Validation of the biological responses of reference drugs in the zebrafish embryo by electrocardiographic analysis and by novel phenotyping tools

Drug toxicities represent a major problem in drug discovery and development; therefore there is a push to develop new technologies to detect these early on. In this thesis I investigated the utility of zebrafish embryos and larvae in evaluating the biological activity of novel compounds and developed new methods for assaying the potential toxic effects of drugs $in$ $vivo$. An electrocardiogram (ECG) recording set-up for zebrafish embryos and larvae was developed to assay drug-induced cardiotoxicity. The set-up was validated by testing drugs known to induce cardiotoxicity in humans in zebrafish larvae. The results obtained were in agreement with those documented in humans demonstrating the utility of the zebrafish larva in detecting drug-induced cardiotoxicity. The zebrafish embryo was also found to be a useful model for probing the biological activity of novel and marketed compounds providing an insight into the relationship between chemical properties and biological effects. Additionally, the assessment of the anti-inflammatory activity of a set of reference drugs revealed that the zebrafish larva also presents a promising model for therapeutic drug screens. Overall, the results described in this thesis show that the zebrafish presents an effective, reliable and rapid model for assessing the biological activity of drugs $in$ $vivo$

Optimisation of Embryonic and Larval ECG Measurement in Zebrafish for Quantifying the Effect of QT Prolonging Drugs

Effective chemical compound toxicity screening is of paramount importance for safe cardiac drug development. Using mammals in preliminary screening for detection of cardiac dysfunction by electrocardiography (ECG) is costly and requires a large number of animals. Alternatively, zebrafish embryos can be used as the ECG waveform is similar to mammals, a minimal amount of chemical is necessary for drug testing, while embryos are abundant, inexpensive and represent replacement in animal research with reduced bioethical concerns. We demonstrate here the utility of pre-feeding stage zebrafish larvae in detection of cardiac dysfunction by electrocardiography. We have optimised an ECG recording system by addressing key parameters such as the form of immobilization, recording temperature, electrode positioning and developmental age. Furthermore, analysis of 3 days post fertilization (dpf) zebrafish embryos treated with known QT prolonging drugs such as terfenadine, verapamil and haloperidol led to reproducible detection of QT prolongation as previously shown for adult zebrafish. In addition, calculation of Z-factor scores revealed that the assay was sensitive and specific enough to detect large drug-induced changes in QTc intervals. Thus, the ECG recording system is a useful drug-screening tool to detect alteration to cardiac cycle components and secondary effects such as heart block and arrhythmias in zebrafish larvae before free feeding stage, and thus provides a suitable replacement for mammalian experimentation

3D finite element electrical model of larval zebrafish ECG signals

Assessment of heart function in zebrafish larvae using electrocardiography (ECG) is a potentially useful tool in developing cardiac treatments and the assessment of drug therapies. In order to better understand how a measured ECG waveform is related to the structure of the heart, its position within the larva and the position of the electrodes, a 3D model of a 3 days post fertilisation (dpf) larval zebrafish was developed to simulate cardiac electrical activity and investigate the voltage distribution throughout the body. The geometry consisted of two main components; the zebrafish body was modelled as a homogeneous volume, while the heart was split into five distinct regions (sinoatrial region, atrial wall, atrioventricular band, ventricular wall and heart chambers). Similarly, the electrical model consisted of two parts with the body described by Laplace’s equation and the heart using a bidomain ionic model based upon the Fitzhugh-Nagumo equations. Each region of the heart was differentiated by action potential (AP) parameters and activation wave conduction velocities, which were fitted and scaled based on previously published experimental results. ECG measurements in vivo at different electrode recording positions were then compared to the model results. The model was able to simulate action potentials, wave propagation and all the major features (P wave, R wave, T wave) of the ECG, as well as polarity of the peaks observed at each position. This model was based upon our current understanding of the structure of the normal zebrafish larval heart. Further development would enable us to incorporate features associated with the diseased heart and hence assist in the interpretation of larval zebrafish ECGs in these conditions

Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016

As mortality rates decline, life expectancy increases, and populations age, non-fatal outcomes of diseases and injuries are becoming a larger component of the global burden of disease. The Global Burden of Diseases, Injuries, and Risk Factors Study 2016 (GBD 2016) provides a comprehensive assessment of prevalence, incidence, and years lived with disability (YLDs) for 328 causes in 195 countries and territories from 1990 to 2016

Global, regional, and national disability-adjusted life-years (DALYs) for 333 diseases and injuries and healthy life expectancy (HALE) for 195 countries and territories, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016

BACKGROUND: Measurement of changes in health across locations is useful to compare and contrast changing epidemiological patterns against health system performance and identify specific needs for resource allocation in research, policy development, and programme decision making. Using the Global Burden of Diseases, Injuries, and Risk Factors Study 2016, we drew from two widely used summary measures to monitor such changes in population health: disability-adjusted life-years (DALYs) and healthy life expectancy (HALE). We used these measures to track trends and benchmark progress compared with expected trends on the basis of the Socio-demographic Index (SDI). METHODS: We used results from the Global Burden of Diseases, Injuries, and Risk Factors Study 2016 for all-cause mortality, cause-specific mortality, and non-fatal disease burden to derive HALE and DALYs by sex for 195 countries and territories from 1990 to 2016. We calculated DALYs by summing years of life lost and years of life lived with disability for each location, age group, sex, and year. We estimated HALE using age-specific death rates and years of life lived with disability per capita. We explored how DALYs and HALE differed from expected trends when compared with the SDI: the geometric mean of income per person, educational attainment in the population older than age 15 years, and total fertility rate. FINDINGS: The highest globally observed HALE at birth for both women and men was in Singapore, at 75·2 years (95% uncertainty interval 71·9-78·6) for females and 72·0 years (68·8-75·1) for males. The lowest for females was in the Central African Republic (45·6 years [42·0-49·5]) and for males was in Lesotho (41·5 years [39·0-44·0]). From 1990 to 2016, global HALE increased by an average of 6·24 years (5·97-6·48) for both sexes combined. Global HALE increased by 6·04 years (5·74-6·27) for males and 6·49 years (6·08-6·77) for females, whereas HALE at age 65 years increased by 1·78 years (1·61-1·93) for males and 1·96 years (1·69-2·13) for females. Total global DALYs remained largely unchanged from 1990 to 2016 (-2·3% [-5·9 to 0·9]), with decreases in communicable, maternal, neonatal, and nutritional (CMNN) disease DALYs offset by increased DALYs due to non-communicable diseases (NCDs). The exemplars, calculated as the five lowest ratios of observed to expected age-standardised DALY rates in 2016, were Nicaragua, Costa Rica, the Maldives, Peru, and Israel. The leading three causes of DALYs globally were ischaemic heart disease, cerebrovascular disease, and lower respiratory infections, comprising 16·1% of all DALYs. Total DALYs and age-standardised DALY rates due to most CMNN causes decreased from 1990 to 2016. Conversely, the total DALY burden rose for most NCDs; however, age-standardised DALY rates due to NCDs declined globally. INTERPRETATION: At a global level, DALYs and HALE continue to show improvements. At the same time, we observe that many populations are facing growing functional health loss. Rising SDI was associated with increases in cumulative years of life lived with disability and decreases in CMNN DALYs offset by increased NCD DALYs. Relative compression of morbidity highlights the importance of continued health interventions, which has changed in most locations in pace with the gross domestic product per person, education, and family planning. The analysis of DALYs and HALE and their relationship to SDI represents a robust framework with which to benchmark location-specific health performance. Country-specific drivers of disease burden, particularly for causes with higher-than-expected DALYs, should inform health policies, health system improvement initiatives, targeted prevention efforts, and development assistance for health, including financial and research investments for all countries, regardless of their level of sociodemographic development. The presence of countries that substantially outperform others suggests the need for increased scrutiny for proven examples of best practices, which can help to extend gains, whereas the presence of underperforming countries suggests the need for devotion of extra attention to health systems that need more robust support. FUNDING: Bill & Melinda Gates Foundation

Nations within a nation: variations in epidemiological transition across the states of India, 1990–2016 in the Global Burden of Disease Study

18% of the world's population lives in India, and many states of India have populations similar to those of large countries. Action to effectively improve population health in India requires availability of reliable and comprehensive state-level estimates of disease burden and risk factors over time. Such comprehensive estimates have not been available so far for all major diseases and risk factors. Thus, we aimed to estimate the disease burden and risk factors in every state of India as part of the Global Burden of Disease (GBD) Study 2016

Optimisation of ECG recording system.

<p>A) Mean ECG interval measurements obtained from 3 dpf larvae following exposure to different ambient temperatures (n = 10), and B) over different developmental periods (2, 3, 4 and 5 days; n = 8).</p

Investigation of effect of electrode position on ECG waveform.

<p>A) Mean ECG interval measurements obtained from 3 dpf larvae at different electrode positions over the heart (n = 10 per position), B) diagram of a 3 dpf larva with numbers identifying the different measurement positions; image created using Adobe Photoshop CS6, C) 1) 2 second raw ECG recording and 2) 2 second raw ECG recording from a 5 dpf larva after moving positive electrode 10 µm forward longitudinally, D) comparison of QTc intervals measured from 5 dpf larvae (n = 3) at starting position (atrium/ventricle boundary) and after movement of electrode longitudinally by 5 µm and 10 µm. Key: 1 =  sinus venosus, 2 =  between atrium and sinus venosus, 3 =  base of atrium, 4 =  apex of atrium, 5 =  between atrium and ventricle, 6 =  base of ventricle, 7 =  apex of ventricle and 8 =  bulbus arteriosus.</p

Detection of electrocardiographic signal.

<p>A) Depiction of an electrocardiogram and associated components, created using Adobe Photoshop CS6, B) 2 second section of a raw ECG recording from a larval zebrafish (3 dpf) without digital filtering, C) digitally filtered version of the ECG recording using low pass filtering to reduce background noise and produce a waveform suitable for analysis, D) analysed trace showing waveform reproducibility and stability during the recording period, with each line representing one cardiac cycle. The dark line in the middle represents the mean of all cycles from a 1 minute record.</p

Exclusion of motion artefact and signal contamination.

<p>A) Mean ECG interval measurements obtained from 3 dpf larvae following motion artefact exclusion using cromakalim drug treatment (n = 5 per concentration), B) parallel invasive and non-invasive recordings (n = 4), C) prior MESAB anaesthesia for 5–10 minutes (n = 10 per concentration), and D) continuous tubocurarine exposure (n = 10 per concentration). Abbreviations: RR = time between the peak of one QRS complex to the next, QT = time of ventricular depolarisation and repolarisation, QTc = QT corrected for heart rate.</p