Rapid histological quantification of muscle fibrosis and lysosomal activity using the HSB colour space

Introduction Fibrosis is a key feature of many chronic myopathic disorders, such as in the muscle-wasting condition, Duchenne muscular dystrophy. Fibrosis disrupts skeletal muscle architecture, limits muscle function, impairs regeneration and might reduce efficacy of therapeutic interventions: quantifying muscle fibrosis is thus of key value in monitoring disease progression (or response to treatment) in both pre-clinical and clinical settings. Fibrosis can be visualised histologically via staining with picrosirius red, but its quantification can be time consuming and subject to investigator bias: a rapid, reliable and user-friendly means of quantifying muscle fibrosis in histological images is currently lacking. Methods We investigated whether the Hue/Saturation/Brightness (HSB) colour-space could be used to quantify connective tissue content in picrosirius red (PSR)-stained muscle sections, using multiple healthy and dystrophic muscles, sampled from two animal models of Duchenne muscular dystrophy (the mdx mouse and the DE50-MD dog), at different ages. Results HSB-based analysis allows muscle fibres, connective tissue and slide background to be readily distinguished in PSR images using only a minimal set of parameters, and correctly identifies fibrotic accumulation under conditions where progressive fibrosis is expected. We have developed an imageJ macro that allows semi-automated high-throughput measurement of fibrotic accumulation, and then further extended our method to demonstrate its validity with another histological stain (acid phosphatase) to quantify lysosomal activity. Conclusions Histological analysis of muscle pathology is challenging and time consuming, especially with large collections of images: our methods permit fibrotic accumulation to be quantified in such collections rapidly and easily in open-source software, with minimal hardware requirements, and the underlying methodology can be readily extended to other colorimetric histopathological stains. ### Competing Interest Statement The authors have declared no competing interest.