Construction method and application of smog disease animal model

Abstract

The invention relates to the technical field of biological medicine, in particular to a construction method and application of a smog disease animal model. The construction method comprises the following steps: (1) carrying out bilateral carotid artery ligation on an experimental animal; and (2) feeding the ligated animals in a low-oxygen environment to obtain the smog disease animal model. According to the construction method provided by the invention, the stable smog disease animal model can be constructed, and the construction method is simple, reliable and easy to implement.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to a method for constructing a moyamoya disease animal model and its application. Background Art

[0002] Moyamoya disease is a chronic progressive cerebrovascular occlusive disease characterized by stenosis or occlusion of the distal ends of bilateral internal carotid arteries (intracranial segments of internal carotid arteries) (or) severe stenosis or even occlusion of the starting ends of anterior cerebral arteries and middle cerebral arteries, accompanied by the formation of puff-like and fine blood vessels at the base of the skull and leptomeninges. The imaging feature is stenosis or occlusion of the distal ends of bilateral internal carotid arteries, accompanied by the formation of puff-like compensatory fine blood vessels at the base of the skull and leptomeninges; moyamoya disease is one of the important causes of ischemic and hemorrhagic strokes; the onset age of moyamoya disease shows a bimodal distribution, with the peak periods being 5-9 years old and 40-45 years old. Moyamoya disease is a proliferative disease, with pathological thickening of the vascular intima, and various microscopic changes are shown in the moyamoya collaterals, including fibrin deposition in the blood vessel wall, rupture of the elastic layer, formation of microaneurysms, etc. However, the etiology and pathogenesis of moyamoya disease are still unclear. Some studies have shown that it may be related to the activation of pro-proliferation pathways, nitric oxide signaling pathways, and disruption of chromatin remodeling, etc. Population studies are limited to exploring genetic susceptibility, risk factors, environmental effects, etc. At present, there is still a lack of reliable research on disease deterioration and reversal of disease progression.

[0003] Due to the unclear etiology and pathogenesis of moyamoya disease, it makes the early prevention and identification of moyamoya disease relatively difficult. Therefore, there is still a lack of effective experimental animal models, the progress in scientific research and surgical treatment is slow, and the development of targeted therapies is in trouble. At present, the attempts to establish experimental models of moyamoya disease involve genetics, immunology, and mechanical methods.

[0004] Given the identification of the human susceptibility gene RNF213 for Moyamoya disease in clinical practice, this has become strong evidence to support the genetic model. The study believes that a decrease in RNF213 expression leads to an increase in endothelial cell proliferation, migration, and tube formation. However, unfortunately, the genetic model has only made progress in zebrafish research. Human genetic studies directly inform the limitations of mutant genes, and this gene mutation usually cannot induce Moyamoya-like histopathological changes or ischemic consequences, and Moyamoya-like lesions have not been successfully simulated in mammals. The study believes that there is a relationship between intimal thickening of the internal carotid artery and the immune response, and many immunological attempts have also been made in the Moyamoya disease model, mainly by systemic or local injection of inflammatory or infectious substances to induce vascular changes in the carotid circulation. As a result, Moyamoya-related histopathological changes can be produced, but usually cannot cause stenosis, occlusion, and ischemic consequences. Mechanical methods mainly involve surgical means. Chronic cerebral ischemia is a hallmark consequence of Moyamoya disease, so the surgical model of chronic hypoperfusion has always been the mainstay for attempting to reveal the intrinsic ischemic response of Moyamoya disease and evaluating the effectiveness of surgical revascularization. The most studied in the past was to use microcoil stenosis or suture / electrocautery ligation of the common carotid artery in mice, rats, and pigs to induce perfusion insufficiency, and multiple paradigms have been described, including unilateral, concurrent bilateral, and staged bilateral ligation. Among them, internal carotid artery stenosis is a promising direction. The researchers placed microcoils on the internal carotid artery of 16-week-old male mice (~26-year-old humans) for 28 days, simulating the stenosis of the distal internal carotid artery and downstream blood vessels in early Moyamoya syndrome, as well as a decrease in the number of anastomoses in the cerebral watershed area. However, this model only simulates the primary vascular lesion and does not simulate typical Moyamoya-like vascular changes and clinical processes.

[0005] Therefore, there is currently no reliable experimental animal model, and there is an urgent need to find a stable, reliable, and easy-to-establish method for establishing a Moyamoya disease model to promote the research on the mechanism and intervention of Moyamoya disease. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a method for constructing a Moyamoya disease animal model and its application. The construction method provided by the present invention has the advantages of stability, reliability, and easy establishment.

[0007] In the first aspect, the present invention provides a method for constructing a Moyamoya disease animal model, and the construction method includes the following steps:

[0008] (1) Bilateral carotid artery ligation is performed on the experimental animals;

[0009] (2) The ligated animals are raised in a hypoxic environment to obtain the Moyamoya disease animal model.

[0010] As a preferred technical solution of the present invention, the experimental animals are mice.

[0011] As a preferred technical solution of the present invention, the experimental animals are male mice at 2-3 months old.

[0012] As a preferred technical solution of the present invention, the method of bilateral carotid artery ligation is the staged ligation method.

[0013] As a preferred technical solution of the present invention, after step (1), it is also necessary to detect the cerebral blood flow, and the successful surgical model is established when the brain shows a chronic hypoperfusion state.

[0014] As a preferred technical solution of the present invention, the oxygen content in the hypoxic environment is 13%.

[0015] As a preferred technical solution of the present invention, the feeding time is 14 days.

[0016] As a preferred technical solution of the present invention, the construction method includes the following steps:

[0017] (1) Male mice at 2-3 months old are subjected to bilateral carotid artery ligation by the staged ligation method. After ligation, the cerebral blood flow is detected, and the mice with a chronic hypoperfusion state in the brain are the mice with successful surgical model establishment;

[0018] (2) One to two days after the successful surgical model establishment, the cerebral blood flow is detected again, and the mice with a chronic hypoperfusion state in the brain are placed in a hypoxic environment with 13% oxygen content for 14 days. After verification, the animal model of moyamoya disease is obtained.

[0019] In the second aspect, the present invention provides an application of the construction method as described in the first aspect in the pathological research of moyamoya disease.

[0020] The technical solutions provided by the embodiments of the present invention have the following advantages compared with the prior art:

[0021] The construction method provided by the present invention can construct a stable animal model of moyamoya disease, and the construction method is simple, reliable and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings herein are incorporated into the specification and form a part of the specification, showing embodiments consistent with the present invention, and are used together with the specification to explain the principles of the present invention.

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

[0024] Figure 1Schematic diagram of the simple structure of the continuous hypoxic chamber described in the embodiments of the present invention;

[0025] Figure 2 Graph showing the comparison of cerebral blood flow changes before and after the surgical modeling in step (1) (after modeling, it is called the BICAO group, referring to animals with only bilateral internal carotid artery ligation) using the laser speckle blood flow imaging system described in the embodiments of the present invention;

[0026] Figure 3 Graph showing the detection results of monitoring the cerebral blood flow status of animals using the laser speckle described in the embodiments of the present invention;

[0027] Figure 4 Statistical graph of the detection results of monitoring the cerebral blood flow status of animals using the laser speckle described in the embodiments of the present invention;

[0028] Figure 5 Photo of the basilar artery of the brain after ink perfusion described in the embodiments of the present invention;

[0029] Figure 6 Comparison graph of hippocampal region slices after ink perfusion described in the embodiments of the present invention;

[0030] Figure 7 Comparison graph of striatum region slices after ink perfusion described in the embodiments of the present invention;

[0031] Figure 8 Comparison graph before and after brain tissue clearing described in the embodiments of the present invention;

[0032] Figure 9 Whole brain fluorescence signal imaging graph of the sample of brain tissue clearing described in the embodiments of the present invention, with a scale of 2000μm;

[0033] Figure 10 Graph of the blood vessel remodeling results described in the embodiments of the present invention, with a scale of 300μm;

[0034] Figure 11 Characterization graph of the formation of abnormal vascular masses in the anterior cerebral artery and posterior cerebral artery regions in the Willis ring region described in the embodiments of the present invention, with scales of 200μm, 300μm, and 700μm;

[0035] Figure 12 Results of the analysis of the abnormal vascular masses described in the embodiments of the present invention Figure 1 , and result graph A corresponds to Figure 11 abnormal vascular mass A, and result graph B corresponds to Figure 11 abnormal vascular mass B;

[0036] Figure 13 Results of the analysis of the abnormal vascular masses described in the embodiments of the present invention Figure 2 ;

[0037] Figure 14 The vascular analysis result of the hippocampal region according to the embodiment of the present invention Figure 1 , scale bar: 150μm;

[0038] Figure 15 The vascular analysis result of the hippocampal region according to the embodiment of the present invention Figure 2 ;

[0039] Figure 16 The vascular analysis result of the striatum according to the embodiment of the present invention Figure 1 , scale bar: 150μm;

[0040] Figure 17 The vascular analysis result of the striatum according to the embodiment of the present invention Figure 2 ;

[0041] Figure 18 The detection result diagram of the vascular anastomosis (anastomotic branch of anterior cerebral artery and middle cerebral artery) of the cerebral cortex according to the embodiment of the present invention, scale bar: 500μm. Detailed implementation manners

[0042] In order to be able to more clearly understand the above objects, features and advantages of the present invention, the solution of the present invention will be further described below. It should be noted that, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0043] Many specific details are set forth in the following description in order to fully understand the present invention, but the present invention can also be implemented in other ways different from those described herein; obviously, the embodiments in the specification are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0044] Model construction is an important research-limited area in the study of moyamoya disease. The previously involved immunological and genetic methods have been proven unable to cause typical moyamoya-like vascular pathology. In contrast, the mechanical method of simulating chronic cerebral hypoperfusion state is an important support in the treatment research of moyamoya disease. However, only using the mechanical method can only simulate primary vascular lesions and still cannot simulate typical moyamoya-like vascular pathology. Previous studies have focused more on improving the surgical methods of the mechanical method, such as internal carotid / common carotid artery stenosis or occlusion, unilateral or bilateral, single ligation or staged ligation, but there is still no reliable and stable model construction method at present. Therefore, the present invention provides a reliable and stable model construction method. The present invention combines hypoxia with mechanical internal carotid artery ligation to simulate stable moyamoya disease model mice, laying a foundation for subsequent basic research.

[0045] In the first aspect, the present invention provides a method for constructing a moyamoya disease animal model, and the construction method includes the following steps:

[0046] (1) Bilaterally ligate the carotid arteries of the experimental animals;

[0047] (2) Raise the ligated animals in a hypoxic environment to obtain the moyamoya disease animal model.

[0048] In the present invention, bilateral ligation of the internal carotid artery can simulate the typical changes of internal carotid artery stenosis in moyamoya disease. At the same time, since hypoxia and hypoxia-responsive signals play an important role in angiogenesis, therefore, by combining the mechanical method with hypoxic environment culture, the present invention can successfully obtain an animal model with moyamoya-like vascular pathology, and the construction method is stable, reliable and easy to succeed.

[0049] As a preferred technical solution of the present invention, the experimental animal is a mouse.

[0050] As a preferred technical solution of the present invention, the experimental animal is a male mouse at 2-3 months old.

[0051] In view of the dysplasia of the anterior cerebral artery and middle cerebral artery, which are branches of the internal carotid artery, accompanied by moyamoya disease, and moyamoya disease is manifested as ischemic and hemorrhagic symptoms, and the vast majority of young patients are prone to ischemia. Therefore, the present invention preferably uses 2-3-month-old mice for experiments, which correspond to the young stage.

[0052] As a preferred technical solution of the present invention, the method of bilateral carotid artery ligation is the staged ligation method.

[0053] Using the staged ligation method can improve the compensatory ability of animals and reduce the mortality of experimental animals. The left internal carotid artery can be ligated first. After the experimental animal wakes up, about 1.5 hours later, the right internal carotid artery is ligated.

[0054] As a preferred technical solution of the present invention, after step (1), it is also necessary to detect the cerebral blood flow. The successful surgical modeling is indicated by a chronic hypoperfusion state in the brain.

[0055] In the present invention, the hypoperfusion state refers to the state where the whole brain blood flow is stabilized at about 30%.

[0056] In the present invention, laser speckle can be used to detect the cerebral blood flow before ligation, immediately after unilateral ligation, 1.5 hours after ligation, and immediately after bilateral ligation, so as to ensure the chronic hypoperfusion state in the animal brain.

[0057] As a preferred technical solution of the present invention, the oxygen content in the hypoxic environment is 13%.

[0058] The present invention finds that an oxygen parameter of 13% can bring about angiogenesis within a controllable range. Therefore, by combining bilateral carotid artery ligation with a 13% hypoxic environment, the present invention can successfully construct a moyamoya disease animal model.

[0059] As a preferred technical solution of the present invention, the feeding time is 14 days.

[0060] As a preferred technical solution of the present invention, the construction method includes the following steps:

[0061] (1) Male mice at 2-3 months old are subjected to bilateral carotid artery ligation by staged ligation. After ligation, cerebral blood flow is detected. Mice with chronic cerebral hypoperfusion in the brain are the mice with successful surgical modeling.

[0062] (2) One to two days after successful surgical modeling, cerebral blood flow is detected again. Mice with chronic cerebral hypoperfusion in the brain are placed in a 13% hypoxic environment and fed for 14 days. After verification, the moyamoya disease animal model is obtained.

[0063] In the second aspect, the present invention provides an application of the construction method as described in the first aspect in the pathological study of moyamoya disease.

[0064] The following is an explanation through specific experiments:

[0065] Animal experiment ethics statement:

[0066] All animal experiments involved in the embodiments of the present invention have been approved by the Institutional Animal Care and Use Committee of Capital Medical University (license number AEEI-2021-058) and are carried out in accordance with ethical requirements. The experimental procedures in this example follow the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0067] For those not specifying specific techniques or conditions in the examples, the conventional techniques or conditions in the art, or the techniques or conditions described in the literature, or the product specifications are followed. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0068] Example

[0069] This example provides a method for constructing a moyamoya disease animal model and the constructed moyamoya disease animal model. At the same time, experimental animals without any treatment are prepared as a control group. The method is as follows:

[0070] (1) Using 2-month-old, male, C57 mice for model construction. First, isolate and ligate the unilateral internal carotid artery. After the animal wakes up from the anesthetized state, about 1.5 h later, ligate the other side. During the operation, laser speckle is used to detect cerebral blood flow before ligation, instantaneously after unilateral ligation, 1.5 hours after ligation, and instantaneously after bilateral ligation to ensure chronic cerebral hypoperfusion in the animal's brain.

[0071] (2) Approximately 30 h after surgical modeling, after reconfirming the low perfusion blood flow in the animal's brain, the experimental animals were placed in a 13% continuous hypoxic chamber for 14 days of continuous treatment. The hypoxic research experiment was carried out in a sealed chamber that could use nitrogen to maintain a specific oxygen concentration balance. The structural schematic diagram is shown in Figure 1 .

[0072] Verification:

[0073] After the hypoxic treatment was completed, methods such as ink staining and brain clearing imaging after lectin injection via the tail vein were used to evaluate the abnormal angiogenesis in the cerebral blood vessels. The specific methods are as follows:

[0074] (1) The changes in cerebral blood flow before and after the surgical modeling in step (1) were observed using a laser speckle flow imaging system. The results are shown in Figure 2 , and it can be seen from Figure 2 that after modeling, the animals showed an obvious state of low perfusion blood flow in the brain, and then they could enter the hypoxic chamber to receive 14 days of continuous hypoxic treatment; among them, BICAO refers to bilateral internal carotid artery ligation.

[0075] (2) During the surgical modeling process, the method of staged bilateral internal carotid artery ligation was adopted. Before modeling, immediately after unilateral modeling, 30 min after unilateral modeling, 1.5 h after unilateral modeling, immediately after bilateral modeling, 30 min after bilateral modeling, and 1.5 h after bilateral modeling, the cerebral blood flow status of the animals was monitored using laser speckle. The results are shown in Figures 3 - 4 , and it can be seen from the figure that after unilateral ligation of the internal carotid artery, the blood flow on the contralateral side of the mouse compensated. At about 1.5 h after modeling, the blood flow on the ipsilateral side of the modeling recovered to more than 90%. At this time, contralateral modeling was performed, which could effectively reduce the mortality rate of animal modeling; 30 min after contralateral modeling, the whole-brain blood flow dropped below 50%. After about 30 h, the whole-brain blood flow stabilized at a low perfusion state of about 30%.

[0076] (3) After 14 days in the hypoxic chamber, the animals were taken out, and the heart apex was perfused with ink to detect angiogenesis. Specifically: 7.5% gelatin was prepared with 0.9% normal saline. The above liquid was used as a solvent, and ink was added to prepare ink with a concentration of 25%. After constant temperature treatment in a 37 °C water bath, after each animal was perfused with 20 mL of normal saline, 10 mL of ink was then perfused. After completing the fixation and dehydration of the brain tissue, 100-μm sections were made with a cryostat, and the angiogenesis in the same brain region of the control group and the model group was monitored under a microscope. The results are shown in Figures 5 - 7 ; among them, Figure 5 is a photo of the basilar artery of the brain after ink perfusion, Figure 6 is a comparison diagram of hippocampal region sections after ink perfusion, Figure 7It is a comparison diagram of striatum region slices after ink perfusion. As can be seen from the figure, compared with the control group, more blood vessels were seen in the distribution sites of the internal carotid artery branches in the striatum and hippocampus in the model group.

[0077] It can be seen from validations (1)-(3) that there are more small blood vessels in the internal carotid artery branch region of the model group.

[0078] (4) Phenotype verification of angiogenesis: It is carried out using tomato lectin (Lectin). Tomato lectin can bind well with glycophorin and Tamm-Horsfall glycoprotein, and effectively bind and label the vascular endothelium (vascular endothelial cells) of rodents.

[0079] Inject 100 μL of Lectin with a concentration of 10 μg / mL into the tail vein. Perform perfusion fixation 30 min after injection: Inject 2% sodium pentobarbital intraperitoneally to deeply anesthetize the mice and perform cardiac perfusion. Perfuse the 1×PBS solution at a speed of 10 mL / min until the blood is completely cleared, and then perfuse pre-cooled 4% paraformaldehyde and fix overnight for 16-24 h. Discard the paraformaldehyde the next day after overnight fixation, and wash the sample three times with 1×PBS solution (more than 10 times the sample volume, with the sample immersed) for 2 hours each time. The washing process is carried out on a shaker at a rotation speed of not less than 60 rpm to thoroughly wash away the residual paraformaldehyde.

[0080] Brain tissue clearing: The experiment has four groups, including the Con group (animals without any treatment), the BICAO group (animals with only bilateral internal carotid artery ligation), the CH group (animals with only 14-day hypoxia at a concentration of 13%), and the BICAO-CH group (animals with bilateral internal carotid artery ligation combined with 14-day hypoxia treatment at a concentration of 13%). Immerse the brain tissues of the above four groups in 8% sodium dodecyl sulfate (SDS), dilute with 0.1 M PBS solution (pH 7.5), and shake at 100 rpm and 37 °C. Replace the cleaning solution every few days until the tissue becomes clean. After clearing, soak in 0.01% PBS and Tween 20 (prepared with PBST) and wash the brain tissue at 37 °C and 100 rpm for 24 hours to complete clearing. The comparison diagrams before and after clearing are shown in Figure 8 .

[0081] Perform whole-brain fluorescence signal imaging (photographed with a light-sheet confocal microscope) on the samples that have completed brain tissue clearing above. The imaging results are shown in Figure 9, and then complete the vascular analysis under the lectin labeling indication. The basic pathological changes of moyamoya disease are as follows: bilateral internal carotid arteries are occluded at the siphon, showing the characteristics of occlusion or stenosis at the end of the internal carotid artery; vascular remodeling is performed on the end of the internal carotid artery (ICA) and its main branches, the anterior cerebral artery (ACA) and the middle cerebral artery (MCA). The results are shown in Figure 10 , in Figure 10 , A is the vascular signal labeled by lectin photographed by a confocal microscope. The selected vascular diagram is identified as B through the surface tool, and the completed vascular remodeling and measurement are C. It can be seen from Figure 9 and Figure 10 that animals in the model group (BICAO-CH) had obvious internal carotid artery stenosis, which was in line with the preliminary pathological changes of moyamoya disease. It should be noted that since the blood vessels in the Willis circle area are all large blood vessels, after losing blood, the blood vessels will become flat, not uniform and saturated tubular vascular signals. Due to the limitations of vascular remodeling technology, the recognition sensitivity of this type of blood vessels is insufficient. Therefore, in the measurement of blood vessel diameter, only the regional signals can be recognized to generate values. That is to say, the obtained values are smaller than the actual blood vessel diameter. However, considering the uniformity of inter-group measurements, the conclusion of internal carotid artery stenosis at the end of the model group is reliable.

[0082] (5) Another typical pathological change of moyamoya disease is the formation of abnormal moyamoya-like blood vessels at the base of the skull, mainly in the anterior cerebral circulation area, and also involving the posterior cerebral circulation. It is an abnormal collateral compensation phenomenon. The representation of the Willis circle area of animals in the model group is shown in Figure 11 , Figure 11 is a representation diagram of the formation of abnormal vascular masses in the anterior cerebral artery and posterior cerebral artery areas of the Willis circle area.

[0083] Further analysis of the abnormal vascular masses can reveal that these blood vessels originate from the same main blood vessel ( Figures 12 - 13 ), and the blood vessels have smaller average diameter, average length, and average volume, and the vascular curvature is close to 1, which is in line with the pathological characteristics of abnormal collateral compensation in moyamoya disease.

[0084] At the same time, in view of the abnormal angiogenesis in moyamoya disease, vascular analysis was performed on the important functional areas of the striatum and hippocampus. The results are shown in Figures 14 - 17 , and the results show that the total number of blood vessels in the model group decreased; the blood vessel changes in the hippocampal area and striatum area were consistent. The average diameter of blood vessels in the model group decreased significantly, and the number of microvessels with a diameter of 1-5 μm increased significantly; the blood vessel length in the model group was shorter, and the total blood vessel volume decreased significantly, which was in line with the phenomenon of abnormal blood vessel generation.

[0085] (6) Detect the vascular anastomosis of the cerebral cortex (anastomotic branches of the anterior cerebral artery and the middle cerebral artery), and the results are shown in Figure 18 . The results show that there is an obvious anastomotic defect in the model group, which provides conditions for the occurrence of ischemia in moyamoya disease.

[0086] It should be noted that in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising a..." does not exclude the presence of additional identical elements in the process, method, article or device comprising the said element.

[0087] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments described herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing an animal model of moyamoya disease, characterized in that, The construction method includes the following steps: (1) Bilateral carotid artery ligation is performed on the experimental animals; (2) The ligated animals are raised in a hypoxic environment to obtain the moyamoya disease animal model.

2. The construction method according to claim 1, wherein The experimental animals are mice.

3. The construction method according to claim 2, wherein The experimental animals are male mice aged 2 - 3 months.

4. The construction method according to claim 1, characterized in that The method of bilateral carotid artery ligation is the staged ligation method.

5. The construction method according to claim 1, wherein After step (1), the cerebral blood flow needs to be detected. If the brain shows a chronic hypoperfusion state, the surgical modeling is considered successful.

6. The construction method according to claim 1, characterized in that, The oxygen content in the hypoxic environment is 13%.

7. The construction method according to claim 1, characterized in that The feeding time is 14 days.

8. The construction method according to any one of claims 1-7, characterized in that, The construction method includes the following steps: (1) Male mice aged 2 - 3 months are subjected to bilateral carotid artery ligation by the staged ligation method. After ligation, the cerebral blood flow is detected. Mice with a chronic hypoperfusion state in the brain are considered successfully modeled by surgery; (2) One to two days after the successful surgical modeling, the cerebral blood flow is detected again. Mice with a chronic hypoperfusion state in the brain are placed in a 13% hypoxic environment and raised for 14 days. After verification, the moyamoya disease animal model is obtained.

9. Application of the construction method according to any one of claims 1 - 8 in the pathological study of moyamoya disease.

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