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

doi:10.21203/rs.3.rs-1972541/v1

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

https://doi.org/10.21203/rs.3.rs-1972541/v1

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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.

skeletal muscle

muscle injury

muscle regeneration

ischaemia

histology.

Skeletal muscle is the most abundant tissue in the body, comprising approximately 40% of total body weight. Structurally, skeletal muscle is a highly organised tissue, which comprises several bundles of muscle fibres surrounded by connective tissue with different names (epimysium, perimysium, endomysium) depending on the location [1] (Fig. 1). Within the skeletal muscle, there is an abundant supply of blood vessels and nerves, which are essential for maintaining the principal muscle functions such as contraction, oxygen delivery and waste removal [2].

Skeletal muscle is also a very dynamic tissue with a strong regenerative capacity in response to injury or disease. Regeneration of fibres mainly relies on resident muscle stem cells (MuSCs), also called satellite cells (SCs), which are localised between the basal lamina and the muscle fibre membrane [3]. SCs typically exist in a quiescent state but following injury SCs become activated, proliferate and give raise to myogenic precursor cells, known as myoblasts, which will differentiate into new myotubes and fuse with damaged myofibers, to ultimately mature into functional myofibers [3]. Muscle injuries can occur as a result of a variety of events [4], including direct trauma or mechanical deformation (such as muscle freezing, contusions, lacerations, and strains), indirect causes (such as ischaemia, neurological damage, underlying chronic diseases), due to genetic disorders (such as muscular dystrophies), or exposure to toxic substances (e.g. cardiotoxins, myotoxins, barium chloride). The disruption of muscle tissue homeostasis due to injury leads to a time course of events that can be summarised in three sequential but overlapping phases (reviewed in [5, 6]). During the degeneration/inflammatory phase, the rupture and necrosis of myofibers leads to an activation of a cascade of events that result in the recruitment of leukocytes, initially neutrophils and then pro-inflammatory CD68+/CD163- (M1) macrophages, involved in the removal of necrotic debris via phagocytosis [6]. Between 2 and 4 days after injury, these macrophages undergo a phenotype swich towards an ‘anti-inflammatory’ non-phagocytic CD68-/CD163+/CD206+ (M2) phenotype, that contribute to the termination of inflammation by secretion of anti-inflammatory cytokines such as interleukin-10. This is followed by a regeneration/repair phase, characterised by the activation, proliferation and differentiation of SCs into myoblasts and then myotubes to replace damaged myofibers. The final phase involves extracellular matrix remodelling and maturation of regenerated myofibers with the recovery of muscle function. In most cases, this system leads to successful muscle repair and regeneration of the injured tissue. For instance, in minor or acute muscle injuries, the activation of the muscle repair and regeneration program often leads to full functional recovery. However, if the duration, frequency and magnitude of the injury is too great, successful regeneration may not be achieved and instead injured tissue is replaced by connective tissue and fat [7].

Over the past years there has been a growing interest in understanding the cellular and molecular mechanisms underlaying skeletal muscle regeneration with the aim of developing novel therapies. In vivo pre-clinical animal models remain the optimal tools for assessing therapeutic efficacy of novel therapeutic products, which often requires a complete morphologic assessment of tissues for treatment group comparisons. Histopathological scoring is a tool by which semi-quantitative data can be obtained from tissues [8]. It usually involves scoring of a lesion’s magnitude on an ordinal scale. Several multiparametric, semi-quantitative standard scoring systems have been introduced previously for histopathological assessment of tissue lesions in different mouse models of disease [9]. However, fewer semi-quantitative scoring systems have been described in the literature for assessing skeletal muscle damage [10–14].

Here, we have developed and validated a novel multiparametric, semi-quantitative scoring system which assesses histopathological parameters known to be present in ischaemic muscle tissue [15–17], with excellent inter- and intra-rater reliability. This scoring system can be used for assessing the degree of muscle ischaemic damage and for treatment group comparison in pre-clinical efficacy studies that use the murine model of hindlimb ischaemia (HLI). The use of a reliable and standardised scoring system for a specific disease model will allow more meaningful comparison of results from different studies and laboratories.

Animals

Male 8–10 weeks old BALB/c nude mice were purchased from Envigo (United Kingdom) and were housed in a licensed preclinical facility at Biomedical Science, at the National University of Galway, with monitoring and support from qualified animal technicians and a veterinary surgeon. Ethical approval was granted by the Institutional Animal Care Research Ethics Committee. Project authorisation was granted by the Health Products Regulatory Authority in Ireland.

Induction of HLI

unilateral HLI was surgically induced in 8–10 weeks old male BALB/c nude mice. Animals were anesthetised with 75mg/kg Ketamine and 0.5mg/kg Dormitor 10 solution injected subcutaneously. The femoral triangle in the left leg was exposed through an incision in the inguinal region and the femoral artery was separated from the femoral vein and nerve by blunt dissection distal to the inguinal ligament. The femoral artery was occluded by placement of sutures at the proximal and distal regions above the proximal caudal femoral artery branch and a cut was made between the ligation sites (Fig. 2). The incision was then closed with interrupted sutures. After this, anaesthesia was partially reverted with Atipamezole (5mg/kg). Mice received analgesia (0.05–0.1 mg/kg of buprenorphine 8-12h for 3 days, and as required thereof) and prophylactic antibiotic (0.1mg/kg of Enrofloxacin/Baytril) was also given once post-operatively. Twenty-eight days after ischaemia induction, animals were humanely euthanised, and the body weight of the mice was recorded.

Dissection and tissue preparation

for each animal the posterior calf muscles (gastrocnemius, soleus and plantaris from the fascia) from ischaemic and non-ischaemic limbs were carefully dissected as previously reported [18], and their weight was recorded. To adjust for small differences in animal size, calf muscle weights were normalized to the total mouse body weight (calf muscle weight [g]/ body weight [g]). Tissue samples were fixed with 10% buffered formalin for 48h and processed for histological analysis using a tissue processor (Leica ASP3000).

Sectioning and staining

calf muscles were embedded in paraffin blocks and transverse sections of 5µm in thickness were obtained. A total of four representative transverse sections across 2 different levels of the muscle were used for analysis. Tissue sections were rehydrated through a series of ethanol grade prior to staining with Haematoxylin and Eosin (H&E) and Mallory’s trichrome staining using standard protocols.

Scoring system

Tissue samples from calf muscles from 70 mice were evaluated. For each calf muscle, four representative tissue sections across two different muscle levels were carefully observed under the microscope using 4X, 10X, 20X and 40X objectives (Olympus Bx43) by three independent operators. Histological analysis is often performed on multiple single frame images taken from the same muscle section (i.e. fields of view) but do not analyse the entire cross-sectional area. However, this assumes that histological features are homogeneous through the muscle cross section. To avoid this, the entire sample was visualised and the maximum (worst) score from across the four sections was provided as a final score for each sample. The histopathological parameters assessed included muscle inflammation, fibrosis, necrosis, adipocyte (fat) infiltration, and muscle fibre degeneration/regeneration. The system of evaluation is described in Additional file 1, which includes a detailed description and sample pictures of muscle tissue for each of the categories and levels. Each parameter was independently scored using an analogue scale of 0 to 3 using a grading sheet (see Additional file 2). Additional significant observations were scored + 1 if present. At the end, a cumulative ischaemia severity score (cISS) for each muscle sample was obtained by the sum of each individual score. We ensured that a range of different score levels (0 to 3) were present in similar frequencies in each of the categories.

Appraisers. There was 2 male and 1 female appraisers, who had good experience visualising histological samples prior to performing the scoring. One appraiser is a qualified consultant pathologist (S.O.H), who would have a wide range of experience in scoring histological specimens from many different tissues. The other two appraisers (C.SN and M.C) are scientists who have experience in observing histological specimens in this study area. The same three appraisers were blinded for the study and evaluated each sample independently. In addition, one observer (C.SN) re-scored the 70 samples following a 4-month washout period for intra-observer analysis. To ensure that the order of data collection would not influence results, each appraiser evaluated all samples in a random order using a pre-defined agreement analysis worksheet.

First consensus scoring meeting. Prior to commencing the study, the three appraisers met to discuss the parameters and criteria of this scoring system. The definitions for each morphological parameter were reviewed and any discrepancy or lack of clarity in the scoring definition was addressed. Several sample slides were examined by the three appraisers and a score was given. This was to ensure that appraisers had some level of agreement prior to commencing the study.

Final conclusion meeting. A meeting between the three appraisers was arranged at the end of the study to discuss the results. As in some cases appraisers differed in their scores,

it was agreed that it was most appropriate to use the median value of the three scores given to each of the individual histological parameters when calculating the cISS. This value was then used to investigate the association between the cISS with a clinical parameter of disease severity such as calf muscle weight.

Statistical analysis. Kendall’s coefficient of concordance (W) (a nonparametric test of rank ordering concordance) was used to measure the degree of association of ordinal assessments made by the three appraisers [19]. Kendall W accounts for the order of the ratings, for instance, a classification of an observation among raters which differs by 2 points is considered more serious than a classification which only differs by 1 point (e.g. severe (score 3) vs mild (score 1) is considered worse than severe (score 3) vs. moderate (score 2)). A perfect agreement is indicated by values of 1, while no agreement is indicated by values of 0. Intra-rater agreement was assessed similarly. Spearman rank-order correlation and linear regression analysis was performed between muscle weights and the cISS. All statistical analyses were performed in Minitab 17 statistical software. Statistical significance was assigned at p-value ≤ 0.05.

Score tool reliability: inter-rater and intra-rater variation for scoring histopathological parameters.

Inter-rater reliability was demonstrated on the basis of a significant level of rank ordering concordance using Kendall’s W [20]. For all the histopathological parameters scored, we estimated inter-rater reliability to be significantly high (Table 1). In most cases, disagreements between the three appraisers were no more than 1 point of difference (e.g. one appraiser gave a score of 3 and another appraiser gave a score of 2). Disagreements among appraisers of > 2 points were minimal (Table 1). Complete disagreements (three appraisers completely disagreed in their scores) were also minimal, i.e. agreement among two appraisers was almost 100% in all the cases (Table 1). Necrosis was the parameter with highest % of agreement, but with lower Kendall’s W. This may be due to having more scores that differed more than 1 point (5/70). Apart from the 5 general histopathological findings assessed, other significant histopathological findings were reported if significant. For instance abnormal haemorrhage was reported in 2/70 samples. Finally, intra-rater reliability was also found to be excellent for all the parameters scored, with Kendall’s W of 0.94 or greater (Table 2).

Table 1

Kendall’s W analysis reflecting inter-rater reliability assessment of histopathological features between three appraisers.
Histopathological parameter	% Complete agreement (95% CI)*	% Agreement between 2 appraisals (95% CI)	% Disagreement > 2 scores (95% CI)	Kendall’s W*
Inflammation	44.3 (32.4; 56.6)	97.1 (90.1; 99.7)	5.7 (1.6; 14.0)	0.923
Fibrosis	51.4 (39.2; 63.6)	100 (94.9; 100)	0.0 (0.0; 5.1)	0.942
Necrosis	58.6 (46.2; 70.2)	98.6 (92.3; 100)	7.1 (2.3; 15.9)	0.771
Degeneration/ Regeneration	47.1 (35.1; 59.5)	94.3 (86.0; 98.4)	7.1 (2.3; 15.9)	0.863
Adipocyte Infiltration	41.4 (29.8; 53.83)	98.6 (92.3; 100)	5.7 (1.6; 14.0)	0.914

CI: confidence interval; W: Kendall’s coefficient of concordance; *all appraisers’ assessments agree with each other. P-values were calculated based on Kendall’s W scores from complete agreements.

Table 2

Kendall’s W analysis reflecting intra-rater reliability assessment of histopathological features by one appraiser after a 4-month washout period.
Histopathological parameter	% Agreement (95% CI)	Kendall’s W
Inflammation	82.7 (71.9; 90.8)	0.978
Fibrosis	85.7 (75.3; 92.9)	0.977
Necrosis	90.0 (80.5; 95.9)	0.951
Degeneration/Regeneration	77.1 (65.5; 86.3)	0.943
Adipocyte Infiltration	88.6 (78.7; 94.9)	0.977

CI: confidence interval; W: Kendall’s coefficient of concordance.

Validation of tissue pathology: clinical measure of disease severity.

Once appraisers scored all the histopathological parameters, the median value for each parameter was calculated. A cISS per muscle sample was calculated by adding up all the individual scores. The cISS score ranged from 0 (score of 0 in all individual parameters) to 15 (score of 3 in all individual parameters). Additional points (+ 1) were added to the final score for every other significant histopathological finding (i.e. presence of abnormal haemorrhage). This total score was used to investigate the association between cISS and an objective clinical parameter of disease severity. Calf muscle weight can be a relevant parameter of tissue pathology. We, and others, have observed significant muscle mass loss after ischaemic injury, most likely secondary to muscle necrosis and fibrosis [21, 22]. We hypothesised that the degree of muscle ischaemic damage would be correlated with the level of muscle weight loss (i.e. higher cISS would be correlated with lower calf muscle weights). In order to investigate the relationship between the cISS score and calf muscle weight, a Spearman rank-order correlation analysis was performed. We found a statistically significant strong negative relationship between cISS scores and calf muscle weights with r-0.86 and 95%CI of -0.92 to -0.77 (p-value 0.000). A linear regression analysis indicated that there was an indirect linear relationship between these two parameters (R²adj 75.7%, p-value 0.000) (Fig. 3).

Ischaemia in skeletal muscle occurs due to insufficient supply of nutrients and oxygen. In patients with peripheral arterial disease (PAD), ischaemia of distal muscles occurs due to the narrowing or occlusion of peripheral arteries due to the build-up of atherosclerotic plaques [23]. Ischaemic calf muscle in PAD patients is characterised by several histopathological changes such as local inflammation, increased fibrosis and inter- and intra-muscle adipocyte content, muscle fibre atrophy, and impaired metabolic function, among others [15, 24]. The most severe manifestation of PAD, namely critical limb ischaemia (CLI), is characterised by rest pain, non-healing ulcers, gangrene, tissue loss and death [23]. In the past years, there has been an increased interest in developing novel therapeutic products aiming to improve tissue perfusion and/or restoration of tissue function in these patients [25]. The mouse model of HLI is considered the most clinically relevant pre-clinical model of PAD, and especially CLI [26], and has been largely used to assess pre-clinical efficacy of cell therapy products such as mesenchymal stromal cells (MSCs) [27–31]. In most cases, a complete morphologic assessment of tissue using a range of histological techniques is performed for treatment group comparisons. However, no standardised tools are used for the assessment of the degree of skeletal muscle damage across all these studies and there exists great variation amongst the histological techniques and quantification methods employed. This may impair inter-study comparability. Semi-quantitative histopathology scoring systems have been previously used to obtain semi-quantitative data from tissue samples [8]. To our knowledge, there are a small number of studies that have described a semi-quantitative scoring system as part of their methodology to assess the level of skeletal muscle damage [10–14]. McCormack et al. described an absolute injury score (i.e. percentage of injury) calculated by dividing the number of injured myocytes by the total myocytes scored within 15 photographed fields (approximately 1000 fibres per animal) [13]. While this scoring system has the advantage of providing quantitative data (e.g. ratio) from a tissue, it does not provide information about other important histopathological parameters such the level of inflammation, fibrosis or others. Erkanh et al. described a histological damage score tool for histological evaluation of tissue sections based on a severity level (0: Normal, 1: Mild, 2: Moderate, 3: Severe) of disorganisation and degeneration of muscle fibres and inflammatory cell infiltration [12]. However, no scoring definitions are provided for each category to guide the observer when performing the scoring [12]. This is likely to result in a reduction of intra and inter-rater repeatability. Carter et al. described a more comprehensive skeletal muscle histopathology scoring system that scores a lesion’s magnitude on an ordinal scale from 0 to 10 [10]. While a score definition is provided for each category to guide the observer during the scoring, each category scores several parameters at once (e.g. the severity of mononuclear cell infiltration, polynuclear cell infiltration, level of fibre necrosis, presence of haemorrhage). In cases when a tissue has multiple lesions, it is preferable to assign its own appropriate scoring system for each parameter [32]. This approach is more sensitive, and results in higher inter-rater repeatability. Also, a large number of ordinal scores may cause difficulty or ambiguity during score assignment and is prone to have reduced repeatability [32]. Indeed, Smajović et al. reported a simplified version of the Carter et al. scoring system to include only 4 levels [11]. Finally, Hardy et. al. described a morphometric semi-quantitative analysis to assess the extent of muscle injury in four different injury models at different timepoints. In this case, a symbol ‘+’ with more or less +’s is given to each morphological parameter depending on the % of tissue affected [14].

Here, we have developed and validated a new semi-quantitative histopathological scoring tool to assess skeletal muscle damage due to ischaemia with excellent intra- and inter-rater reliability. We believe our scoring tool has many advantages over the scoring systems described above. We have used the ‘splitter’ approach, where we have assigned a specific score system to different individual parameters (e.g. inflammation, fibrosis, necrosis, etc.), as it is the preferred approach to use when multiple lesions are present in the same tissue [32]. We also used an ordinal scale with a maximum of 4 score levels for each parameter to describe the severity of the lesion, as it has been previously suggested that 4–5 score levels may be optimal for maximising detection and repeatability [32]. In addition, we have provided a comprehensive description of each score level including representative examples to guide the raters and enhance inter-observer repeatability (See Additional file 1). At the end, a cISS can be calculated by addition of all the individual scores. This can give an overview of the level of skeletal muscle ischaemic damage when taking in consideration all the histopathological parameters examined in the sample. An interpretation of the severity of ischaemic severity in muscle based on the cISS is proposed in Table 3. Also, here we can confirm that there is a similar distribution of each of the proposed levels across all the 70 scored samples, which is considered important when designing a new scoring system (Table 3) [32].

Table 3

Interpretation of the overall level of muscle ischaemia damage samples based on cISS.
cISS	Level of muscle ischaemia damage	Distribution of levels in scored samples
0–1	Normal	24.3%
2–6	Mild	27.1%
7–10	Moderate	24.3%
11–15+	Severe	24.3%

cISS: cumulative ischaemia severity score

How To Use This Tool

We propose that samples should be scored by a minimum of two independent appraisers blinded to the treatments. While adding more appraisers may result in a reduction of the % of agreement, the calculation of cISS may become less biased when there are disagreements, as the cISS can be calculated using the median scores among three appraisers. In the cases where two appraisers score the samples, the two appraisers must discuss the disagreements and agree a final score. Furthermore, if the appraisers do not have experience in this study-area, we recommend achieving some level of training prior to starting the scoring. The National Toxicology Program (NTP) Nonneoplastic Lesion Atlas is a publicly available web-based resource containing images, terminology and guidelines for diagnosis of nonneoplastic lesions in rodents. Overall, the lowest percentage of agreement for a specific morphological category scored by two selected appraisers was 51% (Table 4). Therefore, we recommend reaching, at least, 50% agreement in scoring each morphological parameter among two selected appraisers prior to commencing the study. This will enhance inter-rater reliability. When reporting the results, we propose to report both, individual scores and cISS. Average scores (median) of the different experimental groups can be then compared using non-parametric statistical tests. Thuilliez et. al. work has compiled a glossary of definitions and pictorial examples of histopathological lesions often observed in skeletal muscle of rodents after intramuscular injection that may guide the researchers when reporting histological findings [33]. Finally, while this is a simple tool that requires the use of two routinary histological stains such as H&E and Mallory trichrome stain (Masson’s Trichrome staining is also valid), it can complement other staining and assessments that may be required and be specific to each study aim.

Table 4

Percentage of agreement among two selected appraisers.
Rater Comparison	Rater 1 vs 2	Rater 1 vs 3	Rater 2 vs 3
Inflammation	65.7 (53.4; 76.6)	71.4 (59.4, 81.6)	51.4 (39.2; 63.6)
Fibrosis	70.0 (57.9; 80.0)	71.4 (59.4, 81.6)	58.6 (46.2; 20.2)
Necrosis	74.3 (62.4; 83.9)	55.7 (64.0, 85.2)	62.9 (51.5; 74.2)
Degeneration/Regeneration	65.7 (53.4; 76.6)	65.7 (53.4; 76.6)	57.2 (44.7; 68.9)
Fat Infiltration	58.6 (46.2; 20.2)	70.0 (57.9; 80.0)	54.3 (41.9; 66.3)
Results are expressed as % agreement (95% confidence interval)

Limitations

We caution that Kendall’s W does not imply that any particular appraiser is correct or incorrect, simply whether observers agreed or not. We, however, have validated this tool using a clinical measure of disease severity in these mice, such as calf muscle weight. Muscle wasting and weakness is a common symptom in PAD [34]. We, and others have observed muscle mass loss after ischaemia in rodent, which is most likely secondary to muscle necrosis and fibrosis and can return to baseline levels with regeneration [21, 22]. Spearman rank-order correlation analysis showed a strong and statistically significant negative relationship between the cISS and calf muscle weight. This convincing finding lends credence and scientific merit to our scoring method, which shows a good representation of the pathology of the tissue.

However, there are some parameters that must be taken in consideration prior to using this tool, including the endpoint of the study at which muscles are scored, and the animal strain. Our in vivo study endpoint and assessment has been optimised at 28 days after ischaemia surgery. At this timepoint we have observed significant muscle mass loss compared to the non-ischaemic limb, and also muscle gain due to regeneration, which allows treatment group comparisons (unpublished observations, Sanz-Nogués et al). However, this study endpoint may differ for other studies. One should take caution as the severity of lesions can differ when scoring samples at different endpoints. Also, correlation analysis between skeletal muscle weight and cISS must be done with caution when other timepoints are used, as this was not included in our evaluation. On the other hand, it is widely acknowledged that there are differences between inbred strains of mice to surgically induced HLI [35–38]. For instance, C57BL/6 mice showed significantly better collateral artery formation and limb perfusion, and less tissue damage than BALB/c mice in response to HLI [36–38]. BALB/c mice have significantly lower expression of vascular endothelial growth factor A (VEGF-A), poor collateral artery formation, reduced limb perfusion and impaired recovery [36–38], as well as significantly greater myofiber atrophy, greater apoptosis and attenuated myogenic regulatory gene expression than C57BL/6 mice [35]. In cases when different animal strains and/or study endpoints are utilised, we recommend first evaluating whether the range of lesions present in the samples can be assessed using the lesion severity proposed in this scoring system for each parameter evaluated.

Here, we have developed and validated a novel multiparametric semi-quantitative scoring system that can be used to evaluate the level of ischaemia-induced muscle damage with excellent inter- and intra-rater reliability. We propose that this tool can be used for treatment comparisons in pre-clinical animal models such as the HLI mouse model. Nevertheless, we anticipate that the use of this tool can be extended for assessing muscle damage due to other injuries, as the process of muscle repair and regeneration, as well as the histopathological features evaluated here, can be similar. As publications on validated multiparametric semi-quantitative scoring system for muscle injury are rare we believe that this article is an important contribution to the very limited database of published scoring systems.

cISS

cumulative ischaemia severity score

CLI

critical limb ischaemia

H&E

haematoxylin & eosin

HLI

hindlimb ischaemia

MuSCs

muscle stem cells

MSCs

mesenchymal stromal cells

PAD

peripheral arterial disease

SCs

satellite cells.

VEGF-A

vascular endothelial growth factor A

Ethics approval

All animal experiments were carried out in compliance with the Directive 2010/63/EU. Ethical approval was granted by the Animal Care Research Ethics Committee (ACREC) at the National University of Galway and appropriate individual and project authorisations were granted by the Health Products Regulatory Authority in Ireland (AE19125/P081 and AE19125/P46).

Consent for publication

Not applicable.

Availability of data and materials

The authors declare that the data supporting the findings of this study are available within the article, and its supplementary information files. The raw data generated during and/or analysed during the current study are available from the corresponding author (TOB) upon request.

Competing Interests

T.OB is a founder, director, and equity holder in Orbsen Therapeutics Ltd. The other authors declare no competing interests.

Funding

This publication has emanated from research supported in part by Science Foundation Ireland (SFI) and the European Regional Development Fund (ERDF) under grant number 13/RC/2073_P2) (CSN, TOB), Irish Research Council (IRC) (EPSPD/2016/38) (CSN), and Science Foundation of Ireland (SFI) grant number 18/IF/6252.

Authors’ Contribution

C.SN, M.C and X.Z carried out animal work, tissue processing and staining. C.SN, M.C, S.O.H carried out tissue sample scoring, analysis and interpretation of results. C.SN performed statistical analysis. C.SN, M.C, S.O.H, X.Z and T.OB conceived the study, participate in the design of the study and helped to draft the manuscript. Final manuscript was read and approved by all the authors.

Acknowledgements

Not applicable

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Competing interest reported. T.OB is a founder, director, and equity holder in Orbsen Therapeutics Ltd. The other authors declare no competing interests.

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

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