Construction method of chronic thromboembolic pulmonary hypertension animal model

Abstract

The invention provides a construction method of a chronic thromboembolic pulmonary hypertension animal model, which comprises the following steps: taking 180-200g of adult healthy male SD (Sprague Dawley) rats, and freely taking water and food; a soluble gelatin sponge solution is injected into the jugular vein of the rat; after one day, carrying out subcutaneous injection of a Sugen5416 solution; and allowing the rat to freely ingest water and food for 28 days to obtain the chronic thromboembolic pulmonary hypertension rat 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 chronic thromboembolic 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 (Table 5): (1) PH due to pulmonary arterial hypertension (PAH); (2) PH due to left heart disease; (3) PH due to lung disease and / or hypoxia; (4) PH due to chronic thromboembolic PH (CTEPH) and / or other pulmonary artery obstructive lesions; and (5) PH due to unknown and / or multiple factors.

[0005] CTEPH is the most common type of PH. The incidence of CTEPH reported in different literatures varies greatly. The incidence of CTEPH confirmed by RHC after symptomatic acute pulmonary thromboembolism (PTE) abroad is 0.45% to 6.2%, while data from my country show that the cumulative incidence of CTEPH is about 1.3% 2 years after PTE.

[0006] CTEPH is a disease in which thrombi are insoluble and gradually organized after one or more PTEs, and vascular remodeling occurs, causing progressive increases in pulmonary artery pressure and ultimately leading to right heart failure. However, CTEPH has many pathogenic factors and a complex pathogenesis. It is unlikely that most patients have CTEPH caused by a single factor. Its development is closely related to multiple factors, including abnormal coagulation and fibrinolysis mechanisms, inflammation, genetic susceptibility factors, angiogenesis, and in situ thrombosis. The specific molecular mechanism is still unclear. Some patients have a long-term complication of acute PTE. After acute PTE, thrombi are incompletely dissolved and organized, resulting in a continuous increase in PVR, causing pulmonary vascular remodeling, and ultimately leading to right heart failure.

[0007] The mechanism by which pulmonary embolism or thrombus cannot be dissolved and organized into occlusive fibrous material is still unclear. 63% of CTEPH patients have no history of acute pulmonary embolism. Studies have suggested that abnormalities and damage to pulmonary artery endothelial cells may play an important role in the process of vascular repair. Since CTEPH patients have a heavy disease burden and untreated patients have a very poor prognosis, its pathophysiological mechanism needs to be explored in depth.

[0008] 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.

[0009] Establishing an animal model can effectively promote the study of the pathogenesis of CTEPH and provide basic research for early warning and individualized prevention and treatment of the disease. In order to clarify the molecular mechanism of CTEPH and conduct new pharmacological research, it is crucial to establish an animal model that can completely replicate the natural course of human CTEPH. In recent years, researchers have spent a lot of effort trying to establish a reliable animal model, but there is no stable animal model. Many CTEPH animal models have achieved varying degrees of success, but there is no mature animal model that can replicate the natural course of human CTEPH and the main characteristics of the lesions, especially the process of transformation from acute pulmonary thromboembolism to chronic pulmonary thromboembolism.

[0010] Autologous thrombus injection is an earlier method that has been tried and studied. Specifically, the blood clot prepared in vitro is infused through the animal's jugular vein to directly cause pulmonary embolism. The pulmonary embolism simulated by this method is similar to the process of deep vein thrombosis in the human pulmonary artery. It is relatively easy to perform in rat, dog, and rabbit models, and has also been described in mice with small blood vessels. Animal distress symptoms or hemodynamic changes can be observed immediately after the embolus is injected, but the size, shape, and distribution of the embolus are unclear. The advantage of this method is that it can simulate the process of venous thromboembolism in the human pulmonary artery. However, due to the existence of a powerful fibrinolytic system in animals, autologous thrombi are easy to dissipate and cannot stabilize long-term embolism of the pulmonary artery.

[0011] Other studies on pulmonary embolism have used ligation of the pulmonary artery or embolization of foreign bodies, such as polystyrene microspheres. In a porcine model, a 100-300 μm diameter dextran microsphere was percutaneously inserted into the pulmonary artery four times under fluoroscopic guidance for a period of 1-2 months to establish a CTEPH model. In a canine model, right heart dysfunction was observed after infusion of 100-300 μm dextran microspheres every 3-4 days for several months. In a rat model, a study used three consecutive tail vein injections of fibrinogen / collagen-coated polystyrene microspheres and thrombin; after the third embolization, the rats showed characteristic changes of CTEPH, including a slight increase in right ventricular systolic pressure (<33 mmHg), right ventricular myocyte hypertrophy, pulmonary artery remodeling, increased serum brain natriuretic peptide levels, and thrombus fibrosis.

[0012] The model constructed by foreign body embolism is more stable than that constructed by autologous thrombus embolism, and the operation method is easy. However, its shortcomings are that the existing foreign body embolism uses insoluble substances, which makes it difficult to simulate the characteristics of thrombosis in the pathological and physiological changes of CTEPH, and fails to simulate the mechanism of human thromboembolism in the pulmonary artery, and the discussion on the occurrence and development mechanism of insoluble thrombus is relatively limited.

[0013] Due to the lack of animal models, translational medical research has progressed slowly. Therefore, it is necessary to establish a scalable animal model to accelerate the study of the pathogenesis of CTEPH, fully understand the natural course of the disease, and develop new treatment strategies and intervention targets. The key technical problem that this application hopes to solve is to simulate pulmonary artery obstruction and in situ thrombosis in the pathophysiological changes of CTEPH to the greatest extent. Summary of the invention

[0014] In one aspect, the present application provides a method for constructing an animal model of chronic thromboembolic pulmonary hypertension, comprising:

[0015] Adult healthy male SD rats weighing 180-200 g were taken and allowed to take water and food freely;

[0016] injecting a soluble gelatin sponge solution into the jugular vein of the rat;

[0017] One day later, Sugen5416 solution was injected subcutaneously;

[0018] Thereafter, the rats were allowed to freely take in water and food for 28 days to obtain a rat model of chronic thromboembolic pulmonary hypertension.

[0019] In some embodiments, the concentration of the Sugen5416 solution is 10 mg / mL.

[0020] In some embodiments, the Sugen5416 solution is a Sugen5416 suspension obtained by mixing a Sugen5416 stock solution with an equal amount of physiological saline before use and dissolving it by ultrasonication. The Sugen5416 stock solution is a solution of Sugen5416 dissolved in DMSO at a concentration of 20 mg / mL.

[0021] In some embodiments, the soluble gelatin sponge solution is 1 mL of a 2 mg / mL gelatin sponge solution.

[0022] In some embodiments, the soluble gelatin sponge solution is prepared by mixing 0.4 mL of gelatin sponge stock solution with 0.6 mL of normal saline before use, and the gelatin sponge stock solution is a 5 mg / mL gelatin sponge normal saline solution.

[0023] In some embodiments, the gelatin sponge is injected through the left jugular vein.

[0024] In some embodiments, the method of the present application further comprises detecting right ventricular systolic pressure and right ventricular hypertrophy index in the obtained rat model.

[0025] In yet another aspect, the present application provides use of the above-mentioned model animals in screening drugs.

[0026] This application utilizes a single injection of soluble gelatin sponge, which has the advantages of being simple and easy to operate, and combines the endothelial injury drug Sugen5416 to cause endothelial injury, which can form a CTEPH animal model with a severe phenotype. The method of this application is simple to operate, low cost, suitable for large-scale sample experiments, and the experimental animals are small in size and easy to obtain, without the need for multiple people to cooperate; during the experimental operation, there is no need for repeated injections, the surgical trauma is small, and the experimental animals have a strong tolerance to acute ischemia and hypoxia. It has the characteristics of stable model, easy to replicate, economical and practical, etc., which is conducive to wide promotion and provides a basis for the construction of CTEPH animal models and subsequent drug and device development. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a diagram showing the right ventricular systolic pressure test results of the experimental group and the control group of the embodiment of the present application;

[0028] Figure 2 This is a diagram showing the right ventricular hypertrophy index detection results of the experimental group and the control group of the embodiment of the present application. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Among rodent experimental animals, rats have a systemic blood pressure similar to that of humans, and can tolerate acute ischemia and hypoxia. They have stronger adaptability, survival ability, and anti-infection ability than mice. They are easy to obtain and do not require the cooperation of multiple people. Compared with other medium and large animals, they are reasonably priced. Compared with mice, rats are larger in size and are relatively easy to inject into the jugular vein. However, rats and mice also have the characteristic of excessive fibrinolysis, and injected thrombi are easy to dissolve and disappear. Despite the limitations, considering cost-effectiveness and efficiency, rats are indeed the ideal modeling object for constructing small CTEPH animal models. Current rat models mostly focus on the use of insoluble foreign body emboli or thrombus additives or multi-factor combined attacks to improve the stability of emboli. The rat model can explain some of the molecular mechanisms of the occurrence and development of CTEPH to a certain extent, such as inflammation and endothelial damage.

[0033] The present application uses SD rat (Sprague-Dawley rat) as the basic experimental animal to develop an animal model. SD rat is named after its founder, and was bred by two American biologists Sprague and Dawley in the 1920s. The characteristics of this rat include medium size, docile character and long life span, and the color is generally white. The gentle character and relatively long life span of SD rats make them ideal objects for research experiments and are widely used in various biomedical research, including toxicology, pharmacology, genetics, immunology and other fields. SD rats have strong resistance to diseases, especially strong resistance to respiratory diseases.

[0034] In order to better simulate the pathophysiological state of CTEPH patients, the present application uses a combination of soluble gelatin sponge and endothelial injury drug Sugen5416. Gelatin sponge is a new type of biomaterial composed of gelatin and sponge. It has excellent biocompatibility, degradability, plasticity and transparency. Soluble gelatin sponge is not as easily cleared by the fibrinolytic system as autologous thrombus, and it also overcomes the defect that insoluble emboli such as polystyrene microspheres are difficult to simulate the characteristics of thrombosis in the pathophysiological changes of CTEPH. Sugen5416 is a tyrosine kinase inhibitor and a vascular endothelial growth factor receptor antagonist that can cause damage to the vascular endothelium. The pulmonary vascular bed requires multiple different types of blows. This application uses soluble gelatin sponge to increase the pulmonary circulation load, and at the same time uses the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor Sugen5416 to block the VEGF signaling pathway, causing damage to the vascular endothelium, and establishes a rat animal model that can relatively objectively simulate the pathophysiological process of CTEPH caused by human pulmonary thromboembolism.

[0035] This application utilizes a single injection of soluble gelatin sponge, which has the advantages of being simple and easy to operate, and combines the endothelial injury drug Sugen5416 to cause endothelial injury, which can form a CTEPH animal model with a severe phenotype. The method of this application is simple to operate, low cost, suitable for large-scale sample experiments, and the experimental animals are small in size and easy to obtain, without the need for multiple people to cooperate; during the experimental operation, there is no need for repeated injections, the surgical trauma is small, and the experimental animals have a strong tolerance to acute ischemia and hypoxia. It has the characteristics of stable model, easy to replicate, economical and practical, etc., which is conducive to wide promotion and provides a basis for the construction of CTEPH animal models and subsequent drug and device development.

[0036] 1. Experimental Materials

[0037] 1. Experimental Animals

[0038] SD rats were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. All animal experiments were carried out in accordance with the relevant provisions of the Regulations of the People's Republic of China on the Administration of Laboratory Animals and have been approved by the Animal Ethics Committee of the Clinical Research Institute of the China-Japan Friendship Hospital.

[0039] 2. Reagents

[0040]

[0041] Configuration method:

[0042] Prepare a 20 mg / mL Sugen5416 stock solution: slowly and evenly pour 200 mg of Sugen5416 powder into the DMSO solution, vortex and mix continuously, sonicate to dissolve, and set aside; when using, dilute with physiological saline to 10 mg / mL working solution, sonicate to dissolve, and obtain Sugen5416 solution for standby use.

[0043] On the day of pressure measurement, prepare 25U / mL heparin saline: 12500U heparin sodium injection dissolved in 500mL saline.

[0044] Take 50 mg of gelatin sponge and dissolve it in 10 mL of normal saline to prepare a 5 mg / mL gelatin sponge stock solution, and let it stand overnight for use.

[0045] 3. Instruments

[0046]

[0047]

[0048] 2. Operation steps

[0049] Adult healthy male SD rats weighing 180-200 g were used. The animals were raised according to a normal day and night rhythm, with relative humidity (60-85%) and indoor temperature controlled at 20-25° C. The cages were cleaned regularly, and all rats had free access to water and food.

[0050] The experimental animals were divided into four groups, namely, sham control group (sham), gelatin sponge group (gelfoam), Sugen5416 group (Sugen5416 (10 mg / kg)) and gelatin sponge combined with Sugen5416 group (gelfoam + Sugen5416 (10 mg / kg)).

[0051] 1. Surgical injection of gelatin sponge

[0052] 1) Anesthetize the rats with isoflurane gas;

[0053] 2) Gently fix the anesthetized rat on the operating table, expose the neck, and prepare the skin;

[0054] 3) Carefully shave the hair off the neck and disinfect the skin at the surgical site;

[0055] 4) Make a 0.5 cm long longitudinal incision on the left side of the neck skin, use tissue forceps to bluntly separate layer by layer until the left jugular vein is exposed, and use a 2 mL syringe to draw 0.4 mL of 5 mg / mL gelatin sponge stock solution, and draw an additional 0.6 mL of normal saline for later use;

[0056] 5) Puncture the left jugular vein with an 18G cannula needle, with the needle bevel facing upward and the needle tube parallel to the jugular vein, remove the needle and connect the syringe;

[0057] 6) If the syringe is injected smoothly and there is no extravasation, the puncture is successful. After the air is withdrawn, blood can be seen and the gelatin sponge solution is injected. The chest rise and fall and respiratory rate of the rat are observed to avoid sudden death caused by excessive injection (if shortness of breath or distress occurs, stop the injection);

[0058] 7) After the gelatin sponge is completely injected, the needle is withdrawn, and after pressing with a sterile cotton ball to stop bleeding, a small amount of normal saline is used to rinse the incision to prevent tissue adhesion, followed by suture and disinfection, and iodine is applied to the incision to prevent infection;

[0059] 8) Observe the rat's condition and return it to the cage after it wakes up.

[0060] 2. Sugen5416 injection

[0061] One day after the gelatin sponge injection, the Sugen5416 group and the gelatin sponge combined with Sugen5416 group were subcutaneously injected with a single dose of 10 mg / kg of Sugen5416 suspension.

[0062] The rats were allowed to freely take in water and food, and after 28 days of breeding, a rat model of chronic thromboembolic pulmonary hypertension was obtained, and clinical indicators were tested.

[0063] 3. Model Results

[0064] The obtained chronic thromboembolic pulmonary hypertension rat animal model was tested, which can reproduce the increase in right ventricular systolic pressure (RVSP) in CTEPH patients, while the right ventricular hypertrophy index (RVHI) indicates right ventricular remodeling.

[0065] 1. Right ventricular systolic pressure detection

[0066] 1) Use a Millar catheter connected to a pressure transducer to measure right ventricular systolic pressure;

[0067] 2) The rats were anesthetized with isoflurane gas, fixed in a supine position, the skin was prepared, and the surgical site was disinfected;

[0068] 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;

[0069] 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

[0070] 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.

[0071] Figure 1 The right ventricular systolic pressure (RVSP) of each group is shown, indicating that the gelatin sponge combined with Sugen5416 group has significantly increased right ventricular systolic pressure compared with the sham control group, gelatin sponge group, and Sugen5416 group.

[0072] 2. Evaluation of right ventricular hypertrophy

[0073] RVHI is a commonly used hemodynamic index used to detect pulmonary hypertension models. It can indirectly reflect pulmonary artery pressure and directly reflect the degree of right ventricular hypertrophy.

[0074] 1) The heart was separated from the rat after the lungs were removed, the blood was flushed out with saline, and the atrium and residual blood vessels were cut off;

[0075] 2) The right ventricular wall (RV) is freed from the pulmonary artery outlet, and the rest is the left ventricle

[0076] +ventricular septum tissue (LV+S);

[0077] 3) After the filter paper absorbs the water, weigh each weight;

[0078] 4) According to the formula RVHI = RV / [LV+S], when the pulmonary artery pressure increases, the right ventricle undergoes compensatory hypertrophy due to increased afterload.

[0079] Figure 2The right ventricular hypertrophy index of each group is shown, indicating that the gelatin sponge combined with Sugen5416 group has a significantly increased right ventricular hypertrophy index compared with the sham operation control group, gelatin sponge group, and Sugen5416 group, which represents right ventricular wall hypertrophy, leading to a decrease in right ventricular function, which is similar to the indications of clinical pulmonary hypertension patients.

[0080] 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.

[0081] 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.

[0082] 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 chronic thromboembolic pulmonary hypertension, comprising: Adult healthy male SD rats weighing 180-200 g were taken and allowed to take water and food freely; injecting a soluble gelatin sponge solution into the left jugular vein of the rat; One day later, Sugen5416 solution was injected subcutaneously; Thereafter, the rats were allowed to freely take in water and food for 28 days to obtain a rat model of chronic thromboembolic pulmonary hypertension.

2. The method of claim 1, wherein the concentration of the Sugen5416 solution is 10 mg / mL.

3. The method of claim 2, wherein the Sugen5416 solution is a Sugen5416 suspension obtained by mixing a Sugen5416 stock solution with an equal amount of physiological saline before use and dissolving it by ultrasonication, and the Sugen5416 stock solution is a solution of Sugen5416 dissolved in DMSO at a concentration of 20 mg / mL.

4. The method of claim 1, wherein the soluble gelatin sponge solution is 1 mL of a 2 mg / mL gelatin sponge solution.

5. method as claimed in claim 4, wherein said soluble gelatin sponge solution is prepared by mixing 0.4mL gelatin sponge mother solution with 0.6mL normal saline before use, and said gelatin sponge mother solution is the gelatin sponge normal saline solution of 5mg / mL.

6. The method of claim 1, wherein the gelatin sponge is injected through the left jugular vein.

7. The method according to claim 1, further comprising detecting right ventricular systolic pressure and right ventricular hypertrophy index in the obtained rat model.

8. Use of the rat model obtained by the method of claims 1 to 7 in screening drugs for treating chronic thromboembolic pulmonary hypertension.

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