Method for constructing and analyzing zebra fish heart failure model

Abstract

The invention belongs to the technical field of experimental animal models, and particularly relates to a construction and analysis method of a zebra fish heart failure model. The invention discloses a construction method of a zebra fish heart failure model. The construction method comprises the following steps: S1, pretreating a zebra fish embryo; and S2, putting the zebra fish embryos pretreated in the step S1 into an exposure solution for exposure, thereby completing the construction of the zebra fish heart failure model. According to the construction and analysis method of the zebra fish heart failure model, accurate induction and dynamic monitoring of heart failure phenotypes are achieved through a standardized drug treatment process and a multi-mode heart function evaluation system, and an efficient platform is provided for cardiovascular drug screening and toxicity evaluation. The construction and analysis method of the zebra fish heart failure model further extends to the field of medicine and functional food heart protection efficacy evaluation, and objective grading authentication of food and medicine efficacy is achieved.

Description

Technical Field

[0001] This invention belongs to the field of experimental animal model technology, specifically relating to a method for constructing and analyzing a zebrafish heart failure model. Background Technology

[0002] Cardiovascular disease is the leading cause of death worldwide, and research into its pathological mechanisms and the development of treatment strategies heavily rely on animal models. While traditional mammalian models (such as rat coronary artery ligation) can mimic the human heart failure phenotype, they suffer from inherent drawbacks such as long modeling cycles, high costs, and phenotypic instability, hindering drug development efficiency. More critically, mammalian embryonic heartbeats begin in late pregnancy, making early dynamic monitoring of cardiac function impossible. Existing doxorubicin-induced models have significant limitations: zebrafish possess extremely strong regenerative and self-healing abilities, and transient myocardial damage induced solely by drug withdrawal often recovers rapidly, resulting in a very short effective window for drug evaluation. Furthermore, clinical heart failure is often accompanied by increased peripheral resistance, and purely chemical toxicity models cannot simulate the cardiac decompensation process under high stress.

[0003] Zebrafish (Danio rerio) have become an emerging model for cardiovascular research due to their embryonic transparency, external developmental characteristics, high reproductive rate (more than 200 eggs per week), and remarkable ability to recover their hearts after injury—ventricular contractile activity can be captured as early as 24 hours post-fertilization (hpf), providing a unique window for real-time observation. However, current technologies have significant limitations: the methods for inducing zebrafish heart failure models are inconsistent, and the models lack stability; the evaluation system is fragmented, with most studies relying solely on baseline heart rate or static morphological indicators (such as ventricular area), neglecting key hemodynamic parameters and failing to comprehensively reflect the pathological process of heart failure.

[0004] The technological gap in evaluating the cardiac efficacy of functional foods is particularly prominent: current methods rely on in vitro antioxidant tests or patient questionnaires, which cannot capture dynamic improvements in in vivo cardiac function. Although zebrafish is used sporadically for safety testing, a correlation system between heart failure models and efficacy parameters has not yet been established. This technological deficiency results in a lack of evidence-based medical support for the cardioprotective efficacy claimed by functional foods, hindering the industry's innovative development.

[0005] Doxorubicin can damage cardiomyocytes by inhibiting topoisomerase II and generating reactive oxygen species. The core value of this invention lies in establishing a standardized doxorubicin induction protocol and a multi-parameter dynamic evaluation system, thereby enabling the phenotypic quantification and scalable application of the zebrafish heart failure model. Summary of the Invention

[0006] To address the aforementioned problems, this invention aims to provide a method for constructing and analyzing a zebrafish heart failure model. This method, through a standardized drug treatment process and a multimodal cardiac function assessment system, achieves precise induction and dynamic monitoring of the heart failure phenotype, providing an efficient platform for cardiovascular drug screening and toxicity evaluation. Furthermore, this method extends to the field of evaluating the cardioprotective efficacy of drugs and functional foods, enabling objective grading and certification of food and drug efficacy.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: a method for constructing a zebrafish heart failure model, comprising the following steps: S1. Pre-treatment of zebrafish embryos; S2. The zebrafish embryos obtained from the pretreatment in step S1 are exposed in an exposure solution to complete the construction of the zebrafish heart failure model.

[0008] In one embodiment of the zebrafish heart failure model construction method of the present invention, the zebrafish embryos used in step S1 are zebrafish embryos that have developed to 48 hpf. The present invention selects 48 hpf embryos as the modeling object mainly based on the following considerations: First, at this stage, zebrafish have completed atrioventricular differentiation and possess a complete dynamic structure, which can simulate the systolic and diastolic characteristics of the human heart; second, their circulatory system has established a homeostasis, and various physiological indicators have entered a stable period, enhancing the statistical reliability of the model; third, this stage can effectively eliminate the interference of early developmental toxicity, ensuring the specific presentation of heart failure phenotypes such as pericardial effusion.

[0009] As one embodiment of the method for constructing the zebrafish heart failure model of the present invention, the pretreatment in step S1 includes the following steps: treating zebrafish embryos with 0.001%~0.005% 1-phenyl-2-thiourea (PTU) to remove body color, and demembraning with 0.5~2 mg / mL protease solution.

[0010] In one embodiment of the zebrafish heart failure model construction method of the present invention, the concentration of 1-phenyl-2-thiourea is 0.0045%; and the concentration of the protease solution is 1 mg / mL.

[0011] As one embodiment of the method for constructing the zebrafish heart failure model of the present invention, the treatment time of 1-phenyl-2-thiourea is from 24 hpf to 48 hpf of zebrafish embryos and until the membrane is completely removed.

[0012] As one embodiment of the zebrafish heart failure model construction method of the present invention, the exposure solution in step S2 includes 60-80 μM doxorubicin hydrochloride, 0.1%-0.5% (w / v) methylcellulose, 0.1% (v / v) dimethyl sulfoxide (DMSO) and culture medium.

[0013] The 0.1%-0.5% (w / v) methylcellulose mentioned in this invention refers to a ratio of the mass of methylcellulose to the total volume of the final exposed liquid of 0.1%-0.5%.

[0014] The selection of exposure fluid components in this invention can stabilize the pericardial edema rate in the constructed zebrafish heart failure model, while controlling the mortality rate to <10%.

[0015] As one embodiment of the zebrafish heart failure model construction method of the present invention, the exposure solution in step S2 includes 70 μM doxorubicin hydrochloride, 0.5% (w / v) methylcellulose, 0.1% (v / v) dimethyl sulfoxide and culture medium.

[0016] As one embodiment of the zebrafish heart failure model construction method of the present invention, the culture medium is E3 culture medium. The preparation method of E3 culture medium includes the following steps: weighing 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate and 55 mg of potassium chloride and dissolving them in 10 L of pure water to prepare the medium, with a pH value of 6.5 to 8.5.

[0017] In one embodiment of the zebrafish heart failure model construction method of the present invention, the dynamic viscosity of the exposure solution in step S2 is 1.2-1.5 cP. In this invention, the zebrafish heart failure model can only be constructed effectively when the dynamic viscosity of the exposure solution is maintained within a suitable range. When the dynamic viscosity of the exposure solution is too low, the physical stress is insufficient to overcome the compensatory capacity of the zebrafish heart, resulting in an insignificant heart failure phenotype that is prone to spontaneous remission, making it difficult to form a stable chronic heart failure model. When the dynamic viscosity of the exposure solution is too high, the ejection resistance exceeds the physiological limit, leading to cardiac arrest or severe systemic hypoxia, significantly increasing mortality. Furthermore, excessively high viscosity leads to decreased imaging quality, making it impossible to accurately quantify ventricular area and hemodynamic parameters. Only within the specific viscosity range of 1.2-1.5 cP can the chemical toxicity damage and physical load pressure produce the optimal synergistic effect, forming an ideal heart failure pathological state.

[0018] As one embodiment of the zebrafish heart failure model construction method of the present invention, the exposure conditions in step S2 are exposure to light at 25~30℃ for 20~30 hours.

[0019] As one embodiment of the zebrafish heart failure model construction method of the present invention, the exposure condition in step S2 is to expose to light at 28.5°C for 24 hours.

[0020] The zebrafish heart failure model construction method of this invention breaks through the pathological compensatory limitations of the zebrafish heart by applying dual stresses of physical resistance and chemical toxicity. Theoretical and experimental analysis shows that although the zebrafish heart is damaged under the action of 70 μM doxorubicin alone, it can still remain in the compensatory stage due to its strong physiological regulatory capacity. Core indicators such as cardiac output (CO) usually only show a moderate decrease (maintaining about 60% of the control group). This invention innovatively introduces methylcellulose. From a fluid dynamics perspective, doxorubicin weakens the driving force of myocardial contraction, while methylcellulose increases the resistance to blood ejection. The synergistic effect of the two causes the cardiac load to rapidly exceed the compensation threshold, prompting the model to enter the decompensated stage. This model accurately simulates the vicious cycle of myocardial contractility weakness and increased peripheral resistance in clinical severe heart failure.

[0021] The present invention also claims protection for an analytical method for a zebrafish heart failure model obtained by the construction method described above, wherein the exposed embryo is fixed in a left lateral decubitus position and a video of blood flow in the dorsal aorta is recorded; the average flow velocity (V) and vessel diameter (D) are measured based on the video of blood flow in the dorsal aorta, and the shear stress (τ) and blood flow (U) are calculated.

[0022] As one embodiment of the analysis method described in this invention, the duration of the dorsal aortic blood flow video is ≥10 seconds.

[0023] As one embodiment of the analysis method described in this invention, the exposed embryo is fixed in the left lateral decubitus position, and ventricular pulsation video is recorded. The heart rate (HR) and ventricular pulsation video are analyzed using ViewPoint software, and the fractional area change (FAC), fractional shortening (FS), ejection fraction (EF), and cardiac output (CO) are determined based on the end-diastolic area (EDA), end-systolic area (ESA), and the last frame image of the diastolic / systolic ventricle. Model stability verification: After removing the exposure solution, the model was cultured in normal E3 medium for another 24 hours. The above steps were repeated. If the decrease in cardiac output remained above 60%, the model was considered to have been constructed as an irreversible chronic heart failure model.

[0024] As one embodiment of the analysis method described in this invention, the duration of the ventricular beat video is ≥20 seconds and the frame rate is 120 fps.

[0025] As one embodiment of the analytical method described in this invention, the steps include the direct measurement of the ventricular end-diastolic / end-systolic area and the long and short axis diameters.

[0026] The analytical method of this invention develops a multi-dimensional quantitative system for cardiac function, integrating morphological, contractile function, and hemodynamic parameters. First, using an upright microscope, the embryo is placed in a left lateral decubitus position, with the right side of the embryo positioned above, to image the ventricle from above. High-resolution imaging is then used to capture pericardial edema, ventricular collapse, and venous sinus congestion for morphological observation. Simultaneously, this invention further optimizes the automated analysis process: based on the ViewPoint instrument's video acquisition module, a 20-second recording duration (120 fps frame rate) is set to ensure the capture of ≥10 complete cardiac cycles. ViewPoint software is used to select ventricular regions and analyze heart rate. Through a region selection optimization strategy—that is, using a large ventricular area—the accuracy of heart rate detection is improved.

[0027] At the dynamic level, high-resolution imaging, combined with ImageJ's frame-by-frame analysis function, was used to capture images of the ventricular end-diastolic (and systolic) frames, reducing human error. The long and short axis diameters and area of ​​the ventricle were measured at different time points; the fractional change in ventricular area, fractional shortening, stroke volume, ejection fraction, and cardiac output were calculated using formulas for each parameter. Hemodynamic measurements were performed using a ViewPoint instrument. The measured blood flow velocity and vessel diameter allowed for the calculation of shear stress on the vessel wall and blood flow within the vessel, which are important mechatronic factors affecting the function of the cardiac chambers and vascular endothelial cells. A 10-second video recording of embryonic dorsal aortic blood flow was recorded using the ViewPoint instrument's video acquisition module. The measured average linear velocity and vessel diameter (μm) were used, along with formulas to calculate shear stress and blood flow.

[0028] In the zebrafish heart failure model construction and analysis method of the present invention, compared with the use of other common cardiovascular inducing factors (such as isoproterenol, barium chloride, etc.) for induction, the "doxacin + methylcellulose" system used in the present invention has significant logical advantages in constructing a stable and highly sensitive model: ① Isoproterenol primarily induces tachycardia by activating β-adrenergic receptors, resulting in mostly transient metabolic overload. Due to the strong regenerative capacity of zebrafish, simple functional overload is easily self-healed after drug withdrawal, making it difficult to form a stable chronic heart failure phenotype. However, the combination of doxorubicin and methylcellulose at a certain concentration used in the construction method of this invention not only exhibits clear structural cardiomyocyte toxicity (causing damage through inhibition of topoisomerase II and ROS production) but also effectively prolongs the duration of the corresponding structural cardiomyocyte toxicity. The constructed model possesses characteristics such as phenotype lock-in and difficulty in self-healing, and its model stability is significantly superior to isoproterenol-based models.

[0029] ② As an ion channel blocker, barium chloride's pathological effects are primarily manifested in electrophysiological arrhythmias, with limited impact on the actual ventricular pumping structures (such as contractile force (FAC) and stroke volume (SV)). In contrast, the system of this invention achieves comprehensive pathological simulation from electrophysiological rhythm and mechanical contraction amplitude to global perfusion dynamics. Under the same physical stress, this system produces a more dramatic decrease in cardiac output and effectively avoids the common acute death risk associated with barium chloride drugs, thus providing a broader and more stable detection window for drug evaluation.

[0030] ③ The variation range of the core indicators induced by the system of this invention provides excellent sensitivity for screening functional foods or early candidate drugs with weak protective effects. In contrast, the indicator fluctuation range induced by isoproterenol and barium chloride is narrow, which can easily lead to false negatives in the evaluation results.

[0031] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention proposes a complete zebrafish heart failure model construction and multimodal evaluation system. Addressing the shortcomings of existing technologies, such as insufficient model stability and fragmented evaluation parameters, this system adjusts environmental viscosity by introducing methylcellulose into the induction system. Experiments show that appropriately increasing the viscosity of the culture medium significantly increases the afterload of the zebrafish heart. At this point, the combined use of doxorubicin can trigger a cascade reaction of cardiotoxicity and dynamic inhibition, achieving stable reproduction of the heart failure phenotype. This system innovatively integrates morphological and dynamic evaluation mechanisms: quantifying the ventricular dilation ratio and pericardial edema area; simultaneously capturing heart rate, ventricular area change fraction, shortening fraction, stroke volume, ejection fraction, and cardiac output; measuring the mean blood flow velocity in the dorsal aorta, and further obtaining shear stress and blood flow. A complete process method system from phenotype induction → dynamic monitoring → efficacy quantification is established, providing a high-precision, high-throughput, and low-cost technical platform for cardiovascular drug screening and functional food efficacy certification.

[0032] (2) This invention verified through corresponding experiments that, in the induction group using doxorubicin and methylcellulose, the core indicator of cardiac pumping function, cardiac output (CO), significantly decreased to 5.50±2.3 nL / min, a reduction of 85.36% compared to the control group. Simultaneously, myocardial contractile function was comprehensively impaired, with fractional area change (FAC) and fractional shortening (FS) decreasing by 53.1% and 40.6%, respectively, and stroke volume (SV) decreasing by 64.6%. Hemodynamic parameters exhibited systemic disturbances: mean flow velocity decreased by 62.3%, vascular shear stress (τ) decreased by 53.7%, and blood flow (U) sharply decreased by 75.1%. This model successfully simulated the core characteristics of clinical heart failure—low cardiac output, low blood perfusion, and ventricular systolic dysfunction—confirming its effectiveness and reliability as a platform for heart failure research and the evaluation of drugs and functional foods. Attached Figure Description

[0033] Figure 1 The zebrafish imaging placement method (left lateral position) is shown in this embodiment of the invention.

[0034] Figure 2 This is a schematic diagram of the morphological features of the zebrafish heart failure model in an embodiment of the present invention.

[0035] Figure 3 This is a schematic diagram of the zebrafish ventricle major axis, minor axis, and area measurement region in an embodiment of the present invention.

[0036] Figure 4 This is a schematic diagram of the blood flow measurement area of ​​the dorsal aorta in a zebrafish according to an embodiment of the present invention. Detailed Implementation

[0037] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0039] The preparation method of the E3 culture medium selected in this embodiment of the invention includes the following steps: 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, and 55 mg of potassium chloride are weighed and dissolved in 10 L of pure water to prepare the medium with a pH value of 6.5 to 8.5. All chemicals are of analytical grade.

[0040] Example 1: Construction of a Zebrafish Heart Failure Model (1) Embryo preparation All zebrafish broodstock were housed in a zebrafish farming system, and quality control was performed in accordance with GB / T 39649-2020, "Quality Control of Laboratory Animals and Experimental Fish". Ten pairs of AB strain broodstock were used, with males and females temporarily housed separately in 3L mating boxes. Fertilized eggs with basically synchronized development were obtained using the light-induced method.

[0041] Embryos developed to 24 hpf were selected. 0.0045% 1-phenyl-2-thiourea (PTU) was added to the embryo culture medium (E3) to inhibit the formation of body color in the larvae, and this was continued until the end of the experiment. Embryos developed to 48 hpf were demembraned with protease (1 mg / ml) for drug treatment.

[0042] (2) Drug treatment Preparation of the stock solution of doxorubicin hydrochloride, the inducing drug: First, prepare a 70mM stock solution of doxorubicin hydrochloride (weigh 40.60 mg of doxorubicin hydrochloride → dissolve in 1mL DMSO), and store at 4°C protected from light.

[0043] Preparation of methylcellulose stock solution: Weigh out high-purity methylcellulose powder, dissolve it in E3 medium preheated to 80°C to prepare a 1.0% (w / v) methylcellulose stock solution, stir until completely dissolved and refrigerate overnight to remove bubbles.

[0044] Induction group: Doxorubicin hydrochloride stock solution, methylcellulose stock solution, and E3 medium were mixed according to a preset ratio. Specifically, doxorubicin stock solution was added to E3 medium to achieve a concentration of 70 μM, and 1.0% (w / v) of methylcellulose stock solution was added proportionally and mixed thoroughly to ensure that the final system (i.e., the exposure solution) had a methylcellulose concentration of 0.5% (w / v) and a DMSO concentration of 0.1%, with a final dynamic viscosity of approximately 1.2-1.5 cP. This final mixture was the exposure solution used in the induction group.

[0045] Doxorubicin monotherapy group: E3 medium containing only 70 μM doxorubicin and 0.1% DMSO.

[0046] Blank control group: E3 medium containing only 0.1% DMSO.

[0047] Construction method The obtained embryos were placed in E3 culture medium of the induction group, the doxorubicin-only treatment group, and the blank control group, respectively, and exposed to light at 28.5℃ for 24 hours to complete the construction of the zebrafish heart failure model.

[0048] Example 2: Analysis of a Zebrafish Heart Failure Model (1) Phenotypic Imaging and Detection Morphological assessment: After exposure, the embryos were placed in the left lateral decubitus position and observed under an upright microscope to check for typical heart failure features, such as pericardial edema, ventricular collapse, and venous sinus congestion. Experimental results are as follows: Figure 2 As shown.

[0049] from Figure 2 The experimental results show that the blank control group has a compact ventricular structure and a clear pericardial cavity. Compared with the blank control group, the zebrafish induced by this invention showed obvious pericardial edema, and the pericardial edema was more obvious than that of the group treated with only doxorubicin. This indicates that the construction method of this invention can effectively construct a zebrafish heart failure model.

[0050] (2) Dynamic monitoring of cardiac function The embryo was placed in the left lateral decubitus position, and ventricular contraction videos were recorded under an upright microscope (20 s, 120 fps). Heart rate (HR) was analyzed using ViewPoint software (Heart Beat program), with the selected region covering the entire ventricular area to improve accuracy. The recorded videos were analyzed frame-by-frame using ImageJ to determine the diameter and area of ​​end-diastolic (EDA) and end-systolic (ESA). The core parameters are shown in Table 1, and the experimental results are presented in Table 2.

[0051] Table 1 Formulas for Calculating Cardiac Function Parameters Note: EDF is the end-diastolic frame; ESF is the end-contraction frame; EDA is the end-diastolic area; ESA is the end-contraction area; D d It is the diastolic ventricular diameter; D s It is the systolic ventricular diameter; D L It is the long axis ventricular diameter; D S Short-axis ventricular diameter; EDV is end-diastolic volume; ESV is end-systolic volume. Table 2. Statistical analysis of changes in cardiac function parameters in a zebrafish heart failure model. The experimental results in Table 2 show that, compared with the blank control group, the heart rate of the doxorubicin-only treatment group decreased slightly, myocardial contraction was still compensable, and the shortening fraction and ejection fraction were almost the same as those of the blank control group. However, the induction group treated with doxorubicin and methylcellulose showed significant bradycardia due to high afterload, with a contractility decrease of >50%, indicating deeper damage. The ventricular radial shortening capacity was significantly weaker than that of the doxorubicin-only treatment group, and the single pumping volume was further reduced compared with the doxorubicin-only group, indicating further failure of pumping function. The ejection fraction showed a clear downward trend, and the cardiac output decreased by more than 85%, showing a more significant difference.

[0052] (3) Hemodynamic analysis MicroZebraLab recorded a 10-second video of blood flow in the dorsal aorta, measuring the average flow velocity (V) and vessel diameter (D); it also calculated the shear stress (τ) and blood flow rate (U). The core parameters are shown in Table 3, and the experimental results are presented in Table 4.

[0053] Table 3 Formulas for Calculating Blood Flow Parameters Note: In the shear stress formula, μ is the blood viscosity (dynes / cm). 2 V is the average blood flow velocity (μm / s), and D is the vessel diameter (μm). Shear stress was calculated based on the Poiseuille flow assumption, using a dynamic viscosity of 4 × 10⁻⁶ at 3 dpf.−3 PAS. In the blood flow formula, V is the average blood flow velocity (μm / s), and D is the vessel diameter (μm).

[0054] Table 4. Statistical analysis of blood flow parameters in a zebrafish heart failure model. As can be seen from the experimental results in Table 4, the mean blood flow velocity in the zebrafish heart failure model constructed by using doxorubicin and methylcellulose in the construction method of this invention drops sharply, which can simulate severe hypoperfusion. At the same time, compared with the blank control group, the shear stress loss in the induction group is more severe, the biomechanical environment changes more significantly, and the peripheral perfusion volume changes more significantly.

[0055] The experimental results above indicate that the success of the zebrafish heart failure model is primarily determined by the total cardiac output. When the decrease in cardiac output (CO) and total blood flow (U) exceeds 75% (the experimentally measured decrease in CO was 85.36% and U was 75.1%), it signifies near-total cessation of circulatory function, indicating severe heart failure. Regarding stroke volume (SV) and mean velocity (V) showed a coordinated decrease of 60%-70% (experimental values: SV decrease of 64.6% and V decrease of 62.3%), reflecting severe circulatory insufficiency. The weakening of myocardial contractility was manifested by a significant impairment of 50%-55% in fractional area change (FAC) and wall shear stress (τ) (experimental values: FAC decrease of 53.1% and τ decrease of 53.7%), indicating that the amplitude of myocardial movement had been reduced by more than half. Furthermore, abnormalities in cardiac morphology and rhythm were observed, with a 25%-45% decrease in fractional shortening (FS) and heart rate (HR) (experimental values ​​showed a 40.6% decrease in FS and a 28.9% decrease in HR), indicating impaired radial shortening of the ventricles and a significantly slowed heart rate. Finally, ejection fraction (EF) showed a robust decrease of 10%-15% (experimental value 13.1%), serving as a fundamental indicator for assessing a decline in ejection ratio. These wide fluctuations in these indicators collectively constitute a highly sensitive system for diagnosing heart failure in zebrafish.

[0056] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for constructing a zebrafish heart failure model, characterized in that, Includes the following steps: S1. Pre-treatment of zebrafish embryos; S2. The zebrafish embryos obtained from the pretreatment in step S1 are exposed in an exposure solution to complete the construction of the zebrafish heart failure model.

2. The method for constructing the zebrafish heart failure model as described in claim 1, characterized in that, The zebrafish embryos used in step S1 are zebrafish embryos that have developed to 48 hpf.

3. The method for constructing the zebrafish heart failure model as described in claim 1, characterized in that, The pretreatment described in step S1 includes the following steps: treating zebrafish embryos with 0.001% to 0.005% 1-phenyl-2-thiourea to remove body color, and then demembraning them with a 0.5 to 2 mg / mL protease solution.

4. The method for constructing the zebrafish heart failure model as described in claim 3, characterized in that, The treatment time for the 1-phenyl-2-thiourea was from 24 hpf to 48 hpf for zebrafish embryos and until the membrane was completely removed.

5. The method for constructing the zebrafish heart failure model as described in claim 1, characterized in that, The exposure solution in step S2 includes 60-80 μM doxorubicin hydrochloride, 0.1%-0.5% (w / v) methylcellulose, 0.1% (v / v) dimethyl sulfoxide, and culture medium.

6. The method for constructing the zebrafish heart failure model as described in claim 5, characterized in that, The culture medium is E3 medium, and the preparation method of E3 medium includes the following steps: weigh 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate and 55 mg of potassium chloride and dissolve them in 10 L of pure water to prepare the medium, with a pH value of 6.5 to 8.

5.

7. The method for constructing the zebrafish heart failure model as described in claim 1, characterized in that, The dynamic viscosity of the exposure liquid in step S2 is 1.2-1.5 cP.

8. The method for constructing the zebrafish heart failure model as described in claim 1, characterized in that, The exposure conditions described in step S2 are exposure to light at 25-30°C for 20-30 hours.

9. An analytical method for a zebrafish heart failure model obtained by the construction method as described in any one of claims 1-8, characterized in that, Includes at least one of the following (a)-(b): (a) After exposure, the embryo was fixed in the left lateral decubitus position and the dorsal aorta blood flow video was recorded; the mean flow velocity and vessel diameter were determined based on the dorsal aorta blood flow video, and the shear stress and blood flow were calculated; (b) After exposure, the embryo was fixed in the left lateral decubitus position, and ventricular pulsation video was recorded. The heart rate was analyzed using ViewPoint software and ventricular pulsation video. Based on the end-diastolic area, end-systolic area, and the last frame image of the ventricular pulsation during diastole / systole, the area change fraction, shortening fraction, ejection fraction, and cardiac output were determined. Model stability verification: After removing the exposure solution, continue culturing in normal E3 medium for 24 hours and repeat the above step (b). If the decrease in cardiac output still remains above 60%, it is determined that an irreversible chronic heart failure model has been constructed.

10. The analytical method as described in claim 9, characterized in that, The duration of the ventricular beat video is ≥20 seconds, and the frame rate is 120 fps; The duration of the dorsal aortic blood flow video is ≥10 seconds; Step (b) includes direct measurement of the ventricular end-diastolic / end-systolic area and the long and short axis diameters.

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