A novel method for the quantification of fatty infiltration in skeletal muscle

Nicole K Biltz,Gretchen A Meyer

doi:10.1186/s13395-016-0118-2

Abstract

BackgroundFatty infiltration of the skeletal muscle is a common but poorly understood feature of many myopathies. It is best described in human muscle, where non-invasive imaging techniques and representative histology have been optimized to view and quantify infiltrating fat. However, human studies are limited in their ability to identify cellular and molecular mechanisms regulating fatty infiltration, a likely prerequisite to developing targeted interventions. As mechanistic investigations move to small animals, studies may benefit from new or adapted imaging tools optimized for high resolution and whole muscle quantification.ResultsHere, we describe a novel method to evaluate fatty infiltration, developed for use with mouse muscle. In this methodology, muscle cellular membranes and proteins are removed via decellularization, but fatty infiltrate lipid is spared, trapped in its native distribution in a transparent extracellular matrix construct. This lipid can then be stained with visible or fluorescent dyes and imaged. We present three methods to stain and evaluate lipid in decellularized muscles which can be used individually or combined: (1) qualitative visualization of the amount and 3D spatial distribution of fatty infiltration using visible lipid soluble dye Oil Red O (ORO), (2) quantitative analysis of individual lipid droplet metrics (e.g., volume) via confocal imaging of fluorescent lipid soluble dye boron-dipyrromethene (BODIPY), and (3) quantitative analysis of total lipid content by optical density reading of extracted stained lipid.This methodology was validated by comparing glycerol-induced fatty infiltration between two commonly used mouse strains: 129S1/SvlmJ (129S1) and C57BL/6J (BL/6J). All three methods were able to detect a significant increase in fatty infiltrate volume in the 129S1 muscle compared with that in BL/6J, and methods 1 and 2 additionally described a difference in the distribution of fatty infiltrate, indicating susceptibility to glycerol-induced fatty infiltration is strain-specific.ConclusionsWith more mechanistic studies of fatty infiltration moving to small animal models, having an alternative to expensive non-invasive imaging techniques and selective representative histology will be beneficial. In this work, we present a method that can quantify both individual adipocyte lipids and whole muscle total fatty infiltrate lipid.

Highlights

Fatty infiltration of the skeletal muscle is a common but poorly understood feature of many myopathies
We have developed a novel method for qualitatively and quantitatively analyzing fatty infiltration in muscle by staining and imaging lipid retained in decellularized constructs
This methodology can provide a comprehensive image of the 3D pattern of fatty infiltration in muscle and a quick quantification via Oil Red O (ORO) staining, without the need for access to a cryostat, computed tomography (CT), or magnetic resonance imaging (MRI)

Summary

Introduction

Fatty infiltration of the skeletal muscle is a common but poorly understood feature of many myopathies It is best described in human muscle, where non-invasive imaging techniques and representative histology have been optimized to view and quantify infiltrating fat. Despite its prevalence and association with dysfunction, little is known about the signaling mechanisms that regulate this process of fatty infiltration, and considerable debate continues about its origin and consequences (reviewed in [21]). This is partly due to the fact that the vast majority of studies in this area have been in humans, where causation is difficult to demonstrate. The size scale of small animal models frequently allows and demands different experimental methodology than what is developed for human studies

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