zebra-ii-as-novel-system-to-record-electrophysiological-signals

Zebra II as A Novel System to Record Electrophysiological Signals in Zebrafish

Zebrafish and their mutant lines have been extensively used in biomedical investigations, cardiovascular studies, and drug screening. In the current ...

UC Irvine

Because of its powerful genetics, the adult zebrafish has been increasingly used for studying cardiovascular diseases. Considering its heart rate of ~100 beats per minute at ambient temperature, which is very close to human, we assessed the use of this vertebrate animal for modeling heart rhythm disorders such as sinus arrest (SA) and sick sinus syndrome (SSS). We firstly optimized a protocol to measure electrocardiogram in adult zebrafish. We determined the location of the probes, implemented an open-chest microsurgery procedure, measured the effects of temperature, and determined appropriate anesthesia dose and time. We then proposed an PP interval of more than 1.5 seconds as an arbitrary criterion to define an SA episode in an adult fish at ambient temperature, based on comparison between the current definition of an SA episode in humans and our studies of candidate SA episodes in aged wild-type fish and Tg(SCN5A-D1275N) fish (a fish model for inherited SSS). With this criterion, a subpopulation of about 5% wild-type fish can be considered to have SA episodes, and this percentage significantly increases to about 25% in 3-year-old fish. In response to atropine, this subpopulation has both common SSS phenotypic traits that are shared with the Tg(SCN5A-D1275N) model, such as bradycardia; and unique SSS phenotypic traits, such as increased QRS/P ratio and chronotropic incompetence. In summary, this study defined baseline SA and SSS in adult zebrafish and underscored use of the zebrafish as an alternative model to study aging-associated SSS.

Aging-associated sinus arrest and sick sinus syndrome in adult zebrafish.

Light-sheet fluorescence microscopy (LSFM) provides access to multi-dimensional and multi-scale  imaging of animal models with highly coherent volumetric reconstruction of the tissue morphology, via a focused laser light sheet. The orthogonal illumination and detection LSFM pathways account for minimal photobleaching and deep tissue optical sectioning through different perspective views. Although rotation of the sample and deep tissue scanning constitutes major advantages of LSFM, images may suffer from intrinsic problems within the modality, such as light mismatch of refractive indices between the sample and mounting media and varying quantum efficiency across different depths. To overcome these challenges, we hereby introduce an illumination correction technique integrated with depth detail amelioration to achieve symmetric contrast in large field-of-view images acquired using a low power objective lens. Due to an increase in angular dispersion of emitted light flux with the depth, we combined the dehazing algorithm with morphological operations to enhance poorly separated overlapping structures with subdued intensity. The proposed method was tested on different LSFM modalities to illustrate its applicability on correcting anisotropic illumination affecting the volumetric reconstruction of the fluorescently tagged region of interest.

Correcting anisotropic intensity in light sheet images using dehazing and image morphology.

The zebrafish model has been demonstrated as an ideal vertebrate model system for a diverse range of biological studies. Along with conventional approaches, monitoring and analysis of zebrafish electrocardiogram (ECG) have been utilized for cardio-physiological screening and elucidation. ECG monitoring has been carried out with fish treated with anesthetic drugs, rendering the short period of time in recording the signals (<5 min). In this work, a prolonged sedation system for continuous ECG monitoring of multiple zebrafish was proposed and developed. We built a circulation system to provide prolonged mild anesthesia which allows more consistent and intrinsic ECG measurement. The use of prolonged anesthesia helped reduce the concentration of the anesthetic drug (MS222 or Tricaine) from 200 mg/L to 100 mg/L and even lower; thus, maintaining the integrity of intrinsic ECG. Moreover, heartrate variation during recording was investigated, showing minute changes (±3.2 beats per minute - BPM). The development of this prolonged ECG monitoring system would open the possibility of long-term monitoring for studies such as drug screening and forward genetic screening.

Continuous Electrocardiogram Monitoring in Zebrafish with Prolonged Mild Anesthesia.

Electrocardiogram (ECG) monitoring of the fetus during pregnancy, before and during labor, can provide crucial information for the assessment of fetal well-being and development, as well as labor progress. An out-of-clinics fetal ECG monitoring system may pave the way for instant diagnosis, suggesting immediate intervention, which could help reduce the fetal mortality rate. In this paper, we present an unobtrusive fetal maternal ECG monitoring system which can operate in the home setting. The acquisition of the mother's abdominal ECG is done using the non-contact electrode approach. The extraction of the fetal ECG from the combined fetal/maternal ECG signal is investigated using both Fast Independent Component Analysis (FastICA) and RobustICA algorithms. An accelerometer is integrated for motion artifact detection which would help reduce interferences due to movement. The device also is connected to a cloud server, allowing doctors to access the data in real time.

Home-based mobile fetal/maternal electrocardiogram acquisition and extraction with cloud assistance.

Zebrafish and their mutant lines have been extensively used in cardiovascular studies. In the current study, the novel system, Zebra II, is presented for prolonged electrocardiogram (ECG) acquisition and analysis for multiple zebrafish within controllable working environments. The Zebra II is composed of a perfusion system, apparatuses, sensors, and an in-house electronic system. First, the Zebra II is validated in comparison with a benchmark system, namely iWORX, through various experiments. The validation displayed comparable results in terms of data quality and ECG changes in response to drug treatment. The effects of anesthetic drugs and temperature variation on zebrafish ECG were subsequently investigated in experiments that need real-time data assessment. The Zebra II's capability of continuous anesthetic administration enabled prolonged ECG acquisition up to 1 h compared to that of 5 min in existing systems. The novel, cloud-based, automated analysis with data obtained from four fish further provided a useful solution for combinatorial experiments and helped save significant time and effort. The system showed robust ECG acquisition and analytics for various applications including arrhythmia in sodium induced sinus arrest, temperature-induced heart rate variation, and drug-induced arrhythmia in Tg(SCN5A-D1275N) mutant and wildtype fish. The multiple channel acquisition also enabled the implementation of randomized controlled trials on zebrafish models. The developed ECG system holds promise and solves current drawbacks in order to greatly accelerate drug screening applications and other cardiovascular studies using zebrafish.

A novel wireless ECG system for prolonged monitoring of multiple zebrafish for heart disease and drug screening studies.

Simultaneous monitoring of electrocardiogram (ECG) and electroencephalogram (EEG) in studied animal models requires innovative engineering techniques that can capture minute physiological changes. However, this is often administered with a bulky and/or invasive system that may cause discomfort to animals and signal distortions. Here, we develop an integrated bioelectronic sensing system to provide simultaneous recordings of ECG and EEG in real-time for Xenopus laevis. The microelectrode array (MEA) membrane and the distinct anatomy of Xenopus offer noninvasive multi-modal electrophysiological monitoring with favorable spatial resolution. The system was validated under different environmental conditions, including drug exposure and temperature changes. Under the exposure of Pentylenetetrazol (PTZ), an epilepsy-inducing drug, clear ECG and EEG alterations, including frequent ictal and interictal EEG events, 30 dB average EEG amplitude elevations, abnormal ECG morphology, and heart rate changes, were observed. Furthermore, the ECG and EEG were monitored and analyzed under different temperatures. A decrease in relative power of delta band was observed when cold environment was brought about, in contrast to an increase in relative power of other higher frequency bands while the ECG remained stable. Overall, the real-time electrophysiology monitoring system using the Xenopus model holds potential for many applications in drug screening and remote environmental monitoring.

Microelectrode array membranes to simultaneously assess cardiac and neurological signals of xenopus laevis under chemical exposures and environmental changes.

 quantitative assessment of structural and functional biomarkers is essential for characterizing the pathophysiology of congenital disorders. In this regard, fixed tissue analysis has offered revolutionary insights into the underlying cellular architecture. However, histological analysis faces major drawbacks with respect to lack of spatiotemporal sampling and tissue artifacts during sample preparation. This study demonstrates the potential of light sheet fluorescence microscopy (LSFM) as a non-invasive, 4D (3days + time) optical sectioning tool for revealing cardiac mechano-transduction in zebrafish. Furthermore, we have described the utility of a scale and size-invariant feature detector, for analyzing individual morphology of fused cardiomyocyte nuclei and characterizing zebrafish ventricular contractility.

Article	Year
Aging-associated sinus arrest and sick sinus syndrome in adult zebrafish. PloS one\| PubMed ID: 32401822	2020
Correcting anisotropic intensity in light sheet images using dehazing and image morphology. APL bioengineering\| PubMed ID: 32637858	2020
Continuous Electrocardiogram Monitoring in Zebrafish with Prolonged Mild Anesthesia. Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference\| PubMed ID: 33018541	2020
Home-based mobile fetal/maternal electrocardiogram acquisition and extraction with cloud assistance. The IEEE MTT-S 2019 International Microwave Biomedical Conference (IMBioC 2019) : proceedings : May 06-08, 2019, International Conference Hotel of Nanjing, Nanjing, China. IEEE MTT-S International Microwave Bio Conference (2019 : Nanjin...\| PubMed ID: 34622249	2019
A novel wireless ECG system for prolonged monitoring of multiple zebrafish for heart disease and drug screening studies. Biosensors & bioelectronics\| PubMed ID: 34801796	2022
Microelectrode array membranes to simultaneously assess cardiac and neurological signals of xenopus laevis under chemical exposures and environmental changes. Biosensors & bioelectronics\| PubMed ID: 35490628	2022
Scale space detector for analyzing spatiotemporal ventricular contractility and nuclear morphogenesis in zebrafish. iScience\| PubMed ID: 36034231	2022

Hung Cao

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