阅读说明：本技术 一种颈动脉粥样硬化斑块动物模型的构建方法 (Method for constructing carotid atherosclerotic plaque animal model ) 是由 郭应坤 叶鹏飞 刘灵军 文凌仪 许华燕 谢林均 傅航 陈林 许可 于 2020-05-14 设计创作，主要内容包括：本发明提供了一种颈动脉粥样硬化斑块动物模型的构建方法,属于动物模型构建领域。该构建方法是通过耳中央动脉损伤颈总动脉动脉内膜联合高脂饲料喂养造模。本发明成功构建了颈动脉粥样硬化斑块动物模型。该构建方法经兔耳中央动脉入路造模,创伤小、操作简便、动物存活率高、造模成功率高且其他因素影响小,克服了现有技术中构建颈动脉粥样硬化斑块动物模型存在的问题,可用于筛选治疗动脉粥样硬化斑块药物等,对于颈动脉粥样硬化的研究具有重要意义。(The invention provides a method for constructing a carotid atherosclerotic plaque animal model, and belongs to the field of animal model construction. The construction method is to combine the injury of the central artery of the ear with the endarterium of the common carotid artery and feed for molding with high fat feed. The invention successfully constructs the carotid atherosclerotic plaque animal model. The construction method is used for model building through rabbit ear central artery access, has the advantages of small wound, simple and convenient operation, high animal survival rate, high model building success rate and small influence of other factors, overcomes the problems in the prior art for building a carotid atherosclerotic plaque animal model, can be used for screening drugs for treating atherosclerotic plaque and the like, and has important significance for research on carotid atherosclerosis.)

1. A method for constructing a carotid atherosclerotic plaque animal model is characterized by comprising the following steps: it is formed by combining central auricular artery injury and common carotid artery intima with high-fat feed.

2. The construction method according to claim 1, characterized in that: the high-fat feed feeding is started the first day after the injury of the carotid artery intima;

preferably, the high-fat feed is fed for 8 weeks for 3 times a day, and the amount of the high-fat feed is 15g/kg body weight each time;

more preferably, the temperature and humidity in the room are constant and the water is freely drunk when the high-fat feed is fed.

3. The construction method according to claim 2, wherein: the high-fat feed is prepared by adding 1% of cholesterol by mass into a basic feed.

4. The construction method according to any one of claims 1 to 3, characterized in that: the animal used in the animal model is a rabbit; preferably, New Zealand white rabbits are used.

5. The construction method according to any one of claims 1 to 3, characterized in that: the steps for damaging the common carotid artery intima through the ear central artery are as follows: taking a micro catheter, sending the micro catheter into the central artery of the rabbit ear under the guidance of a micro guide wire, sequentially passing the micro catheter through the superficial temporal artery and the external carotid artery under the guidance of the micro guide wire, finally sending the micro catheter to the common carotid artery, and dilating the common carotid artery by using a balloon catheter.

6. The construction method according to claim 5, wherein: the steps for damaging the common carotid artery intima through the ear central artery are as follows:

(1) the puncture needle punctures the central artery of the rabbit ear, exits the needle core and enters the needle sheath;

(2) the micro-guide wire enters the central artery of the rabbit ear from the puncture needle sheath and exits from the puncture needle sheath;

(3) the micro catheter is sent into the central artery of the rabbit ear under the guidance of the micro guide wire, and then sequentially passes through the superficial temporal artery and the external carotid artery under the guidance of the micro guide wire, and finally is sent to the common carotid artery;

(4) and (4) reserving the micro guide wire, withdrawing the micro catheter, sending the balloon catheter into the common carotid artery under the guidance of the micro guide wire, filling the balloon, and dragging the balloon catheter.

7. The construction method according to claim 6, wherein:

in the step (1), the puncture needle is a 24G puncture needle;

and/or, in the step (1), heparin anticoagulation is injected during puncture;

and/or, in the step (2), the micro guide wire is a micro guide wire with the diameter of 0.014 cm;

and/or, in step (3), the microcatheter is a 1.8F microcatheter;

and/or, in step (4), the balloon catheter is a 3.0mm by 2cm balloon catheter;

and/or, in the step (4), the balloon catheter is sent to a common carotid artery to 4-6 vertebral bodies of cervical vertebra;

and/or, in step (4), the filling balloon is a balloon filled with an iodine contrast agent.

8. The construction method according to claim 7, wherein: the filling balloon is filled with iodine contrast agent for three times;

preferably, the balloon is inflated to 12-14KPa pressure for 60s each time, and the balloon catheter is dragged back and forth under the pressure of the balloon;

more preferably, the interval of each filling of the balloon is 60 s; and/or, the back-and-forth dragging of the balloon catheter is 1 time back dragging of the balloon catheter after each balloon filling.

9. Use of the model constructed by the construction method according to any one of claims 1 to 8 in screening drugs for treating atherosclerotic plaques;

preferably, the atherosclerotic plaque is a carotid atherosclerotic plaque.

10. A method of screening a drug for treatment of atherosclerotic plaques, comprising: the method comprises the following steps:

a. constructing an animal model of carotid atherosclerotic plaques according to the construction method of any one of claims 1 to 8;

b. administering the candidate to the carotid atherosclerotic plaque animal model of step a;

c. evaluating potential drugs for treating atherosclerotic plaques in a carotid atherosclerotic plaque animal model;

preferably, the atherosclerotic plaque is a carotid atherosclerotic plaque.

Technical Field

The invention belongs to the field of animal model construction, and particularly relates to a construction method of a carotid atherosclerotic plaque animal model.

Background

Atherosclerosis is a disease in which a lipid layer like wax is deposited on the wall of an artery, so that the elasticity of the artery is reduced and the lumen is narrowed. When these waxy deposits form together, they are called atherosclerotic plaques. These plaques are flush with the intima of the blood vessel at an early stage, usually running longitudinally along the vessel, and then if continued, slowly protrude inwardly on the vessel wall, causing a narrowing of the inner diameter of the artery to varying degrees; if there is an inducing factor, a certain portion of the arterial plaque ruptures, and a thrombus forms to block the blood vessel. Carotid atherosclerosis is related to a plurality of cardiovascular and cerebrovascular diseases, wherein the carotid atherosclerosis is closely related to cerebral ischemic stroke, and the mechanisms of causing the cerebral ischemic stroke are various, mainly: firstly, atheromatous plaques are continuously enlarged, and blood vessels are directly blocked; plaque rupture, the ruptured plaque embolizing a distal blood vessel; the surface of the broken or unbroken plaque is rough, the blood platelet and the blood coagulation factor are activated to form thrombus, and emboli fall off; and fourthly, the carotid stenosis reduces the perfusion pressure at the far end, so that the blood supply in watershed areas is insufficient, and marginal infarction or low-perfusion infarction is formed.

At present, drugs for preventing and treating atherosclerosis become a popular research, and most of research needs to establish atherosclerotic plaque models when screening drugs for treating atherosclerosis. At present, the model of the atherosclerotic plaque is established mainly by using rabbits as animal models, and a plurality of methods for preparing the model are available, such as a carotid adventitia implanted silicone rubber ring method, a balloon injury method, an electrical stimulation method, an air modeling method and the like. Wherein, the operation wound of the implanted silicone rubber ring method is large, and the survival rate of animals is low; the electrical stimulation method has multiple procedures, complicated operation, long time consumption and uncertain feasibility; the air modeling method has large difference of endangium damage degree because of no determined gas flow, and the complete blockage of the carotid artery inevitably causes the histological change of acute ischemia of brain tissues and blood vessels, thereby artificially changing the cerebral hemodynamics. The balloon injury method is a method which is widely used at present, but most of the methods are constructed in thoracic aorta, abdominal aorta, femoral artery or carotid artery of animals, and the problems of large wound, difficult operation, low survival rate of animals and the like exist. The method is a great obstacle to relevant research on atherosclerosis, a new modeling method of the carotid atherosclerotic plaque animal model is found to reduce animal trauma, reduce influence of other factors, simplify operation, improve animal survival rate and improve modeling success rate, and the method has important significance to research on atherosclerosis.

Disclosure of Invention

The invention aims to provide a method for constructing a carotid atherosclerotic plaque animal model.

The invention provides a method for constructing a carotid atherosclerotic plaque animal model, which is used for molding by combining central auricular artery injury and carotid artery intima with high-fat feed feeding.

Further, the high-fat diet feeding is started the first day after the injury of the carotid artery intima;

preferably, the high-fat feed is fed for 8 weeks for 3 times a day, and the amount of the high-fat feed is 15g/kg body weight each time;

more preferably, the temperature and humidity in the room are constant and the water is freely drunk when the high-fat feed is fed.

Further, the high-fat feed is prepared by adding 1% by mass of cholesterol into a basal feed.

Further, the animal model uses animals that are rabbits; preferably, New Zealand white rabbits are used.

Further, the step of damaging the common carotid artery intima through the ear central artery is as follows: taking a micro catheter, sending the micro catheter into the central artery of the rabbit ear under the guidance of a micro guide wire, sequentially passing the micro catheter through the superficial temporal artery and the external carotid artery under the guidance of the micro guide wire, finally sending the micro catheter to the common carotid artery, and dilating the common carotid artery by using a balloon catheter.

Further, the step of damaging the common carotid artery intima through the ear central artery is as follows:

(1) the puncture needle punctures the central artery of the rabbit ear, exits the needle core and enters the needle sheath;

(2) the micro-guide wire enters the central artery of the rabbit ear from the puncture needle sheath and exits from the puncture needle sheath;

(3) the micro catheter is sent into the central artery of the rabbit ear under the guidance of the micro guide wire, and then sequentially passes through the superficial temporal artery and the external carotid artery under the guidance of the micro guide wire, and finally is sent to the common carotid artery;

(4) and (4) reserving the micro guide wire, withdrawing the micro catheter, sending the balloon catheter into the common carotid artery under the guidance of the micro guide wire, filling the balloon, and dragging the balloon catheter.

Further, the air conditioner is provided with a fan,

in the step (1), the puncture needle is a 24G puncture needle;

and/or, in the step (1), heparin anticoagulation is injected during puncture;

and/or, in the step (2), the micro guide wire is a micro guide wire with the diameter of 0.014 cm;

and/or, in step (3), the microcatheter is a 1.8F microcatheter;

and/or, in step (4), the balloon catheter is a 3.0mm by 2cm balloon catheter;

and/or, in the step (4), the balloon catheter is sent to a common carotid artery to 4-6 vertebral bodies of cervical vertebra;

and/or, in step (4), the filling balloon is a balloon filled with an iodine contrast agent.

Further, the inflating balloon is inflated three times with an iodine contrast agent;

preferably, the balloon is inflated to 12-14KPa pressure for 60s each time, and the balloon catheter is dragged back and forth under the pressure of the balloon;

more preferably, the interval of each filling of the balloon is 60 s; and/or, the back-and-forth dragging of the balloon catheter is 1 time back dragging of the balloon catheter after each balloon filling.

The invention also provides application of the model constructed by the construction method in screening drugs for treating atherosclerotic plaques;

preferably, the atherosclerotic plaque is a carotid atherosclerotic plaque.

The invention also provides a method for screening a medicament for treating atherosclerotic plaques, which comprises the following steps:

a. constructing a carotid atherosclerotic plaque animal model according to the construction method;

b. administering the candidate to the carotid atherosclerotic plaque animal model of step a;

c. evaluating potential drugs for treating atherosclerotic plaques in a carotid atherosclerotic plaque animal model;

preferably, the atherosclerotic plaque is a carotid atherosclerotic plaque.

The invention successfully constructs the carotid atherosclerotic plaque animal model. The construction method is used for model building through rabbit ear central artery access, has the advantages of small wound, simple and convenient operation, high animal survival rate, high model building success rate and small influence of other factors, overcomes the problems in the prior art for building a carotid atherosclerotic plaque animal model, can be used for screening drugs for treating atherosclerotic plaque and the like, and has important significance for research on carotid atherosclerosis.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a graph of central arteriopuncture of rabbit ears: a is to puncture the central artery of the rabbit ear on the right side by a 24G puncture needle; b, a 0.014cm micro-guide wire enters the central artery of the rabbit ear from a puncture needle sheath; c and D are withdrawing the puncture needle sheath, and sending the 1.8F micro-catheter into the central artery of the rabbit ear under the guidance of the micro-guide wire.

FIG. 2 is a balloon catheter dilating a rabbit common carotid artery; a is the level of a saccule catheter positioned at the cervical vertebra 4-6 of the right common carotid; b is balloon filling with iodine contrast (white arrow) and right common carotid artery dilated.

Fig. 3 is a DSA imaging normal blood vessel anatomical image of new Zealand white rabbit head and neck without molding treatment, wherein the DSA imaging normal blood vessel anatomical image includes central auricular artery, common carotid artery, middle cerebral artery and submandibular artery: a is the left lateral position; and B is the prone position.

Fig. 4 is DSA imaging before and after balloon dilatation of rabbit common carotid artery: a is a visualization before balloon dilatation and about 2.222mm in diameter, and B is a visualization after balloon dilatation and about 2.822mm in diameter.

Fig. 5 shows DSA imaging vessel diameter of bilateral common carotid artery of white rabbit in new zealand.

FIG. 6 is a transverse axial scan image of the carotid atherosclerotic plaque animal model T1 WI: a is a horizontal axis common scanning image of T1 WI; b is a transverse axial enhancement scanning image of T1WI after 7min of gadolinium contrast agent injection.

FIG. 7 is a general observation picture of common carotid artery on both sides of a new Zealand white rabbit successfully modeled by dissection.

Fig. 8 is a HE staining optical lens image of a rabbit common carotid artery section: a is an experimental group; b is the self-control group.

Detailed Description

The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.