Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Abstract

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.

Figure 1:. Example Oil Red O (ORO) staining of decellularized muscles:

ORO-stained muscles 14 days post injection with saline (SAL), cardiotoxin (CTX) or glycerol (GLY). Mouse extensor digitorum longus (EDL) and tibialis anterior (TA) muscles have little IMAT (red spheres) with saline treatment (A), but accumulate IMAT in response to CTX (B) and GLY (C) treatment. Note complete decellularization and ORO washout evidenced by distinct ORO-positive lipid droplets in a transparent white muscle background. Scale bars = 500 μm.

Figure 2:. Examples of poor ORO staining results:

Incomplete decellularization or incomplete ORO clearance leads to semi-opaque pink/red background. Compared with the transparent white background of fully decellularized mouse diaphragm muscle (A), incomplete decellularization is characterized by light pink/red fiber tracks (B), and incomplete ORO clearance is characterized by diffuse pink/red background (C). Scale bars: upper panels, 1 mm; lower panels, 500 μm.

Figure 3:. Examples of individual lipid droplet identification with fluorescent BODIPY staining and confocal microscopy:

Individual BODIPY stained lipid droplets can be identified and quantified in decellularized muscles using confocal microscopy. Individual slices through a confocal stack show lipid droplets in plane as bright green ellipses and lipid droplets out of plane as fainter shapes (A; blue arrows). Thresholding combined with watershed object segmentation and region of interest (ROI) identification can map BODIPY-stained ROIs (B). Thresholding may miss some fainter lipid droplets (B; pink double arrow), requiring identification by hand (C). Watershed segmentation may group several lipid droplets together (B; red asterisk), requiring deletion of the ROI and re-estimation by hand (D). The same lipid droplet will be identified in multiple slices requiring image registration (E) to delete the duplicate ROIs. Scale bars = 100 μm.