A zebrafish-based platform for comprehensive analysis of the external environment impact on the cardiovascular and muscular systems
According to the World Health Organization, 80% of diseases can be attributed to poor water quality. Fish and aquatic organisms are among the first to be affected by pollutants. Heavy metals, organic pollutants, agricultural chemicals can significantly disrupt the development of basic systems, reduce immune function, and affect overall population size and biodiversity. Thus, the fish welfare serves as a biological indicator of water pollution and the overall environmental health. As the role of aquaculture and fisheries in providing global food security is becoming increasingly clear, timely toxicity monitoring is mandatory for reducing fish loss and preventing consumer intoxication. Existing biotest methods is quite robust, but time-consuming, elaborate, and as a rule invasive, or even lethal techniques required expensive equipment, significant computing power, rely on the only adult fish analysis and etc. The issue Practice addressed is the lack of non-invasive, rapid, accurate, reliable, and cost-effective instruments for the comprehensive analysis of environmental pollutants impact on a variety of fish species at early developmental stages. To address this issue, we have developed a unique platform that combines equipment, software, and a methodology to assess the cardiovascular and muscular systems of fish at different developmental stages in a single format. The proposed approach unsure studies in a non-invasive, in vivo, low-stressed, label-free, rapid, and typically automated manner, with high performance and spatial resolution and clarity and ease of data interpretation. It is an easy, cost-effective and informative technique based on the digital analysis of microscopic video frames of optical transparency tissues of most fish species at early developmental stages. We employed optical diagnostic techniques, specifically photoplethysmography, videocapillaroscopy, and Mueller-matrix imaging, for qualitative and quantitative assessments of body abnormalities. The designed approach provides visualization and quantification of body parameters, such as heart rate, blood flow velocity, vascular and muscular maps, etc., using a commonly available and cost-effective bright-field microscope, high-speed polarizing camera, and bespoke image processing software. The platform is designed to monitor harm to health caused by current pollution or climate change to fish populations, as well as to assess the potential muscle and cardiovascular toxicity of new substances or various stressors impact. Toxicity studies can be carried out on model organisms, such as zebrafish (Danio rerio), which have been approved as one of the models for human diseases. Both commercial and wild-caught fish species can be used for ongoing climate and environmental monitoring. Quantitative criteria for the allow for screening and objectively determining the severity of disorders in the cardiovascular and musculoskeletal systems caused by toxicants.
Climate And Environmental Technologies
Technologies for the healthy environment
The Practice is a platform that includes equipment, software and methodology. Since all three components have already been developed and approved by authors, the practice replication involves assembling the equipment and, if necessary, organizing the maintenance of fish (for conducting toxicological testing on model objects). The manufacturing and assembly of the equipment may take up to two months, and a week is required for training the staff. The organization of fish maintenance is a more complex process, thus, equipping an aquarium room and bringing fish to sexual maturity may take up to six months.
One of the key benefits of the platform is its potential for a wide use. This is particularly relevant in areas with large water bodies close to agricultural land, manufacturing facilities, and etc.
Numerous techniques are considered as well-established for imaging organs and tissues of fishes at early developmental stages for toxicology study. Although histological and immunohistochemical, PCR analysis, atomic force microscopy, mass spectrometry, and fluorescent volumetric imaging of samples provide reliable and ground truth measurements, they require euthanasia and sample fixation. Intravital studies, e.g. confocal Raman spectroscopy, photoacoustic microscopy, and optical coherence tomography often exclude sample survival after measurement due to the duration and conditions of the study. Confocal fluorescence microscopy, scanning light-sheet microscopy, and hyperspectral light-sheet microscopy stand out for their minimally invasive nature, enabling volumetric in vivo visualization of tissue samples, demand expensive equipment and substantial computing power, with many methods requiring the introduction of fluorophores and dyes into the organism. Ultrasonic diagnostics allows to obtain 3D tissues images, evaluate cardiac contractility of both embryos and adult fish and conduct blood flow velocity measurement, but is limited in spatial resolution, penetration death and requires plenty of time for measurements which complicates detailed and efficient cardiovascular and muscular diagnostics. Existing biotest methods in environmental monitoring primarily rely on the assessment of current toxicants levels in adult fish tissue. They do not indicate toxicant effect on aquatic life welfare, and have lower sensitivity when assessing the environmental status or toxicity of individual substances. The main advantages of the proposed approach compared to the listed approaches, which for the first time are inherent in a single method, are: - accuracy, reliability and exhaustiveness of the measurements (an approved model object Danio rerio, a joint study of cardiovascular and muscular systems, high spatial resolution, a large number and imaging capability of diagnosed parameters) - versatility (appropriate for most fish species and some other aquatic organisms anywhere in the world), and therefore platform is suitable for worldwide distribution - quantitative (in addition to vessel and muscle maps, a set of significant hemodynamic parameters are provided, as well as cross-polarization microscopy measurements), which means the objectivity of assessment and unambiguity of criteria. - the cost-effectiveness of implementation, as well as the absence of the need for consumable materials (the setup based on a standard microscope, a high-speed camera, and original software), indicates the possibility for widespread distribution in practice. - non-invasive, in vivo and low-impact study that provides unique and significant insights into the functioning of systems and enables long-term, reliable experiments. - automation, productivity, rapidness and therefore timely large-scale screening.
The Practice have been reported in the media, including Russian Science Foundation publications, the Russian Academy of Sciences' news outlets, and various newspapers and online resources. The team has also received recognition at international conferences, receiving awards such as national prizes for young researchers, awards from the Ministry of Education, and international awards related to engineering and instrumentation. Detailed data provided in Appendix 3.
The project team has 14 publications in Scopus/WOS journal, including 9 in Q1-Q2, 4 patents, 21 presentations at international conferences, which describe and discuss the results of the Practice. Detailed data provided in Appendix 4.
