별 웰에서 200마이크로리터의 이소프로판올로 옮기는 것으로 시작합니다. 플레이트를 두드려 용액을 저어줍니다. 그리고 세포가 제거된 근육 현탁액에 빨간색 구체가 보이지 않을 때까지 현미경으로 피펫 팁으로 근육을 기계적으로 분쇄합니다.다시 말하지만, 75마이크로리터를 플레이트의 두 개의 깨끗한 웰로 옮기?...

비디오: 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.