A method for constructing an atherosclerotic plaque thrombosis model
A simple and reproducible model of atherosclerotic plaque thrombosis was constructed by feeding ApoE-/- mice with a high-fat diet and carotid artery constriction, combined with intraperitoneal injection of LPS. This model solves the problem of unreliability in existing technologies and achieves low-cost and efficient simulation of atherosclerotic plaque thrombosis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-17
- Publication Date
- 2026-04-10
AI Technical Summary
Existing technologies lack simple, reproducible, and clinically relevant models of atherosclerotic plaque thrombosis. In particular, ApoE-/- mice cannot be induced to form atherosclerotic plaque thrombosis using conventional methods, and high-dose LPS spray devices are difficult to promote and apply.
A carotid artery constriction ApoE-/- mice fed a high-fat diet were used to induce atherosclerotic plaque thrombosis by intraperitoneal injection of low-dose LPS (0.5-1 mg/kg), which was then used to construct a carotid artery constriction model.
A simple and reproducible model of atherosclerotic plaque thrombosis was successfully constructed. The incidence of thrombosis under low-dose LPS stimulation was comparable to that of high-dose spray devices, simulating the pathological process associated with clinical endotoxemia and reducing experimental costs and time.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of animal model construction, in particular to a method for constructing an atherosclerotic plaque thrombosis model. BACKGROUND
[0002] Atherosclerosis is one of the most common cardiovascular diseases in humans, and atherosclerotic thrombosis can cause cardiovascular events such as myocardial infarction and cerebral infarction. Inflammation is the main factor leading to atherosclerotic thrombosis, and lipopolysaccharide (LPS) can induce endotoxemia, which is one of the main causes of inflammation in the body. However, due to species differences, ApoE - / - Mice cannot spontaneously form atherosclerotic thrombosis, even if they are injected intraperitoneally with a high dose of LPS (3mg / kg), they cannot induce atherosclerotic plaque thrombosis. There are few reports on animal models of atherosclerotic plaque thrombosis, and in 2016, Jen Erh Jaw et al. reported that LPS (3mg / kg) administered through a spray device into the lungs of mice can induce ApoE - / - Mice with atherosclerotic plaque thrombosis in the brachiocephalic artery (Lung exposure to lipopolysaccharide causes atherosclerotic plaque destabilisation. Eur Respir J. 2016). However, the atherosclerotic thrombosis they showed in the paper is not a typical atherosclerotic plaque, and there are few research papers citing their experimental method on the Web of Science website. In addition, using a spray device to administer drugs through the lungs requires special equipment and operation, which is difficult to popularize and apply. The laboratory has also failed to reproduce the results reported. Moreover, the 3mg / kg dose of LPS is relatively high and not very close to clinical endotoxemia. Therefore, there is currently a lack of a simple and repeatable atherosclerotic thrombosis model related to endotoxemia that is closer to the clinic. SUMMARY
[0003] The purpose of the present application is to overcome the shortcomings of the prior art and provide a method for constructing an atherosclerotic plaque thrombosis model that is simple to operate, highly repeatable, and closer to the clinic.
[0004] To achieve the above object, the technical scheme adopted by the present application is as follows:
[0005] A method for constructing an atherosclerotic plaque thrombosis model, comprising the following steps:
[0006] 1) Select ApoE - / - Mice fed with high-fat feed and subjected to carotid artery narrowing;
[0007] 2) intraperitoneal injection of LPS, i.e. to obtain an animal model of atherosclerotic plaque thrombosis.
[0008] In the method for constructing the animal model of atherosclerotic plaque thrombosis as described above, preferably the mice in step 1) are male or female mice.
[0009] In the method for constructing the animal model of atherosclerotic plaque thrombosis as described above, preferably the mice in step 1) are 15-20 weeks old.
[0010] In the method for constructing the animal model of atherosclerotic plaque thrombosis as described above, preferably the high-fat feed has the following mass ratio: casein 22.18%, L-cystine 0.334%, corn starch 23.51%, malt dextrin 7.87%, sucrose 13.794%, cellulose 5.54%, soybean oil 2.774%, cocoa oil 17.19%, sodium ammonium 0.29%, mixed mineral S100200 200.55%, calcium hydrogen phosphate 1.44%, calcium carbonate 0.61%, potassium citrate 1.834%, vitamin mixture 0.110%, choline tartrate 0.224%, cholesterol 1.25%, sodium cholate 0.50%. The high-fat feed has the following heat ratio: protein 19.97%, fat 39.85%, carbohydrate 40.18%, total 4.51 kcal / g.
[0011] In the method for constructing the animal model of atherosclerotic plaque thrombosis as described above, preferably the high-fat feed is fed to the mice in step 1) for 8-12 weeks.
[0012] In the method for constructing the animal model of atherosclerotic plaque thrombosis as described above, preferably the ApoE - / - mouse is a neck constriction ApoE - / - mouse for 3-4 weeks.
[0013] In the method for constructing the animal model of atherosclerotic plaque thrombosis as described above, preferably the neck constriction in step 1) is right neck constriction or left neck constriction.
[0014] In the method for constructing the animal model of atherosclerotic plaque thrombosis as described above, preferably the intraperitoneal injection of LPS is at a dose of 0.5-1 mg / kg.
[0015] The present application also provides the use of a neck constriction ApoE - / - mouse fed with high-fat feed for preparing an animal model, for preparing an animal model of atherosclerotic plaque thrombosis. The neck constriction ApoE - / -The mouse is injected intraperitoneally with LPS to obtain an animal model of atherosclerotic plaque thrombosis.
[0016] The present application has the advantages of:
[0017] The present application constructs an ApoE - / - The mouse is injected intraperitoneally with LPS to obtain an animal model of atherosclerotic plaque thrombosis. Compared with the prior art, which uses a spray device to spray 3mg / kg LPS into the lungs of a mouse, the construction method provided by the present application is simple to operate and has strong repeatability. The method can induce atherosclerotic plaque thrombosis at a low dose of 1mg / kg LPS, has a thrombosis rate comparable to the prior art, and can more closely simulate the formation of atherosclerotic thrombosis related to clinical endotoxemia. The method can significantly reduce the cost and time of modeling experiments, has strong repeatability, and is more practical. BRIEF DESCRIPTION OF DRAWINGS
[0018] The present application provides a method for constructing an animal model of atherosclerotic plaque thrombosis. Figure 1 Detection results of LPS-induced atherosclerotic plaque thrombosis at the carotid artery constriction site. (A) Schematic diagram of the structure of the carotid artery constriction site; (B) HE staining and immunohistochemical staining of platelet markers (vWF and CD41) showing stage I atherosclerotic thrombosis; (C) Atherosclerotic thrombosis rate of different experimental groups. DETAILED DESCRIPTION
[0019] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the content disclosed in the present application, and these equivalent forms also fall within the scope of the claims attached hereto.
[0020] The present application provides a method for constructing an animal model of atherosclerotic plaque thrombosis, which selects a high-fat diet-fed ApoE - / -The mouse is injected with LPS in the abdominal cavity to obtain an animal model of atherosclerotic plaque thrombosis. Experimental results show that the construction method of the present application can successfully induce atherosclerotic plaque thrombosis at a very low dose of 0.5 mg / kg of LPS stimulation, and the incidence of atherosclerotic thrombosis can be increased to more than 60% when the dose of LPS stimulation is increased to 1 mg / kg. Compared with the prior art method of spraying 3 mg / kg of LPS through the lungs of the mouse, the present application is not only simple to operate and highly reproducible, but also can obtain a thrombosis incidence comparable to the prior art at a low dose of 1 mg / kg of LPS stimulation, which is more close to the formation of atherosclerotic thrombosis related to clinical endotoxemia and better simulates the clinical pathological process.
[0021] The ApoE - / - mouse is a commonly used ApoE - / - model animal, and the ApoE - / - mouse can be purchased commercially, for example, a 15-20-week-old male ApoE - / - knockout mouse with right carotid artery stenosis fed with high-fat feed can be obtained from Shanghai South Model Organism Technology Co., Ltd. - / - The ApoE - / - mouse is subjected to carotid artery stenosis, and the ApoE - / - mouse is subjected to left and right carotid artery stenosis. Alternatively, the ApoE - / - mouse is subjected to carotid artery stenosis in the following manner: the mouse is anesthetized with isoflurane, and the mouse is fixed in a supine position after anesthesia; the mouse is shaved with a depilatory cream, and the neck is disinfected with 75% alcohol; the mouse is incised along the median line of the neck to expose and separate the right common carotid artery; at about 3 mm below the bifurcation of the internal and external carotid arteries, the right common carotid artery is tightly tied with a 6-0 surgical suture line together with a fine needle with a diameter of 150 μm, and then the fine needle is withdrawn, the skin is sutured, and the right carotid artery stenosis is completed. Those skilled in the art can also select different sources of ApoE - / - knockout mice subjected to carotid artery stenosis, which are fed with high-fat feed about 6 weeks before stenosis and about 4 weeks after stenosis, to construct the model according to the principle of the present application, and the ApoE - / - mice are subjected to 0.5-1 mg / kg of LPS intraperitoneal injection stimulation to obtain an animal model of atherosclerotic plaque thrombosis. The methods for constructing the atherosclerotic plaque thrombosis model of the ApoE
[0022] Example 1 Construction and identification of lipopolysaccharide (LPS) induced atherosclerotic plaque thrombosis model
[0023] 1. Method
[0024] Model construction method: 16-week-old male apolipoprotein E knockout mice (ApoE - / - ) which have been constructed and fed with high-fat diet for 10 weeks and right carotid artery stenosis for 4 weeks are selected for modeling. Different concentrations of LPS are injected intraperitoneally to induce carotid atherosclerotic thrombosis. After 24 hours, the mice are anesthetized for sample collection and pathological examination.
[0025] Basic feed: mass ratio of casein 14%, corn starch 49.6, maltodextrin 12.5%, sucrose 10%, cellulose 5%, soybean oil 4%, L-cystine 0.18%, mixed mineral S10022M 3.5%, mixed vitamin V10037 1%, hydrogen tartrate 0.25%. The basic feed has a heat ratio of protein 14.7%, fat 9.4%, and carbohydrate 75.9%, with a total of 3.9 kcal / g.
[0026] High-fat feed: mass ratio of casein 22.18%, L-cystine 0.334%, corn starch 23.51%, maltodextrin 7.87%, sucrose 13.794%, cellulose 5.54%, soybean oil 2.774%, cocoa oil 17.19%, sodium ammonium 0.29%, mixed mineral S10020 0.55%, calcium hydrogen phosphate 1.44%, calcium carbonate 0.61%, potassium citrate 1.834%, vitamin mixture 0.110%, choline tartrate 0.224%, cholesterol 1.25%, sodium cholate 0.50%. The high-fat feed has a heat ratio of protein 19.97%, fat 39.85%, and carbohydrate 40.18%, with a total of 4.51 kcal / g.
[0027] Lipopolysaccharide: purchased from sigma company, item number L2630.
[0028] ApoE - / - mice with right carotid artery stenosis refer to ApoE - / - mice with right carotid artery stenosis rate of 60-80%, stenosis rate = (DCC-DMC) / DCC x 100%, where DMS represents the diameter of the most stenotic region of the carotid artery lumen, and DCC represents the normal carotid artery lumen diameter.
[0029] 2. Results
[0030] According to the above method, we set up the following groups: no carotid artery stenosis + LPS (3 mg / kg) group: select no carotid artery stenosis ApoE - / - mice, after 10 weeks of high-fat diet, intraperitoneal injection of LPS (3 mg / kg);
[0031] Carotid artery stenosis 30 days group: select high-fat diet for 10 weeks of right carotid artery stenosis ApoE - / - mice;
[0032] Carotid artery stenosis 30 days + LPS (0.5 mg / kg) group: select high-fat diet for 10 weeks of right carotid artery stenosis ApoE - / - mice, intraperitoneal injection of LPS (0.5 mg / kg);
[0033] Carotid artery stenosis 30 days + LPS (1 mg / kg) group: select high-fat diet for 10 weeks of right carotid artery stenosis ApoE - / - mice, intraperitoneal injection of LPS (1 mg / kg).
[0034] Each group of 20, using HE staining, immunohistochemical staining and other pathological methods to detect carotid artery I segment, brachiocephalic trunk and aortic root of atherosclerotic plaque thrombosis. HE staining showed that after intraperitoneal injection of LPS, carotid artery stenosis 30 days + LPS (0.5 mg / kg) group and carotid artery stenosis 30 days + LPS (1 mg / kg) group mice I segment atherosclerotic plaque thrombosis Figure 1 ). vWF (platelet marker) and CD41 (platelet marker) immunohistochemical staining further proved that I segment atherosclerotic plaque thrombosis Figure 1 ), while the brachiocephalic trunk and aortic root were not found atherosclerotic thrombosis. As shown in Figure 1 C, when the intraperitoneal injection of LPS dose increased from 0.5 mg / kg to 1 mg / kg, the incidence of atherosclerotic thrombosis increased significantly, in carotid artery stenosis 30 days + LPS (1 mg / kg) group, the incidence of atherosclerotic thrombosis was more than 60%.
[0035] In summary, in ApoE - / - mice atherosclerosis carotid artery stenosis model, intraperitoneal injection of LPS successfully induced I segment atherosclerotic plaque thrombosis, and the incidence of atherosclerotic thrombosis in LPS model animals of different doses was different. While the ApoE - / - mice atherosclerosis model even if injected with high dose of LPS cannot form atherosclerotic plaque thrombosis.
[0036] The above merely describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and supplements can be made without departing from the method of the present application, and these improvements and supplements should also be considered as the protection scope of the present application.
Claims
1. A method for constructing a model of atherosclerotic plaque thrombosis, characterized in that, Includes the following steps: 1) Select ApoE with a carotid artery stenosis rate of 60-80% fed a high-fat diet. - / - Mice; 2) Intraperitoneal injection of LPS to obtain an animal model of atherosclerotic plaque thrombosis.
2. The method for constructing the atherosclerotic plaque thrombosis model according to claim 1, characterized in that, In step 1), the mouse is either male or female.
3. The method for constructing the atherosclerotic plaque thrombosis model according to claim 1, characterized in that, The mice in step 1) are 15-20 weeks old.
4. The method for constructing the atherosclerotic plaque thrombosis model according to claim 3, characterized in that, In step 1), the animal was fed a high-fat diet for 8-12 weeks.
5. The method for constructing the atherosclerotic plaque thrombosis model according to claim 4, characterized in that, In step 1), ApoE with a carotid artery narrowing rate of 60-80% is used. - / - Mice refer to ApoE mice with carotid artery constriction for 3-4 weeks - / - Mice.
6. The method for constructing the atherosclerotic plaque thrombosis model according to claim 1, characterized in that, In step 1), the narrowing is either the right carotid artery or the left carotid artery.
7. The method for constructing an atherosclerotic plaque thrombosis model according to claim 1, characterized in that, In step 2), the intraperitoneal injection of LPS is 0.5-1 mg / kg.