Coronary artery non-obstructive myocardial ischemia animal model and preparation method thereof

Abstract

The invention relates to the technical field of medical experiments, and particularly discloses a coronary artery non-obstructive myocardial ischemia animal model and a preparation method thereof. The preparation method of the coronary artery non-obstructive myocardial ischemia animal model comprises the following steps: providing an experimental animal; injecting sodium laurate and isoprenaline into coronary artery branches of the heart of the experimental animal to obtain the coronary artery non-obstructive myocardial ischemia animal model. The coronary artery non-obstructive cardiac ischemia animal model is prepared by injecting sodium laurate and isoproterenol into different branches of the coronary artery, so that the problem that non-obstructive myocardial ischemia is difficult to realize by an existing myocardial ischemia model is solved; and the method can realize non-obstructive myocardial ischemia at different positions of the heart in a targeted manner, and has controllability.

Description

Technical Field

[0001] The present application relates to the field of medical experimental technology, and in particular to an animal model of non-obstructive coronary artery myocardial ischemia and a preparation method thereof. Background Art

[0002] Non-obstructive coronary ischemia is a chronic coronary syndrome that is increasingly recognized as an important cause of adverse cardiovascular death and outcomes, including myocardial infarction and heart failure with preserved ejection fraction. Most patients with non-obstructive coronary ischemia have abnormal epicardial coronary vascular function and coronary microvascular dysfunction on invasive coronary function testing. Coronary microvascular dysfunction can coexist with obstructive epicardial coronary disease, diffuse non-obstructive epicardial coronary disease, and coronary vasospasm. Epicardial vasospasm can occur in normal coronary arteries without atherosclerotic plaques on intravascular imaging. Although all precipitating factors are currently unknown, cardiometabolic risk factors and endothelial dependence that increases oxidative stress and inflammation are associated with microvascular damage, non-obstructive coronary ischemia, and coronary microvascular dysfunction.

[0003] Coronary artery non-obstructive myocardial ischemia is an early link of coronary heart disease and myocardial infarction. The study of coronary artery non-obstructive myocardial ischemia plays a vital role in the early diagnosis and early treatment of coronary heart disease. The study of various mechanisms of coronary artery non-obstructive myocardial ischemia is a hot topic. However, there is a lack of animal models of coronary artery non-obstructive myocardial ischemia. The existing models of myocardial ischemia include myocardial ischemia-reperfusion models, myocardial infarction models or transgenic mouse models. Myocardial ischemia-reperfusion models and myocardial infarction models are studies of obstructive myocardial ischemia through operations such as vascular ligation or balloon dilatation. Even if the constraints are released after ligation, the goal of non-obstructive myocardial ischemia cannot be achieved. The myocardial ischemia caused by transgenic mice and other models focuses on gene knockout, and it is impossible to explore the mechanism in combination with other genetic mice, and it can only be limited to mice. There is no method for establishing non-obstructive myocardial ischemia for large animals such as rats, rabbits, and zebra pigs. Therefore, the existing technology needs to be improved. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide an animal model of coronary artery non-obstructive myocardial ischemia and a preparation method thereof, aiming to solve the problem that the existing myocardial ischemia model cannot achieve non-obstructiveness.

[0005] The technical solution of this application is as follows:

[0006] In a first aspect of the present application, a method for preparing an animal model of non-obstructive coronary artery myocardial ischemia is provided, comprising: providing an experimental animal; injecting sodium laurate and isoproterenol into the coronary artery branches of the heart of the experimental animal to prepare the animal model of non-obstructive coronary artery myocardial ischemia.

[0007] Optionally, the experimental animal includes one of a mouse, a rat, a rabbit, a zebra, and a pig.

[0008] Optionally, the coronary artery branches include at least one of the left anterior descending artery, the left circumflex artery, and the right circumflex artery.

[0009] Optionally, the sodium laurate and isoproterenol are dissolved in anhydrous ethanol or water, the concentration of the sodium laurate is 10-50 mg / mL, and the concentration of the isoproterenol is 50-100 mg / mL.

[0010] Optionally, sodium laurate and isoproterenol are injected into the coronary artery branches of the heart of the experimental animal using a microneedle device; wherein the sodium laurate and isoproterenol are loaded on the microneedle array.

[0011] Optionally, the method for preparing the non-obstructive coronary artery myocardial ischemia animal model further comprises: evaluating the prepared non-obstructive coronary artery myocardial ischemia animal model; the evaluation method comprises performing electrocardiogram analysis and heart tissue staining analysis on the non-obstructive coronary artery myocardial ischemia animal model.

[0012] The second aspect of the present application provides an animal model of non-obstructive coronary artery myocardial ischemia prepared by the method for preparing an animal model of non-obstructive coronary artery myocardial ischemia according to the first aspect of the present application.

[0013] In a third aspect of the present application, a microneedle device is provided, the microneedle device comprising a circular bottom surface, a structural fence and a microneedle array, the structural fence is arranged around the periphery of the circular bottom surface, and the microneedle array is arranged on a side of the bottom surface that is in the same extension direction as the structural fence;

[0014] The diameter of the circular bottom surface is 375-500 μm, the height of the structural fence is 1000-2000 μm, the microneedle array includes 15 microneedles arranged evenly, the height of the microneedles is 800-1000 μm, and the distance between two adjacent microneedles is 1.5 mm.

[0015] In the fourth aspect of the present application, a microneedle device model is prepared using a 3D printing method, PDMS and the microneedle device model are used for inverting to obtain a microneedle device mold, sodium metasilicate and isopropyl epinephrine are added to the microneedle device mold, and the mold is stored at -20°C for 2 hours to obtain the microneedle device.

[0016] The fifth aspect of the present application provides a use of the microneedle device of the third aspect of the present application in preparing an animal model of non-obstructive coronary artery myocardial ischemia.

[0017] Beneficial effects of the present application: The present application injects sodium laurate into different branches of the coronary arteries of the heart, wherein sodium laurate causes the formation of thrombi in the microcirculation layer of the injection site, and isoproterenol causes the rapid contraction of myocardial cells to cause myocardial ischemia, thereby obtaining an animal model of non-obstructive myocardial ischemia in the coronary arteries, thereby solving the problem that it is difficult to achieve non-obstructive myocardial ischemia in the existing myocardial ischemia models, and the method can achieve non-obstructive myocardial ischemia in different locations of the heart (including blood vessels and myocardial cells) in a targeted manner, and is controllable. In addition, the method is not limited to mice, and can also be used for large animals such as rats, rabbits, zebras, and pigs, and is of great significance for the study of the inducing factors of non-obstructive myocardial ischemia in the coronary arteries. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for describing the embodiments are briefly introduced below.

[0019] Figure 1 A schematic diagram of a process for preparing an animal model of non-obstructive coronary artery myocardial ischemia provided in an embodiment of the present application;

[0020] Figure 2 A schematic diagram of the structure of a microneedle device provided in an embodiment of the present application;

[0021] Figure 3 A schematic diagram of a process for preparing a microneedle device according to an embodiment of the present application;

[0022] Figure 4 The electrocardiogram of the animal model of non-obstructive coronary artery myocardial ischemia provided in the embodiments of the present application;

[0023] Figure 5 This is a picture of the iron alum hematoxylin and eosin staining of heart tissue provided in the examples of the present application;

[0024] Figure 6 This is a Masson staining image of heart tissue provided in the examples of this application. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0026] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in the field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0027] See also Figure 1 The present application provides a method for preparing an animal model of non-obstructive myocardial ischemia in coronary arteries, comprising:

[0028] Providing experimental animals;

[0029] Sodium silicate and isoproterenol were injected into the coronary artery branches of the experimental animals to produce an animal model of non-obstructive coronary artery myocardial ischemia.

[0030] Sodium metasilicate can cause the formation of blood clots at the microcirculation level of cardiac tissue, and at the same time can damage vascular endothelial cells and the integrity of arteriovenous circulation. Isoproterenol causes rapid contraction of myocardial cells, leading to myocardial ischemia, thereby achieving non-obstructive ischemia of the coronary arteries.

[0031] In some embodiments, the experimental animal includes one of mice, rats, rabbits, zebras, and pigs. Compared with the prior art of achieving obstructive myocardial ischemia through operations such as vascular ligation or balloon dilatation, or constructing a myocardial ischemia model through transgenic technology, the preparation method of the present application can be applied to different animals.

[0032] In some embodiments, the coronary artery branches include at least one of the left anterior descending artery, the left circumflex artery, and the right circumflex artery. For example, a sodium silicate solution may be injected into one of the left anterior descending artery, the left circumflex artery, and the right circumflex artery to construct a local non-obstructive myocardial ischemia animal model, or a sodium silicate solution may be injected into at least two of the left anterior descending artery, the left circumflex artery, and the right circumflex artery to construct a complete non-obstructive myocardial ischemia animal model.

[0033] In some embodiments, sodium laurate and isoproterenol are dissolved in anhydrous ethanol or water. Wherein, the concentration of sodium laurate is 10-50 mg / mL, for example, the concentration of sodium laurate is 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL or 50 mg / mL, etc. The concentration of isoproterenol is 50-100 mg / mL. For example, the concentration of isoproterenol is 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL or 100 mg / mL, etc.

[0034] In some embodiments, sodium laurate and isoproterenol are injected into the myocardial tissue of the experimental animal using a microneedle device, wherein the concentration of sodium laurate loaded on the microneedle array is 1 mg / kg, and the concentration of isoproterenol is 10 mg / kg.

[0035] Microneedle devices such as Figure 2 As shown, it generally includes a circular bottom surface, a structural fence and a microneedle array. The structural fence is arranged around the periphery of the bottom surface, and the microneedle array is arranged on the side of the bottom surface that is in the same extension direction as the structural fence; sodium metasilicate and isoproterenol are loaded on the microneedle array.

[0036] Further, the diameter of the circular bottom surface is 375-500 μm, the height of the construction fence is 1000-2000 μm, the microneedle array includes 15 uniformly arranged microneedles, the height of the microneedles is 800-1000 μm, and the spacing between two adjacent microneedles is 1.5 mm. For example, the diameter of the circular bottom surface is 375 μm, 400 μm, 425 μm, 450 μm, 475 μm or 500 μm, etc., the height of the construction fence is 1000 μm, 1200 μm, 1400 μm, 1500 μm, 1600 μm, 1800 μm or 2000 μm, etc., and the height of the microneedle is 800 μm, 850 μm, 900 μm, 950 μm or 1000 μm, etc. When sodium laurate and isoproterenol are loaded onto the microneedle array, the structural fence having a certain height facilitates the sodium laurate and isoproterenol to remain on the microneedle device.

[0037] In some embodiments, see Figure 3 , the preparation method of the microneedle device comprises the steps of:

[0038] The 3D printing technology is used to print a microneedle device model using a high temperature resistant photosensitive resin (HTL) as a raw material; PDMS and a microneedle film are used for molding to obtain a microneedle device mold, and the microneedle device mold includes a microneedle mold and a backing; a certain amount of sodium silicate and isoproterenol are weighed and dissolved in anhydrous ethanol or water according to the set concentration to obtain a drug solution; the drug solution is added to the microneedle mold, and centrifuged at 3000 rpm for 3 minutes to ensure that the needle tip part of the microneedle device is evenly filled with the drug solution; the drug solution is added to the backing, which is covered on the microneedle mold, and then stored at -20°C for 2 hours; the microneedle device formed by the solidification of the drug solution is demolded and stored at -80°C for standby use.

[0039] Since the size of the microneedle device is at the micron level, 3D printing technology can more accurately prepare microneedle devices of specified specifications.

[0040] It should be noted that when the microneedle device is needed, a sticky material can be added to the side of the circular surface away from the microneedle array to serve as a gripping portion to facilitate taking the microneedle device. A clamping tool that can stably abut the microneedle device can also be used to clamp it.

[0041] In some embodiments, the method for preparing the non-obstructive coronary artery myocardial ischemia animal model further comprises: evaluating the prepared non-obstructive coronary artery myocardial ischemia animal model. The evaluation method comprises performing electrocardiogram analysis and cardiac tissue staining analysis on the non-obstructive coronary artery myocardial ischemia animal model. The cardiac tissue staining analysis comprises Heidenhain staining analysis and Masson staining analysis.

[0042] The present application also provides an animal model of non-obstructive coronary artery myocardial ischemia prepared by the method for preparing the animal model of non-obstructive coronary artery myocardial ischemia as described above.

[0043] The following is further described by means of specific examples.

[0044] Example 1 Preparation of microneedle device

[0045] (1) Using 3D printing technology and high temperature resistant photosensitive resin (HTL) as raw material, a microneedle device model with a diameter of 300 μm, a height of 800 μm and a structural fence with a height of 2000 μm on the outside was printed, wherein the microneedle array in the microneedle device model includes 15 microneedles, the height of the microneedles is 800 μm, and the distance between two adjacent microneedles is 1.5 mm.

[0046] (2) Use PDMS casting to construct a microneedle device mold, including a microneedle mold and a backing.

[0047] (3) Weigh 32 mg of sodium laurate and 600 mg of isoproterenol, add them to 1 mL of pure water, and heat them in a 37°C water bath to completely dissolve. Pipette 100uL of the solution and add it to the surface of the microneedle mold; centrifuge the microneedle mold at 3000rpm for 3 minutes to allow the microneedle tip of the microneedle mold to be evenly filled with the solution; then take 100μL of the solution to fill the backing, cover the backing on the microneedle mold, and then store it at -20°C for 30 minutes. After the solution solidifies to form a microneedle device, remove the mold, and store the removed microneedle device at -80°C, and transfer it to liquid nitrogen for storage before use.

[0048] Example 2 Preparation of animal model of non-obstructive coronary artery myocardial ischemia

[0049] (1) Prepare SD rats. First, anesthetize the rats with isoflurane gas. Then weigh the rats. Anesthetize the rats with 2.5% afodine anesthetic (1-2 mg / g). Apply pressure to the rats' nails to determine whether the rats are deeply anesthetized.

[0050] (2) The rat was fixed in supine position on a 37° constant temperature heating pad. The hair in the chest area was shaved with an epilator. The surgical area on the left side of the chest was disinfected with iodine 2-3 times.

[0051] (3) Use cotton balls to clean the oral cavity of the SD rat to ensure that the oral cavity is clean, the airway is well exposed, and there is no excessive mucus. Press the root of the tongue to expose the airway opening, carefully and gently insert the tracheal tube into the trachea, and then connect the ventilator. Observe the tidal volume and the symmetrical rise and fall of the chest on both sides (which is consistent with the ventilator frequency). If the trachea is inserted into the esophagus, remove the trachea as soon as possible. Connect the small animal ECG system.

[0052] (4) After the animal's breathing becomes stable, cut the skin, free the muscles, expose the ribs, open the chest at the third intercostal space, use a chest expander to expand the intercostal space (be careful not to touch the lungs), expose the surgical field, and tear open the pericardium on the surface of the heart. At this time, the thymus and left atrial appendage can be clearly seen.

[0053] (5) Use tweezers to pick open the thymus, find the left atrial appendage, open the left atrial appendage with a cotton swab, and find the main coronary artery branches of the heart (left anterior descending branch, left circumflex branch, right circumflex branch). The microneedle device prepared in Example 1 is added with a viscous material on one side away from the microneedle array, and the viscous material is used as a handheld part. The microneedle device is taken out from liquid nitrogen and placed in the position (left anterior descending branch, left circumflex branch, right circumflex branch) where lauric acid and isoproterenol need to be injected within 10s. Inject until the microneedle melts and the handheld part of the microneedle device falls off, and a non-obstructive myocardial ischemia model is obtained. In this process, the small animal electrocardiogram waveform is observed in real time, and the electrocardiogram changes are mainly in the ST segment horizontal type, or the downward type is depressed by more than or equal to 0.1mV, and it lasts for 2min. Ventricular tachycardia may occur, and the electrocardiogram can be restored to the electrocardiogram performance before the operation after the needle is pulled out of the myocardium.

[0054] (6) Close the chest as quickly as possible and suture the ribs and skin. The ribs should be sutured tightly to avoid pneumothorax. Remove the ventilator and check whether the chest width rises or falls. Ensure that the animal recovers spontaneous breathing and obtain an animal model of non-obstructive coronary artery myocardial ischemia.

[0055] Example 3 Evaluation of the Coronary Artery Non-Obstructive Myocardial Ischemia Animal Model

[0056] (1) The non-obstructive coronary artery ischemic animal model prepared in Example 2 was used to analyze the heart using small animal ultrasound. The results were as follows: Figure 4 As shown. Figure 4 It can be seen that the animal model of non-obstructive coronary myocardial ischemia did not show obvious movement abnormalities at each stage of strain, and the electrocardiogram had a horizontal ST segment, and the downward depression was greater than or equal to 0.1 mg.

[0057] (2) The heart tissues in the animal model of non-obstructive coronary artery myocardial ischemia prepared in Example 2 were subjected to Heidenhain staining and Masson staining. The results of Heidenhain staining are shown in Figure 2. Figure 5 The results of Masson staining are shown in Figure 6 As shown. Figure 5 It can be seen that the heart tissue has obvious myocardial ischemia. Figure 6 It can be seen that myocardial infarction did not occur in the heart tissue, which further indicates that the coronary artery non-obstructive myocardial ischemia animal model was successfully prepared in the examples of the present application.

[0058] In summary, the present application injects sodium laurate into different branches of the coronary arteries of the heart, sodium laurate causes the formation of thrombi in the microcirculation layer of the injection site, and isoproterenol causes rapid contraction of myocardial cells to cause myocardial ischemia, thereby preparing an animal model of non-obstructive myocardial ischemia in the coronary artery, solving the problem that it is difficult to achieve non-obstructive myocardial ischemia in the existing myocardial ischemia model, and the method can achieve non-obstructive myocardial ischemia in different locations of the heart (including blood vessels and myocardial cells) in a targeted manner, and is controllable. In addition, the method is not limited to mice, and can also be used for large animals such as rats, rabbits, zebras, pigs, etc., and is of great significance to the study of the inducing factors of non-obstructive myocardial ischemia in the coronary artery.

[0059] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.

Claims

1. A method for preparing an animal model of non-obstructive coronary artery myocardial ischemia, characterized in that: include: Providing experimental animals; Sodium laurate and isoproterenol were injected into the coronary artery branches of the hearts of the experimental animals to prepare the coronary artery non-obstructive myocardial ischemia animal model.

2. The method for preparing an animal model of non-obstructive coronary artery myocardial ischemia according to claim 1, characterized in that: The experimental animal includes one of mice, rats, rabbits, zebras, and pigs.

3. The method for preparing an animal model of non-obstructive coronary artery myocardial ischemia according to claim 1, characterized in that: The coronary artery branches include at least one of the left anterior descending artery, the left circumflex artery, and the right circumflex artery.

4. The method for preparing an animal model of non-obstructive coronary artery myocardial ischemia according to claim 1, characterized in that: The sodium laurate and isoproterenol are dissolved in anhydrous ethanol or water, the concentration of the sodium laurate is 10-50 mg / mL, and the concentration of the isoproterenol is 50-100 mg / mL.

5. The method for preparing an animal model of non-obstructive coronary artery myocardial ischemia according to claim 1, characterized in that: injecting sodium laurate and isoproterenol into the coronary artery branches of the heart of the experimental animal using a microneedle device; Wherein, the sodium laurate and isoproterenol are loaded on the microneedle array.

6. The method for preparing an animal model of non-obstructive coronary artery myocardial ischemia according to claim 1, characterized in that: The method for preparing the non-obstructive coronary artery myocardial ischemia animal model further comprises: evaluating the prepared non-obstructive coronary artery myocardial ischemia animal model; The evaluation method comprises performing electrocardiographic analysis and heart tissue staining analysis on the coronary artery non-obstructive myocardial ischemia animal model.

7. An animal model of non-obstructive coronary artery myocardial ischemia prepared by the method for preparing an animal model of non-obstructive coronary artery myocardial ischemia according to any one of claims 1 to 6.

8. A microneedle device, characterized in that The microneedle device comprises a circular bottom surface, a structural fence and a microneedle array, wherein the structural fence is arranged around the periphery of the circular bottom surface, and the microneedle array is arranged on the side of the bottom surface which is in the same extension direction as the structural fence; The diameter of the circular bottom surface is 375-500 μm, the height of the structural fence is 1000-2000 μm, the microneedle array includes 15 microneedles arranged evenly, the height of the microneedles is 800-1000 μm, and the distance between two adjacent microneedles is 1.5 mm.

9. A method for preparing the microneedle device according to claim 8, characterized in that: Includes steps: A microneedle device model was prepared by a 3D printing method, and PDMS and the microneedle device model were used for inverting to obtain a microneedle device mold. Sodium laurate and isopropyl epinephrine were added to the microneedle device mold, and the mold was stored at -20°C for 2 hours to obtain the microneedle device.

10. Use of the microneedle device according to claim 8 in preparing an animal model of non-obstructive coronary artery myocardial ischemia.

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