CN108739556A - Application of photochemically-induced zebra fish model with cerebral arterial thrombosis - Google Patents

Abstract

The invention discloses the application of a photochemically induced zebrafish ischemic stroke model, and the telencephalon tissue is used as an identification index of the zebrafish ischemic stroke model. The present invention utilizes the principle of photochemically induced thrombus formation, injects rose bengal solution into the zebrafish intraperitoneal cavity, and irradiates for a short period of time to cause complete local infarction in the cerebral cortex of the zebrafish, and establishes the method of triggering rose bengal to release singlet state under light irradiation. The in vivo animal model of blood coagulation caused by oxygen damage to vascular endothelial cells effectively simulates the pathological process of ischemic stroke. According to the experimental results of the observation indicators, the telencephalon region of the zebrafish has significant looseness and infarction, and the zebrafish has swimming behaviors such as turning in circles and standing upright. The construction method is simple and appropriate, has a high success rate of the model, is easy to promote, has high sensitivity to telencephalon tissue injury, and has good repeatability, and can be used as a practical tool index for screening vascular protection and thrombolytic therapy after cerebral infarction.

Description

Application of a photochemically induced ischemic stroke model in zebrafish

technical field

The invention relates to the application of a photochemically induced zebrafish ischemic stroke model, belonging to the field of medical animal model research.

Background technique

Stroke, commonly known as "stroke", is an acute disease caused by disease in the blood vessels that supply blood to the brain. Ischemic stroke is the disorder of blood supply to local brain tissue caused by various reasons, leading to ischemic and hypoxic lesions and necrosis of brain tissue, which in turn leads to clinically corresponding neurological deficits ^[1] .

Stroke is a common and refractory cardiovascular and cerebrovascular disease that seriously endangers human health and life safety. Together with tumor diseases and respiratory diseases, it is one of the three diseases with the highest human mortality rate, and it is also the most developed among the three diseases. The disease with the fastest recovery, the slowest recovery, the most death, and the most severe disability. China is the largest stroke country in the world, with a new case of stroke every 12 seconds, and the incidence of stroke is increasing at a rate of 8.7% per year. The "China Cardiovascular Disease Report 2016" was released, and the results of national health service surveys showed that from 1993 to 2013, the prevalence of cerebrovascular diseases in China showed an overall upward trend, and the annual recurrence rate of ischemic stroke was as high as 17.7%. The number of patients is about 13 million, and the economic burden caused by stroke to China is as high as 40 billion yuan per year ^[2] .

The drugs butylphthalide and edaravone, which have been approved for marketing in China and are widely used in the clinical treatment of ischemic stroke, both exhibit hepatotoxicity and nephrotoxicity when taken for a long time ^[3] , so facing such severe ischemic stroke The development of new, safe and effective drugs for the treatment of ischemic stroke is of great significance.

At present, various methods such as surgical ligation, laser injury, and chemical substance induction can successfully induce ischemic stroke models in mice, rats, and rabbits. Due to the large number of animals required, complex experimental operations, long period, high cost, and poor model reproducibility, this type of animal model is only suitable for the effect evaluation of a small number of drugs, and is not yet suitable for large-scale screening of drugs. . Therefore, it is particularly important to prepare and construct animal disease models with simple methods, large throughput, short cycle time, high modeling success rate, and good reproducibility.

Compared with traditional mammalian models such as rodents and mice, the zebrafish animal model has the advantages of low feeding cost, convenient observation, easy to achieve high-throughput screening, and can effectively simulate the pathological characteristics of human diseases. It has become a recognized ideal drug Screening models. Now the team of XingeYu and Yang V.Li have established the ischemic stroke model of adult zebrafish by hypoxic environment induction method and photochemical induction method, both of which can cause cerebellum infarction in zebrafish and induce zebrafish In the ischemic stroke model, DEDTC compounds were screened out to significantly reduce the neuroprotective effect of infarct size in cerebellar brain slices of zebrafish ^{[4, 5, 6]} . Among them, in the process of inducing hypoxic environment to zebrafish, it caused the ischemia and hypoxia of the zebrafish systemic cardiovascular and cerebrovascular system, not just the ischemia process of the brain, which is quite different from the clinical pathogenesis . The identification indicators of the above-mentioned zebrafish ischemic stroke models are all in the cerebellum of the zebrafish brain tissue, and the infarct area ratio of the brain tissue slices is used as an indicator of the degree of brain injury, and changes in the zebrafish telencephalon tissue are not described. Pudur Jagadeeswaran's research team tried to establish a zebrafish thrombus model in a variety of ways, such as ferric chloride-induced thrombus model, phenylhydrazine-induced thrombus model and laser injury-induced thrombus model ^[7] . Ferric chloride triggers the outbreak of free radicals and induces a severe thrombosis reaction, making it difficult to observe the antithrombotic effect of the drug. Induction of thrombus with phenylhydrazine has not been successfully achieved. Laser damage can induce thrombus formation within tens of seconds, and the evaluation of the effect of antithrombotic drugs can only be identified from the size and shape of the thrombus. None of the above identification indicators of the zebrafish thrombus model involved changes in the zebrafish brain tissue. Domestic Hangzhou Huante Biological Co., Ltd. optimized the zebrafish development stage, treatment time and compound dosage, and successfully induced the zebrafish juvenile thrombus model with phenylhydrazine solution. The identification indicators were heart red blood cell staining as thrombus formation rate, and zebrafish tail vein thrombus fluorescence rate. , also did not mention changes in zebrafish brain organization. Lin Ruichao's research group in China used epinephrine solution to induce platelet fluorescent transgenic zebrafish Tg(CD41:GFP) juveniles to observe thrombus formation in the zebrafish tail veins. The zebrafish thrombus models mentioned above are only suitable for the screening of antithrombotic drugs, and all of them involve changes in zebrafish brain tissue.

Contents of the invention

The technical problem to be solved by the present invention is to provide the application of a photochemically induced ischemic stroke model that can cause infarct damage in the telencephalon of zebrafish.

In order to solve the problems of the technologies described above, the technical scheme adopted in the present invention is as follows:

Application of a photochemically induced zebrafish ischemic stroke model, using telencephalon tissue as the identification index of the zebrafish ischemic stroke model.

Wherein, the photochemically induced zebrafish ischemic stroke model is prepared according to the following steps:

(1) Carry out anesthesia treatment to zebrafish;

(2) photochemical induction treatment;

(3) The zebrafish brain was stained.

In step (1), the described anesthesia treatment of zebrafish refers to the selection of adult zebrafish of the wild-type strain with normal development, using 0.16mg/ml tricaine aminobenzoate ethyl methylsulfonate (MS -222) for anesthesia.

In step (2), the photochemical induction treatment refers to that according to the body weight of the zebrafish, the dose of rose bengal solution is 100 μg/g, and the total dosage is not more than 20 μl. In tricaine-aminobenzoic acid ethyl ester methanesulfonate, the entire optic roof region of the head was exposed to the light source for 20 minutes. Except the head region, the rest of the parts were covered by tinfoil paper. Transfer to a normal container to recover for 24 hours, and place the container in a dark room for observation.

Wherein, the rose bengal solution is prepared as follows: weigh 2 mg of rose bengal powder, dissolve in 1 ml of 0.9% (9 mg/ml) physiological saline, add 5 μl of 1M sodium hydroxide, vortex until completely dissolved, The prepared concentration is 2 mg/ml, and then diluted 1:1 to 1 mg/ml for later use. The working concentration of the rose bengal solution used for the photosensitization inducer is 1 mg/ml.

In step (3), the dyeing treatment is to use 2% (ie 20 mg/ml) 2,3,5-triphenyltetrazolium chloride (TTC) for dyeing.

Wherein, the 2% TTC (2,3,5-triphenyltetrazolium chloride) solution is configured as follows: Weigh 0.1g TTC powder, dissolve it in 5ml PBS (phosphate buffered saline, pH= In 7.2-7.4), shake and vortex until completely dissolved. The concentration is 20mg/ml, and the mass fraction of solute is 2%. Each sample needs 200μl 2% TTC solution for staining, and it is prepared according to the number of samples.

Image processing was performed on the infarct area of the zebrafish telencephalon tissue, and the white stained area of the telencephalon tissue and the total surface area of the telencephalon tissue were recorded, and the ratio of the infarct area was used as an indicator of the severity of brain injury in ischemic stroke, and the calculation method was as follows:

Infarct area ratio%=(white staining area of telencephalon tissue in model group/total surface area of telencephalon tissue in model group)×100%.

Wherein, the identification index also includes the observation record of swimming behavior.

Beneficial effect: adopting the method of the present invention, through the intraperitoneal injection of rose bengal solution in zebrafish, light irradiation in a short period of time causes complete partial infarction in the cerebral cortex area of zebrafish, and establishes that rose bengal releases singlet oxygen to damage blood vessels under light irradiation The in vivo animal model of coagulation caused by endothelial cells effectively simulates the pathological process of ischemic stroke. According to the experimental results of the observation indicators, the telencephalon region of the zebrafish has significant looseness and infarction, and the zebrafish has swimming behaviors such as turning in circles and standing upright. The construction method is simple and appropriate, the model has a high success rate, a short period, high sensitivity to telencephalon tissue damage, and good repeatability, and can be used as a practical tool index for screening vascular protection and thrombolytic therapy after cerebral infarction.

Description of drawings

Figure 1 shows the TTC staining results of the brain slices of the cerebellum 24 hours after the photochemical induction of zebrafish.

Figure 2 shows the TTC staining of the whole brain tissue of the zebrafish 24 hours after photochemical induction, and the staining of the telencephalon tissue is shown by the blue circle.

Detailed ways

The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the content described in the embodiments is only for illustrating the present invention, and should not and will not limit the present invention described in the claims.

Example:

1. Anesthetize the zebrafish

Adult zebrafish of a wild-type strain with normal development were selected and anesthetized with 0.16 mg/ml tricaine aminobenzoate ethyl methylsulfonate (MS-222).

2. Photochemical Induction Treatment

According to the body weight of each zebrafish, the dose of rose bengal solution was injected into its abdomen at a dose of 100 μg/g, and the total injection volume did not exceed 20 μl. The control group was injected with an equal volume of 0.9% normal saline. After 10 minutes, the zebrafish was transferred to a solution containing 75 mg/l tricaine anthranilate ethyl ester methanesulfonate, and the entire optic tectum area of the head was exposed to the light source for 20 minutes, except the head area, The rest of the parts were covered with tinfoil, and after irradiation, they were transferred to normal containers for recovery, and the containers had to be placed in a dark room for observation.

24 hours after recovery induced by light irradiation, the zebrafish were anesthetized and sacrificed immediately after behavioral observation and recording, and the indicators of zebrafish brain tissue were identified.

The rose bengal solution used above was prepared as follows: Weigh 2 mg of rose bengal powder, dissolve it in 1 ml of 0.9% normal saline, add 5 μl of 1M sodium hydroxide, vortex until completely dissolved, and configure the concentration to be 2 mg/ml, then Dilute it 1:1 to 1 mg/ml for later use.

The working concentration of the rose bengal solution used for the photosensitization inducer is 1 mg/ml.

The experimental animals are pure-line wild-type adult zebrafish.

3. Staining the zebrafish brain

(1) Preparation of 2% TTC (2,3,5-triphenyltetrazolium chloride) solution

Weigh 0.1g of TTC powder, dissolve it in 5ml of PBS (phosphate buffered saline, PH=7.2-7.4), vortex and vortex until completely dissolved, configure the concentration as 20mg/ml, solute mass fraction as 2%, and stain each sample 200μl 2% TTC solution is required, and it is prepared according to the sample quantity.

(2) 2% TTC (2,3,5-triphenyltetrazolium chloride) staining

2% TTC staining observation and treatment method: put the zebrafish recovered from irradiation in 0.16 mg/ml tricaine aminobenzoate ethyl methylsulfonate anesthetized to unconsciousness, and fix it on the operating table with tacks Above, cut from the back about 1cm away from the head, dissect the whole zebrafish brain under a stereomicroscope and immediately freeze it at -20°C for 20min, then place it in a water bath at 37°C for 30min in the dark, and transfer it to 4 Fix in % paraformaldehyde overnight, take pictures and image, and use image processing software to quantify the infarct area, record the white stained area of the telencephalon tissue and the total surface area of the telencephalon tissue, and use the ratio of the infarct area as the severity of brain injury in ischemic stroke The index of is calculated as follows:

Infarct area ratio%=(white staining area of telencephalon tissue in model group/total surface area of telencephalon tissue in model group)×100%. 4. Model establishment and application evaluation results

It can be seen from Figure 1 that after staining with 2% TTC (2,3,5-triphenyltetrazolium chloride), the normal tissue area is red, the cerebral infarction area tissue is white, and both the blank control group and the model group are In the brain slices of the cerebellar region of zebrafish, there was no infarction in the blank control group after staining, and there was no obvious pale infarction in the brain tissue of the model group, and the two were very close and indistinguishable.

It can be seen from Figure 2 that the blue circle shows the telencephalon tissue area of zebrafish, the telencephalon tissue of the blank control group showed red without infarction, the telencephalon tissue of the model group showed significant loosening and infarction, and the positive control group The zebrafish telencephalon tissue in the group was relatively intact and stained pink or red, and the infarction phenomenon was significantly reduced. The quantitative analysis of the above pictures was realized by the picture processing software Image.J 6.0.

Compared with the preparation method and application of the existing models, the present invention has the following advantages:

1. Compared with the existing photochemically induced zebrafish ischemic stroke identification index, the present invention believes that telencephalon tissue is a more sensitive and stable identification index of cerebral infarction phenomenon, and can be used as an indicator of zebrafish ischemic stroke phenotype. One of the evaluation indicators.

Through research, it has been nearly ten years of research to establish a thrombus formation model based on zebrafish. Pudur Jagadeeswaran's research team tried to establish a zebrafish thrombus model using a variety of methods, such as ferric chloride-induced thrombus model, phenylhydrazine-induced thrombus model and laser injury-induced thrombus model. Ferric chloride triggers the outbreak of free radicals and induces a severe thrombosis reaction, making it difficult to observe the antithrombotic effect of the drug. Induction of thrombus with phenylhydrazine has not been successfully achieved. Laser damage can induce thrombus formation within tens of seconds, and the evaluation of the effect of antithrombotic drugs can only be identified from the size and shape of the thrombus. None of the above identification indicators of the zebrafish thrombus model involved changes in the zebrafish brain tissue. Domestic Hangzhou Huante Biological Co., Ltd. optimized the zebrafish development stage, treatment time and compound dosage, and successfully induced the zebrafish juvenile thrombus model with phenylhydrazine solution. The identification indicators were heart red blood cell staining as thrombus formation rate, and zebrafish tail vein thrombus fluorescence rate. , also did not mention changes in zebrafish brain organization. Lin Ruichao's research group in China used adrenaline solution to induce platelet fluorescent transgenic zebrafish Tg(CD41:GFP) juveniles, and observed thrombus formation in the tail veins of zebrafish. The zebrafish thrombus models mentioned above are only suitable for the screening of antithrombotic drugs, and all of them involve changes in zebrafish brain tissue.

In addition, the teams of XingeYu and Yang V.Li have published 3 papers based on the animal model of zebrafish ischemic stroke. They have established the ischemic stroke model of adult zebrafish by using hypoxic environment induction method and photochemical induction method. Both can cause infarction in the cerebellum of zebrafish, and in the hypoxic environment-induced zebrafish ischemic stroke model, DEDTC compounds have the neuroprotective effect of significantly reducing the infarct size in cerebellar brain slices of zebrafish ^{[4, 5, 6]} . Among them, in the process of inducing hypoxic environment to zebrafish, it caused the ischemia and hypoxia of the zebrafish systemic cardiovascular and cerebrovascular system, not just the ischemia process of the brain, which is quite different from the clinical pathogenesis . The identification indicators of the above-mentioned zebrafish ischemic stroke models are all in the cerebellum of the zebrafish brain tissue, and the infarct area of the brain tissue slice is used as an indicator of the degree of brain injury, and changes in the zebrafish telencephalon tissue are not described.

Our laboratory used photochemical induction to construct a zebrafish ischemic stroke model. After 24 hours of photochemical induction, it was found that the infarct changes in the cerebellar slices of the zebrafish were very insignificant (Fig. 1 ). After repeated attempts to adjust the intensity of induction modeling of zebrafish to different degrees, no obvious infarction phenomenon in the brain slices of the cerebellum region could be found. At the same time, it was found that the telencephalon of the zebrafish brain tissue was loose and infarcted (as shown in Figure 2). Photochemically induced ischemic stroke in zebrafish can cause damage to the telencephalon region in the zebrafish brain tissue first, and the loose damage to the telencephalon tissue affects the motor behavior and perception ability of the zebrafish, causing the zebrafish to appear circling, standing upright and other nerves. Dysfunctional swimming behavior, and thus the telencephalon region, is currently the most sensitive indicator of the extent of brain damage after ischemic stroke in zebrafish.

2. Compared with the existing photochemically induced zebrafish ischemic stroke research content, the present invention believes that the looseness of the telencephalon tissue affects the swimming behavior of the zebrafish neurological dysfunction, and the telencephalon tissue can be regarded as a zebrafish based on the histological level. The identification indicators of the fish ischemic stroke model will help to further study the role of the telencephalon in the process of neurological recovery and regeneration.

The adult zebrafish telencephalon ventricle can generate new neurons like the mammalian subependymal region, so it can be used as a model for the study of neurogenesis and neuron regeneration. The zebrafish telencephalon plays an important role in spatial memory, and the zebrafish telencephalon injury model has been used to screen related therapeutic drugs. In this experiment, it was found that the zebrafish telencephalon tissue loosened after photochemical induction and coexisted with the swimming behavior of zebrafish neurological dysfunction. Therefore, it is believed that the histological level of the zebrafish telencephalon can be used as an identification of neurological recovery and regeneration index.

references:

[1] Feng Jiachun, Zhu Yu, Rao Mingli, et al. Experimental study on the protective effect of cistrobolase on global cerebral ischemia-reperfusion injury [J]. Journal of Stroke and Neurological Diseases, 2009, 13(1): 9- 10.

[2] "China Cardiovascular Disease Report 2016" released [J]. Journal of Practical Cardiology, 2017, (4).

[3] Zhang Yang, Shi Limin, Cheng Sheng, etc. Retrospective study on the renal safety of Edaravone injection [J]. Journal of Adverse Drug Reactions, 2015, 17(6): 401-411.

[4] Yu X, Li YV. Zebrafish as an alternative model for hypoxic ischemic brain damage. Int J Physiol Pathophysiol Pharmacol 2011; 3: 88-96.

[5] Yu X, Li YV. Zebrafish (Danio rerio) Developed as an Alternative Animal Model for Focal Ischemic Stroke. Acta Neurochirurgica Supplement 2016, 121: 115-118.

[6] Yu X, Li YV. Neuroprotective effect of zinc chelator DEDTC in azebrafish (Danio rerio) model of hypoxic brain injury. Zebrafish 10(1): 30-35.

[7] Pudur Jagadeeswaran, Mairia Carrillo, Uvaraj P. Laser-Induced Thrombosis in Zebrafish. Methods Cell Biology. 2011, 101: 197-203.

Claims (8)

1. a kind of application of photochemical induction zebra fish cerebral arterial thrombosis model, which is characterized in that using akrencephalon tissue as spot The identification of indicator of horse fish cerebral arterial thrombosis model.

2. application according to claim 1, which is characterized in that the photochemical induction zebra fish cerebral arterial thrombosis mould Type is prepared in accordance with the following steps：

(1) anaesthetic treatment is carried out to zebra fish；

(2) photochemical induction is handled；

(3) dyeing processing is carried out to zebra fish brain.

3. application according to claim 2, which is characterized in that described to carry out anaesthetic treatment to zebra fish in step (1) Refer to selecting normotrophic wild-type lines Adult Zebrafish, uses 0.16mg/ml tricaine aminobenzene acid adding ethylacrylate methyls Sulfonate is anaesthetized.

4. application according to claim 2, which is characterized in that in step (2), the photochemical induction, which is handled, refers to, The use of rose-red solution dosage is 100 μ g/g according to the weight of zebra fish, total dosage is no more than 20 μ l, will after handling 10min Zebra fish is transferred in the aminobenzene acid adding ethylacrylate methyl sulfonate of tricaine containing 75mg/l, its entire head optic tectum region is sudden and violent It is exposed under light source and irradiates 20min, in addition to head zone, remaining position is covered by masking foil, is transferred them to after irradiation normal Restore for 24 hours in container.

5. application according to claim 4, which is characterized in that the rose-red solution is prepared as follows to be obtained： Rose-red powder 2mg is weighed, is dissolved in 0.9% physiological saline of 1ml, is added 5 μ l 1M sodium hydroxides, oscillation is vortexed to complete molten, matches A concentration of 2mg/ml is set, then 1: 1 to be diluted to 1mg/ml spare by it.

6. application according to claim 2, which is characterized in that in step (3), dyeing processing is to use 2,3,5- Triphenyltetrazolium chloride is dyed.

7. application according to claim 1, which is characterized in that carried out at image to zebra fish akrencephalon tissue infarct area Reason records akrencephalon tissue white stained area and akrencephalon tissue surface gross area, using infarct size ratio as ischemic cerebral apoplexy The middle index that cerebral injury severity occurs, computational methods are as follows：

Infarct size ratio %=(model group akrencephalon tissue white stained area/model group akrencephalon tissue surface gross area) × 100%.

8. application according to claim 1, which is characterized in that the identification of indicator further includes swimming behavior observation note Record.