Optical transparency and label-free vessel imaging of zebrafish larvae in shortwave infrared range as a tool for prolonged studying of cardiovascular system development

Maciag M., Wnorowski A., Mierzejewska M., Plazinska A.

Cardiotoxicity remains the most common reason for failure during drug development. Recently, the zebrafish (Danio rerio) model has emerged for the evaluation of drug-dependent cardiotoxicity and for the identification of cardioprotective molecules. However, it remains unknown how closely the zebrafish-based results may be translated to humans. To tackle this issue, we established embryonic zebrafish models of doxorubicin-, adrenaline- and terfenadine-induced cardiotoxicity with unified dosing regimen which eventually enabled head-to-head comparison of the drugs. Subsequently, we determined whether human cardioprotective medications - dexrazoxane, metoprolol, carvedilol and valsartan - are able to manage heart dysfunction in zebrafish. Our results indicated that doxorubicin, adrenaline and terfenadine elicited overt signs of cardiotoxicity in fish, and we further showed that the blockade of the renin-angiotensin system and, to a lesser extent, β-adrenergic system, ameliorated the heart disease in zebrafish. From the drug development standpoint, our work opens the possibility to determine the cardiovascular properties of tested compounds using the rapid and affordable zebrafish model.

Yang D., Yuan Z., Yang Z., Hu M., Liang Y.

Zebrafish is an important animal model, which is used to study development, pathology, and genetic research. The zebrafish skin model is widely used in cutaneous research, and angiogenesis is critical for cutaneous wound healing. However, limited by the penetration depth, the available optical methods are difficult to describe the internal skin structure and the connection of blood vessels between the skin and subcutaneous tissue. By a homemade high-resolution polarization-sensitive optical coherence tomography (PS-OCT) system, we imaged the polarization contrast of zebrafish skin and the zebrafish skin vasculature with optical coherence tomography angiography (OCTA). Based on these OCT images, the spatial distribution of the zebrafish skin vasculature was described. Furthermore, we monitored the healing process of zebrafish cutaneous wounds. We think the high-resolution PS-OCT system will be a promising tool in studying cutaneous models of zebrafish.

Huisken J., Weber M., Graves A., Schlaeppi A.

Bowley G., Kugler E., Wilkinson R., Lawrie A., Eeden F., Chico T.J., Evans P.C., Noël E.S., Serbanovic‐Canic J.

Lai K.P., Gong Z., Tse W.K.

• Use of zebrafish model for environmental studies. • Application of transgenic technology for environmental toxicity researches. • Application of integrated omics to understand the toxic mechanism. • Use of zebrafish model for transgenerational studies. The production of large amounts of synthetic industrial and biomedical compounds, together with environmental pollutants, poses a risk to our ecosystem and induces negative effects on the health of wildlife and human beings. With the emergence of the global problem of chemical contamination, the adverse biological effects of these chemicals are gaining attention among the scientific communities, industry, governments, and the public. Among these chemicals, endocrine disrupting chemicals (EDCs) are regarded as one of the major global issues that potentially affecting our health. There is an urgent need of understanding the potential hazards of such chemicals. Zebrafish have been widely used in the aquatic toxicology. In this review, we first discuss the strategy of transgenic lines that used in the toxicological studies, followed by summarizing the current omics approaches (transcriptomics, proteomics, metabolomics, and epigenomics) on toxicities of EDCs in this model. We will also discuss the possible transgenerational effects in zebrafish and future prospective of the integrated omics approaches with customized transgenic organism. To conclude, we summarize the current findings in the field, and provide our opinions on future environmental toxicity research in the zebrafish model.

Chen X., Li Y., Yao T., Jia R.

As a promisingin vivotool for cancer research, zebrafish have been widely applied in various tumor studies. The zebrafish xenograft model is a low-cost, high-throughput tool for cancer research that can be established quickly and requires only a small sample size, which makes it favorite among researchers. Zebrafish patient-derived xenograft (zPDX) models provide promising evidence for short-term clinical treatment. In this review, we discuss the characteristics and advantages of zebrafish, such as their transparent and translucent features, the use of vascular fluorescence imaging, the establishment of metastatic and intracranial orthotopic models, individual pharmacokinetics measurements, and tumor microenvironment. Furthermore, we introduce how these characteristics and advantages are applied other in tumor studies. Finally, we discuss the future direction of the use of zebrafish in tumor studies and provide new ideas for the application of it.

Lane S., More L.A., Asnani A.

Purpose of review: Both traditional and novel cancer therapies can cause cardiovascular toxicity in patients. In vivo models integrating both cardiovascular and cancer phenotypes allow for the study of on- and off-target mechanisms of toxicity arising from these agents. The zebrafish is the optimal whole organism model to screen for cardiotoxicity in a high throughput manner, while simultaneously assessing the role of cardiotoxicity pathways on the cancer therapy’s antitumor effect. Here we highlight established zebrafish models of human cardiovascular disease and cancer, the unique advantages of zebrafish to study mechanisms of cancer therapy-associated cardiovascular toxicity, and finally, important limitations to consider when using the zebrafish to study toxicity. Recent findings: Cancer therapy-associated cardiovascular toxicities range from cardiomyopathy with traditional agents to arrhythmias and thrombotic complications associated with newer targeted therapies. The zebrafish can be used to identify novel therapeutic strategies that selectively protect the heart from cancer therapy without affecting antitumor activity. Advances in genome editing technology have enabled the creation of several transgenic zebrafish lines valuable to the study of cardiovascular and cancer pathophysiology. Summary: The high degree of genetic conservation between zebrafish and humans, as well as the ability to recapitulate cardiotoxic phenotypes observed in patients with cancer, make the zebrafish an effective model to study cancer therapy-associated cardiovascular toxicity. Though this model provides several key benefits over existing in vitro and in vivo models, limitations of the zebrafish model include the early developmental stage required for most high-throughput applications.

Huang Y., Chen Z., Meng Y., Wei Y., Xu Z., Ma J., Zhong K., Cao Z., Liao X., Lu H.

Famoxadone-cymoxanil is a new protective and therapeutic fungicide, but little research has been done on it or its toxicity in aquatic organisms. In this study, we used zebrafish to investigate the cardiotoxicity of famoxadone-cymoxanil and the potential mechanisms involved. Zebrafish embryos were exposed to different concentrations of famoxadone-cymoxanil until 72 h post-fertilization (hpf), then changes of heart morphology in zebrafish embryos were observed. We also detected the levels of oxidative stress, myocardial-cell proliferation and apoptosis, ATPase activity, and the expression of genes related to the cardiac development and calcium-signaling pathway. After famoxadone-cymoxanil exposure, pericardial edema, cardiac linearization, and reductions in the heart rate and cardiac output positively correlated with concentration. Although myocardial-cell apoptosis was not detected, proliferation of the cells was severely reduced and ATPase activity significantly decreased, resulting in a severe deficiency in heart function. In addition, indicators of oxidative stress changed significantly after exposure of the embryos to the fungicide. To better understand the possible molecular mechanisms of cardiovascular toxicity in zebrafish, we studied the transcriptional levels of cardiac development, calcium-signaling pathways, and genes associated with myocardial contractility. The mRNA expression levels of key genes in heart development were significantly down-regulated, while the expression of genes related to the calcium-signaling pathway (ATPase [ atp2a1 ], cardiac troponin C [ tnnc1a ], and calcium channel [ cacna1a ]) was significantly inhibited. Expression of klf2a , a major endocardial flow-responsive gene, was also significantly inhibited. Mechanistically, famoxadone-cymoxanil toxicity might be due to the downregulation of genes associated with the calcium-signaling pathway and cardiac muscle contraction. Our results found that famoxadone-cymoxanil exposure causes cardiac developmental toxicity and severe energy deficiency in zebrafish. • Famoxadone-cymoxanil exposure can cause cardiac developmental toxicity in zebrafish. • Famoxadone-cymoxanil exposure can induce disordering cardiogenesis during the window period of cardiogenesis. • Famoxadone-cymoxanil exposure can induce oxidative stress and reduce cell proliferation in zebrafish heart. • Famoxadone-cymoxanil exposure significantly inhibited ATPase activity.

Benslimane F.M., Zakaria Z.Z., Shurbaji S., Abdelrasool M.K., Al-Badr M.A., Al Absi E.S., Yalcin H.C.

This work is supported by Qatar National Research Fund (QNRF),National Priority Research Program under grant number NPRP 10-0123-170222.

Wenz R., Conibear E., Bugeon L., Dallman M.

The availability of transparent zebrafish mutants (eitherTraNac:trab6/b6; nacw2/w2orcasper: roya9/a9; nacw2/w2) for live imaging studies together with the ease of generating transgenic lines are two of the strengths of the zebrafish model organism. The fact that transparentcasper(roya9/a9;nacw2/w2)and silvernacre(nacw2/w2)mutants are indistinguishable by eye at early stages (1-5 days post-fertilization; dpf) means many fish must be raised and later culled if they are not transparent. To identify translucent mutants early and easily at the early larval stage (≤5 dpf) before they are classified as protected animals, we developed a simple screening method using standard fluorescence microscopy. We estimate that this procedure could annually save 60,000 animals worldwide.

Machikhin A.S., Burlakov A.B., Volkov M.V., Khokhlov D.D.

We report on the noninvasive method for in vivo study of fish's cardiovascular system, that is, the heart and the structure of vessels that carry blood throughout the body. The proposed approach is based on combined photoplethysmographic and videocapillaroscopic microscopic imaging and enables noncontact two-dimensional mapping of blood volume changes. We demonstrate that the obtained data allows precise measurements of heartbeat, blood flow velocity and other important parameters (see Videos S1 and S2). To validate the developed image processing technique, we have carried out multiple experiments on zebrafish-a well-proven informative model organism widely used to understand cardiac development. The proposed approach may be effective for the study of cardiovascular system formation and functioning as well as the impact of various influencing factors on them.

Marcos-Vidal A., Vaquero J.J., Ripoll J.

Light in the near infrared has advantageous properties such as reduced scattering and higher depth of penetration that make it ideal for optical imaging either in transmission or in fluorescence. This chapter gives an overview of the physics behind light propagation, the optical properties of biological tissues and finally presents the potential benefits that this part of the spectrum can offer to imaging.

Haindl R., Deloria A.J., Sturtzel C., Sattmann H., Rohringer W., Fischer B., Andreana M., Unterhuber A., Schwerte T., Distel M., Drexler W., Leitgeb R., Liu M.

We present a dual modality functional optical coherence tomography and photoacoustic microscopy (OCT-PAM) system. The photoacoustic modality employs an akinetic optical sensor with a large imaging window. This imaging window enables direct reflection mode operation, and a seamless integration of optical coherence tomography (OCT) as a second imaging modality. Functional extensions to the OCT-PAM system include Doppler OCT (DOCT) and spectroscopic PAM (sPAM). This functional and non-invasive imaging system is applied to image zebrafish larvae, demonstrating its capability to extract both morphological and hemodynamic parameters in vivo in small animals, which are essential and critical in preclinical imaging for physiological, pathophysiological and drug response studies.

Gierten J., Pylatiuk C., Hammouda O.T., Schock C., Stegmaier J., Wittbrodt J., Gehrig J., Loosli F.

Accurate quantification of heartbeats in fish models is an important readout to study cardiovascular biology, disease states and pharmacology. However, dependence on anaesthesia, laborious sample orientation or requirement for fluorescent reporters have hampered the use of high-throughput heartbeat analysis. To overcome these limitations, we established an efficient screening assay employing automated label-free heart rate determination of randomly oriented, non-anesthetized medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos in microtiter plates. Automatically acquired bright-field data feeds into an easy-to-use HeartBeat software with graphical user interface for automated quantification of heart rate and rhythm. Sensitivity of the assay was demonstrated by profiling heart rates during entire embryonic development. Our analysis revealed rapid adaption of heart rates to temperature changes, which has implications for standardization of experimental layout. The assay allows scoring of multiple embryos per well enabling a throughput of >500 embryos per 96-well plate. In a proof of principle screen for compound testing, we captured concentration-dependent effects of nifedipine and terfenadine over time. Our novel assay permits large-scale applications ranging from phenotypic screening, interrogation of gene functions to cardiovascular drug development.

Meng H., Liang J., Zheng X., Zhang K., Zhao Y.

Cardiovascular agents are among the most frequently prescribed pharmaceuticals worldwide. They are widely detected in aquatic ecosystems, while their ecotoxicological implications are rarely explored. Here, by the use of a new developed high-throughput zebrafish embryo screening approach, we systematically assessed the cardiovascular disruptive effects of 32 commonly used cardiovascular agents at environmental relevant concentrations and above (0.04, 0.2 and 1 µM). Multiple endpoints, including cardiac output, heart rate and blood flow, were quantified via customized video analysis approaches. Among the 32 agents, simvastatin and lovastatin exhibited the strongest toxicities to fish embryos, and the lethal doses were observed at 0.2 µM and 1 µM. Beta-blockers such as atenolol and metoprolol significantly decreased heart rates by up to 15% and 12% and increased blood flows by up to 14% and 14%, respectively, at concentrations as low as 0.04 µM. Several hypertension/hyperlipidemia medications such as pravastatin and enalapril led to significant inhibition of heart rates (up to 14% and 16% decreases, respectively) as well as slightly decreases of the cardiac outputs and blood flows. In addition, a tentative risk assessment clearly demonstrated that some compounds such as atenolol, metoprolol and bezafibrate pose considerable risks to aquatic organisms at environmental or slightly higher than surface water concentrations. Our results provided novel insights into understanding of the potential risks of cardiovascular agents and contributed to their environmental hazard ranking.

Krylov V.V., Lukyanov T.F., Korzhevina V.I., Machikhin A.S., Guryleva A.V., Tchougounov V.K., Burlakov A.B.

Machikhin A.S., Titov S.A., Huang C., Guryleva A.V., Burlakov A.B., Zykova L.A., Bukova V.I.

Udayakumar S., Metkar S.K., Girigoswami A., Deepika B., Janani G., Kanakaraj L., Girigoswami K.

Amyloids, with their β-sheet-rich structure, contribute to diabetes, neurodegenerative diseases, and amyloidosis by aggregating within diverse anatomical compartments. Insulin amyloid (IA), sharing structural resemblances with amyloids linked to neurological disorders, acts as a prototype, while compounds capable of degrading these fibrils hold promise as therapeutic agents for amyloidosis intervention. In this research, liposomal nanoformulated iota carrageenan (nCG) was formulated to disrupt insulin amyloids, demonstrating about a 17-20 % higher degradation efficacy compared to conventional carrageenan through thioflavin T fluorescence, dynamic light scattering analysis, and turbidity quantification. The biocompatibility of the nCG and nCG-treated insulin amyloids was evaluated through MTT assay, live-dead cell assay on V79 cells, and hemolysis testing on human blood samples to establish their safety for use in vivo. Zebrafish embryos were utilized to assess in vivo biocompatibility, while adult zebrafish were employed to monitor the degradation capacity of IA post subcutaneous injection, with fluorescence emitted by the fish captured via IVIS. This demonstrated that the formulated nCG exhibited superior anti-amyloid efficacy compared to carrageenan alone, while both materials demonstrated biocompatibility. Furthermore, through docking simulations, an exploration was conducted into the molecular mechanisms governing the inhibition of the target protein pancreatic insulin by carrageenan.

Wang Y., Wang S., Wang Y., Gao P., Wang L., Wang Q., Zhang Y., Liu K., Xia Q., Tu P.

The rhizome of Corydalis decumbens is a traditional Chinese medicine commonly utilized in the clinical treatment of acute ischemic stroke. Numerous phytochemical and biological investigations have demonstrated that protoberberine alkaloids from C. decumbens exhibit diverse pharmaceutical activities against various diseases. Sinometumine E (SE), a protoberberine alkaloid isolated from C. decumbens for the first time, is characterized by a complex 6/6/6/6/6/6 hexacyclic skeleton. In the current study, we investigated the protective effects of SE on endothelial cell injury and its angiogenesis effects in zebrafish. The results suggested that SE showed significant anti-ischemic effects on OGD/R-induced HBEC-5i and HUVECs cell ischemia/reperfusion injury model. Furthermore, it promoted angiogenesis in PTK787-induced, MPTP-induced, and atorvastatin-induced vessel injury models of zebrafish, while also suppressing hypoxia-induced locomotor impairment in zebrafish. Transcriptome sequencing analysis provided a sign that SE likely to promotes angiogenesis through the HIF-1/VEGF signaling pathway to exert anti-ischemic effects. Consistently, SE modulated several genes related to HIF-1/VEGF signal pathway, such as hif-1, vegf, vegfr-2, pi3k, erk, akt and plcγ. Molecular docking analysis revealed that VEGFR-2 exhibited high binding affinity with SE, and western blot analysis confirmed that SE treatment enhanced the expression of VEGFR-2. In conclusion, our study profiled the angiogenic activities of SE in vitro and in vivo. The key targets and related pathways involved in anti-ischemic effects of SE, shedding light on the pharmacodynamic components and mechanisms of Corydalis decumbens, and provides valuable insights for identifying effective substances for the treatment of ischemic stroke.

Machikhin A., Guryleva A., Chakraborty A., Khokhlov D., Selyukov A., Shuman L., Bukova V., Efremova E., Rudenko E., Burlakov A.

AbstractToxic environmental pollutants pose a health risk for both humans and animals. Accumulation of industrial contaminants in freshwater fish may become a significant threat to biodiversity. Comprehensive monitoring of the impact of environmental stressors on fish functional systems is important and use of non‐invasive tools that can detect the presence of these toxicants in vivo is desirable. The blood circulatory system, by virtue of its sensitivity to the external stimuli, could be an informative indicator of chemical exposure. In this study, microscopic photoplethysmography‐based approach was used to investigate the cardiac activity in broad whitefish larvae (Coregonus nasus) under acute exposure to cadmium and phenol. We identified contamination‐induced abnormalities in the rhythms of the ventricle and atrium. Our results allow introducing additional endpoints to evaluate the cardiac dysfunction in fish larvae and contribute to the non‐invasive evaluation of the toxic effects of industrial pollutants on bioaccumulation and aquatic life.

Slavin A.E., Guryleva A.V., Bukova V.I., Shuman L.A., Burlakov A.B.

Deng Y., Hu T., Chen J., Zeng J., Yang J., Ke Q., Miao L., Chen Y., Li R., Zhang R., Xu P.

ABSTRACT Heart rate is a crucial physiological indicator for fish, but current measurement methods are often invasive or require delicate manipulation. In this study, we introduced two non-invasive and easy-to-operate methods based on photoplethysmography, namely reflectance-type photoplethysmography (PPG) and remote photoplethysmography (rPPG), which we applied to the large yellow croaker (Larimichthys crocea). PPG showed perfect synchronization with electrocardiogram (ECG), with a Pearson's correlation coefficient of 0.99999. For rPPG, the results showed good agreement with ECG. Under active provision of green light, the Pearson's correlation coefficient was 0.966, surpassing the value of 0.947 under natural light. Additionally, the root mean square error was 0.810, which was lower than the value of 1.30 under natural light, indicating not only that the rPPG method had relatively high accuracy but also that green light may have the potential to further improve its accuracy.

Chaoul V., Dib E., Bedran J., Khoury C., Shmoury O., Harb F., Soueid J.

Neurological diseases, including neurodegenerative and neurodevelopmental disorders, affect nearly one in six of the world’s population. The burden of the resulting deaths and disability is set to rise during the next few decades as a consequence of an aging population. To address this, zebrafish have become increasingly prominent as a model for studying human neurological diseases and exploring potential therapies. Zebrafish offer numerous benefits, such as genetic homology and brain similarities, complementing traditional mammalian models and serving as a valuable tool for genetic screening and drug discovery. In this comprehensive review, we highlight various drug delivery techniques and systems employed for therapeutic interventions of neurological diseases in zebrafish, and evaluate their suitability. We also discuss the challenges encountered during this process and present potential advancements in innovative techniques.