Construction method of heart failure combined pulmonary arterial hypertension animal model

Abstract

The invention provides a construction method of a heart failure combined pulmonary hypertension animal model. The construction method comprises an ejection fraction retention type heart failure combined pulmonary hypertension animal model and an ejection fraction reduction type heart failure combined pulmonary hypertension animal model.

Description

Technical Field

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

[0002] Pulmonary hypertension (PH) refers to a clinical and pathophysiological syndrome caused by changes in pulmonary vascular structure or function due to a variety of heterogeneous diseases (causes) and different pathogenesis, resulting in increased pulmonary vascular resistance and pulmonary artery pressure, which then develops into right heart failure or even death.

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

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

[0005] Pulmonary hypertension associated with left heart disease (PH-LHD) is a common type of pulmonary hypertension, which can be associated with various left heart diseases, such as valvular heart disease, heart failure, congenital or acquired left heart inflow or outflow obstruction, and hereditary cardiomyopathy. PH caused by left heart disease is mainly due to abnormal increase in pulmonary artery pressure caused by left heart systolic and diastolic dysfunction and / or left heart valve disease. Its pathophysiological characteristics are increased left heart filling pressure, obstructed pulmonary venous return, increased pulmonary venous pressure, and secondary increase in pulmonary artery pressure. The prevalence of PH-LHD accounts for about 1 / 3 of pulmonary hypertension. At the same time, pulmonary hypertension is a common complication of left heart disease, which is usually related to the severity of left heart disease. Up to 80% of heart failure patients may develop PH-LHD. Once combined with pulmonary hypertension, it can lead to aggravated symptoms and poor prognosis.

[0006] There is little evidence for the treatment of PH-LHD. To solve this practical clinical problem, it is urgent to study the pathogenesis of pulmonary hypertension caused by left heart disease, which requires the construction of an animal model that can simulate the development of the disease.

[0007] Animal models play an important role in clinical research. They can be used to study the pathogenesis of diseases, test the safety and efficacy of new drugs, and explore treatments. By using animal models, we can better understand the physiological and pathological processes of diseases, evaluate potential treatments, evaluate the toxicity, efficacy, and pharmacokinetics of new drugs, and study the metabolism and pharmacokinetic properties of drugs in vivo. We can also conduct preliminary tests on new drugs before clinical trials, better understand the mechanism of action of drugs in vivo, evaluate their safety and efficacy, and provide important data for further clinical trials. It is one of the important tools for researching and developing new treatments.

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

[0009] The model of heart failure with preserved ejection fraction is mature. It is usually constructed by using a high-fat diet combined with L-NAME. The animal disease model fits the clinical pathological characteristics of human heart failure and can be well used for preclinical research on heart failure with preserved ejection fraction. However, given the differences in the mechanisms of pulmonary hypertension caused by different types of heart failure, there is no mouse model of heart failure with preserved ejection fraction combined with pulmonary hypertension. The only remaining rat model has defects such as the degree of fit of the disease development process is not as good as that of mice, high cost, and difficulty in constructing genotypes.

[0010] Other large animal models are expensive, require strict surgery, are unstable, and have problems such as poor fit between the disease model and the development of human diseases.

[0011] The mouse model and the rat model each have their own characteristics and advantages. Compared with the rat model, the mouse model has lower cost and more complete supporting scientific research tools (such as complete gene-edited mice, easier to purchase antibodies, etc.). The severity of the disease is relatively high, while the rat model has the advantages of larger size and easier surgical operation than the mouse model.

[0012] Due to the differences in mouse strains, modeling ages, and modeling protocols, the various existing experimental protocols lead to large differences in experimental effects and the results cannot be reproduced. For rat models, the parameters of the experimental protocol, such as animal weight, age, modeling details, modeling time, and evaluation methods, are not unified during the modeling process, resulting in inconsistent modeling effects.

[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] On the one hand, the present application provides a method for constructing an animal model of heart failure with preserved ejection fraction and pulmonary hypertension, which comprises: taking male C57BL / 6N mice at a starting time point and allowing them to freely ingest 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; 6, 8, 10, 12, and 14 weeks after the starting time point, respectively, subcutaneously injecting the mice with Sugen5416 solution; 15 weeks after the starting time point, obtaining a mouse model of heart failure with preserved ejection fraction and pulmonary hypertension.

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

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

[0017] On the other hand, the present application provides a method for constructing an animal model of heart failure with reduced ejection fraction and pulmonary hypertension, which comprises: taking male C57BL / 6N mice, using a 27G catheter to ligate the aortic arch; allowing the mice to freely consume water and food for 12 weeks, and obtaining a mouse model of heart failure with reduced ejection fraction and pulmonary hypertension.

[0018] In some embodiments, the method further comprises performing ultrasound examination on the mouse at week 8 and week 12. In some embodiments, the method further comprises measuring right ventricular systolic pressure under gas anesthesia at week 12. In some embodiments, the method further comprises killing the mouse at week 12, and detecting the right ventricular hypertrophy index and the degree of vascular remodeling of the left lung of the mouse, and further comprises detecting the weight of ventricle + septum tissue, right ventricular weight, heart weight, heart weight to body weight ratio, right ventricular weight / tibia length ratio, heart weight / tibia length ratio, left ventricle plus septum tissue weight / tibia length ratio of the mouse.

[0019] This application uses mice to replace rats with special genotypes, as well as large animals such as pigs, dogs, and cats, to construct disease models with different heart failure phenotypes combined with pulmonary hypertension, which provides a research basis for heart failure combined with pulmonary hypertension and greatly reduces the cost of animal model construction. In addition, this application uses a non-invasive echocardiography evaluation scheme and an invasive interventional evaluation scheme to jointly evaluate the severity of the disease, accurately evaluate the left heart lesions and right ventricular pressure of mice, and better simulate human heart failure combined with pulmonary hypertension.

[0020] Compared with the shortcomings of current large animal models, the present application has the following advantages: low model cost, stable model, and good fit to the development of human diseases, but due to the pulmonary vascular characteristics of mice themselves, they are not susceptible to pulmonary hypertension and the severity of the disease is average, but through the construction of a comprehensive evaluation system, the scoring system can be refined to construct an evaluation system suitable for the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the dosing time points of the modeling group in Example 1 of the present application;

[0022] Figure 2 The right ventricular systolic pressure result diagram of the modeling group and the control group in Example 1 of the present application;

[0023] Figure 3 A comparison chart of anatomical indicators between the modeling group and the control group in Example 1 of the present application;

[0024] Figure 4 This is a comparison chart of ultrasound indicators between the modeling group and the control group in Example 2 of the present application;

[0025] Figure 5 A comparison chart of anatomical indicators between the modeling group and the control group in Example 2 of the present application; and

[0026] Figure 6 This is a comparison chart of the staining images and indicators of the modeling group and the control group in Example 2 of the present application. DETAILED DESCRIPTION

[0027] In order to more clearly understand the above-mentioned purposes, features and advantages of the present application, the specific implementation methods of the present application are described below in conjunction with the accompanying drawings. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0028] In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present application is not limited by the specific embodiments disclosed below. Some embodiments of the present application are described below in conjunction with the accompanying drawings.

[0029] Rodents, mainly represented by mice, are regarded as "standard experimental animals". These animals have controllable innate genetic traits, acquired breeding conditions, microbial carrying conditions, nutritional requirements and environmental factors, which can ensure the reliability, accuracy, uniformity, repeatability and comparability of experimental results. Although they are small in size, thin in vascular walls, and require high experimental technical operations, they are currently the most ideal animal model species because of their clear genetic background, significant and stable model traits, quality and specifications that can be freely selected to a certain extent, and the corresponding detection reagents are comprehensive and reasonably priced. In addition, the diversity of mouse strains and genotypes also provides a guarantee for the subsequent optimization and enrichment of this model.

[0030] Example 1: Combination of high-fat diet, L-NAME and Sugen5416 to establish an animal model of heart failure with preserved ejection fraction and pulmonary hypertension

[0031] Male C57BL / 6N mice (weight 22g-25g) were divided into a control group (control), a Sugen5416 injection group (sugen), a high-fat diet combined with L-NAME group (HFD+L-NAME), and a high-fat diet combined with L-NAME injection Sugen5416 group (HFD+L-NAME+sugen). The mice were kept at a constant temperature of 25°C and a room humidity of 50% using a 12 / 12 light and night cycle.

[0032] The mice in the control group and the group injected with Sugen5416 alone were given a normal diet. The mice in the high-fat diet combined with L-NAME group and the high-fat diet combined with L-NAME injected Sugen5416 group were given a high-fat diet, and L-NAME (N-nitro-L-arginine methyl ester hydrochloride) was added to the drinking water at a concentration of 0.5%. Six weeks later, the mice in the group injected with Sugen5416 alone and the group injected with high-fat diet combined with L-NAME injected Sugen5416 were subcutaneously administered Sugen5416 for the first time, with a dose of 20 mg / kg, once every two weeks, for a total of five times. The use of Sugen5416 reagent is to slowly and evenly pour 200 mg of Sugen5416 powder into the DMSO solution, and constantly vortex to mix, ultrasonically dissolve, and prepare a Sugen5416 stock solution with a concentration of 20 mg / mL; when used, dilute it with normal saline to 10 mg / mL working solution, ultrasonically dissolve, and obtain Sugen5416 solution for standby use.

[0033] Attached Figure 1 This is a schematic diagram of the dosing time points. The obtained animal model was tested at the 15th week.

[0034] 1. Pulmonary artery pressure testing

[0035] 1) Use a Millar catheter connected to a pressure transducer to measure pulmonary artery pressure;

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

[0037] 3) Make a longitudinal incision on the right neck skin, bluntly separate the jugular vein above the right clavicle, gently clamp the proximal end of the jugular vein with a vascular clamp, pick up the jugular vein with an 8 / 0 suture needle, cut a "V"-shaped incision with microscissors, insert the catheter tip into the vein, and fix the catheter. The standard is that there is no bleeding at the insertion site and the catheter can rotate freely;

[0038] 4) The PowerLab physiological recorder records and evaluates the position of the catheter based on the waveform displayed in the system; usually, after the catheter is inserted about 1 cm, it is almost the position where the superior vena cava and axillary vein meet, and the catheter insertion angle can be adjusted; then the catheter is rotated counterclockwise and inserted about 1-1.5 cm toward the superior vena cava and right atrial opening; then the catheter is rotated clockwise and slowly withdrawn about 0.5

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

[0040] Figure 2 The right ventricular systolic pressure (RVSP) of each group is shown, indicating that the high-fat diet combined with L-NAME injection Sugen5416 group (HFD+L-NAME+sugen) significantly increased RVSP relative to the control group (control).

[0041] 2. Anatomical indicators

[0042] 1) After drawing arterial blood through the apex of the heart under gas anesthesia, the mice were killed, the pulmonary circulation was lavaged with heparinized saline, and then the lungs were taken out, the right lung was quickly frozen with liquid nitrogen, and the left lung was perfused with formalin;

[0043] 2) The heart was separated from the mouse after lung removal, the blood was washed out with 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, free the right ventricular wall (RV) from the pulmonary artery outlet, and the rest is the left ventricle + ventricular septum (LV + S). After drying with filter paper, weigh each part;

[0045] 4) Take the right hind limb tibia and measure the tibia length (TL) with a vernier caliper;

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

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

[0048] C57BL / 6N mice (weight 22g-25g) were selected and divided into a modeling group (TAC) and a control group (SHAM, sham operation), and the weight and ear tags were weighed and recorded. A 12 / 12 light and night cycle was used, and the mice were free to drink water and eat. The breeding temperature was constant at 25°C and the room humidity was 50%.

[0049] 1. Ligation of the aortic arch

[0050] 1.2% tribromoethanol was injected intraperitoneally at 0.12ml / 10g for anesthesia. After 3-5 minutes, the depth of anesthesia was verified: the mouse's heartbeat and breathing were even, the muscles were relaxed, the limbs were inactive, the whiskers did not respond to touch, and the pedal reflex disappeared. The mouse was tied to the dissection table. The mouse's neck was first degreased with alcohol cotton balls, disinfected with iodine twice, and then deiodinated with alcohol after drying. When disinfecting with alcohol and iodine, proceed from the center to the surrounding areas, and do not wipe repeatedly once.

[0051] Use ophthalmic scissors to cut the skin along the midline of the mouse neck to the second rib (the wound is about 1.2 cm). Bluntly separate the subcutaneous tissue, fat, thyroid gland, superficial fascia, platysma, deep fascia and anterior tracheal muscle. After exposing the trachea, find the left and right common carotid arteries. Use ophthalmic scissors to cut the first sternum, use the sternum hook to fix the cut sternum, use ophthalmic forceps to bluntly separate the thymus and expose the aortic arch.

[0052] Aortic arch ligation: Use 7-0 silk suture and 27G catheter to ligate the aortic arch. After ligation, quickly remove the catheter.

[0053] Use a needle holder to suture with 5-0 surgical sutures, and suture the first sternum and epidermis in sequence. Irradiate with a warm light, observe vital signs such as breathing and heartbeat, and keep the mice in a barrier after they have fully recovered their normal activities.

[0054] The above steps were repeated to complete the modeling of all mice in the modeling group. The steps of the control group mice were exactly the same as those of the modeling group except that the aortic arch was not ligated.

[0055] 2. Ultrasonic testing

[0056] At 8 and 12 weeks after modeling, all mice were depilated from the chest and abdomen and underwent chest ultrasound, including M-mode ultrasound and Doppler ultrasound. The ventricular short axis shortening fraction (FS) was calculated as an indicator of cardiac pumping function, the pulmonary artery blood flow velocity was measured, the pulmonary artery acceleration time (PAT) and pulmonary artery acceleration time / pulmonary artery ejection time (PAT / PET) were calculated, and the tricuspid annular systolic displacement (TAPSE) was measured. Figure 4 The data comparison between the modeling group and the control group is shown, indicating that the modeling group produced a significant data reduction relative to the control group at 12 weeks, and the comparison between the modeling group at 12 weeks and 8 weeks also reached a significant level.

[0057] After the collection of ultrasonic indexes was completed, the right ventricular pressure of the mouse was measured using a Miller catheter under gas anesthesia as in Example 1. After the mouse was killed, the lung circulation was lavaged with heparin saline and the left lung was perfused with formalin as in Example 1, the heart was isolated, and the physiological indexes were measured. Figure 5 The data of the modeling group and the control group are shown. The left ventricle + septum tissue (LV + S), right ventricle (RV), heart weight (HW) and heart weight to body weight ratio (HW / BW) of the modeling group are significantly increased. At the same time, the (left ventricle + septum tissue) / tibia length ratio (LV + S / TL), right ventricle / tibia length ratio (RV / TL), heart weight / tibia length ratio (HW / TL) are also significantly increased compared with the sham operation.

[0058] After the left lung was fully fixed, it was embedded and sectioned, and α-SMA immunohistochemical staining was performed to calculate the degree of vascular remodeling. Figure 6 The staining images and data comparisons of the modeling group and the control group are shown. The ratio of the media thickness to the vascular radius (media thickness / r%) and the ratio of the media thickness to the vascular cross-sectional area (media thickness / CSA%) in the modeling group were significantly increased. Media thickness / r% reflects the thickness of the vascular wall relative to the vascular radius. An increase in this value indicates that the media thickness is larger relative to the vascular radius, which is associated with vascular sclerosis, reduced vasodilation capacity, and increased vascular resistance; an increase in media thickness / CSA% indicates that the media layer of the vascular wall accounts for a higher proportion of the total cross-sectional area, indicating a significant thickening of the vascular wall. This thickening affects the overall structure and function of the blood vessels, leading to obstructed blood flow and increased blood pressure.

[0059] In this application, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance. Unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be an indirect connection through an intermediate medium, or it can be internal communication between two elements. For those of ordinary skill in the art, the specific meanings of the above terms in this application can be understood according to the specific circumstances.

[0060] In the description of this specification, the description of the terms "one embodiment", "some embodiments", "specific embodiments", "some implementation schemes", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or implementation scheme of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or implementation scheme. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or implementation schemes in a suitable manner.

[0061] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for constructing an animal model of heart failure with preserved ejection fraction combined with pulmonary hypertension, comprising: At the starting time point, male C57BL / 6N mice were taken and allowed to freely consume a high-fat diet and L-NAME drinking water containing 0.5% N-nitro-L-arginine methyl ester hydrochloride; 6, 8, 10, 12, and 14 weeks after the start time point, the mice were subcutaneously injected with Sugen5416 solution; Fifteen weeks after the starting time point, a mouse model of heart failure with preserved ejection fraction and pulmonary hypertension was obtained.

2. The method of claim 1, wherein the amount of the Sugen5416 solution is 20 μg / g mouse body weight.

3. The method of claim 1, further comprising measuring right ventricular systolic pressure under anesthesia at week 15.

4. The method according to claim 1, further comprising killing the mice at week 15, and detecting the right ventricular hypertrophy index and vascular remodeling degree of the mice.

5. The method as claimed in claim 4 further comprises detecting the right ventricle weight / tibia length ratio, heart weight / tibia length ratio, and left ventricle plus ventricular septum tissue weight / tibia length ratio of the mouse.

6. A method for constructing an animal model of heart failure with reduced ejection fraction combined with pulmonary hypertension, comprising: Male C57BL / 6N mice were taken and the aortic arch was ligated using a 27G catheter; The mice were allowed to freely consume water and food for 12 weeks to obtain a mouse model of heart failure with reduced ejection fraction and pulmonary hypertension.

7. The method of claim 6, further comprising performing ultrasound examination on the mice at weeks 8 and 12.

8. The method of claim 7, further comprising measuring right ventricular systolic pressure under anesthesia at week 12.

9. The method as claimed in claim 8, further comprising killing the mice at the 12th week, and detecting the right ventricular hypertrophy index and the degree of vascular remodeling of the left lung of the mice, and further comprising detecting the ventricle + septum tissue weight, right ventricular weight, heart weight, heart weight to body weight ratio, right ventricular weight / tibia length ratio, heart weight / tibia length ratio, left ventricle plus septum tissue weight / tibia length ratio of the mice.

10. Use of the mouse model obtained by the method of claims 1 to 9 in screening drugs for treating heart failure with preserved ejection fraction and pulmonary hypertension or heart failure with reduced ejection fraction and pulmonary hypertension.

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