Method for constructing an animal model of heart failure combined with pulmonary arterial hypertension

Abstract

Provided are methods for constructing animal models of heart failure combined with pulmonary arterial hypertension, including an animal model of heart failure combined with pulmonary arterial hypertension with preserved ejection fraction and an animal model of heart failure combined with pulmonary arterial hypertension with reduced ejection fraction.

Description

Methods for constructing an animal model of heart failure complicated with pulmonary hypertension Technical Field

[0001] This application generally relates to the field of animal model construction technology, and more specifically, to a method for constructing an animal model of heart failure combined with pulmonary hypertension. Background Technology

[0002] Pulmonary hypertension (PH) is a clinical and pathophysiological syndrome caused by changes in the structure or function of pulmonary blood vessels due to various heterogeneous diseases (causes) and different pathogenesis mechanisms, resulting in increased pulmonary vascular resistance and pulmonary artery pressure, which may eventually lead to right heart failure or even death.

[0003] Based on current evidence-based medicine, the Pulmonary Embolism and Pulmonary Vascular Disease Group of the Chinese Medical Association's Respiratory Disease Branch and the Pulmonary Embolism and Pulmonary Vascular Disease Working Committee of the Chinese Medical Doctor Association's Respiratory Physician Branch organized multidisciplinary experts in respiratory and critical care medicine, cardiology, rheumatology, imaging, basic medicine, and evidence-based medicine to develop the "Guidelines for the Diagnosis and Treatment of Pulmonary Hypertension in China (2021 Edition)". The hemodynamic definition of pulmonary hypertension is a mean pulmonary artery pressure (mPAP) ≥ 25 mmHg (1 mmHg = 0.133 kPa) measured by right heart catheterization (RHC) at sea level and at rest.

[0004] Clinically, PH is classified into 5 categories: (1) pulmonary arterial hypertension (PAH); (2) PH caused by left heart disease; (3) PH caused by lung disease and / or hypoxia; (4) chronic thromboembolic pulmonary hypertension (CTEPH) and / or PH caused by other pulmonary artery obstruction; (5) PH caused by unknown and / or multiple factors.

[0005] Pulmonary hypertension associated with left ventricular disease (PH-LHD) is a common type of pulmonary hypertension that can occur alongside various left ventricular diseases, such as valvular heart disease, heart failure, congenital or acquired left ventricular inflow or outflow tract obstruction, and hereditary cardiomyopathy. PH caused by left ventricular disease is mainly due to abnormally elevated pulmonary artery pressure caused by left ventricular systolic and diastolic dysfunction and / or left ventricular valvular disease. Its pathophysiological characteristics include elevated left ventricular filling pressure, obstruction of pulmonary venous return, and elevated pulmonary venous pressure, which in turn leads to secondary elevated pulmonary artery pressure. The prevalence of PH-LHD is approximately one-third of all pulmonary hypertension cases. Simultaneously, pulmonary hypertension is a common complication of left ventricular disease, usually related to the severity of the disease; up to 80% of heart failure patients may develop PH-LHD. Once pulmonary hypertension is present, it can lead to worsening symptoms and a poor prognosis.

[0006] There is limited evidence for PH-LHD treatment. To address this real-world clinical problem, it is urgent to investigate the pathogenesis of pulmonary hypertension caused by left ventricular disease, which necessitates the development of animal models that can simulate disease progression.

[0007] Animal models play a crucial role in clinical research. They are used to study the pathogenesis of diseases, test the safety and efficacy of new drugs, and explore treatment methods. By using animal models, we can better understand the physiological and pathological processes of diseases, evaluate potential treatments, assess the toxicity, efficacy, and pharmacokinetics of new drugs, study the metabolic and pharmacokinetic properties of drugs in vivo, conduct preliminary testing of new drugs before clinical trials, better understand the mechanisms of drug action in vivo, assess their safety and efficacy, and provide important data for further clinical trials. Animal models are one of the most important tools for researching and developing new treatment methods.

[0008] Previous animal models only included idiopathic pulmonary hypertension or left heart disease, and there was no mouse model of pulmonary hypertension caused by left heart disease, so it was impossible to accurately simulate the occurrence and development of the disease in patients.

[0009] Preservative-ejection-fraction (PEF) heart failure models are well-established, typically constructed using a high-fat diet combined with L-NAME. These animal models closely resemble the clinicopathological characteristics of human heart failure and are well-suited for preclinical research on PEF. However, given the differences in the mechanisms of pulmonary hypertension resulting from different types of heart failure, there is currently no mouse model of PEF combined with pulmonary hypertension. The existing rat model suffers from drawbacks such as less close alignment with disease progression compared to mice, higher cost, and difficulty in genotyping.

[0010] Other large animal models are costly, require strict surgical procedures, and suffer from instability and limitations in accurately reflecting the progression of human diseases.

[0011] Mouse and rat models each have their own characteristics and advantages. Compared with rat models, mouse models have lower costs, more complete supporting research tools (such as well-developed gene-edited mice, easier to purchase antibodies, etc.), and relatively higher disease severity. Compared with mouse models, rat models have advantages such as larger body size and easier surgical operation.

[0012] Due to differences in mouse strains, modeling age, and modeling protocols, existing experimental protocols result in significant variations in experimental outcomes and make the results unreproducible. For rat models, parameters such as animal weight, age, modeling details, modeling time, and evaluation methods are inconsistent during the modeling process, leading to inconsistent modeling results.

[0013] This application proposes for the first time a mouse disease model that can simulate pulmonary hypertension caused by left heart disease, providing a suitable research tool for studying the occurrence, development, and intervention of such diseases. Summary of the Invention

[0014] In one aspect, this application provides a method for constructing an animal model of heart failure with preserved ejection fraction complicated with pulmonary hypertension, comprising: taking male C57BL / 6N mice at the initial time point and allowing them to freely consume a high-fat diet and L-NAME drinking water, wherein the L-NAME drinking water contains 0.5% N-nitro-L-arginine methyl ester hydrochloride; injecting the mice subcutaneously with Sugen5416 solution at initial time points of 6, 8, 10, 12, and 14 weeks; and obtaining a mouse model of heart failure with preserved ejection fraction complicated with pulmonary hypertension 15 weeks after the initial time point.

[0015] In some embodiments, the amount of the Sugen5416 solution is 20 μg / g mouse body weight.

[0016] In some embodiments, the method further includes measuring right ventricular systolic pressure under anesthesia at week 15. In some embodiments, the method further includes euthanizing the mice at week 15 and measuring the right ventricular hypertrophy index and vascular remodeling. In some embodiments, the method further includes measuring the right ventricular weight / tibia length ratio, heart weight / tibia length ratio, and left ventricular plus interventricular septum tissue weight / tibia length ratio of the mice.

[0017] In another aspect, this application provides a method for constructing an animal model of heart failure with reduced ejection fraction complicated with pulmonary hypertension, which includes: taking male C57BL / 6N mice and ligating the aortic arch using a 27G catheter; allowing the mice to freely ingest water and food for 12 weeks to obtain a mouse model of heart failure with reduced ejection fraction complicated with pulmonary hypertension.

[0018] In some embodiments, the method further includes ultrasound examination of the mice at weeks 8 and 12. In some embodiments, the method further includes measuring right ventricular systolic pressure under anesthesia at week 12. In some embodiments, the method further includes euthanizing the mice at week 12 and measuring the right ventricular hypertrophy index and the degree of vascular remodeling in the left lung, and also includes measuring the weight of the ventricle plus interventricular septum tissue, right ventricular weight, heart weight, heart weight to body weight ratio, right ventricular weight / tibia length ratio, heart weight / tibia length ratio, and left ventricular plus interventricular septum tissue weight / tibia length ratio.

[0019] This application uses mice instead of rats with specific genotypes, as well as larger animals such as pigs, dogs, and cats, to construct disease models of heart failure phenotypes complicated with pulmonary hypertension. This provides a research basis for heart failure complicated with pulmonary hypertension and significantly reduces the cost of animal model construction. Furthermore, this application uses a combination of non-invasive echocardiography and invasive interventional assessment protocols to evaluate disease severity, accurately assess left ventricular lesions and right ventricular pressure in mice, and better simulate heart failure complicated with pulmonary hypertension.

[0020] Compared with the shortcomings of current large animal models, this application has the following advantages: the model cost is low, the model is stable, and it can fit the human disease development process well. However, due to the pulmonary vascular characteristics of mice, they are not prone to pulmonary hypertension, and the severity of the disease is generally not high. But through the construction of a comprehensive evaluation system, the scoring system can be refined, and an evaluation system suitable for this application can be constructed. Attached Figure Description

[0021] Figure 1 is a schematic diagram of the drug administration time points of the model group in Embodiment 1 of this application;

[0022] Figure 2 shows the right ventricular systolic pressure results of the model group and the control group in Example 1 of this application;

[0023] Figure 3 is a comparison of anatomical indicators between the model group and the control group in Embodiment 1 of this application;

[0024] Figure 4 is a comparison chart of ultrasound indicators between the model group and the control group in Embodiment 2 of this application;

[0025] Figure 5 is a comparison of anatomical parameters between the model group and the control group in Embodiment 2 of this application; and

[0026] Figure 6 is a comparison of staining images and indicators of the model group and the control group in Example 2 of this application. Detailed Implementation

[0027] To better understand the above-mentioned objectives, features, and advantages of this application, specific embodiments according to this application are described below with reference to the accompanying drawings. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0028] Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application may also be implemented in other ways different from those described herein, and therefore, the scope of protection of this application is not limited to the specific embodiments disclosed below. Some embodiments of this application are described below with reference to the accompanying drawings.

[0029] Rodents, primarily represented by mice, are considered "standard laboratory animals." These animals possess controllable innate genetic traits, reproductive conditions, microbial carriage status, nutritional requirements, and environmental factors, ensuring the reliability, accuracy, uniformity, reproducibility, and comparability of experimental results. Although their small size, thin blood vessel walls, and demanding experimental techniques make them ideal animal models due to their well-defined genetic background, significant and stable model traits, degree of freedom in quality and specifications selection, comprehensive range of diagnostic reagents, and reasonable prices. Furthermore, the diversity of rodent strains and genotypes provides a guarantee for the subsequent optimization and enrichment of this model.

[0030] Example 1: An animal model of heart failure with preserved ejection fraction complicated by pulmonary hypertension was constructed by combining a high-fat diet, L-NAME, and Sugen5416.

[0031] Male C57BL / 6N mice (weighing 22g-25g) were randomly divided into a control group, a group receiving only Sugen5416 injection (sugen), a group on a high-fat diet combined with L-NAME (HFD+L-NAME), and a group on a high-fat diet combined with L-NAME and Sugen5416 injection (HFD+L-NAME+sugen). A 12 / 12 light / dark cycle was used, and the mice were kept at a constant temperature of 25℃ with 50% humidity.

[0032] Mice in the control group and the Sugen5416-injected group were fed a normal diet. Mice in the high-fat diet combined with L-NAME group and the high-fat diet combined with L-NAME-injected Sugen5416 group were fed a high-fat diet, with L-NAME (N-nitro-L-arginine methyl ester hydrochloride) added to their drinking water at a concentration of 0.5%. Six weeks after the Sugen5416-injected group and the high-fat diet combined with L-NAME-injected Sugen5416 group, Sugen5416 was administered subcutaneously for the first time at a dose of 20 mg / kg, given every two weeks for a total of five administrations. The Sugen5416 reagent was prepared by slowly and evenly pouring 200 mg of Sugen5416 powder into DMSO solution, continuously vortexing and sonicating to aid dissolution, to prepare a 20 mg / mL Sugen5416 stock solution. Before use, the stock solution was diluted with physiological saline to a 10 mg / mL working solution, dissolved by sonication, and then prepared for use.

[0033] Figure 1 shows a schematic diagram of the drug administration time points. The animal model obtained was tested at week 15.

[0034] 1. Pulmonary artery pressure measurement

[0035] 1) Pulmonary artery pressure was measured using a Millar catheter connected to a pressure transducer;

[0036] 2) Anesthetize mice with isoflurane gas, fix the mice in a supine position, prepare the skin, and disinfect the surgical site;

[0037] 3) Make a longitudinal incision in the skin of the right neck, bluntly dissect the jugular vein above the right clavicle, gently clamp the proximal end of the jugular vein with a hemostat, lift the jugular vein with an 8 / 0 suture needle, make a "V" shaped incision with microscissors, insert the catheter tip into the vein, fix the catheter, and ensure that there is no bleeding at the insertion site and the catheter can rotate freely.

[0038] 4) Record the data using a PowerLab physiological recorder and assess the catheter position based on the waveforms displayed on the system. Typically, after inserting the catheter approximately 1 cm, it is roughly at the confluence of the superior vena cava and axillary vein; consider adjusting the catheter insertion angle at this point. Then, rotate the catheter counterclockwise and insert it approximately 1-1.5 cm towards the superior vena cava and the opening of the right atrium. Finally, rotate the catheter clockwise and slowly withdraw it approximately 0.5 cm.

[0039] 1 cm, then continue insertion; when a weak right atrial waveform is displayed, continue to advance the catheter 1-2 cm into the right ventricle; then slowly rotate the catheter and advance it 1-2 cm into the pulmonary artery under the influence of blood flow; when a typical pulmonary artery pressure waveform is observed, fix the catheter, allow the waveform to stabilize for 3 minutes, and then collect pulmonary artery pressure data.

[0040] Figure 2 shows the right ventricular systolic pressure (RVSP) of each group, indicating a significant increase in RVSP in the high-fat diet combined with L-NAME injection of Sugen5416 (HFD+L-NAME+sugen) group compared to the control group.

[0041] 2. Anatomical indicators

[0042] 1) After drawing arterial blood through the apex of the heart under gas anesthesia, the mice were euthanized, and the pulmonary circulation was lavaged with heparinized saline. Then the lungs were removed, the right lung was flash-frozen with liquid nitrogen, and the left lung was perfused with formalin.

[0043] 2) After the lungs were removed from the mice, the heart was separated, the blood was rinsed off with physiological saline, the water was absorbed with filter paper, and the heart weight (HW) was measured.

[0044] 3) Cut off the atrium and residual blood vessels, and free the right ventricular wall (RV) from the pulmonary artery outlet. The rest is the left ventricle + interventricular septum tissue (LV+S). After absorbing the moisture with filter paper, weigh each part separately.

[0045] 4) Take another tibia of the right hind limb and measure the tibia length (TL) using calipers;

[0046] 5) Calculate the right ventricle / tibia length ratio (RV / TL), heart weight / tibia length ratio (HW / TL), (left ventricle + interventricular septum tissue) / tibia length ratio (LV+S / TL), and right ventricle / (left ventricle + interventricular septum tissue) mass ratio (RV / LV+S) according to the formula.

[0047] Figure 3 shows a comparison of anatomical data among the groups. The high-fat diet combined with L-NAME injection of Sugen5416 (HFD+L-NAME+sugen) group showed significant increases in RV / TL, HW / TL, and LV+S / TL compared to the control group, indicating heart failure complicated by pulmonary hypertension. RV / TL mainly reflects the degree of right ventricular thickening, used to demonstrate right ventricular involvement after pulmonary hypertension; LV+S / TL mainly reflects the degree of left ventricular thickening, used to demonstrate the pathological characteristics of ventricular hypertrophy in left ventricular failure; HW / TL is used to demonstrate the degree of cardiac remodeling after heart failure complicated by pulmonary hypertension. Example 2: An animal model of heart failure with reduced ejection fraction complicated by pulmonary hypertension was constructed by ligating the aortic arch.

[0048] C57BL / 6N mice (weighing 22g-25g) were selected and divided into a model group (TAC) and a control group (SHAM, sham surgery). Weight and ear tags were recorded. A 12 / 12 light / dark cycle was used, and mice had free access to water and food. The rearing temperature was kept constant at 25℃, and the room humidity was 50%.

[0049] 1. Ligation of the aortic arch

[0050] Anesthetize the mouse with 1.2% tribromoethanol via intraperitoneal injection at a rate of 0.12 ml / 10 g. After 3-5 minutes, verify the required depth of anesthetic effect: the mouse's heartbeat and breathing are regular, muscles are relaxed, limbs are inactive, whiskers show no tactile response, and the pedal reflex is absent. Then, restrain the mouse to the dissection table. First, defatt the mouse's neck with an alcohol swab, then disinfect twice with iodine, allowing it to dry before removing the iodine with alcohol. When disinfecting with alcohol and iodine, proceed from the center outwards, wiping only once and not repeatedly.

[0051] Using ophthalmic scissors, make an incision along the midline of the mouse's neck down to the second rib (approximately 1.2 cm). Bluntly dissect the subcutaneous tissue, fat, thyroid gland, superficial fascia, platysma, deep cervical fascia, and anterior tracheal muscles. After exposing the trachea, locate the left and right common carotid arteries. Use ophthalmic scissors to cut open the first sternum, fix the cut sternum with a sternal hook, and bluntly dissect the thymus with ophthalmic forceps to expose the aortic arch.

[0052] Aortic arch ligation: The aortic arch is ligated using 7-0 sutures and a 27G catheter. After ligation, the catheter is quickly removed.

[0053] Using a needle holder and 5-0 surgical sutures, the first sternum and epidermis were sutured sequentially. The mice were then exposed to a warm light, and their vital signs, such as respiration and heartbeat, were observed. After the mice fully recovered to normal activity, they were housed within a protective barrier.

[0054] Repeat the above steps to complete the modeling of all modeling mice. The control group mice are exactly the same as the modeling group except that the aortic arch is not ligated.

[0055] 2. Ultrasonic testing

[0056] At weeks 8 and 12 of modeling, all mice underwent hair removal of the chest and abdomen, followed by chest ultrasound, including M-mode and Doppler ultrasound. The fractional shortening (FS) of the ventricle was calculated as an indicator of cardiac pumping function. Pulmonary artery blood flow velocity was measured, and pulmonary artery acceleration time (PAT) and the ratio of PAT to pulmonary ejection time (PAT / PET) were calculated. Tricuspid annular systolic displacement (TAPSE) was also measured. Figure 4 shows a comparison of data between the modeling group and the control group, indicating that the modeling group showed significant data reductions relative to the control group at week 12, and the comparison between week 12 and week 8 within the modeling group itself was also statistically significant.

[0057] After collecting ultrasound data, the right ventricular pressure of mice was measured using a Miller catheter under air anesthesia, similar to Example 1. After sacrifice, the mice underwent pulmonary lavage with heparinized saline and formalin perfusion of the left lung, just as in Example 1. The heart was then isolated, and physiological parameters were measured. Figure 5 shows a comparison of data between the model group and the control group. The left ventricle plus interventricular septum (LV+S), right ventricle (RV), heart weight (HW), and heart weight to body weight ratio (HW / BW) were all significantly increased in the model group. Simultaneously, the ratio of (left ventricle plus interventricular septum) to tibia length (LV+S / TL), the ratio of right ventricle to tibia length (RV / TL), and the ratio of heart weight to tibia length (HW / TL) were also significantly increased compared to the sham surgery.

[0058] After the left lung was fully fixed, it was embedded and sectioned for α-SMA immunohistochemical staining to assess the degree of vascular remodeling. Figure 6 shows the staining images and data comparison between the model group and the control group. The ratio of media thickness to vessel radius (media thickness / r%) and the ratio of media thickness to vessel cross-sectional area (media thickness / CSA%) were significantly increased in the model group. Media thickness / r% reflects the thickness of the vessel wall relative to the vessel radius; an increased value indicates a larger media thickness relative to the vessel radius, which is associated with vascular sclerosis, reduced vasodilatory capacity, and increased vascular resistance. An increased media thickness / CSA% indicates that the media layer accounts for a higher proportion of the total cross-sectional area of ​​the vessel wall, demonstrating significant thickening of the vessel wall. This thickening affects the overall structure and function of the blood vessel, leading to blood flow obstruction and increased blood pressure.

[0059] In this application, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance. Unless otherwise expressly specified and limited, the terms "installation," "connection," "linking," "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0060] In the description of this specification, the terms "one embodiment," "some embodiments," "specific embodiments," "some implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or implementation of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or implementation. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or implementations.

[0061] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for constructing an animal model of pulmonary hypertension caused by left ventricular heart failure with preserved ejection fraction, comprising: Male C57BL / 6N mice were selected at the initial time point and allowed free access to a high-fat diet and L-NAME drinking water containing 0.5% N'-nitro-L-arginine methyl ester hydrochloride. At 6, 8, 10, 12, and 14 weeks after the initial time point, the mice were subcutaneously injected with 20 μg / g of mouse body weight of Sugen5416 solution. At 15 weeks after the initial time point, a mouse model of pulmonary hypertension caused by heart failure with preserved ejection fraction was obtained. Right ventricular systolic pressure was measured under anesthesia at week 15. The mice were then sacrificed, and the right ventricular hypertrophy index, degree of vascular remodeling, right ventricular weight / tibia length ratio, heart weight / tibia length ratio, and left ventricular plus interventricular septum tissue weight / tibia length ratio were detected.

2. Use of the mouse model obtained by the method of claim 1 in screening drugs for the treatment of pulmonary hypertension caused by left ventricular heart failure with preserved ejection fraction.

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