Animal model of ischemia with non-obstructive coronary arteries and preparation method therefor

Abstract

An animal model of ischemia with non-obstructive coronary arteries and a preparation method therefor, relating to the technical field of medical experiments. The preparation method for the animal model of ischemia with non-obstructive coronary arteries comprises: providing an experimental animal; and injecting sodium laurate and isoproterenol into coronary artery branches of the heart of the experimental animal to obtain an animal model of ischemia with non-obstructive coronary arteries. By injecting sodium laurate and isoproterenol into different coronary artery branches of the heart to prepare the animal model of ischemia with non-obstructive coronary arteries, the problem in existing myocardial ischemia models that non-obstructive myocardial ischemia is difficult to achieve is solved, and the method can achieve non-obstructive myocardial ischemia at different positions of the heart in a targeted manner and is controllable.

Description

An animal model of non-obstructive coronary myocardial ischemia and its preparation method Technical Field

[0001] This application relates to the field of medical experimental technology, and in particular to an animal model of non-obstructive coronary myocardial ischemia and its preparation method. Background Technology

[0002] Non-obstructive coronary myocardial ischemia is a chronic coronary syndrome increasingly recognized as a significant cause of adverse cardiovascular death and outcomes, including myocardial infarction and heart failure with preserved ejection fraction. Most patients with non-obstructive coronary myocardial ischemia are found to have epicardial coronary vascular dysfunction and coronary microvascular dysfunction during invasive coronary functional testing. Coronary microvascular dysfunction can coexist with obstructive epicardial coronary artery disease, diffuse non-obstructive epicardial coronary artery disease, and coronary vasospasm. Epicardial vasospasm can also occur in normal coronary arteries without atherosclerotic plaques on intravascular imaging. While all precipitating factors are currently unclear, cardiometabolic risk factors and endothelium-dependent factors increasing oxidative stress and inflammation are associated with microvascular damage, non-obstructive coronary myocardial ischemia, and coronary microvascular dysfunction.

[0003] Non-obstructive coronary myocardial ischemia is an early stage of coronary heart disease (CHD) and myocardial infarction, and its research plays a crucial role in the early diagnosis and treatment of CHD. The mechanisms of various aspects of non-obstructive coronary myocardial ischemia are a hot research topic; however, there is a lack of animal models for this purpose. Existing models include myocardial ischemia-reperfusion models, myocardial infarction models, and transgenic mouse models. Myocardial ischemia-reperfusion and myocardial infarction models achieve obstructive myocardial ischemia through surgical procedures such as vascular ligation or balloon dilation, but even after ligation and release of restraint, the goal of non-obstructive myocardial ischemia cannot be achieved. Transgenic mouse models focus on gene knockout, making it impossible to explore mechanisms in conjunction with other gene-modified mice, and are limited to mice. There are no methods for establishing non-obstructive myocardial ischemia in larger animals such as rats, rabbits, and zebra pigs. Therefore, current technologies need further improvement. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this application is to provide an animal model of non-obstructive myocardial ischemia of the coronary artery and its preparation method, in order to solve the problem that existing myocardial ischemia models cannot achieve non-obstruction.

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

[0006] A first aspect of this application provides a method for preparing an animal model of non-obstructive coronary myocardial ischemia, comprising: providing an experimental animal; and injecting sodium lysinate and isoproterenol into the coronary artery branches of the heart of the experimental animal to obtain an animal model of non-obstructive coronary myocardial ischemia.

[0007] Optionally, the experimental animals include one of mice, rats, rabbits, zebras, and pigs.

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

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

[0011] Optionally, the method for preparing the animal model of non-obstructive coronary myocardial ischemia further includes: evaluating the obtained animal model of non-obstructive coronary myocardial ischemia; the evaluation method includes performing electrocardiogram analysis and cardiac tissue staining analysis on the animal model of non-obstructive coronary myocardial ischemia.

[0012] A second aspect of this application provides an animal model of coronary artery non-obstructive myocardial ischemia prepared by the method of the first aspect of this application.

[0013] A third aspect of this application provides a microneedle device, the microneedle device comprising a circular bottom surface, a structural fence, and a microneedle array, the structural fence surrounding the outer periphery of the circular bottom surface, and the microneedle array disposed on the bottom surface on a side extending in the same direction as the structural fence.

[0014] The diameter of the circular base is 375-500μm, the height of the structural fence is 1000-2000μm, and the microneedle array includes 15 microneedles arranged uniformly, with a height of 800-1000μm and a spacing of 1.5mm between adjacent microneedles.

[0015] In a fourth aspect of this application, a microneedle device phantom is prepared using a 3D printing method, and a microneedle device mold is obtained by casting using PDMS and the microneedle device phantom. Sodium ruthenium silicate and isopropanol adrenaline are added to the microneedle device mold and stored at -20°C for 2 hours to obtain the microneedle device.

[0016] The fifth aspect of this application provides the application of the microneedle device of the third aspect of this application in the preparation of an animal model of non-obstructive coronary myocardial ischemia.

[0017] The beneficial effects of this application are as follows: This application establishes an animal model of non-obstructive myocardial ischemia by injecting sodium laurate into different branches of the coronary arteries. Sodium laurate induces thrombus formation at the injection site's microcirculation level, while isoproterenol causes rapid contraction of myocardial cells, leading to myocardial ischemia. This solves the problem of existing myocardial ischemia models failing to achieve non-obstructive myocardial ischemia. Furthermore, this method can specifically induce non-obstructive myocardial ischemia at different locations in the heart (including blood vessels and myocardial cells), and is controllable. In addition, this method is not limited to mice; it can also be used in large animals such as rats, rabbits, zebras, and pigs, which is of great significance for the study of factors inducing non-obstructive myocardial ischemia in the coronary arteries. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0019] Figure 1 is a flowchart illustrating the method for preparing an animal model of non-obstructive coronary myocardial ischemia provided in the embodiments of this application;

[0020] Figure 2 is a schematic diagram of the microneedle device provided in an embodiment of this application;

[0021] Figure 3 is a schematic flowchart of the microneedle device fabrication method provided in the embodiments of this application;

[0022] Figure 4 is an electrocardiogram of an animal model of non-obstructive myocardial ischemia provided in an embodiment of this application;

[0023] Figure 5 is a staining image of cardiac tissue with iron alum hematoxylin and eosin provided in the embodiments of this application;

[0024] Figure 6 is a masson staining image of cardiac tissue provided in an embodiment of this application. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings and examples. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of this application. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0026] It should be noted that if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on enabling those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed in this application.

[0027] Please refer to Figure 1. This application embodiment provides a method for preparing an animal model of non-obstructive coronary myocardial ischemia, including:

[0028] Provide laboratory animals;

[0029] A non-obstructive myocardial ischemia model of the coronary arteries was established by injecting sodium lysinate and isoproterenol into the coronary artery branches of the heart of experimental animals.

[0030] Sodium silicate can cause thrombus formation in the microcirculation layer of cardiac tissue, and at the same time, it can damage vascular endothelial cells and disrupt the integrity of arteriovenous circulation. Isoproterenol causes myocardial cells to contract rapidly, leading to myocardial ischemia, thereby achieving non-obstructive ischemia of the coronary arteries.

[0031] In some embodiments, the experimental animals include one of mice, rats, rabbits, zebras, and pigs. Compared with existing technologies that achieve obstructive myocardial ischemia through surgical procedures such as vascular ligation or balloon dilation, or that construct myocardial ischemia models through transgenic technology, the preparation method of this 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 localized non-obstructive myocardial ischemia animal model can be constructed by injecting sodium lysinate solution into one of the left anterior descending artery, the left circumflex artery, or the right circumflex artery can be injected into at least two of the left anterior descending artery, the left circumflex artery, or the right circumflex artery to construct a complete non-obstructive myocardial ischemia animal model.

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

[0034] In some embodiments, a microneedle device is used to inject sodium lunise silicate and isoproterenol into the myocardial tissue of experimental animals. The concentration of sodium lunise silicate loaded on the microneedle array is 1 mg / kg, and the concentration of isoproterenol is 10 mg / kg.

[0035] The microneedle device, as shown in Figure 2, generally includes a circular bottom surface, a structural fence, and a microneedle array. The structural fence surrounds the outer periphery of the bottom surface, and the microneedle array is located on the bottom surface on the same side as the structural fence. Sodium lysate and isoproterenol are loaded onto the microneedle array.

[0036] Furthermore, the diameter of the circular base is 375-500 μm, the height of the constructed fence is 1000-2000 μm, and the microneedle array includes 15 uniformly arranged microneedles with a height of 800-1000 μm and a spacing of 1.5 mm between adjacent microneedles. For example, the diameter of the circular base is 375 μm, 400 μm, 425 μm, 450 μm, 475 μm, or 500 μm, the height of the constructed fence is 1000 μm, 1200 μm, 1400 μm, 1500 μm, 1600 μm, 1800 μm, or 2000 μm, and the height of the microneedles is 800 μm, 850 μm, 900 μm, 950 μm, or 1000 μm. When loading sodium lysine and isoproterenol onto a microneedle array, a structural fence of a certain height facilitates the retention of sodium lysine and isoproterenol on the microneedle device.

[0037] In some embodiments, referring to Figure 3, the fabrication method of the microneedle device includes the following steps:

[0038] Using 3D printing technology, a microneedle device mold was printed using high-temperature photosensitive resin (HTL) as the raw material. A microneedle device mold was obtained by casting using PDMS and the microneedle membrane, comprising a microneedle mold and a backing. A certain amount of sodium lysinate and isoproterenol were weighed according to a set concentration and dissolved in anhydrous ethanol or water to obtain a drug solution. This drug solution was added to the microneedle mold and centrifuged at 3000 rpm for 3 minutes to ensure the needle tips of the microneedle device were evenly filled with the drug solution. The drug solution was added to the backing and placed on top of the microneedle mold, then stored at -20°C for 2 hours. The solidified microneedle device was then demolded and stored at -80°C for later use.

[0039] Since the size of microneedle devices is in the micrometer range, 3D printing technology can be used to more accurately produce microneedle devices of specified dimensions.

[0040] It should be noted that when using the microneedle device, an adhesive material can be added to the side of the circular surface facing away from the microneedle array to serve as a grip, making it easier to pick up the microneedle device. Alternatively, a clamping tool that can stably contact the microneedle device can be used to hold it.

[0041] In some embodiments, the method for preparing an animal model of non-obstructive coronary myocardial ischemia further includes evaluating the prepared animal model. The evaluation methods include electrocardiographic analysis and cardiac tissue staining analysis of the animal model. The cardiac tissue staining analysis includes heidenhain staining and Masson staining.

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

[0043] The following specific examples will provide further details.

[0044] Example 1: Fabrication of Microneedle Device

[0045] (1) Using 3D printing technology, a microneedle device mold with a diameter of 300μm, a height of 800μm and an outer perimeter with a structural fence of 2000μm is printed using high temperature resistant photosensitive resin (HTL). The microneedle array in the microneedle device mold includes 15 microneedles with a height of 800μm and a spacing of 1.5mm between two adjacent microneedles.

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

[0047] (3) Weigh 32 mg of sodium laurylate and 600 mg of isoproterenol and add them to 1 mL of pure water. Heat in a water bath at 37 °C until completely dissolved. Add 100 μL of the solution to the surface of the microneedle mold. Centrifuge the microneedle mold at 3000 rpm for 3 min to ensure that the microneedle tips are evenly filled with the solution. Then take another 100 μL of the solution to fill the backing and place the backing on the microneedle mold. Then store it at -20 °C for 30 min. After the solution solidifies to form a microneedle device, remove the mold. Store the microneedle device obtained after removal at -80 °C and transfer it to liquid nitrogen for storage before use.

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

[0049] (1) Prepare SD rats. First, anesthetize the rats with isoflurane gas, then weigh them, and anesthetize the rats with 2.5% afodin anesthetic (1-2 mg / g). Determine whether the rats have entered deep anesthesia by applying pressure to the rats' nails.

[0050] (2) Fix the rat and place it supine on a 37° constant temperature heating pad. Use a hair removal tool to shave the hair in the chest area. Disinfect the surgical area on the left side of the anterior chest with iodine solution and wipe it 2-3 times.

[0051] (3) Clean the oral cavity of the SD rat with a cotton ball to ensure it is clean, the airway is well exposed, and there is no excessive mucus. Hold the base of the tongue to expose the airway opening, and carefully and gently insert the endotracheal tube into the trachea. Then connect the ventilator and observe the tidal volume and the symmetrical rise and fall of the chest on both sides (and consistent with the ventilator frequency). If the endotracheal tube is inserted into the esophagus, it needs to be removed as soon as possible. Connect the small animal's electrocardiogram system.

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

[0053] (5) Use forceps to open the thymus, locate the left atrial appendage, and use a cotton swab to lift the left atrial appendage to find the main coronary artery branches (left anterior descending branch, left circumflex branch, and right circumflex branch). Add adhesive material to the side of the microneedle device prepared in Example 1 away from the microneedle array, use the adhesive material as a handle, remove the microneedle device from liquid nitrogen, and place it at the location where laurate and isoproterenol need to be injected (left anterior descending branch, left circumflex branch, and right circumflex branch) within 10 seconds, and inject them. The handle of the microneedle device will fall off the moment the microneedles melt, thus obtaining a non-obstructive myocardial ischemia model. During this process, the electrocardiogram waveform of the small animal is observed in real time. The electrocardiogram changes are mainly ST segment horizontal or downward depression greater than or equal to 0.1mV, lasting for 2 minutes, which may cause ventricular tachycardia. After the needle is removed from the myocardium, the electrocardiogram can return to the preoperative electrocardiogram.

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

[0055] Example 3: Evaluation of an animal model of non-obstructive coronary myocardial ischemia

[0056] (1) The coronary artery non-obstructive myocardial ischemia animal model prepared in Example 2 was used to analyze its heart using small animal ultrasound. The results are shown in Figure 4. As can be seen from Figure 4, the coronary artery non-obstructive myocardial ischemia animal model did not show obvious motion abnormalities in each stage of strain. The electrocardiogram showed ST segment horizontal type and downward type depression greater than or equal to 0.1 mg.

[0057] (2) The heart tissue from the coronary artery non-obstructive myocardial ischemia animal model prepared in Example 2 was analyzed by heidenhain staining and Masson staining. The results of heidenhain staining are shown in Figure 5, and the results of Masson staining are shown in Figure 6. As can be seen from Figure 5, obvious myocardial ischemia was observed in the heart tissue. As can be seen from Figure 6, no myocardial infarction was observed in the heart tissue, thus demonstrating that the coronary artery non-obstructive myocardial ischemia animal model was successfully prepared in this application example.

[0058] In summary, this application establishes an animal model of non-obstructive coronary artery myocardial ischemia by injecting sodium laurate into different branches of the coronary arteries. Sodium laurate induces thrombus formation at the injection site's microcirculation level, while isoproterenol causes rapid contraction of cardiomyocytes, leading to myocardial ischemia. This solves the problem of existing myocardial ischemia models failing to achieve non-obstructive myocardial ischemia. Furthermore, this method can specifically induce non-obstructive myocardial ischemia at different locations in the heart (including blood vessels and cardiomyocytes), offering controllability. Moreover, this method is not limited to mice and can be used in large animals such as rats, rabbits, zebras, and pigs, making it significant for research on the inducing factors of non-obstructive coronary artery myocardial ischemia.

[0059] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing an animal model of non-obstructive coronary myocardial ischemia, characterized in that, include: Provide laboratory animals; Sodium lysine and isoproterenol were injected into the coronary artery branches of the heart of the experimental animals to create an animal model of non-obstructive myocardial ischemia of the coronary arteries.

2. The method for preparing an animal model of non-obstructive coronary myocardial ischemia according to claim 1, characterized in that, The experimental animals include one of the following: mouse, rat, rabbit, zebra, or pig.

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

5. The method for preparing an animal model of non-obstructive coronary myocardial ischemia according to claim 1, characterized in that, Sodium lysine and isoproterenol were injected into the coronary artery branches of the heart of the experimental animal using a microneedle device. The sodium lunozide and isoproterenol are loaded onto the microneedle array.

6. The method for preparing an animal model of non-obstructive coronary myocardial ischemia according to claim 1, characterized in that, The method for preparing the animal model of non-obstructive coronary myocardial ischemia further includes: evaluating the obtained animal model of non-obstructive coronary myocardial ischemia; The assessment method includes electrocardiographic analysis and cardiac tissue staining analysis of the animal model of coronary artery non-obstructive myocardial ischemia.

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

8. A microneedle device, characterized in that, The microneedle device includes a circular bottom surface, a structural fence, and a microneedle array. The structural fence surrounds the outer periphery of the circular bottom surface, and the microneedle array is disposed on the bottom surface on the same side as the structural fence extending in the same direction. The diameter of the circular base is 375-500μm, the height of the structural fence is 1000-2000μm, and the microneedle array includes 15 microneedles arranged uniformly, with a height of 800-1000μm and a spacing of 1.5mm between adjacent microneedles.

9. A method for preparing the microneedle device according to claim 8, characterized in that, Including the following steps: The microneedle device phantom was prepared using 3D printing. A mold was made using PDMS and the microneedle device phantom to obtain the microneedle device mold. Sodium ruthenium silicate and isopropanol adrenaline were added to the microneedle device mold and stored at -20°C for 2 hours to obtain the microneedle device.

10. The application of the microneedle device according to claim 8 in the preparation of an animal model of non-obstructive myocardial ischemia of the coronary arteries.

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