CN110916841A

CN110916841A - Preparation method of mouse thrombotic stroke model

Info

Publication number: CN110916841A
Application number: CN201811100745.XA
Authority: CN
Inventors: 畅君雷; 马寅仲; 何岍妍; 尹美芳; 刘翠莲
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2018-09-20
Filing date: 2018-09-20
Publication date: 2020-03-27

Abstract

The invention provides a preparation method of a mouse thrombotic stroke model, which comprises the following steps: anesthetizing the mouse, tying the common carotid artery with suture, separating and permanently tying the external carotid artery and the pterygopalatine artery, and separating the initial part of the internal carotid artery; placing the common carotid artery into a groove of a blood vessel electric shock clamp of a thrombus generation instrument for forming thrombus by electric stimulation; taking down the electric shock clamp, clamping the distal end of the common carotid artery, crushing thrombus by using a micro forceps, recovering the pulsation of the artery and impacting the crushed thrombus; and (3) releasing the artery clamp, driving thrombus into the internal carotid artery by using forceps, immediately clamping and closing the proximal end of the common carotid artery, and opening all the artery clamps after the time to form the target embolism model. The mouse thrombotic stroke model can be applied to the research on medicaments, treatment methods and pathogenesis for stroke treatment.

Description

Preparation method of mouse thrombotic stroke model

Technical Field

The invention relates to a preparation method of an animal cerebral apoplexy model, in particular to a preparation method of a novel mouse thrombotic cerebral apoplexy model and application of the prepared mouse thrombotic cerebral apoplexy model in thrombolysis research of cerebral thrombosis.

Background

Stroke, also known as stroke or cerebrovascular accident, is a common and frequently encountered disease that seriously threatens human health. Recent data show that about 270 million new stroke people occur in China every year, the new stroke speed is increased at the rate of nearly 9% every year, and the new stroke is the first cause of death and disability of adult residents in China. Cerebral apoplexy is divided into ischemic stroke and hemorrhagic stroke, wherein ischemic stroke is the most common type of cerebral stroke clinically (the incidence rate accounts for about 8 percent), and the ischemic stroke is mainly caused by various emboli in blood (such as mural thrombus in the heart, atherosclerotic plaque and the like) entering cerebral arteries along with blood flow to block blood vessels, so cerebral tissue in the artery blood supply area is subjected to ischemic necrosis, and focal neurological impairment is further caused (Hankey GJ.

At present, the treatment method of cerebral arterial thrombosis is mainly drug thrombolysis. Recombinant tissue-type plasminogen activator (rtPA) is the only thrombolytic drug currently approved by the FDA for ischemic stroke, but the effective treatment window is narrow, only 3.5-4.5h, and the use of the thrombolytic drug beyond this time is easy to cause cerebral hemorrhage and thus is life-threatening. Due to the above situation, only about 1.6% of patients in China have the condition of receiving thrombolytic therapy after admission. Therefore, the animal model is used for simulating the attack characteristics of the stroke, and the research and development of therapeutic drugs are of great significance.

In order to clarify the pathogenesis of stroke and research and develop a medicament for treating stroke, people invent a series of animal models of stroke in the last forty years. The use of animal models has no alternative importance in the development of therapeutic methods and drugs for stroke, because: (1) the etiology and pathological features of human stroke are various and complex, and the variable of the attack can be controlled by using an experimental animal model, so that the research on the pathogenesis becomes continuous. (2) Many molecular, genetic, biochemical or pathological examinations require a certain amount of brain tissue, which is extremely difficult to achieve in clinical procedures. (3) Pathological changes in the very early stage of stroke often cannot be observed by clinical imaging technology, so that the study can be only carried out by animal models. (4) It is difficult to mimic the vascular structure and the pathological course of reperfusion in animal models using in vitro cell models. Although many important advances have been made by scholars in preclinical studies of stroke, a large number of preclinical studies have not produced corresponding stroke therapeutic agents, and currently, except rtPA, few neuroprotective agents have been widely used internationally. In order to solve the above problems, Stroke Therapy and enterprise round-table conference (STAIR) stipulate strict standards for preclinical Stroke research and urgent needs for new Stroke models. As a good model of stroke, clinical thrombosis and embolism processes should be simulated as much as possible, with complications of stroke onset (Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz SI, LoEH, Group S. update of the stroke thermal adaptive diagnostic routines, Stroke. 2009; 40(6): 2244-.

The current rodent focal stroke animal models can be divided into five categories: a wire embolectomy, a craniotomy, a photocoagulation, a local coagulation and a thromboembolism. The wire-embolus method is currently the most commonly used model of cerebral ischemia. The model is usually an embolic model formed by inserting a nylon wire of appropriate diameter from the external carotid artery through the common carotid artery into the middle cerebral artery to occlude blood flow. The method has small invasion to animals, and the inserted embolus can completely block blood supply of middle artery, so as to cause stable cerebral infarction volume and form stable nerve and motor dysfunction. However, the method is not consistent with the formation and development process of clinical cerebral apoplexy, and is not suitable for the research and development of thrombolytic drugs.

Craniotomy is a procedure in which the middle artery on the surface of the white matter of the brain is directly exposed through a craniotomy, and then permanent ischemia is formed through electrocoagulation, or blood flow is blocked for a certain time by using instruments such as an artery clamp. Such methods have the advantages of the ability to form stable infarct volumes, low mortality of the animals, and high success rates of modeling due to the direct observation of whether the middle artery is occluded. But due to craniotomy, physical damage is easily caused to the white matter of the brain near the middle artery. In addition, surgical procedures can affect intracranial pressure and blood brain barrier function, thereby increasing the variability in behavioral scores.

The photocoagulation method is based on the injection of a photosensitizing dye (Rose bengal, erythrosin B) and then, without craniotomy, irradiating the MCA with a laser of a specific wavelength to activate platelets to form a thrombus by causing peroxidation damage to endothelial cells within the MCA's blood vessels. The method is less invasive, can form stable infarct volume, and has low mortality. However, this method produces a large amount of oxygen radicals in cerebral vessels, and thus ischemic injury and angioedema (extracellular edema) are rapidly formed, with fewer penumbra. Therefore, the stroke development of the method is different from the clinical condition, and the method has certain influence on the evaluation of the drug effect.

The local coagulation method employs a surgical operation similar to the wire-embolus method, in which a microcatheter containing an coagulant (endothelin-1 or thrombin) is inserted into the middle artery, and a specific amount of the coagulant is injected to initiate coagulation. The method simulates primary cerebral embolism, and is also a low-invasive molding mode with low mortality. Thrombolysis studies can be performed on thrombi formed by this method, but the ischemia time is difficult to determine because of the phenomenon that blood flow rapidly decreases and gradually rises back to a stable state within hours after injection of the coagulant (which may be related to the in vivo metabolism of the coagulant). In addition, because endothelin receptors are also expressed in neurons and astrocytes, neurite outgrowth and gliogenesis can be caused after injection, thereby affecting experimental results.

The thromboembolism method results in the occlusion of the cerebral vessels by creating fresh or dry blood clots in vitro, and then injecting the clots through a catheter into the middle artery. At present, two types of emboli are mainly used in animal experiments, namely, the plastic emboli are uniform in size and can form a relatively stable cerebral ischemia model; secondly, the thrombus formed in vitro by using the rat autoblood. The method simulates non-primary apoplexy, and the thrombus blocked in the brain can be used for evaluating the drug effect of the thrombolytic drug. The pathological process approaches the clinical actual condition, however, because the thrombus is formed in vitro, the formation and the aging process of the thrombus in vivo cannot be simulated. Thrombus is not easy to adhere to the vessel wall when being guided into the vessel, so that the change of the infarct volume and the infarct area and even the thrombus autolysis phenomenon are easy to cause, and the method is not suitable for the research of the drug thrombolysis time window.

The above model features are summarized in table 1.

TABLE 1 comparison of advantages and disadvantages of common rodent models of cerebral apoplexy

The advantages and disadvantages of the existing stroke modeling method are as described above. There are two main problems to summarize: not conforming to the actual clinical pathological course and failing to perform the thrombolysis time window study.

Disclosure of Invention

The invention aims to overcome the defects that the stroke modeling method in the prior art is not in accordance with the actual clinical pathological process and the like, and provides a mouse stroke model which can simulate the pathological process of thrombosis and embolism processes of non-primary stroke, and the model is suitable for the time window research of thrombolytic drugs of stroke.

In order to achieve the above objects, in one aspect, the present invention provides a method for preparing a mouse model of thrombotic stroke, the method comprising:

anesthetizing a mouse, tying a slipknot at the distal end of a common carotid artery by using a suture, separating and permanently tying the proximal end of an external carotid artery, and separating the initial part of an internal carotid artery;

and (3) placing the common carotid artery into a groove of a blood vessel electric shock clamp of the thrombus generation instrument for two times of electric stimulation, wherein the current intensity is 0.3-0.4 mA, continuously performing electric stimulation for 50-80 sec for the first time to form 100% blockage, and then loosening the electric shock clamp to form a black thrombus with the length of 2-3 mm. After the first electrical stimulation is finished, the artery loses pulsation, the electric shock clamp is taken down, the micro forceps are used for crushing thrombus, the artery recovers pulsation and impacts the crushed thrombus, and at the moment, the common carotid artery slipknot is opened to enable the thrombus to enter the internal carotid artery;

and (3) beating the distal end of the common carotid artery to be alive again, putting the common carotid artery into an electric shock clamp, performing second electric stimulation with the same current intensity for 150-180 sec, taking down the electric shock clamp to form a black thrombus with the length of 3-5 mm, crushing the thrombus by using the micro forceps by using the same method after the second electric stimulation is ended, restoring the pulsation of the artery, impacting the broken thrombus, opening the common carotid artery to enable the thrombus to enter the internal carotid artery. Immediately clamping and closing the proximal end of the common carotid artery for 5-10 min after the thrombus enters the internal carotid artery, opening the artery clamp after the time is up, and suturing the neck skin to form the target embolism model.

According to a specific embodiment of the present invention, in the method for preparing the mouse model of thrombotic stroke according to the present invention, the mouse is a C57BL/6 mouse.

According to a specific embodiment of the present invention, in the method for preparing a mouse model of thrombotic stroke according to the present invention, the mouse is a male mouse with a body weight of 20-30g at 6-8 weeks of age.

According to the specific embodiment of the invention, in the preparation method of the mouse thrombotic stroke model, when the mouse is anesthetized, a gas anesthesia machine is used for anesthesia, and the blood flow of the middle artery on the operation side is monitored by using a laser Doppler blood flow instrument.

According to the specific embodiment of the invention, in the preparation method of the mouse thrombotic stroke model, the common carotid artery is knotted by using suture threads as follows:

fixing the anesthetized mouse in supine position on a mouse plate, longitudinally cutting the skin of the neck, stripping the carotid sheath and vagus nerve, reducing the blood pressure, exposing the common carotid artery, and tying a slipknot by using a suture.

According to the specific embodiment of the invention, in the preparation method of the mouse thrombotic stroke model, after the proximal end of the common carotid artery is clamped and all the artery clamps are opened, the blood flow value is reduced to be less than 30% of the reference value, and the model is regarded as forming the target embolism model.

On the other hand, the invention also provides application of the mouse thrombotic stroke model obtained by the preparation method in preclinical research, a treatment method and/or pathogenesis research of stroke treatment drugs. Preferably, the stroke therapeutic drug is a thrombolytic drug.

On the other hand, the invention also provides application of the mouse thrombotic stroke model obtained by the preparation method in the research of a stroke treatment method.

On the other hand, the invention also provides application of the mouse thrombotic stroke model obtained by the preparation method in the research of stroke pathogenesis

In some embodiments of the present invention, the method for preparing a mouse model of thrombotic stroke of the present invention comprises:

and (3) placing the common carotid artery into a groove of a blood vessel electric shock clamp of the thrombus generation instrument for two times of electric stimulation, wherein the current intensity is 0.3-0.4 mA, and the electric stimulation is continuously carried out for the first time for 50-80 sec, so that a black thrombus with the length of 2-3 mm is formed. After the electrical stimulation is finished, the artery loses pulsation, the electric shock clamp is taken down, the micro forceps are used for crushing thrombus, and the artery recovers pulsation and impacts the crushed thrombus. At this point, the common carotid artery knot is opened, and the thrombus enters the internal carotid artery. Continuously performing electric stimulation for 150-180 sec for the second time to form a black thrombus with the length of 3-5 mm, and enabling the thrombus to enter the internal carotid artery by using the same method after the electric stimulation is finished. Immediately clamping and closing the proximal end of the common carotid artery for 5-10 min after the thrombus enters the internal carotid artery, opening the artery clamp after the time is up, and suturing the neck skin to form the target embolism model.

The invention has the beneficial effects that:

the model has a molding success rate of over 75 percent, if the subsequent thrombolysis operation is not carried out, the mortality rate of animals in 48h is lower than 10 percent, and mice successfully molded can show obvious neurological and motor dysfunction, such as adduction of forelimbs on one side, inclination of the body to one side and even rolling, and the like. Infarct foci with stable location and size were observed by TTC staining. Compared with a rat thrombotic stroke model, the model has more stable model building success rate and pathological symptoms, so that the model is suitable for researching the pathological mechanism and therapeutic drugs of stroke. In addition, because a large number of transgenic animal strains exist in the C57BL/6 mouse, and the transgenic animal strains are combined with the model, the relevant research can be better carried out, which is a special advantage that the rat model does not have.

The mouse stroke model has all typical pathological features of stroke, and the pathological features can be relieved by timely thrombolytic therapy, so that the mouse stroke model is the mouse stroke model which is closest to clinical pathological features at present. In addition, the invention is suitable for preclinical research of cerebral apoplexy treatment medicines, particularly thrombolytic medicines.

Drawings

FIG. 1 is a schematic diagram of a process for preparing a mouse model of thrombotic stroke.

Fig. 2A-2C show general features of a mouse model of thrombotic stroke.

Fig. 3A-3C show the effect of thrombolytic using rtPA on cerebral blood flow in a mouse model of thrombotic stroke.

Fig. 4A-4B show the effect of thrombolysis using rtPA on the neurological score of the mouse model of thrombotic stroke and on the ability of the forepaws.

Fig. 5A-5B show the effect of thrombolytic using rtPA on the volume of cerebral infarction in a mouse model of thrombotic stroke.

Detailed Description

The following detailed description is provided for the purpose of illustrating the embodiments and the advantageous effects thereof, and is not intended to limit the scope of the present disclosure. The method, in which specific conditions are not specified, in the following examples is generally carried out according to the conventional procedures in the art.

Example 1

In the embodiment, a method for constructing a mouse thrombotic stroke model and application of the method to relevant preclinical research are provided. The specific operation is as follows:

1. preparation process of mouse thrombotic stroke model

The preparation process of the mouse thrombotic stroke model is shown in figure 1 and mainly comprises the following steps:

male C57BL/6 mice weighing 20-30g were anesthetized using a gas anesthesia machine (2.5% induction, 1.5% maintenance) and the blood flow in the medial artery on the operative side was monitored using a laser Doppler flow meter.

Fixing the mouse on a mouse board in a supine position, longitudinally cutting the skin of the neck with the length of about 1cm, stripping the carotid sheath and vagus nerve, reducing the blood pressure, exposing the common carotid artery with the length of about 5mm, and tying a slipknot by using a suture; separating and permanently ligating the external carotid artery and the pterygopalatine artery; the internal carotid artery initiation was isolated.

The common carotid artery is placed in a groove of a blood vessel electric shock clamp (a small animal thrombosis generator, Shandong Yiyan science and technology Co., Ltd.) to be electrically stimulated twice, the current intensity is 0.3-0.4 mA, the electric shock clamp is loosened after 100% blockage is formed by first continuous electric stimulation for 50-80 sec, and black thrombus with the length of 2-3 mm is formed. After the electrical stimulation is finished, the artery loses pulsation, the electric shock clamp is taken down, the micro forceps are used for crushing thrombus, and the artery recovers pulsation and impacts the crushed thrombus. At this point, the common carotid artery knot is opened, and the thrombus enters the internal carotid artery. Continuously performing electric stimulation for the second time for 150-180 sec until a black thrombus with the length of about 3-5 mm is formed. The artery lost pulsation after the second electrical stimulation was completed. The electric shock clip is taken down, the distal end of the common carotid artery is closed by the artery clip, and the thrombus is crushed by using the micro forceps (the head width is 0.1mm), at the moment, the blood vessel becomes uniform gray, and meanwhile, the artery is seen to recover the pulsation and impact the crushed thrombus. And (4) releasing the artery clamp, driving the thrombus into the internal carotid artery by using forceps, and immediately clamping and closing the proximal end of the common carotid artery for 5-10 min. And opening the artery clamp after the time, and if the blood flow value is reduced to be less than 30% of the reference value, determining that the target embolism model is formed.

The molding success rate of the embodiment is 75% (15/20), if no subsequent thrombolysis operation is performed, the mortality rate of the animal is 5% (1/20) within 48h, and the mice successfully molded can show obvious neurological and motor dysfunction, such as adduction of one forelimb, inclination of the body to one side and even rolling, and the like. Infarct foci with stable location and size were observed by TTC staining.

2. Pathological characteristics of mouse thrombotic stroke model

(1) C57BL/6 male mice weighing 20-30g at 6-8 weeks of age were randomly divided into rtPA and solvent control groups, and the common carotid artery thrombus was produced by two electrical stimulations using a current intensity of 0.4mA according to the method described above, the first electrical stimulation was continued for 60sec, and the second electrical stimulation was continued for 150 sec. After the formation of thrombus, rtPA was continuously infused for 30min at a dose of 10mg/kg, and the tail of the solvent control group was infused with physiological saline in the same manner, after completion, the upper and lower ends of the common carotid artery were ligated, and blood vessels (containing thrombus) of the same length were cut off and weighed. The vessels were weighed and placed in 4% paraformaldehyde, and tissue sections were made and HE stained, observed using an upright microscope and photographed.

(2) C57BL/6 male mice with the weight of 20-30g and the age of 6-8 weeks are taken and randomly divided into rtPA and solvent control groups, a mouse thrombotic stroke model is prepared according to the method, and the percentage change of left and right cerebral blood flow and middle artery blood flow before and after modeling is detected by adopting a laser speckle and laser Doppler blood flow instrument respectively. 60min after molding, continuously infusing rtPA for 30min according to the dose of 10mg/kg, infusing physiological saline into a solvent control group according to the same way, and detecting the percentage change of cerebral blood flow and middle artery blood flow before and after thrombolysis.

Fig. 2A-2C show general features of the mouse model of thrombotic stroke in this example. Wherein, fig. 2A: carotid thrombosis formed by direct current electric shock and thrombolysis of rtPA. After the electric shock to the common carotid artery, a black thrombus with the length of about 3-5 mm can be observed. After intravenous infusion of rtPA, the common carotid thrombus disappeared. Suggesting that the thrombus produced by this method can be dissolved by the infused rtPA. FIG. 2B: carotid thrombosis and human brain thrombosis formed by direct current electric shock were observed by HE staining. Human brain thrombus has a similar tissue structure to mouse cerebral thrombus, but contains more nuclei. One point to be explained is: human brain thrombus is soaked in 4% paraformaldehyde for a long time, and a tissue sample does not contain blood vessels, so that certain gaps and cracks appear in thrombus tissue after the preparation of the tablet. FIG. 2C: the carotid thrombus is crushed to block the cerebral vessels.

Fig. 3A-3C show the effect of thrombolytic using rtPA on cerebral blood flow in a mouse model of thrombotic stroke. Wherein, fig. 3A: the laser speckle is used for observing the cerebral blood flow conditions of a normal mouse, a thrombotic cerebral apoplexy mouse and a thrombolytic mouse. After the thrombus was crushed, a significant reduction in cerebral blood flow was observed, and this was significantly improved after thrombolysis. The model is shown to be capable of carrying out thrombolysis research. FIG. 3B: the change in mid-arterial blood flow after embolization and thrombolysis was observed before mice surgery using a laser doppler flow meter. Before thrombolysis, the cerebral blood flow is reduced to 30% before molding, and after thrombolysis, the cerebral blood flow is increased back to about 60%, further explaining that the model can be used for related research of thrombolysis. FIG. 3C: the stability of the mid-arterial blood flow was observed before the mouse surgery, within 3 hours after completion of embolization, using a laser doppler rheometer.

3. Behavioural detection before and after thrombolysis of mouse thrombotic stroke model

In this example, mortality was counted 24 hours after molding and two behavioural tests were performed:

(1) the neuroscience score was based on the observation methods of Bederson and Belvyev et al: lifting the mouse tail, and stretching the two forelimbs of the normal mouse forwards symmetrically. If the shoulder pronation and forelimb adduction occur, the score is given according to the severity. Normal extension of bilateral forelimbs was rated 0 points; adducted only to the contralateral forelimb, no other symptoms, scored 1 point; the grasping force of the contralateral forelimb is obviously reduced by horizontally drawing the rat tail and is rated as 2 points; the mouse placed on the plane can move freely, and the inclination of the limb to the contralateral side when the mouse tail is horizontally pulled is rated as 3 points; mice were rated 4 points when placed on a flat surface with only contralateral rotation or tumbling; there was no autonomic activity when placed on a flat surface and death was scored as 5 points within 24 hours.

(2) The gripping ability of the front paw of the rat is observed by adopting a suspension experiment: the rat front paw was simultaneously hung on the grip strength detector. 1 minute: both claws can be hung; and 2, dividing: both claws can be hung, but the difference of the gripping force is obvious; and 3, dividing: only one claw can be hooked.

Fig. 4A-4B show the effect of thrombolysis using rtPA on the neurological function score (fig. 4A) and on the forepaw capacity (fig. 4B) of a mouse model of thrombotic stroke. After thrombolysis, the nerve and motor functions of the mouse are obviously improved, and thrombolysis is successful. The model is suggested to be suitable for thrombolysis research.

4. Cerebral infarction volume detection

After behavioral testing, mice were sacrificed by cervical dislocation after anesthesia, and whole brains were rapidly frozen at-40 ℃ and sectioned. The first knife is positioned at the midpoint of the connecting line of the anterior brain pole and the visual cross; the second knife is at the visual cross part; the third knife is arranged at the position of the funnel handle; the fourth knife is arranged between the funnel handle and the tail pole of the rear blade, and the thickness of each blade is 2 mm. Brain tissue sections were incubated with 0.5% TTC solution at 37 ℃ for 15min and then with 4% paraformaldehyde for 20 min. And finally, placing the slices in sequence and respectively shooting photos of the front and back surfaces of the slices. When data are processed, image analysis software image J is used for processing and calculating the average infarct area on the front side and the back side of each brain slice, the average infarct area is multiplied by the thickness of each brain slice by 2mm, and the infarct areas of all the brain slices of each animal are multiplied by the thickness to be added, so that the cerebral infarct volume is obtained. Infarct volume was expressed as a percentage of the cerebral hemisphere and was calculated as follows to eliminate the effects of cerebral edema.

Fig. 5A-5B show the effect of thrombolytic using rtPA on the volume of cerebral infarction in a mouse model of thrombotic stroke. Wherein, fig. 5A is a photograph of the front and back of a brain slice; fig. 5B shows the calculation result of the cerebral infarction volume processed and calculated by image analysis software image J. The result shows that the cerebral infarction focus with uniform position and size appears in the brain tissue of the mouse after modeling, which indicates the stability of modeling, and the infarction focus is obviously reduced after rtPA thrombolysis, further indicating that the model is suitable for the relevant research of brain tissue injury before and after thrombolysis.

Claims

1. A preparation method of a mouse thrombotic stroke model comprises the following steps:

anesthetizing a mouse, tying a slipknot at the far-end of the common carotid artery by using a suture, separating and permanently tying the near-heart end of the external carotid artery, and separating the initial part of the internal carotid artery;

placing the common carotid artery into a groove of a blood vessel electric shock clamp of a thrombus generation instrument for two times of electric stimulation, wherein the current intensity is 0.3-0.4 mA, continuously carrying out electric stimulation for 50-80 sec for the first time to form 100% blockage, then loosening the electric shock clamp to form a section of black thrombus with the length of 2-3 mm, crushing the thrombus by using a micro forceps, recovering pulsation of the artery, impacting the crushed thrombus, opening a common carotid artery slipknot to enable the thrombus to enter an internal carotid artery;

and (3) beating the distal end of the common carotid artery into a slipknot again, putting into an electric shock clamp, using the same current intensity to shock for 150-180 sec, taking off the electric shock clamp to form a black thrombus with the length of 3-5 mm, using a micro forceps to crush the thrombus, recovering the pulsation of the artery, impacting a broken thrombus, opening the common carotid artery slipknot, immediately clamping and closing the proximal end of the common carotid artery for 5-10 min after the thrombus enters the internal carotid artery, opening the artery clamp after the time, suturing the skin of the neck, and forming a target embolism model.

2. The method of claim 1, wherein the mouse is a C57BL/6 mouse.

3. The method according to claim 1 or 2, wherein the mouse is a male mouse having a body weight of 20-30g at 6-8 weeks of age.

4. The preparation method according to claim 1, wherein the mouse is anesthetized by a gas anesthesia machine, and the blood flow of the middle artery on the operation side is monitored by a laser Doppler blood flow meter.

5. The method for preparing according to claim 1, wherein the common carotid artery is knotted with suture as follows:

6. The method according to claim 1, wherein the occlusion of the proximal carotid artery and the opening of all the arterial clamps reduce the blood flow to less than 30% of the baseline value, and the occlusion model is considered to be the target.

7. Use of a mouse model of thrombotic stroke obtained by the method of any one of claims 1 to 6 in preclinical studies, methods of treatment and/or pathogenesis of stroke therapeutics.

8. The use of claim 7, wherein the stroke treatment drug is a thrombolytic drug.

9. Use of the mouse model of thrombotic stroke obtained by the method of any one of claims 1 to 6 in the study of a method of stroke therapy.

10. Use of the mouse model of thrombotic stroke obtained by the preparation method according to any one of claims 1 to 6 in the study of stroke pathogenesis.