CN111544149A - Method for constructing carotid atherosclerotic plaque animal model - Google Patents

Abstract

The invention provides a method for constructing a carotid atherosclerotic plaque animal model, which belongs to the field of animal model construction. The construction method is to establish a model by feeding the intima of the common carotid artery injuring the central auricular artery and feeding it with a high-fat diet. The present invention successfully constructs the carotid atherosclerotic plaque animal model. The construction method is used for modeling through the central artery of the rabbit ear, which has the advantages of small trauma, simple operation, high animal survival rate, high modeling success rate and little influence of other factors, and overcomes the problems of constructing carotid atherosclerotic plaque animals in the prior art. The problems existing in the model can be used to screen drugs for the treatment of atherosclerotic plaque, etc., which is of great significance for the study of carotid atherosclerosis.

Description

A method for constructing a carotid atherosclerotic plaque animal model

technical field

The invention belongs to the field of animal model construction, and in particular relates to a construction method of an animal model of carotid atherosclerosis plaque.

Background technique

Atherosclerosis is a disease in which a layer of wax-like lipids is deposited on the arterial wall, which reduces the elasticity of the arteries and narrows the lumen. When these wax-like deposits form piece by piece, they are called atherosclerotic plaques. These plaques are flush with the vascular intima in the early stage, and usually distribute longitudinally along the blood vessels. Then, if they continue to progress, the plaques will slowly protrude inward on the vessel wall, causing varying degrees of stenosis in the inner diameter of the artery; if there are predisposing factors, When part of the plaque in the artery ruptures, a blood clot forms and blocks the blood vessel. Carotid atherosclerosis is related to many cardiovascular and cerebrovascular diseases, among which it is closely related to ischemic stroke. There are many mechanisms that cause ischemic stroke, mainly as follows: (1) The continuous increase of atherosclerotic plaques directly 2) The plaque ruptures, and the ruptured plaque embolizes the distal blood vessel; 3) The ruptured or unruptured plaque has a rough surface, platelets and coagulation factors are activated, thrombus is formed, and the emboli fall off; 4) Stenosis of the carotid artery makes the distal Perfusion pressure decreases, resulting in insufficient blood supply to the watershed area, resulting in marginal zone infarcts or hypoperfusion infarcts.

At present, drugs for preventing and treating atherosclerosis have become a hot research topic, and most studies need to establish atherosclerotic plaque models when screening drugs for treating atherosclerosis. At present, the establishment of atherosclerotic plaque models mainly uses rabbits as animal models. There are many methods for preparing models, such as carotid adventitia implantation of silicone rubber rings, balloon injury, electrical stimulation, and air modeling. Wait. Among them, the implantation of a silicone rubber ring has a large surgical trauma and a low animal survival rate; the electrical stimulation method has many procedures, complicated operations, time-consuming, and its feasibility is still under discussion; the air modeling method does not have a definite gas flow rate, and the degree of damage to the vascular intima The difference is large, and the complete occlusion of the carotid artery will inevitably lead to the histological changes of acute ischemia in the brain tissue and the blood vessel itself, and artificially change the cerebral hemodynamics. Balloon injury is the most commonly used method at present. However, most of them are constructed in the thoracic aorta, abdominal aorta, femoral artery or carotid artery of animals, which also has problems such as large trauma, difficult operation, and low animal survival rate. This is a great obstacle to atherosclerosis-related research. To find a new carotid atherosclerotic plaque animal model modeling method to reduce animal trauma, reduce the influence of other factors, simplify operation, and improve animal survival rate , to improve the success rate of modeling, is of great significance for the study of atherosclerosis.

SUMMARY OF THE INVENTION

The purpose of the present invention is 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 established by injuring the common carotid artery intima of the central ear artery and feeding with high-fat feed.

Further, the high-fat feed feeding is started on the first day after the intima of the common carotid artery is injured;

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

More preferably, when the high-fat feed is fed, the indoor temperature and humidity are constant, and drinking water is free.

Further, in the high-fat feed, 1% cholesterol is added to the basic feed.

Further, the animal used in the animal model is a rabbit; preferably a New Zealand white rabbit.

Further, the step of injuring the intima of the common carotid artery through the central ear artery is as follows: take a microcatheter, send it into the central artery of the rabbit ear under the guidance of the microguide wire, and then insert the microcatheter under the guidance of the microguide wire. Pass through the superficial temporal artery, the external carotid artery in turn, and finally send it to the common carotid artery, and then use a balloon catheter to dilate the common carotid artery.

Further, the step of damaging the intima of the common carotid artery through the central auricular 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 at the same time;

(2) Insert the micro-guide wire from the puncture needle sheath into the central artery of the rabbit ear, and exit the puncture needle sheath;

(3) The microcatheter is sent into the central artery of the rabbit ear under the guidance of the micro-guide wire, and then the micro-catheter is passed through the superficial temporal artery and the external carotid artery in turn under the guidance of the micro-guide wire, and finally sent to the common carotid artery;

(4) Retain the micro-guide wire and exit the micro-catheter, under the guidance of the micro-guide wire, send the balloon catheter into the common carotid artery, fill the balloon, and drag the balloon catheter.

further,

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

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

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

And/or, in step (3), described microcatheter is the microcatheter of 1.8F;

And/or, in step (4), the balloon catheter is a balloon catheter of 3.0mm*2cm;

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

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

Further, the filling balloon is filled with iodine contrast agent three times;

Preferably, each time the balloon is inflated to a pressure of 12-14KPa and maintained for 60s, and the balloon catheter is dragged back and forth while maintaining the pressure in the balloon;

More preferably, the interval for each filling of the balloon is 60s; and/or, the dragging the balloon catheter back and forth is dragging the balloon catheter back and forth once after each filling of the balloon.

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

Preferably, the atherosclerotic plaque is carotid atherosclerotic plaque.

The present invention also provides a method for screening and treating atherosclerotic plaque drugs, comprising the following steps:

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

b. Apply the candidate to the carotid atherosclerotic plaque animal model described in step a;

c. Evaluation of potential drugs for the treatment of atherosclerotic plaques with animal models of carotid atherosclerotic plaques;

Preferably, the atherosclerotic plaque is carotid atherosclerotic plaque.

The present invention successfully constructs the carotid atherosclerotic plaque animal model. The construction method is used for modeling through the central artery of the rabbit ear, which has the advantages of small trauma, simple operation, high animal survival rate, high modeling success rate and little influence of other factors, and overcomes the problems of constructing carotid atherosclerotic plaque animals in the prior art. The problems existing in the model can be used to screen drugs for the treatment of atherosclerotic plaque, etc., which is of great significance for the study of carotid atherosclerosis.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1 shows the puncture through the central artery of the rabbit ear: A is the puncture of the right central artery of the rabbit ear with a 24G puncture needle; B is the insertion of a 0.014cm micro-guide wire from the needle sheath of the puncture needle into the central artery of the rabbit ear; C and D are the exit from the puncture The needle sheath was inserted, and the 1.8F microcatheter was introduced into the central artery of the rabbit ear under the guidance of the microguide wire.

Figure 2 shows the balloon catheter to dilate the common carotid artery of the rabbit; A shows the balloon catheter is located at the level of the right common carotid artery at the level of the cervical vertebrae 4-6; artery.

Figure 3 shows the anatomical images of normal blood vessels in the head and neck of New Zealand white rabbits without modeling treatment. ① is the central auricular artery, ② is the common carotid artery, ③ is the middle cerebral artery, and ④ is the submandibular artery: A is the left side Recumbent position; B is prone position.

Figure 4 shows the DSA imaging before and after balloon dilation of the rabbit common carotid artery: A is angiography before balloon dilation, with a diameter of about 2.222 mm, and B is angiography after balloon dilation, with a diameter of about 2.822 mm.

Figure 5 shows the vessel diameters of bilateral common carotid arteries in New Zealand white rabbits.

Figure 6 is a T1WI transverse axial scan image of an animal model of carotid atherosclerotic plaque according to the present invention: A is a T1WI transverse axial normal scan image; B is a T1WI transverse axial enhanced scan image 7 minutes after gadolinium contrast agent injection.

Figure 7 is a general observation picture of the common carotid arteries on both sides of the New Zealand white rabbits that were successfully anatomically modeled.

Figure 8 is the HE staining light microscope image of the rabbit common carotid artery section: A is the experimental group; B is the self-control group.

Detailed ways

The raw materials and equipment used in the specific embodiments of the present invention are all known products, which are obtained by purchasing commercially available products.

Example 1. Construction of the carotid atherosclerotic plaque animal model of the present invention

1. Experimental animals

Healthy male New Zealand white rabbits, each weight (2.1 ± 0.63 kg), rabbit age (5.0 ± 0.12) months, single-cage feeding, constant indoor temperature and humidity, free access to water, 12 hours day and night, by the West China Clinical Medical College of Sichuan University animal Provided by the Experimental Middle School.

2. Construction of an animal model of common carotid atherosclerotic plaque in rabbits

2.1 Balloon catheter injury intima of rabbit common carotid artery

(1) Preoperative preparation

The experimental animals were fed with high-fat diet for 4 weeks in advance, and they were fasted but not allowed to water for 12 hours before surgery.

(2) Operation steps

Tiletamine hydrochloride and zolazepam hydrochloride were injected intramuscularly for anesthesia at a dose of 30-40 mg/kg. After about 5-10 minutes, the New Zealand white rabbit entered an anesthetized state. The rabbit hair on the back of the ear was shaved to fully expose the central artery of the rabbit ear. The rabbit was fixed on the operating table of the digital subtractionangiography (DSA) machine in the left lateral position, the rabbit ears were disinfected with alcohol, and the surface skin of the central artery of the rabbit ear was rubbed back and forth with a cotton swab, and the force was slightly heavy to expand the central artery of the rabbit ear. The puncture needle was used to puncture the central artery of the right rabbit ear. After puncture, the needle core was withdrawn and the needle sheath was inserted at the same time. Bright red blood was seen flowing out. Heparin was injected into the needle sheath for anticoagulation (injection of 2 mL, 15 units/mL). Then, a 0.014cm micro-guide wire was inserted into the central artery of the rabbit ear from the needle sheath of the puncture needle, exited from the needle sheath, and then a small opening was slightly opened in the skin at the puncture point with a surgical blade, about 2 mm; the 1.8F micro-catheter was placed in the Under the guidance of the wire, it was sent into the central artery of the rabbit ear. The microcatheter was both a catheter and a vascular sheath. The elbow of the micro-guide wire was turned to the back (back direction), and the micro-catheter was passed through the right side in turn under the guidance of the micro-guide wire. Superficial temporal artery, external carotid artery, common carotid artery. Retain the micro-guide wire and exit the micro-catheter, and then send the 3.0mm*2cm balloon catheter into the common carotid artery (the balloon catheter is sent into the common carotid artery under the guidance of the micro-guide wire) to the level of the 4-6 cervical vertebrae . Inflate the balloon with iodine contrast agent to a pressure of 12-14KPa for 60 s, repeat three times with an interval of 60 s each time, and maintain the pressure in the balloon to drag the balloon catheter back and forth once each time, and drag the balloon catheter back and forth 3 times in total.

Then observe whether the dilation of the common carotid artery (intimal injury of the common carotid artery) is successful: withdraw the balloon catheter, send the microcatheter to the common carotid artery through the microguide wire, connect the microcatheter to the high pressure syringe for angiography, the flow rate is 1ml/s, and the flow rate is 3ml . The angiography confirmed that the common carotid artery was successfully dilated, the microcatheter and the microguide wire were taken out, the puncture point was compressed to stop the bleeding, and the interventional modeling process was ended. The steps in the surgical procedure are shown in Figures 1 and 2.

(3) Postoperative care

Intramuscular injection of gentamicin was given for 3 days to prevent infection, and the mental state, diet and activity of the experimental animals were closely observed.

2.2 High-fat feed for experimental animals

The experimental rabbits with carotid artery intimal injury after interventional operation were fed with high-fat diet (1% cholesterol + basal diet) for 8 weeks, three times a day, starting from the second day of interventional operation, with a feed amount of 15g/kg body weight each time , the feed continued to increase according to the proportion of body weight, still single-cage feeding, constant indoor temperature and humidity, free drinking water, 12h day and night. After 8 weeks, the rabbit carotid atherosclerotic plaque animal model was successfully established.

A total of 20 New Zealand white rabbits are modeled in the present invention, and the survival rate is 85% after modeling.

3. Modeling results

In the experiment, the side (left side) of the common carotid artery intima of each modeled New Zealand white rabbit without balloon catheter was used as the control group.

3.1 Vascular anatomy of New Zealand white rabbit head and neck DSA imaging

New Zealand white rabbits without modeling treatment were placed on the DSA operating table in the left lateral position and prone position respectively, the microcatheter tube head was placed at the common carotid artery, and DSA imaging was performed to obtain the normal blood vessels of the rabbit head and neck DSA imaging. Anatomical images, as shown in Figure 3.

3.2 DSA imaging results before and after balloon dilation of the rabbit common carotid artery

All modeled New Zealand white rabbits were subjected to DSA imaging before and after balloon dilation of the rabbit common carotid artery, and the diameter was measured. The diameter of the blood vessels after the rabbit common carotid artery was 2.84±0.18mm, and the diameter of the blood vessels after balloon dilatation of the rabbit common carotid artery increased significantly (P<0.01). Figure 4 shows the diameter of DSA imaging before and after balloon dilation of the rabbit common carotid artery for the rabbit with model number 18.

3.3 DSA imaging results after balloon catheter injury of common carotid artery intima combined with high-fat diet

All rabbits were subjected to DSA imaging after the intima of the common carotid artery was injured by balloon catheter combined with high-fat diet for 8 weeks. During imaging, not only the injured common carotid artery was observed (the common carotid artery that was successfully modeled, which is the experimental group) , but also to observe the self-control group. The test results show that: the diameter of the common carotid artery in the experimental group of the present invention is (2.75±0.25) mm, the diameter of the common carotid artery in the control group is (2.26±0.11) mm, and the diameter of the blood vessels in the experimental group is significantly higher than that in the control group (P< 0.01). Among them, the diameters of the left common carotid artery (self-control group) and the right common carotid artery (experimental group) of the rabbit model numbered No. 15 are shown in FIG. 5 .

3.4 MRI findings

Figure 6A is the T1WI transverse axial normal scan image of the New Zealand white rabbit (experimental group) after modeling; Figure 6B is the T1WI transverse axis enhanced scan image 7 minutes after gadolinium contrast agent injection, the results show the neck of the experimental group modeling The common arterial wall was thickened and significantly enhanced after injection of gadolinium-based contrast media (white arrows). In the control group, the common carotid artery wall was not thickened, but was slightly enhanced in the gadolinium contrast agent.

3.5 General observation

The New Zealand white rabbits that were successfully modeled were dissected, and the common carotid arteries on both sides of the rabbits were grossly observed. The observation showed that the common carotid artery wall of the experimental group was obviously thickened, stiff and inelastic, and was yellowish-white (indicated by the white arrow in Figure 7); the common carotid artery wall of the control group was thin, soft and elastic, and was pink (Figure 7). 7 shown by the red arrow). The diameter of the common carotid artery in the experimental group was significantly thicker than that in the control group, and the elasticity was significantly worse.

3.6 Optical microscope observation

Figure 8 is the HE-stained light microscope image of the rabbit common carotid artery section. It can be seen from Fig. 8 that the intima of the common carotid artery in the experimental group (Fig. 8A) is thickened and uneven, as shown by the red "*" in Fig. 8A, the lumen is narrowed, the endothelial cells are not completely arranged, and there are a lot of foam cells Aggregation, a large number of smooth muscle cells infiltrated, disorderly arrangement; media thickening, more foam cells, smooth muscle cells disorderly arrangement. In the self-control group (Fig. 8B), the intima of the common carotid artery of the rabbits was smooth, without thickening or foam cells, and the smooth muscle cells in the media were neatly arranged.

The above results indicate that the carotid atherosclerotic plaque animal model of the present invention was successfully constructed.

In conclusion, the present invention has successfully constructed an animal model of carotid atherosclerotic plaque. The construction method is used for modeling through the central artery of the rabbit ear, which has the advantages of small trauma, simple operation, high animal survival rate, high modeling success rate and little influence of other factors, and overcomes the problems of constructing carotid atherosclerotic plaque animals in the prior art. The problems existing in the model can be used to screen drugs for the treatment of atherosclerotic plaque, etc., which is of great significance for the study of carotid atherosclerosis.

Claims (10)

1. A method for constructing a carotid atherosclerotic plaque animal model, characterized in that: it is a model created by injuring the common carotid artery intima combined with high-fat feed by the central auricular artery. 2. The construction method according to claim 1, wherein the high-fat feed is fed on the first day after the intima of the common carotid artery is damaged; Preferably, the high-fat feed is fed for 8 weeks, three times a day, and the amount of each high-fat feed is 15 g/kg body weight; More preferably, when the high-fat feed is fed, the indoor temperature and humidity are constant, and drinking water is free. 3 . The construction method according to claim 2 , wherein the high-fat feed is made by adding 1% cholesterol in the basic feed. 4 . 4 . The construction method according to claim 1 , wherein the animal used in the animal model is a rabbit; preferably a New Zealand white rabbit. 5 . The construction method according to any one of claims 1 to 3, wherein the step of injuring the intima of the common carotid artery through the central auricular artery is as follows: taking a microcatheter and sending it under the guidance of a microguide wire Enter the central artery of the rabbit ear, and under the guidance of the micro-guide wire, the micro-catheter is passed through the superficial temporal artery, the external carotid artery in turn, and finally sent to the common carotid artery, and then the common carotid artery is dilated with a balloon catheter. 6. The construction method according to claim 5, wherein the step of injuring the intima of the common carotid artery through the central auricular 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 at the same time; (2) Insert the micro-guide wire from the puncture needle sheath into the central artery of the rabbit ear, and exit the puncture needle sheath; (3) The microcatheter is sent into the central artery of the rabbit ear under the guidance of the micro-guide wire, and then the micro-catheter is passed through the superficial temporal artery and the external carotid artery in turn under the guidance of the micro-guide wire, and finally sent to the common carotid artery; (4) Retain the micro-guide wire and exit the micro-catheter, under the guidance of the micro-guide wire, send the balloon catheter into the common carotid artery, fill the balloon, and drag the balloon catheter. 7. construction method according to claim 6 is characterized in that: In step (1), the puncture needle is a 24G puncture needle; And/or, in step (1), heparin anticoagulation is injected during the puncture; And/or, in step (2), the micro-guide wire is a micro-guide wire with a diameter of 0.014cm; And/or, in step (3), described microcatheter is the microcatheter of 1.8F; And/or, in step (4), the balloon catheter is a balloon catheter of 3.0mm*2cm; And/or, in step (4), the balloon catheter is sent into the common carotid artery to the vertebral bodies of cervical vertebrae 4-6; And/or, in step (4), the filling balloon is filled with iodine contrast agent. 8. The construction method according to claim 7, wherein the filling balloon is to fill the balloon three times with iodine contrast agent; Preferably, each time the balloon is inflated to a pressure of 12-14KPa and maintained for 60s, and the balloon catheter is dragged back and forth while maintaining the pressure in the balloon; More preferably, the interval for each filling of the balloon is 60s; and/or, the dragging the balloon catheter back and forth is dragging the balloon catheter back and forth once after each filling of the balloon. 9. Use of the model constructed by the construction method according to any one of claims 1 to 8 in screening a drug for treating atherosclerotic plaque; Preferably, the atherosclerotic plaque is carotid atherosclerotic plaque. 10. A method for screening a drug for the treatment of atherosclerotic plaque, comprising the steps of: a. Construct a carotid atherosclerotic plaque animal model according to the construction method of any one of claims 1 to 8; b. Apply the candidate to the carotid atherosclerotic plaque animal model described in step a; c. Evaluation of potential drugs for the treatment of atherosclerotic plaques with animal models of carotid atherosclerotic plaques; Preferably, the atherosclerotic plaque is carotid atherosclerotic plaque.

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