Development and Validation of a Multiparametric Semiquantitative Scoring System for the Histopathological Assessment of Ischaemia Severity in Skeletal Muscle

Abstract

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 preclinical animal model. Tissue ischaemia was surgically induced in mice by ligation and excision of the femoral artery. Calf muscles were carefully dissected, prepared for histological analysis, and scored for inflammation, fibrosis, necrosis, adipocyte infiltration, and muscle fibre degeneration/regeneration. Kendall’s coefficient of concordance (W) showed a very good agreement between the appraisers when scoring each individual histological feature: inflammation (W = 0.92, p ≤ 0.001 ), fibrosis (W = 0.94, p ≤ 0.001 ), necrosis (W = 0.77, p ≤ 0.001 ), adipocyte infiltration (W = 0.91, p ≤ 0.001 ), and fibre degeneration/regeneration (W = 0.86, p ≤ 0.001 ). Intrarater agreement was also excellent (W = 0.94 or more, p ≤ 0.001 ). There was a statistically significant negative association between the level of muscle ischaemia damage and the calf muscle weight and skeletal muscle fibre diameter. Here, we have developed and validated a new multiparametric, semiquantitative scoring system for assessing skeletal muscle damage due to ischaemia, with excellent inter- and intrarater reproducibility. This scoring system can be used for assessing treatment efficacy in preclinical models of hind limb ischaemia.

miR-1, miR-133a, and miR-29b and Skeletal Muscle Fibrosis in Chronic Limb-Threatening Ischaemia.

“…47 Histologically, ischaemic muscle presented severe inflammation, necrosis and fibrosis with significant loss of muscle fibre integrity, which is consistent with human data [48][49][50] . Mild inflammation in the absence of obvious disturbance of myofiber architecture was observed in the contralateral non-ischaemic limb of HLI mice, which disappear by 28 days post-HLI surgery 38 . This is an interesting observation, which may be important to take into consideration when undertaking preclinical studies using this animal model.…”

“…were purchased from Janvier Labs, (France) and were housed in the Bio-Resource Unit (BRU) at the University of Galway, with monitoring and support provided by qualified animal technicians and a veterinary surgeon. Unilateral hindlimb ischaemia (HLI) was induced in BALB/c nude mice by ligation of the femoral artery distal to the deep femoral artery, as previously described by our group 38 . The laser Doppler perfusion imager (MoorLDI V6.0, Moor Instruments, Axminster, UK) was used to confirm drop in blood flow perfusion after HLI surgery as previously described 38 .…”

“…Unilateral hindlimb ischaemia (HLI) was induced in BALB/c nude mice by ligation of the femoral artery distal to the deep femoral artery, as previously described by our group 38 . The laser Doppler perfusion imager (MoorLDI V6.0, Moor Instruments, Axminster, UK) was used to confirm drop in blood flow perfusion after HLI surgery as previously described 38 . Blood flow was measured in the soles of both feet, before (Pre-) and immediately after HLI surgery (Post-), and 7 days post-surgery, at which point, mice were humanely euthanised and their body weights were recorded.…”

miR-1, miR-133a, miR-29b and Skeletal Muscle Fibrosis in Chronic Limb-Threatening Ischaemia.

“…The mice received analgesia (0.05-0.1 mg/kg of buprenorphine 8-12h for three days, and as required thereafter) and prophylactic antibiotic (0.1 mg/kg of Enro oxacin/Baytril) was also given once post-operatively. The laser Doppler perfusion imager (MoorLDI V6.0, Moor Instruments, Axminster, UK) was used to con rm drop in blood ow perfusion after HLI surgery as previously described 40 . Blood ow was measured in the soles of both feet, before (Pre-) and immediately after HLI surgery (Post-), and 7 days post-surgery, at which point, mice were humanely euthanised.…”

“…At day 7 post-HLI, mice were euthanised by overdose of anaesthesia (225mg/kg ketamine and 1.5 mg/kg medetomidine solution injected subcutaneously) followed by cervical dislocation, and their body weights were recorded Induction of HLI and assessment of limb function. Unilateral hindlimb ischaemia (HLI) was induced in BALB/c nude mice by ligation of the femoral artery distal to the deep femoral artery, as previously described by our group 40 . Animals were anaesthetised with 75mg/kg ketamine and 0.5 mg/kg medetomidine (Domitor 10) solution injected subcutaneously.…”