Development and validation of a multiparametric semi-quantitative scoring system for the histopathological assessment of ischaemia severity in skeletal muscle.

Abstract

Abstract Background. Skeletal muscle is one of the most abundant and dynamic tissues of the body, with a strong regenerative capacity. Muscle injuries can occur as a result of a variety of events, including tissue ischaemia. Lower limb ischaemia occurs when there is an insufficient nutrient and oxygen supply, often caused by stenosis of the arteries due to atherosclerosis. The aim of this study was to develop and validate a multiparametric scoring tool for assessing ischaemia severity in skeletal muscle in a commonly used pre-clinical animal model.Methods. Tissue ischaemia was surgically induced in mice by ligation and excision of the femoral artery. Calf muscles were carefully dissected and prepared for histological analysis and scored for inflammation, fibrosis, necrosis, adipocyte (fat) infiltration and muscle fibre degeneration/regeneration. Tissue samples from calf muscles from 70 mice were scored by three independent blinded operators. Kendell’s coefficient of concordance (W) was used to measure agreement among the three observers and for intra-rater agreement. Spearman’s rank-order correlation was used to assess the association between the cumulative ischaemia severity score (cISS) and calf muscle weights.Results. Kendall’s W showed a very good agreement between the appraisers when scoring each individual histological feature: inflammation (W = 0.92 p-value 0.000), fibrosis (W = 0.94 p-value 0.000), necrosis (W = 0.77 p-value 0.000), adipocyte infiltration (W = 0.91 p-value 0.000) and fibre degeneration/regeneration (W = 0.86 p-value 0.000). Intra-rater agreement was also excellent (W = 0.94 or more, p-value 0.000). There was a statistically significant negative direct association between the level of muscle ischaemia damage (defined by the cISS) and the calf muscle weight (r= -0.86; R2adj.= 75.7% p-value 0.000).Conclusion. Here, we have developed and validated a new multiparametric, semi-quantitative scoring system for assessing skeletal muscle damage due to ischaemia, with excellent inter- and intra-rater reproducibility. This scoring system can be used for assessing treatment efficacy in a pre-clinical model of hindlimb ischaemia.