A new web-based method for automated analysis of muscle histology

Abstract

BackgroundDuchenne Muscular Dystrophy is an inherited degenerative neuromuscular disease characterised by rapidly progressive muscle weakness. Currently, curative treatment is not available. Approaches for new treatments that improve muscle strength and quality of life depend on preclinical testing in animal models. The mdx mouse model is the most frequently used animal model for preclinical studies in muscular dystrophy research. Standardised pathology-relevant parameters of dystrophic muscle in mdx mice for histological analysis have been developed in international, collaborative efforts, but automation has not been accessible to most research groups. A standardised and mainly automated quantitative assessment of histopathological parameters in the mdx mouse model is desirable to allow an objective comparison between laboratories.MethodsImmunological and histochemical reactions were used to obtain a double staining for fast and slow myosin. Additionally, fluorescence staining of the myofibre membranes allows defining the minimal Feret’s diameter. The staining of myonuclei with the fluorescence dye bisbenzimide H was utilised to identify nuclei located internally within myofibres. Relevant structures were extracted from the image as single objects and assigned to different object classes using web-based image analysis (MyoScan). Quantitative and morphometric data were analysed, e.g. the number of nuclei per fibre and minimal Feret’s diameter in 6 month old wild-type C57BL/10 mice and mdx mice.ResultsIn the current version of the module “MyoScan”, essential parameters for histologic analysis of muscle sections were implemented including the minimal Feret’s diameter of the myofibres and the automated calculation of the percentage of internally nucleated myofibres. Morphometric data obtained in the present study were in good agreement with previously reported data in the literature and with data obtained from manual analysis.ConclusionsA standardised and mainly automated quantitative assessment of histopathological parameters in the mdx mouse model is now available. Automated analysis of histological parameters is more rapid and less time-consuming. Moreover, results are unbiased and more reliable. Efficacy of therapeutic interventions, e.g. within the scope of a drug screening or therapeutic exon skipping, can be monitored. The automatic analysis system MyoScan used in this study is not limited exclusively to dystrophin-deficient mice but also represents a useful tool for applications in the research of other dystrophic pathologies, various other skeletal muscle diseases and degenerative neuromuscular disorders.

Sarcolemmal loss of active nNOS (Nos1) is an oxidative stress‐dependent, early event driving disuse atrophy

“…Serial consecutive cryosections were assayed using indirect peroxidase immunohistochemistry with mouse monoclonal antibodies (mAbs) specific for distinct myosin heavy chain (My) isoforms to identify fiber‐type populations, as previously described . Myofiber size was evaluated in human biopsies by measuring the minimal Feret's diameter, which allows obliquely oriented myofibers to be considered too as its value is independent of orientation , whereas in rat muscles, myofiber cross‐sectional area (CSA) was measured. All measurements were performed independently by two investigators using a computerized interactive method (ImageJ, NIH, Bethesda, MD, USA).…”

“…In fact, T8 biopsies did not show myofiber atrophy . Nevertheless, all the samples were evaluated de novo using a fiber orientation‐independent parameter , and sarcolemmal nNOS activity was investigated using modified NADPH‐diaphorase (NADPH‐d) histochemistry . The experimental animal model of muscle disuse and simulated microgravity – the tail‐suspended rat – was then used to validate the results and to inhibit in vivo nNOS activity after short unloading periods, to provide mechanistic evidence of the participation of the active enzyme in atrophy initiation of unloaded myofibers.…”

Goals and practicalities of immunoblotting and immunohistochemistry: A guide for submission to the British Journal of Pharmacology

“…We expect that blinding during image analysis stages is routinely conducted, perhaps by anonymisation of images or through use of automated computer‐based processes. Where automated methods have been used to limit bias, full details of the parameters set for this automation should be provided. Automated segmentation methods are available using freely available software such as ImageJ ( http://imagej.net/Principles) or MyoScan (Pertl et al, ). If, for some reason, these approaches cannot be undertaken, then a justification must be included in the manuscript. Often, focussed regions of antigen expression are present within the section/culture/cell.…”

SMASH – semi-automatic muscle analysis using segmentation of histology: a MATLAB application

“…The software also includes built-in image editing to manually inspect and manipulate fiber boundaries. Fully automated fiber size determination as well as fiber types and CNFs may be possible with adequate image acquisition [ 9 , 20 ]. However, these newly designed fully automated programs are not yet available [ 9 ] and/or have a significant cost [ 20 ].…”