A method for constructing a rat model simulating the clinical characteristics of peripheral artery disease and an evaluation method thereof

Abstract

The present invention provides a method for constructing a rat model simulating the clinical characteristics of peripheral arterial disease, comprising the following steps: Step 1: A unilateral femoral artery of a diabetic rat is ligated with an Ameroid constrictor ring, and the rat is raised for a period of time; Step 2: The femoral artery of the rat surviving after being raised in Step 1 is ligated. In Step 1 and Step 2, the ligation of the Ameroid constrictor ring and the femoral artery ligation are both located on the same side femoral artery of the rat. The present invention establishes a chronic limb ischemia animal model conforming to the clinical characteristics of peripheral arterial disease. The present invention intends to induce type 2 diabetes in rats by feeding them with a high-fat and high-sugar diet, use an Ameroid constrictor ring on the femoral artery to cause progressive limb ischemia, and then ligate the femoral artery to cause vascular occlusion to simulate critical limb ischemia. The "staged" chronic limb ischemia in diabetic rats provides a suitable animal model for the basic research of peripheral arterial disease.

Description

Technical Field

[0001] The present invention belongs to the field of research on peripheral arterial diseases and is used for basic research on chronic limb ischemia. In particular, it relates to a method for constructing a rat model simulating the clinical characteristics of peripheral arterial diseases and an evaluation method therefor. Background Art

[0002] Peripheral arterial disease significantly impairs motor ability, increases the risk of amputation, and seriously affects the quality of life and life safety of patients. The formation of collateral circulation is crucial for saving limb blood flow. Therapeutic angiogenesis strategies mainly based on gene amplification of angiogenic factors have been proven to significantly improve angiogenesis in chronically ischemic limbs of experimental animals, but the clinical translation effect is not good, suggesting that existing animal models are insufficient to simulate the peripheral ischemic state, including resistance to angiogenic signals. Therefore, it is of great practical significance to explore a chronic limb ischemia animal model that conforms to the clinical characteristics of peripheral arterial diseases.

[0003] In previous studies, the chronic limb ischemia models used mainly caused complete limb ischemia in a short time by ligating or excising the femoral artery. However, 35 days later, the limb blood flow recovered to 84% of the baseline value, which does not conform to the characteristics of impaired angiogenesis in ischemic limbs of patients with peripheral arterial diseases. The chronic ischemia induced by the Ameroid constrictor ring can simulate the gradual stenosis of arteries caused by atherosclerotic plaque accumulation, but the degree of ischemia is relatively mild, the blood flow recovery is faster, and it cannot induce the critical limb ischemia stage of complete vascular occlusion.

[0004] Diabetes is rapidly becoming the most important risk factor for peripheral arterial disease. Scholars believe that a typical animal model simulating human peripheral arterial disease should include atherosclerosis, risk factors for peripheral arterial disease, and gradually acquired chronic ischemia. Therefore, theoretically, establishing a "staged" chronic limb ischemia animal model that combines the high-risk factor of diabetes and includes progressive limb ischemia and acute vascular occlusion is more in line with the clinical characteristics of peripheral arterial diseases. Summary of the Invention

[0005] The present invention provides a method for constructing a rat model simulating the clinical characteristics of peripheral arterial diseases and an evaluation method therefor in view of the deficiencies of the prior art.

[0006] To achieve the above object, the present invention provides the following technical solution. A method for constructing a rat model simulating the clinical characteristics of peripheral arterial diseases includes the following steps:

[0007] Step 1: Place an Ameroid constrictor ring around the unilateral femoral artery of a diabetic rat and raise it for a period of time;

[0008] Step 2: Ligate the femoral artery of the rats that survived after being raised in Step 1. In Step 1 and Step 2, both the Ameroid constrictor ring ligation and the femoral artery ligation are located on the same femoral artery of the rats.

[0009] Step 3: Raise the rats in Step 2 for a period of time, continuously monitor the changes in skin blood flow of the rats, and measure the motor function and ischemia degree of the rats to obtain a model that conforms to the clinical characteristics of patients with peripheral artery disease.

[0010] Optionally, in Step 1, after ligating the femoral artery of the diabetic rats with an Ameroid constrictor ring, raise them with a standard diet for 14 days.

[0011] Optionally, in Step 3, raise the rats in Step 2 with a standard diet for 28 days.

[0012] Optionally, the method for raising the diabetic rats includes the following steps:

[0013] Step 1: Continuously feed healthy rats with a high-sugar and high-fat diet for a period of time.

[0014] Step 2: Inject 40 mg / kg of streptozotocin into the abdominal cavity of the rats obtained in Step 1. When the fasting blood glucose of the rats is measured to be ≥16.7 mmol / L, it indicates that the diabetic rat model is successfully established.

[0015] Step 3: Continuously feed the diabetic rats in Step 2 with a standard diet for a period of time and observe the changes in their fasting blood glucose to determine the stability of the model.

[0016] Optionally, in Step 1, the healthy rats need to be continuously fed with a high-sugar and high-fat diet for more than 28 days. In Step 3, the diabetic rats need to be continuously fed with a standard diet for more than 28 days.

[0017] Optionally, in Step 1, the raw materials of the high-sugar and high-fat diet are 10% lard, 2.5% cholesterol, 1.0% cholate, 20% sucrose, and 66.5% standard diet.

[0018] A method for evaluating a rat model simulating the clinical characteristics of peripheral artery disease, used to verify a method for constructing a rat model simulating the clinical characteristics of peripheral artery disease.

[0019] Step (1): Use the constructed rat model that conforms to the clinical characteristics of patients with peripheral artery disease as the experimental group, and simultaneously complete the construction of the control group. Among them, the control group includes the following three groups:

[0020] Group 1: Unilateral femoral artery of healthy rats was ligated with an Ameroid constrictor ring (ethylene oxide sterilized), and the ipsilateral femoral artery was exposed again for ligation after 14 days, and the rats were continuously fed with standard feed for 28 days;

[0021] Group 2: Unilateral femoral artery of healthy rats was ligated and the rats were fed with standard feed for 42 days;

[0022] Group 3: Unilateral femoral artery of healthy rats was ligated with an Ameroid constrictor ring (ethylene oxide sterilized) and the rats were fed with standard feed for 42 days;

[0023] Step (2): Laser Doppler flowmetry was used to measure the limb skin blood flow in the experimental group and the control group of rats;

[0024] Step (3): Motor function and degree of ischemia of the rats in the experimental group and the control group were measured;

[0025] Step (4): Based on the limb blood flow change trend graph, motor function change and degree of ischemia of the rat model, it was evaluated whether the rat model was successfully constructed.

[0026] Optionally, in the step (4), the standard for judging the successful construction of the rat model is that the blood flow of the limb skin measured by laser Doppler flowmetry is less than 0.5.

[0027] In summary, the present invention establishes a chronic limb ischemia animal model that conforms to the clinical characteristics of peripheral artery disease. The present invention intends to induce type 2 diabetes in rats by feeding with high-fat and high-sugar feed, use an Ameroid constrictor ring on the femoral artery to cause progressive limb ischemia, and then ligate the femoral artery to cause vascular occlusion to simulate critical limb ischemia. The "phased" chronic limb ischemia of diabetic rats provides a suitable animal model for the basic research of peripheral artery disease.

[0028] The advantages of the present invention are

[0029] 1. The "phased" chronic limb ischemia model of diabetic rats involved in the present invention fully considers the three major characteristics of chronic limb ischemia in patients with peripheral artery disease, namely: atherosclerosis, risk factors of peripheral artery disease, and gradually acquired chronic ischemia. Theoretically, this animal model is more in line with the clinical characteristics of patients with peripheral artery disease, overcomes the drawbacks of existing chronic limb ischemia animal models, and has certain innovation and practical necessity.

[0030] 2. Improve the preparation method of the existing chronic limb ischemia rat model, which can provide a suitable animal model for the future basic research of peripheral artery disease and has strong applicability. Brief Description of the Drawings

[0031] Figure 1In the present invention, it is the process of constructing a rat model simulating the clinical characteristics of peripheral arterial disease.

[0032] Figure 2 In the experimental group, it is the measurement of the limb skin blood flow of the rat model simulating the clinical characteristics of peripheral arterial disease in a double-layer glass cage at a constant temperature of 37 °C at 0 d, 14 d, and 42 d.

[0033] Figure 3 It is the trend chart of the changes in the limb skin blood flow of the rat model in each group, including Group C1, Group C2, Group C3, and Group E.

[0034] Figure 4 They are the Tarlov scoring scale and the modified ischemia scoring form.

[0035] Figure 5 It is the limb movement and limb activity of the rat model in each group, including Group C1, Group C2, Group C3, and Group E. Detailed implementation mode

[0036] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0037] Reference Figures 1-5 , a method for constructing a rat model simulating the clinical characteristics of peripheral arterial disease and its evaluation method include

[0038] (1) Induction of type 2 diabetes in rats

[0039] After feeding with a high-sugar and high-fat diet (10% lard, 2.5% cholesterol, 1.0% sodium cholate, 20% sucrose, 66.5% standard feed) for 28 d, streptozotocin at 40 mg / kg was intraperitoneally injected. When the fasting blood glucose ≥ 16.7 mmol / L, the establishment of the rat diabetes model was successful. After the model was established, the standard feed was continued to be fed for 28 d to observe the stability of the model.

[0040] (2) Establishment of a rat "phased" chronic limb ischemia model

[0041] Phase 1: After the rats were anesthetized, the skin was incised in the inguinal region, the left femoral artery was dissected, and an Ameroid constrictor ring (sterilized by ethylene oxide) of appropriate size was ligated and fixed outside the blood vessel lumen, with the diameter of the femoral artery lumen not being affected as appropriate, and the incision was sutured.

[0042] Phase 2: After 14 d, the ipsilateral femoral artery was exposed again for ligation, paying attention to preserving the femoral nerve (local application of lidocaine to reduce pain), and continuing to feed with the standard feed for 28 d.

[0043] (3) Verification of model preparation

[0044] ① Blood flow signal: At 0d, 14d, and 42d, the limb skin blood flow was measured by a laser Doppler blood flowmeter in a double-layer glass cage at a constant temperature of 37°C and quantified;

[0045] ② Measurement of motor function and degree of ischemia: At 0d, 14d, and 42d, the Tarlov and modified ischemia scoring scales were used, and the specific scores were as Figure 5 .

[0046] Furthermore, the experiments conducted in the above steps were set as Group E, and according to the operation steps of Group E, three experimental groups were set up. The specific experimental methods are as follows.

[0047] Group C1:

[0048] (1) Blank control for rat feeding

[0049] Rats were fed with standard feed for 28 days, and then injected with normal saline intraperitoneally to detect the blood glucose of the rats to determine that the rats did not suffer from diabetes; then they were continued to be fed with standard feed for 28 days, and the blood glucose changes of the rats were continuously observed to ensure that the blood glucose of the rats was within the normal range.

[0050] (2) Establishment of a rat model of chronic limb ischemia

[0051] After the rats were anesthetized, the skin was incised in the groin area, the left femoral artery was dissected, and a suitable-sized Ameroid constrictor ring (sterilized by ethylene oxide) was ligated and fixed outside the blood vessel lumen, with the diameter of the femoral artery lumen not being affected as appropriate. The incision was sutured, and the rats were fed with standard feed for 14 days. Then, the ipsilateral femoral artery was exposed again for ligation, and the rats were continued to be fed with standard feed for 28 days.

[0052] (3) Verification of model preparation

[0053] ① Blood flow signal: At 0d, 14d, and 42d, the limb skin blood flow was measured by a laser Doppler blood flowmeter in a double-layer glass cage at a constant temperature of 37°C and quantified;

[0054] ② Measurement of motor function and degree of ischemia: At 0d, 14d, and 42d, the Tarlov and modified ischemia scoring scales were used, and the specific scores were as Figure 5 .

[0055] Group C2:

[0056] (1) Blank control for rat feeding,

[0057] The rats were fed with standard feed for 28 days, and then intraperitoneally injected with normal saline to detect the blood glucose of the rats to determine that the rats did not suffer from diabetes. Then, they were continuously fed with standard feed for 28 days, and the blood glucose changes of the rats were continuously observed to ensure that the blood glucose of the rats was within the normal range.

[0058] (2) Establishment of a rat model of chronic limb ischemia

[0059] After the rats were anesthetized, the skin was incised in the inguinal region, the left femoral artery was dissected, the femoral artery was ligated, and the femoral nerve was preserved. The rats were continuously fed with standard feed for 42 days.

[0060] (3) Verification of model preparation

[0061] ① Blood flow signal: At 0 d, 14 d, and 42 d, the limb skin blood flow was measured with a laser Doppler flowmeter in a double-layer glass cage at a constant temperature of 37 °C and quantified.

[0062] ② Measurement of motor function and degree of ischemia: At 0 d, 14 d, and 42 d, the Tarlov and modified ischemia scoring scales were used, and the specific scores were as follows Figure 5 .

[0063] Group C3

[0064] (1) Blank control for rat feeding

[0065] The rats were fed with standard feed for 28 days, and then intraperitoneally injected with normal saline to detect the blood glucose of the rats to determine that the rats did not suffer from diabetes. Then, they were continuously fed with standard feed for 28 days, and the blood glucose changes of the rats were continuously observed to ensure that the blood glucose of the rats was within the normal range.

[0066] (2) Establishment of a rat model of chronic limb ischemia

[0067] After the rats were anesthetized, the skin was incised in the inguinal region, the left femoral artery was dissected, and a suitable-sized Ameroid constrictor ring (sterilized with ethylene oxide) was ligated and fixed outside the blood vessel lumen, with the diameter of the femoral artery lumen not being affected as appropriate. The incision was sutured, and the rats were fed with standard feed for 42 days.

[0068] (3) Verification of model preparation

[0069] ① Blood flow signal: At 0 d, 14 d, and 42 d, the limb skin blood flow was measured with a laser Doppler flowmeter in a double-layer glass cage at a constant temperature of 37 °C and quantified.

[0070] ② Measurement of motor function and degree of ischemia: At 0 d, 14 d, and 42 d, the Tarlov and modified ischemia scoring scales were used, and the specific scores were as follows Figure 5 .

[0071] By comparing Group C1, Group C2, Group C3 and Group E, the following conclusions can be drawn:

[0072] * Compared with Group C1, C2 and C3, the blood perfusion index of the lower limb on the operative side of Group E rats decreased at 42 days (P<0.05).

[0073] * Compared with Group C1, C2 and C3, the Tarlov score and the lower limb ischemia score on the operative side of Group E rats both decreased at 42 days (P<0.05).

[0074] Therefore, the method provided by the present invention for simulating peripheral artery disease in rats has a higher degree of coincidence.

[0075] Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

Claims

1. A method for constructing a rat model simulating the clinical characteristics of peripheral artery disease, characterized in that, It includes the following steps: Step 1: Place an Ameroid constrictor ring around the unilateral femoral artery of a diabetic rat and raise it for a period of time; the feeding method of the diabetic rat includes the following steps: Step 1: Continuously feed healthy rats with a high-sugar and high-fat diet for a period of time; Step 2: Inject 40 mg / kg of streptozotocin into the abdominal cavity of the rats obtained in Step 1. When the fasting blood glucose of the rats is measured to be ≥ 16.7 mmol / L, it indicates that the diabetic rat model is successfully established; Step 3: Continuously feed the diabetic rats obtained in Step 2 with a standard diet for a period of time and observe the changes in the fasting blood glucose of the rats to determine the stability of the model; Step 2: Ligate the femoral artery of the rats that survived after being raised in Step 1. Among them, in Step 1 and Step 2, the Ameroid constrictor ring placement and femoral artery ligation are both located on the same side femoral artery of the rat; Step 3: Raise the rats in Step 2 for a period of time, continuously monitor the changes in skin blood flow of the rats, and measure the motor function and degree of ischemia of the rats to obtain a rat model that conforms to the clinical characteristics of patients with peripheral arterial disease.

2. The construction method of a rat model simulating the clinical characteristics of peripheral artery disease according to claim 1, wherein, In Step 1, after placing the Ameroid constrictor ring around the femoral artery of the diabetic rat, raise it with a standard diet for 14 days.

3. The method for constructing a rat model simulating the clinical characteristics of peripheral arterial disease according to claim 1, wherein, In Step 3, raise the rats in Step 2 with a standard diet for 28 days.

4. The construction method of a rat model simulating the clinical characteristics of peripheral arterial disease according to claim 1, characterized in that, In Step 1, healthy rats need to be continuously fed with a high-sugar and high-fat diet for 28 days. In Step 3, diabetic rats need to be continuously fed with a standard diet for 28 days.

5. The construction method of a rat model simulating the clinical characteristics of peripheral arterial disease according to claim 1, wherein, In Step 1, the raw materials of the high-sugar and high-fat diet are 10% lard, 2.5% cholesterol, 1.0% cholate, 20% sucrose, and 66.5% standard diet.

6. A method for evaluating a rat model simulating the clinical characteristics of peripheral arterial disease, used to verify a method for constructing a rat model simulating the clinical characteristics of peripheral arterial disease as described in claims 1-5, characterized in that Step (1): Use the constructed rat model that conforms to the clinical characteristics of patients with peripheral arterial disease as the experimental group, and simultaneously complete the construction of the control group; among them, the control group includes the following three groups: The first group: Place an Ameroid constrictor ring around the unilateral femoral artery of a healthy rat, expose the ipsilateral femoral artery again for ligation after 14 days, and continue to raise it with a standard diet for 28 days; The second group: Ligate the unilateral femoral artery of a healthy rat and raise it with a standard diet for 42 days; The third group: Place an Ameroid constrictor ring around the unilateral femoral artery of a healthy rat and raise it with a standard diet for 42 days; Step (2): Measure the limb skin blood flow of the rats in the experimental group and the control group with a laser Doppler flowmeter; Step (3): Measure the motor function and degree of ischemia of the rats in the experimental group and the control group; Step (4): Combine the limb blood flow change trend graph, motor function change situation, and degree of ischemia of the rat model to evaluate whether the rat model is successfully constructed.

7. A method for evaluating a rat model simulating clinical characteristics of peripheral arterial disease according to claim 6, characterized in that In Step (4), the standard for judging that the rat model is successfully constructed is that the blood flow of the limb skin measured by the laser Doppler flowmeter is less than 0.5.

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