Method for constructing zebra fish developmental angiogenesis defect-cerebral hemorrhage model

Abstract

The invention provides a construction method of a zebra fish developmental angiogenesis defect-cerebral hemorrhage model based on quintozene. Specifically, zebra fish embryos which normally develop 5-6 hours after fertilization (5-6 hpf) are exposed in a quintozene solution, and the zebra fish embryos are cultured and molded in an incubator. The invention also provides an application of quintozene in preparation of an angiogenesis defect and cerebral hemorrhage research reagent, and an application of a zebra fish developmental angiogenesis defect-cerebral hemorrhage model based on quintozene in high-throughput screening of therapeutic drugs. While the application field of quintozene is expanded, powerful improvement is provided for a modeling method of developmental angiogenesis defect-cerebral hemorrhage and a related drug screening method.

Description

Technical Field

[0001] The present invention relates to the field of animal models, and particularly to a method for inducing a zebrafish developmental angiogenesis defect-intracerebral hemorrhage model by using quintozene. Background Art

[0002] Quintozene is a fungicide and a substance commonly detected in vegetables, traditional Chinese medicines, environmental media (such as soil, river water, groundwater, ocean), and human plasma.

[0003] Intracerebral hemorrhage caused by developmental angiogenesis defect (hereinafter referred to as developmental intracerebral hemorrhage) is a cerebrovascular disease that can induce severe neurological dysfunction, mainly caused by vascular leakage due to immature or malformed blood vessels in the brain region. Developmental intracerebral hemorrhage mainly occurs in neonates and infants, especially premature neonates. Approximately 20-40% of premature infants will have spontaneous intracerebral hemorrhage in the brain when born before 30 gestational weeks. This spontaneous intracerebral hemorrhage is considered to be the result of incomplete development of cerebral blood vessels, weak blood vessel walls, hemodynamic changes, and blood system diseases. Animal models of developmental intracerebral hemorrhage are helpful for deeply understanding the impact of angiogenesis and stability during development on intracerebral hemorrhage and are important tools for evaluating treatment strategies and drug efficacy. Currently, common animal models of developmental intracerebral hemorrhage mainly focus on simulating intracerebral hemorrhage caused by angiogenesis defects by methods such as gene knockout, signal pathway interference, and chemical drug intervention in mice, rats, and rabbits. For example, mice or rats with gene knockout of Ccm1, Ccm2, Ccm3, etc. have defects in the generation of blood vessel wall structure during the development of cerebral blood vessels, resulting in fragile blood vessels and prone to rupture and bleeding; mice lacking Angiopoietin-1 (Ang-1) or Tie2 will lead to incomplete blood vessel development, causing angiogenesis defects and unstable blood vessel structure, resulting in rupture and bleeding of cerebral blood vessels; neonatal rabbits (such as at the P1 stage) are intravenously or subcutaneously injected with VEGF inhibitor drugs to cause incomplete blood vessel development and blood vessel rupture, simulating pathological conditions such as intracerebral hemorrhage; by injecting or infusing chemical substances (such as calcium chloride, levodopa, etc.) into early neonatal mice (such as at the P1 stage) to simulate intraventricular hemorrhage caused by angiogenesis defects or blood vessel rupture; intraperitoneal injection of bactericidal factors ELANE and CXCL16 into pregnant mice during pregnancy will disrupt the integrity of angiogenesis and cause hemorrhage in the lateral ventricles of neonatal mice. Although these animal models are currently useful tools for studying the mechanism of developmental intracerebral hemorrhage and evaluating drug efficacy, they are all established based on mouse, rat, and rabbit animal models, and they mainly have the disadvantages of high cost, long test cycle, difficult visualization of pathological changes, difficult to evaluate a large number of individuals (high throughput) simultaneously, complex methods, and difficult operation. Therefore, there is an urgent need to design a developmental intracerebral hemorrhage model that is easy to screen drug efficacy, low-cost, fast, visual, high-throughput, and easy to operate.

[0004] The genomic sequence of zebrafish has a homology of up to 87% with that of humans, and the molecular and cellular mechanisms of development are very similar to those of mammals, which can effectively simulate the pathological characteristics of human diseases. For example, the conservation in the development of zebrafish brain blood vessels and the biological processes shared with other vertebrates (including humans) have made it a mature in vivo model in the fields of vascular biology, drug safety, and pharmacological efficacy evaluation. Compared with traditional animal models (such as mice, rats, rabbits, etc.), zebrafish have the advantages of low cost, rapid growth and development (most of the body's tissues and organs are functionally developed within 3 days after the fish eggs are born, including blood vessels), visualization (the body of zebrafish is transparent during the embryonic period, and with fluorescent labeling of tissues and organs, it can be directly used for pathological observation, such as blood vessels), high-throughput processing (strong reproductive ability, a pair of male and female fish can produce 60 - 100 fish eggs each time), easy and simple operation, etc.

[0005] Therefore, the technical problem to be solved by the present invention is: to provide a method for constructing a zebrafish developmental angiogenesis defect - intracerebral hemorrhage model based on quintozene, so as to solve the problems existing in the prior art, such as expensive modeling cost, long test cycle, difficult visualization of pathological changes, difficult evaluation of a large number of individuals (high-throughput) at the same time, complex method, and difficult operation. Summary of the Invention

[0006] Based on the zebrafish model, the present invention discovers and constructs a zebrafish developmental angiogenesis defect - intracerebral hemorrhage model based on quintozene.

[0007] In the first aspect of the present invention, a method for constructing a zebrafish developmental angiogenesis defect - intracerebral hemorrhage model based on quintozene is provided, including the following steps:

[0008] S1: Prepare zebrafish embryos with normal development 5 - 6 hours (5 - 6hpf) after fertilization;

[0009] S2: Expose the zebrafish embryos in S1 to a quintozene solution and culture them in an incubator for modeling;

[0010] S3: Characterize the model completed in S2.

[0011] In some embodiments, the method for preparing zebrafish embryos in step S1 is: Put normally cultured zebrafish into a mating tank in a ratio of male to female of 1:1 or 1:2, and separate them with a partition for one night. The next day, remove the partition barrier, and the male and female zebrafish start to mate and lay eggs. Collect the embryos within 30 minutes after egg laying, and use a pipette to remove dead and unfertilized eggs, feces and other sundries; then rinse with pure water 3 times. Finally, place them in an incubator at 28 - 28.5 °C.

[0012] In some embodiments, the quintozene solution in step S2 is a quintozene solution prepared with zebrafish embryo culture water. Preferably, the concentration of the quintozene solution is 0.01 - 1.0 mg / L; more preferably, the concentration of the quintozene solution is 0.1 - 0.8 mg / L; further preferably, the concentration of the quintozene solution is 0.3 - 0.6 mg / L.

[0013] In some embodiments, the culturing and modeling conditions in the incubator in step S2 are: culturing at 28 - 28.5 °C for 2.8 - 3.5 days, and changing the liquid every 22 - 24 h.

[0014] In some embodiments, the characterization in step S3 includes detecting the angiogenesis and bleeding conditions in the zebrafish brain using a fluorescence microscope.

[0015] In some embodiments, the detection of the angiogenesis and bleeding conditions in the zebrafish brain includes detecting the defect in the formation of the central artery vascular plexus in the brainstem, and the leakage and accumulation of blood cells in the brainstem tissue resulting in intracerebral hemorrhage.

[0016] In some embodiments, the formation defect is mainly reflected in the reduction in the number of sprouts (budding) of the central artery blood vessels in the brainstem, the decrease in the growth length, and the increase in fragility or permeability.

[0017] In the second aspect of the present invention, there is provided a zebrafish developmental angiogenesis defect - intracerebral hemorrhage model constructed using quintozene.

[0018] In some embodiments, the model is constructed using the method of the first aspect.

[0019] In the third aspect of the present invention, there is provided an application of quintozene in the preparation of research reagents for angiogenesis defects and intracerebral hemorrhage.

[0020] In some embodiments, the quintozene can induce a defect in the formation of the central artery vascular plexus in the zebrafish brainstem, thereby causing the leakage and accumulation of blood cells in the brainstem tissue resulting in intracerebral hemorrhage.

[0021] In some embodiments, the formation defect is mainly reflected in the reduction in the number of sprouts (budding) of the central artery blood vessels in the brainstem, the decrease in the growth length, and the increase in fragility or permeability.

[0022] In the fourth aspect of the present invention, there is provided an application of a zebrafish developmental angiogenesis defect - intracerebral hemorrhage model based on quintozene in high - throughput screening of therapeutic drugs, characterized in that the therapeutic drugs have the potential to protect the growth and development of cerebral blood vessels and rescue developmental intracerebral hemorrhage.

[0023] In the fifth aspect of the present invention, a method for high-throughput screening of therapeutic drugs using a zebrafish developmental angiogenesis defect-intracerebral hemorrhage model based on quintozene is provided, characterized in that during the screening process, a potential therapeutic drug is added to the zebrafish developmental angiogenesis defect-intracerebral hemorrhage model, and then it is cultured in an incubator at 28-28.5 °C for 2.8-3.5 days; the culture medium is changed every 22-24 hours. Observation and statistics of intracerebral hemorrhage and vascular pathology are carried out to screen out drugs with the potential to protect the growth and development of cerebral blood vessels and rescue developmental intracerebral hemorrhage.

[0024] The beneficial effects achieved by the present invention are as follows:

[0025] (1) Using zebrafish to construct a developmental angiogenesis defect-intracerebral hemorrhage model has low test costs, a short cycle, and an easy and simple operation method. The dynamic changes of intracerebral hemorrhage and cerebral vascular pathology can be directly observed in real time.

[0026] (2) The present invention discovers for the first time that after treatment with quintozene, it can induce defects in the formation of the central artery vascular plexus in the zebrafish brainstem, which in turn leads to blood cell leakage and accumulation in the brainstem tissue, causing intracerebral hemorrhage; the formation defects are mainly reflected in the reduction in the number of sprouts (budding) of the central artery blood vessels in the brainstem, the decrease in growth length, and the increase in fragility or permeability. This discovery has opened up the application value of quintozene in the field of research reagents for angiogenesis defects and intracerebral hemorrhage.

[0027] (3) It provides a feasible method basis for realizing high-throughput screening of drugs with the potential to protect the growth and development of cerebral blood vessels and rescue developmental intracerebral hemorrhage. Brief Description of the Drawings

[0028] Figure 1 Schematic diagram of high-throughput screening of therapeutic drugs based on the zebrafish developmental angiogenesis defect-intracerebral hemorrhage model.

[0029] Figure 2 Effects of quintozene solutions with different concentrations on the total mortality of zebrafish embryos; where the exposure concentrations are 0 (Control), 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10 mg / L respectively, and the mortality rate is represented by the mean ± standard deviation of the total mortality of three parallels in the same group (20-25 embryos in each parallel) after 2.8-3.5 days of exposure.

[0030] Figure 3Cerebral hemorrhage and cerebrovascular angiogenesis defects induced by exposure to quintozene at different concentrations in zebrafish; among which, A is a representative image of cerebral hemorrhage in the left field of view of the zebrafish head under bright field; B is a representative image of cerebral hemorrhage in the dorsal field of view of the zebrafish head after labeling red blood cells with o-dianisidine under bright field; C is a representative image of the overall angiogenesis status and cerebral hemorrhage in the dorsal field of view of zebrafish with green fluorescence-labeled vascular endothelial cells (Flk1: GFP) and red fluorescence-labeled blood cells (gata1: DsRed); D is the cerebral hemorrhage ratio of zebrafish after exposure to quintozene in the implementation case; the cerebral hemorrhage ratio is shown as the mean ± standard deviation of three parallels in the same group (20-25 embryos in each parallel); *** indicates a significant difference with P value < 0.001. fb, mb, and hb represent the positions of the forebrain, midbrain, and hindbrain respectively; red arrows are used to indicate cerebral hemorrhage in the brain; yellow arrows are used to indicate the phenomenon of failed angiogenesis connection on the brain surface.

[0031] Figure 4 Quintozene induces cerebral hemorrhage at the hindbrain position of zebrafish; among which, A shows the main hemorrhage positions throughout the body of zebrafish under bright field; B shows the hemorrhage ratios at various positions (forebrain, midbrain, hindbrain, and trunk) of zebrafish induced by high-concentration (0.6 mg / L) quintozene in the implementation case. Green arrows represent the main hemorrhage positions.

[0032] Figure 5 Quintozene induces cerebral hemorrhage in the zebrafish brainstem and defects in the formation of central arteries of the brainstem (CtAs); among which, A is a representative image of the right field of view of the central artery plexus of the zebrafish brainstem with red fluorescence-labeled vascular endothelial cells (kdrl: mCcherry); B is a representative image of the dorsal field of view of the central artery plexus of the zebrafish brainstem; C is the length of the central artery plexus of the brainstem statistically derived from Figure B; D is the number of branches of the central artery of the brainstem statistically derived from Figure B; E is a representative image of the cerebral hemorrhage in the zebrafish brainstem and the angiogenesis status of the central artery of the brainstem with green fluorescence-labeled vascular endothelial cells (Flk1: GFP) and red fluorescence-labeled blood cells (gata1: DsRed); F is the angiogenesis status of the central artery of the brainstem in zebrafish at 40 hours post-fertilization (40 hpf) under black-and-white imaging effect (RAW) with red fluorescence-labeled vascular endothelial cells (kdrl: mCcherry). BA represents the basilar artery of the zebrafish brain; PHBC represents the primitive hindbrain channel (vein) of the zebrafish brain; yellow arrows indicate the state of failed connection of the central artery of the brainstem induced by low-concentration (0.3 mg / L) quintozene in the implementation case; the enlarged local image shows the budding (sprouting) landmark state of the central artery of the brainstem; red arrows indicate the budding (sprouting) central artery of the brainstem. Detailed implementation methods

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0034] In the following experimental methods of the embodiments, unless otherwise specified, they are all conventional methods; in the following test materials used in the embodiments, unless otherwise specified, they are all obtained from commercial channels.

[0035] Example 1

[0036] Construction of a zebrafish developmental angiogenesis defect-intracerebral hemorrhage model based on quintozene

[0037] Zebrafish cultured normally were placed in a mating tank at a ratio of male to female of 1:1 or 1:2 the night before, and separated by a partition. The next morning, the partition barrier was removed, and the male and female fish began to mate and lay eggs. Embryos were collected within 30 minutes after spawning, and dead and unfertilized eggs, feces and other sundries were removed with a pipette; then rinsed 3 times with pure water. Finally, placed in an incubator at 28 - 28.5 °C.

[0038] Under a microscope, zebrafish embryos with normal development 5 - 6 hours post-fertilization (5 - 6 hpf) were selected and transferred into a six-well plate containing quintozene solutions with different concentrations (0.3 mg / L, 0.6 mg / L) prepared with embryo culture water; at the same time, a control group (incubated with the same volume of embryo culture water) was set; there were three parallels in each group; placed in an incubator at 28 - 28.5 °C for 2.8 - 3.5 days, and the liquid was changed every 22 - 24 hours.

[0039] Using the bright-field imaging effect of a fluorescence microscope (Leica M205 FA, Germany), the intracerebral hemorrhage of zebrafish after quintozene exposure and the blood clots accumulated in the brain labeled with o-dianisidine were recorded.

[0040] Using zebrafish co-labeled with green fluorescence (Flk1:GFP, labeling vascular endothelial cells) and red fluorescence (gata1:Dsred, labeling blood cells), and zebrafish labeled with red fluorescence (kdrl:mCherry, labeling vascular endothelial cells), the intracerebral hemorrhage status and angiogenesis of zebrafish after quintozene exposure were recorded by laser confocal microscopy (Leica SP8, Germany).

[0041] The mortality rate and intracerebral hemorrhage ratio were statistically analyzed by Graphpad Prism 8.0 software.

[0042] The generated length and number of branches of the recorded central arteries of the brainstem (CtAs) were quantified using ImageJ (version 1.51k) software; then statistical analysis was performed using Graphpad Prism 8.0 software.

[0043] Example 2

[0044] High-throughput screening of therapeutic drugs based on a zebrafish developmental angiogenesis defect-intracerebral hemorrhage model

[0045] As shown in the Figure 1 appendix, zebrafish embryos co-labeled with normal green fluorescence (Flk1: GFP, labeling vascular endothelial cells) and red fluorescence (gata1: Dsred, labeling blood cells) at 5-6 hours post-fertilization (hpf) and zebrafish embryos labeled with red fluorescence (kdrl: mCherry, labeling vascular endothelial cells) were selected; embryos were sub-packaged into six-well plates at a quantity of 20-25 per group; zebrafish embryo culture medium containing quintozene (concentration 0.3-0.6 mg / L) was added to the six-well plates and exposed to the zebrafish embryos as the model group; at the same time, drugs 1, 2, 3... were added on the basis of the model group; then the well plate was placed in an incubator at 28-28.5 °C and cultured for 2.8-3.5 days; the medium was changed every 22-24 h. Finally, observations and statistics of intracerebral hemorrhage and vascular pathology were performed to screen out drugs with the potential to protect the growth and development of cerebral blood vessels and rescue developmental intracerebral hemorrhage.

[0046] Test Example 1

[0047] Effect of quintozene solutions at different concentrations on the total mortality of zebrafish embryos

[0048] Before implementing the protocol of Example 1, the changes in the total mortality of quintozene at different concentrations (0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10 mg / L) during continuous exposure to zebrafish at 5-6 hpf for 2.8-3.5 days were recorded and statistically analyzed to determine the exposure range of quintozene that would not cause death and had obvious intracerebral hemorrhage characteristics. The results are as Figure 2 shown, the LC 50 of quintozene was 1.670 mg / L.

[0049] Test Example 2

[0050] Characterization of intracerebral hemorrhage and cerebrovascular angiogenesis defects induced by quintozene in zebrafish

[0051] Furthermore, the exposure concentrations of quintozene were determined to be 0.3 mg / L (low concentration) and 0.6 mg / L (high concentration). As Figure 3As shown, low and high concentrations can induce intracerebral hemorrhage rates of 15% and 56.8% in 20 - 25 embryos respectively ( Figure 3 D); bright - field microscopy can observe the significant characteristics of intracerebral hemorrhage in zebrafish embryos ( Figure 3 A red arrow). To confirm the intracerebral hemorrhage clot, after quintozene exposure, o - dianisidine was further used to specifically label red blood cells; the results showed that quintozene induced obvious accumulated blood clots in the zebrafish brain ( Figure 3 B). A transgenic zebrafish line co - labeled with green fluorescence (Flk1: GFP, labeling vascular endothelial cells) and red fluorescence (gata1: Dsred, labeling blood cells) was exposed to quintozene ( Figure 3 C); the results showed that a large number of blood cells that were not present in blood vessels accumulated in the zebrafish brain, and disconnections or failures were observed in the characteristics of the blood vessels on the brain surface ( Figure 3 C yellow arrow).

[0052] Test Example 3

[0053] Identification of the specific sites of quintozene - induced intracerebral hemorrhage in zebrafish

[0054] As Figure 4 shown, the overall hemorrhage characteristics of zebrafish after quintozene induction ( Figure 4 A green arrow) show that the hindbrain (brainstem) is the main hemorrhage site. The hemorrhage rates at the forebrain, midbrain, hindbrain, and trunk positions were statistically analyzed respectively ( Figure 4 B), and the results showed that the hemorrhage probability at the hindbrain (brainstem) position was much higher than that of other parts.

[0055] Test Example 4

[0056] Characterization of quintozene - induced brainstem hemorrhage and defects in the generation of central trunk arteries (CtAs) in zebrafish

[0057] The hemorrhage phenomenon in the brainstem is predictably closely related to the damage of brainstem - related blood vessels. The blood vessels in the brainstem mainly include the basilar artery (BA), primordial hindbrain channels (PHBCs, veins), and central trunk arteries (CtAs) connecting BA and PHBCs. The central trunk artery vascular plexus is mainly used to irrigate the brainstem nerve tissue and provide it with oxygen and various nutrients.

[0058] Detect the changes in BA, PHBCs, and CtAs at the hindbrainstem position induced by quintozene. After zebrafish labeled with red fluorescence (kdrl: mCherry, labeling vascular endothelial cells) were induced by low - concentration quintozene, the central trunk artery vascular plexus was significantly lacking, and severely lacking or even lost after high - concentration induction ( Figure 5A). To quantify this angiogenesis defect, further obtain the visual field of the vascular status at the dorsal brainstem position of zebrafish ( Figure 5 B). The results showed that quintozene had little effect on the basilar artery (BA) and primary hindbrain channels (PHBCs, veins), but significantly induced the clustering defect of the central arteries of the brainstem (CtAs) Figure 5 B). Through quantification, the generation defect of the central artery vascular cluster in the brainstem was mainly reflected in the reduction of its growth length Figure 5 C) and the decrease in the number of branches Figure 5 D). The generation defect of the central artery vascular cluster in the brainstem can be predicted to be associated with the generation status of early CtAs. At 40 hours post-fertilization (40 hpf) of zebrafish embryos exposed to quintozene, it was observed that the number of germinating (budding) vessels of the central arteries of the brainstem induced by quintozene was significantly less than that of the control group Figure 5 F red arrow). This maximally explains the reasons for the reduction in the growth length and the number of branches of the central arteries of the brainstem.

[0059] Furthermore, to verify the association between the angiogenesis defect of the central arteries of the brainstem induced by quintozene and brainstem hemorrhage, zebrafish co-labeled with green fluorescence (Flk1: GFP, labeling vascular endothelial cells) and red fluorescence (gata1: Dsred, labeling blood cells) were exposed to quintozene; the results showed that in the control group, blood cells filled the entire central artery vascular cluster of the brainstem, while in the brainstem after quintozene induction, there were almost no blood cells in the lumen of the central arteries of the brainstem, but a large number of blood cells accumulated in the non-vascular area at the level of the central arteries of the brainstem Figure 5 E). The results indicate that quintozene leads to blood cell leakage and accumulation in the brainstem tissue (brainstem hemorrhage) by inducing the generation defect of the central artery vascular cluster in the zebrafish brainstem. This generation defect is mainly reflected in the reduction of the number of germinating (budding) vessels, the decrease in growth length, and the increase in fragility or permeability of the central arteries of the brainstem.

[0060] Unless otherwise defined, all professional and scientific terms used in this text have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described in this text are for illustrative purposes only and do not limit the content of this application.

Claims

1. A method for constructing a zebrafish developmental angiogenesis defect-cerebral hemorrhage model based on pentachloronitrobenzene, characterized in that: The following steps are involved: S1: Preparation of normal developing zebrafish embryos at 5-6 hours post fertilization (5-6 hpf); S2: The zebrafish embryos in S1 were exposed to pentachloronitrobenzene solution and cultured in an incubator to establish the model; S3: Characterize the model completed in S2.

2. The construction method according to claim 1, characterized in that: The preparation method of zebrafish embryos in step S1 is as follows: normally cultured zebrafish are placed in a mating tank in a ratio of 1:1 or 1:2 between male and female, and are separated by partitions and raised overnight; the next day, the partition barrier is removed, and the male and female zebrafish begin to mate and lay eggs; the embryos are collected within 30 minutes after spawning, and debris is removed with a straw; then they are rinsed with pure water 3 times and placed in an incubator at 28-28.5°C.

3. The construction method according to claim 1, characterized in that: The pentachloronitrobenzene solution in step S2 is a pentachloronitrobenzene solution prepared with zebrafish embryo culture water.

4. The construction method according to claim 3, characterized in that: The concentration of the pentachloronitrobenzene solution is 0.01-1.0 mg / L; preferably, 0.1-0.8 mg / L; more preferably, 0.3-0.6 mg / L.

5. The construction method according to claim 1, characterized in that: The conditions for culturing and modeling in the incubator in step S2 are: culturing at 28-28.5° C. for 2.8-3.5 days, and changing the medium every 22-24 hours.

6. The construction method according to claim 1, characterized in that: The characterization in step S3 includes detecting angiogenesis and bleeding in the zebrafish brain using a fluorescence microscope.

7. A zebrafish developmental angiogenesis defect-cerebral hemorrhage model based on pentachloronitrobenzene, characterized in that: The model is constructed using the method described in any one of claims 1-6.

8. Use of pentachloronitrobenzene in the preparation of reagents for the study of angiogenesis defects and cerebral hemorrhage; preferably, the pentachloronitrobenzene can induce a defect in the formation of vascular clusters in the central artery of the zebrafish brainstem, thereby causing blood cell leakage and accumulation in the brainstem tissue to cause cerebral hemorrhage; more preferably, the pentachloronitrobenzene-induced defect in the formation of vascular clusters in the central artery of the zebrafish brainstem is mainly manifested in a decrease in the number of sprouts (sprouting), a decrease in the growth length, and an increase in fragility or permeability of the central artery of the brainstem.

9. The use of the pentachloronitrobenzene-based zebrafish developmental angiogenesis defect-cerebral hemorrhage model in high-throughput screening of therapeutic drugs according to claim 7, characterized in that: The drug has the potential to protect the growth and development of cerebral blood vessels and rescue developmental cerebral hemorrhage.

10. A method for high-throughput screening of therapeutic drugs using the zebrafish developmental angiogenesis defect-cerebral hemorrhage model based on pentachloronitrobenzene according to claim 7, characterized in that: During the screening process, potentially effective drugs are added to the zebrafish developmental angiogenesis defect-cerebral hemorrhage model, which is then cultured in a 28-28.5° C. incubator for 2.8-3.5 days; the medium is changed every 22-24 hours, and observations and statistics of cerebral hemorrhage and vascular pathology are performed to screen out drugs with the potential to protect cerebral vascular growth and development and rescue developmental cerebral hemorrhage.

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