estimation of total lipid volume from decellularized muscles by lipid extraction

Quantification of Skeletal Muscle Fatty Infiltration via Decellularization

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.

Author Spotlight: Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration  

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

We are interested in crosstalk between contracting muscle and the adipocytes that form between muscle fibers in injury and disease called intramuscular adipose tissue, or IMAT. Genetic engineering in cells in mice is a powerful tool for mechanistically dissecting fat muscle crosstalk. Analysis of signaling via transcriptomics and proteomics identifies mediators, and then biomaterial based drug delivery can intervene in these pathways.One of the primary challenges is isolating and quantifying IMAT adipocytes. Especially in small animal models, the IMAT signal is washed out by whole muscle analysis like transcriptomics or lipidomics, and quantifying IMAT by noninvasive imaging suffers from similar washout, as most voxels contain a mixture of muscle and IMAT. We recently used this technique described in this article to demonstrate that IMAT directly impairs muscle contractility.With precise measures of IMAT deposition, we showed that it does not just replace contractile material, but impedes a contraction in the remaining lean muscle. This protocol addresses challenges with quantifying IMAT. The current standard to measure IMAT in small animals is single section histology, which is not comprehensive or non-invasive imaging, which is expensive and low resolution.We believe this has limited our understanding of IMAT muscle crosstalk. This technique provides a comprehensive and inexpensive method for assessing IMAT by qualitative visualization and multi-scale quantification. We hope this technique's simplicity will encourage more investigators to measure IMAT in their animal models.Also, this technique will provide a higher precision tool for the investigators interested in IMAT to uncover more subtle relationships between IMAT adipocytes and other cells.

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  

Begin by transferring the decellularized skeletal muscles stained with ORO to 200 microliters of isopropanol in the individual wells of a 96 well plate. Agitate the solution by tapping the plate. And mechanically crush the muscle with a pipette tip under a microscope until no red spheres can be seen in the decellularized muscle suspension.Again, mix the cell suspension in each well by pipetting up and down before transferring 75 microliters to two clean wells of the plate. Cover the plate and read the absorbance of the duplicate 75 microliter wells using a spectrophotometer or a plate reader.

estimation-total-lipid-volume-from-decellularized-muscles-lipid

Research

JoVE Journal

Biology

258 Views.  Washington University in St. Louis. Begin by transferring the decellularized skeletal muscles stained with ORO to 200 microliters of isopropanol in the individual wells of a 96 well plate. Agitate the solution by tapping the plate. And mechanically crush the muscle with a pipette tip under a microscope until no red spheres can be seen in the decellularized muscle suspension.Again, mix the cell suspension in each well by pipetting up and down before transferring 75 microliters to two clean wells of the plate. Cover the pl...