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Neutrophils and macrophages are crucial effectors and modulators of repair and regeneration following myocardial infarction, but they cannot be easily observed in vivo in mammalian models. Hence many studies have utilized larval zebrafish injury models to examine neutrophils and macrophages in their tissue of interest. However, to date the migratory patterns and ontogeny of these recruited cells is unknown. In this study, we address this need by comparing our larval zebrafish model of cardiac injury to the archetypal tail fin injury model. Our in vivo imaging allowed comprehensive mapping of neutrophil and macrophage migration from primary hematopoietic sites, to the wound. Early following injury there is an acute phase of neutrophil recruitment that is followed by sustained macrophage recruitment. Both cell types are initially recruited locally and subsequently from distal sites, primarily the caudal hematopoietic tissue (CHT). Once liberated from the CHT, some neutrophils and macrophages enter circulation, but most use abluminal vascular endothelium to crawl through the larva. In both injury models the innate immune response resolves by reverse migration, with very little apoptosis or efferocytosis of neutrophils. Furthermore, our in vivo imaging led to the finding of a novel wound responsive mpeg1+ neutrophil subset, highlighting previously unrecognized heterogeneity in neutrophils. Our study provides a detailed analysis of the modes of immune cell migration in larval zebrafish, paving the way for future studies examining tissue injury and inflammation.

No abstract available.

Additional file 1. Supplementary material including 4 additional data tables and 4 additional figures as referenced in the results section.

Increasing scientific interest in the zebrafish as a model organism across a range of biomedical and biological research areas raises the need for the development of in vivo imaging tools appropriate to this subject. Development of the embryonic and early stage forms of the subject can currently be assessed using optical based techniques due to the transparent nature of the species at these early stages. However this is not an option during the juvenile and adult stages when the subjects become opaque. Magnetic resonance imaging (MRI) techniques would allow for the longitudinal and non-invasive assessment of development and health in these later life stages. However, the small size of the zebrafish and its aquatic environment represent considerable challenges for the technique. We have developed a suitable flow cell system that incorporates a dedicated MRI imaging coil to solve these challenges. The system maintains and monitors a zebrafish during a scan and allows for it to be fully recovered. The imaging properties of this system compare well with those of other preclinical MRI coils used in rodent models. This enables the rapid acquisition of MRI data which are comparable in terms of quality and acquisition time. This would allow the many unique opportunities of the zebrafish as a model organism to be combined with the benefits of non-invasive MRI.

p { margin-bottom: 0.25cm; direction: ltr; color: rgb(0, 0, 0); line-height: 120%; background: transparent none repeat scroll 0% 0%; }p.western { font-family: "Liberation Serif", "Times New Roman", serif; font-size: 12pt; }p.cjk { font-family: "Source Han Sans CN Regular"; font-size: 12pt; }p.ctl { font-family: "Lohit Devanagari"; font-size: 12pt; } Researchers interested in the heart have long used a wide range of imaging techniques to see, understand and quantify changes throughout heart development, repair and, in certain species, regeneration. Two major challenges in imaging the heart are the contrasting problems of high-frequency heart beating and low-frequency morphological changes. We have previously demonstrated how using prospective optical gating, in combination with light-sheet microscopy, can allow the synchronised capture of 3D images of the in vivo beating zebrafish heart. However, prospective optical gating alone is limited to snapshots of the heart at chosen target heartbeat phases and only over the scale of tens of minutes. We have now developed hybrid prospective-retrospective optical gating technologies that we are using in combination with light-sheet microscopy to enable a range of 3D+time and 3D-timelapse imaging experiments. Here we will demonstrate several key areas where we have begun to exploit these technologies to further describe and understand cardiac function and dynamics. By incorporating these non-invasive optical gating methods with micro particle image velocimetry (μPIV) we can achieve 3D+time resolved imaging of blood flow in the beating zebrafish heart. We use red blood cells as natural tracer particles from which we obtain instantaneous measurements of velocity from light-sheet images. Statistically combining and analysing this data, we can begin to both quantify and fully understand fluid-structure interaction in the developing zebrafish heart. Further, our hybrid prospective-retrospective optical gating technology allows us to carry out 24+ hour, in vivo, 3D-timelapse imaging of the computationally 'frozen' heart across developmental stages, e.g. heart looping, and throughout injury response and repair. Imaging across these timescales is not possible with prospective optical gating alone and phase-locked timelapse imaging is not possible using retrospective optical gating alone: only with our hybrid system are such longitudinal studies possible. Our hybrid prospective-retrospective optical gating system allows researchers to study and understand cardiac development and repair without the use of chemicals or optogenetics to stop or modify the natural heart beating. Combining these powerful techniques produces a hybrid prospective-retrospective optical gating microscopy framework that we are now beginning to use to fully describe cardiac development, dynamics and repair as never before accomplished. Presented at Frontiers in Bioimaging, 2018-06-28, Glasgow, UK.Yellow frames indicate animations/videos.

How do you use imaging to analyse the development of the heart, which not only changes shape but also undergoes constant, high-speed, quasi-periodic changes? We have integrated ideas from prospective and retrospective optical gating to capture long-term, phase-locked developmental time-lapse videos. In this paper we demonstrate the success of this approach over a key developmental time period: heart looping, where large changes in heart shape prevent previous prospective gating approaches from capturing phase- locked videos. We use the comparison with other approaches to in vivo heart imaging to highlight the importance of collecting the most appropriate data for the biological question.Presented at SPIE BiOS, 2018, San Francisco, California, United States. Paper 104991F.Yellow frames indicate animations/videos.

p { margin-bottom: 0.25cm; direction: ltr; color: rgb(0, 0, 0); line-height: 115%; text-align: left; background: transparent none repeat scroll 0% 0%; }p.western { font-family: "Times New Roman", serif; font-size: 12pt; }p.cjk { font-family: "Times New Roman"; font-size: 12pt; }p.ctl { font-family: "Times New Roman"; font-size: 12pt; }a:link { color: rgb(0, 0, 255); text-decoration: underline; } Two major challenges in imaging the living, beating heart are the contrasting problems of high-frequency heart beating and lo\w-frequency morphological changes. Overcoming these problems is essential to quantifying physiological changes throughout heart development, repair and, in certain species, regeneration. We have previously demonstrated how using prospective optical gating, in combination with light-sheet microscopy, can allow the synchronised capture of 3D images of the in vivo beating zebrafish heart [1]. However, prospective optical gating alone is limited to snapshots of the heart at chosen target heartbeat phases and only over the scale of tens of minutes. We have now developed an adaptive prospective optical gating system that we are using in combination with light-sheet microscopy to enable a range of 3D-timelapse imaging experiments [2]. Here we will demonstrate this technology, highlight the benefits of reduced phototoxicity and demonstrate key areas where we have begun to exploit these technologies to further describe and understand cardiac function and dynamics. Our adaptive prospective optical gating technology allows us to carry out 48+ hour, in vivo, 3D-timelapse imaging of the computationally 'frozen' heart across developmental stages, e.g. heart looping and trabeculation, and throughout injury response and repair. Imaging across these timescales is not possible with retrospective optical gating techniques, which lead to significant phototoxic responses: only with our adaptive prospective optical gating system are such longitudinal studies possible. Our adaptive prospective optical gating system allows researchers to study and understand cardiac development and repair with minimal photoresponse and without the use of chemicals or optogenetics to stop or modify the natural heart beating.Presented at Advances in Photonic Tools and Techniques for Life Sciences, Glasgow, UK.

Aims:\ud \ud We investigated which patients with heart failure (HF) should receive specialist palliative care (SPC) by first creating a definition of need for SPC in patients hospitalised with HF using patient‐reported outcome measures (PROMs) and then testing this definition using the outcome of days alive and out of hospital (DAOH). We also evaluated which baseline variables predicted need for SPC and whether those with this need received SPC.\ud Methods and results:\ud \ud PROMs assessing quality of life (QoL), symptoms, and mood were administered at baseline and every 4 months. SPC need was defined as persistently severe impairment of any PROM without improvement (or severe impairment immediately preceding death). We then tested whether need for SPC, so defined, was reflected in DAOH, a measure which combines length of stay, days of hospital re‐admission, and days lost due to death. Of 272 patients recruited, 74 (27%) met the definition of SPC needs. These patients lived one third fewer DAOH than those without SPC need (and less than a quarter of QoL‐adjusted DAOH). A Kansas City Cardiomyopathy Questionnaire (KCCQ) summary score of

Aims: \ud Heart failure may occur following acute myocardial infarction, but with the use of high-sensitivity cardiac troponin assays we increasingly diagnose patients with minor myocardial injury. Whether troponin concentrations remain a useful predictor of heart failure in patients with acute coronary syndrome is uncertain.\ud \ud Methods and results: \ud We identified all consecutive patients (n = 4748) with suspected acute coronary syndrome (61 ± 16 years, 57% male) presenting to three secondary and tertiary care hospitals. Cox-regression models were used to evaluate the association between high-sensitivity cardiac troponin I concentration and subsequent heart failure hospitalization. C-statistics were estimated to evaluate the predictive value of troponin for heart failure hospitalization. Over 2071 years of follow-up there were 83 heart failure hospitalizations. Patients with troponin concentrations above the upper reference limit (URL) were more likely to be hospitalized with heart failure than patients below the URL (118/1000 vs. 17/1000 person years, adjusted hazard ratio: 7.0). Among patients with troponin concentrations

Objective: To evaluate how selection of patients for high sensitivity cardiac troponin testing affects the diagnosis of myocardial infarction across different healthcare settings.Design: Prospective study of three independent consecutive patient populations presenting to emergency departments.Setting: Secondary and tertiary care hospitals in the United Kingdom and United States.Participants: High sensitivity cardiac troponin I concentrations were measured in 8500 consecutive patients presenting to emergency departments: unselected patients in the UK (n=1054) and two selected populations of patients in whom troponin testing was requested by the attending clinician in the UK (n=5815) and the US (n=1631). The final diagnosis of type 1 or type 2 myocardial infarction or myocardial injury was independently adjudicated.Main outcome measures: Positive predictive value of an elevated cardiac troponin concentration for a diagnosis of type 1 myocardial infarction.Results: Cardiac troponin concentrations were elevated in 13.7% (144/1054) of unselected patients, with a prevalence of 1.6% (17/1054) for type 1 myocardial infarction and a positive predictive value of 11.8% (95% confidence interval 7.0% to 18.2%). In selected patients, in whom troponin testing was guided by the attending clinician, the prevalence and positive predictive value were 14.5% (843/5815) and 59.7% (57.0% to 62.2%) in the UK and 4.2% (68/1631) and 16.4% (13.0% to 20.3%) in the US. Across both selected patient populations, the positive predictive value was highest in patients with chest pain, with ischaemia on the electrocardiogram, and with a history of ischaemic heart disease.Conclusions: When high sensitivity cardiac troponin testing is performed widely or without previous clinical assessment, elevated troponin concentrations are common and predominantly reflect myocardial injury rather than myocardial infarction. These observations highlight how selection of patients for cardiac troponin testing varies across healthcare settings and markedly influences the positive predictive value for a diagnosis of myocardial infarction.

Increasing scientific interest in the zebrafish as a model organism across a range ofbiomedical and biological research areas raises the need for the development of in vivoimaging tools appropriate to this subject. Development of the embryonic and early stageforms of the subject can currently be assessed using optical based techniques due to thetransparent nature of the species at these early stages. However this is not an optionduring the juvenile and adult stages when the subjects become opaque. MagneticResonance Imaging (MRI) techniques would allow for the longitudinal and non-invasiveassessment of development and health in these later life stages. However, the small sizeof the zebrafish and its aquatic environment represent considerable challenges for thetechnique. We have developed a suitable flow cell system that incorporates a dedicatedMRI imaging coil to solve these challenges. The system maintains and monitors a zebrafishduring a scan and allows for it to be fully recovered. The imaging properties of this systemcompare well with those of other preclinical MRI coils used in rodent models. This enablesthe rapid acquisition of MRI data which is comparable in terms of quality and acquisitiontime. This would allow the many unique opportunities of the zebrafish as a modelorganism to be combined with the benefits of non-invasive MRI.KEYWORDS: Zebrafish, MRI, solenoid, in vivo, flowcell, physiological monitoringCorresponding Author:Dr William

Neutrophilic inflammation is tightly regulated, and subsequently resolves to limit tissue damage and promote repair. When the timely resolution of inflammation is dysregulated, tissue damage and disease results. One key control mechanism is neutrophil apoptosis, followed by apoptotic cell clearance by phagocytes such as macrophages. Cyclin-dependent kinase (CDK) inhibitor drugs induce neutrophil apoptosis in vitro, and promote resolution of inflammation in rodent models. Here, we present the first in vivo evidence, using pharmacological and genetic approaches, that CDK9 is involved in the resolution of neutrophil-dependent inflammation. Using live cell imaging in zebrafish with labelled neutrophils and macrophages, we show that pharmacological inhibition, morpholinomediatedknockdown and CRISPR/cas9-mediated knockout of CDK9 enhances inflammationresolution by reducing neutrophil numbers via induction of apoptosis after tailfin injury. Importantly, knockdown of the negative regulator La-related protein 7 (LaRP7) increased neutrophilic inflammation. Our data show that CDK9 is a possible target for controlling resolution of inflammation.

CDK9 is a known regulator of cellular transcription, growth and proliferation. Small molecule inhibitors are currently being developed and assessed in clinical trials as anti-cancer drugs. The zebrafish embryo provides an ideal model to explore the effects of CDK9 inhibition in-vivo. This has not been adequately explored previously at the level of a whole organism. We have compared and contrasted the effects of pharmacological and molecular inhibition of CDK9 on somatic growth, apoptosis and cellular proliferation in zebrafish larvae between 0 to 120 hours post fertilisation (hpf) using flavopiridol, a selective CDK9 antagonist, and CDK9-targeting morpholino. We demonstrate that the inhibition of CDK9 diminishes cellular proliferation and increases apoptosis. Subsequently, it affects somatic growth and development of a number of key embryonic structures including the brain, heart, eye and blood vessels. For the first time, we have localized CDK9 at a subcellular level in whole-mounted larvae. This works shows, at a high-throughput level, that CDK9 clearly plays a fundamental role in early cellular growth and proliferation.

Glucocorticoids (GCs) in utero influence embryonic development with consequent programmed effects on adult physiology and pathophysiology and altered susceptibility to cardiovascular disease. However, in viviparous species, studies of these processes are compromised by secondary maternal influences. The zebrafish, being fertilised externally, avoids this problem and has been used here to investigate the effects of transient alterations in GC activity during early development. Embryonic fish were treated either with dexamethasone (a synthetic GC), an antisense GC receptor (GR) morpholino (GR Mo) or hypoxia for the first 120 hours post fertilisation (hpf); responses were measured during embryonic treatment or later, post-treatment, in adults. All treatments reduced cortisol levels in embryonic fish to similar levels. However, morpholino and hypoxia treated embryos showed delayed physical development (slower hatching and straightening of head-trunk angle, shorter body length), less locomotor activity, reduced tactile responses and anxiogenic activity. In contrast, dexamethasone treated embryos showed advanced development and thigmotaxis but no change in locomotor activity or tactile responses. Gene expression changes were consistent with increased (dexamethasone) and decreased (hypoxia, GR Mo) GC activity. In adults, stressed cortisol values were hypoxia pre-treatments. Other responses were similarly differentially affected. In three separate tests of behaviour, dexamethasone programmed fish appeared “bolder” than matched controls whereas Mo and hypoxia pre-treated fish were unaffected or more reserved. Similarly the dexamethasone group but not the Mo or hypoxia groups were heavier, longer and had a greater girth than controls. Hyperglycaemia and expression of GC responsive gene (pepck) were also increased in the dexamethasone group. We conclude that GC activity controls many aspects of early life growth and development in the zebrafish and that, like other species, manipulating GC status pharmacologically, physiologically or genetically in early life leads to programmable metabolic and behavioural traits in adulthood.