A User-Friendly Approach for Routine Histopathological and Morphometric Analysis of Skeletal Muscle Using CellProfiler Software

Valerio Laghi,Alessio Torcinaro,Francesca De Santa,Valentina Ricci

doi:10.3390/diagnostics12030561

Abstract

Adult skeletal muscle is capable of active and efficient differentiation in the event of injury in both physiological and pathological conditions, such as in Duchenne muscular dystrophy (DMD). DMD is characterized by different features, such as continuous cycles of degeneration/regeneration, fiber heterogeneity, chronic inflammation and fibrosis. A well-defined and standardized approach for histological and morphometric analysis of muscle samples is necessary in order to measure and quantify specific regenerative parameters in myopathies. Indeed, non-automatic methods are time-consuming and prone to error. Here, we describe a simple automatized computational approach to quantify muscle parameters with specific pipelines to be run by CellProfiler software in an open-source and well-defined fashion. Our pipelines consist of running image-processing modules in CellProfiler with the aim of quantifying different histopathological muscle hallmarks in mdx mice compared to their wild-type littermates. Specifically, we quantified the minimum Feret diameter, centrally nucleated fibers and the number of macrophages, starting from multiple images. Finally, for extracellular matrix quantification, we used Sirius red staining. Collectively, we developed reliable and easy-to-use pipelines that automatically measure parameters of muscle histology, useful for research in myobiology. These findings should simplify and shorten the time needed for the quantification of muscle histological properties, avoiding challenging manual procedures.

Highlights

Skeletal muscle is a highly dynamic and plastic organ, able to respond to environmental changes and characterized by complete functional recovery upon perturbations such as endurance exercise, overload or muscle injury [1]
We present the data obtained by using a pipeline, which we named MyoProfiler, to measure minimum Feret diameter (MFD), centrally nucleated fibers (CNFs), peripherally nucleated fibers (PNF), cell localization and the number of macrophages in muscle sections from mdx mice compared with wild-type ones
Once we had successfully identified and segmented both muscle fibers and nuclei, we focused on CNF and PNF detection

Summary

Introduction

Skeletal muscle is a highly dynamic and plastic organ, able to respond to environmental changes and characterized by complete functional recovery upon perturbations such as endurance exercise, overload or muscle injury [1]. The dystrophindeficient mouse (C57BL/10ScSn-DMDmdx /J), referred to as mdx mouse, represents the most frequently used animal model to study DMD, the pathology is less severe in this animal compared to DMD patients [5,6]. In both cases, this fatal myopathy leads to continuous cycles of degeneration and regeneration, resulting in high heterogeneity in fiber size and distribution as well as an increase in centrally nucleated fibers (CNFs) [7]. Another key feature of DMD is chronic inflammation, resulting in persistent inflammatory cell infiltration, mainly macrophages, upon the degeneration of myofibers, accompanied by irreversible extracellular matrix deposition (ECM), leading to fibrosis [8,9]

Methods

Discussion

Conclusion