A method for constructing a chronic thromboembolic pulmonary hypertension animal model

Abstract

Provided is a method for constructing a chronic thromboembolic pulmonary hypertension animal model, comprising: taking 180-200 gram adult healthy male SD rats, allowing the rats to freely take water and food; injecting a soluble gelatin sponge solution into the jugular vein of the rats; after 1 day, subcutaneously injecting a Sugen5416 solution; then allowing the rats to freely take water and food for 28 days, to obtain a chronic thromboembolic pulmonary hypertension rat model.

Description

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 chronic thromboembolic 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 (Table 5): (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 of unknown and / or multifactorial causes.

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

[0006] CTEPH is a disease caused by the incomplete dissolution and gradual organization of thrombi following one or more PTEs, leading to vascular remodeling, progressive elevation of pulmonary artery pressure, and ultimately right ventricular failure. However, CTEPH has multiple causative factors and a complex pathogenesis. Most patients are unlikely to develop CTEPH due to a single factor; its development is closely related to various factors, including abnormalities in coagulation and fibrinolysis mechanisms, inflammation, genetic susceptibility, angiogenesis, and in situ thrombosis. The specific molecular mechanisms remain unclear. In some patients, it is a long-term complication of acute PTE. Incomplete dissolution and organization of thrombi following acute PTE lead to a sustained increase in pulmonary artery pressure (PVR), causing pulmonary vascular remodeling and ultimately right ventricular failure.

[0007] The mechanism by which pulmonary embolism or thrombus fails to dissolve and organizes into occlusive fibrous material remains unclear. 63% of CTEPH patients have no history of acute pulmonary embolism. Studies suggest that pulmonary artery endothelial cell abnormalities and damage may play an important role in vascular repair. Due to the heavy disease burden of CTEPH and the extremely poor prognosis of untreated patients, further investigation into its pathophysiological mechanisms is necessary.

[0008] 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.

[0009] Establishing animal models can effectively advance research on the pathogenesis of CTEPH, providing a foundation for early warning and personalized prevention and treatment. To elucidate the molecular mechanisms of CTEPH and develop new pharmacological methods, establishing animal models that can fully replicate the natural progression of human CTEPH is crucial. In recent years, researchers have devoted considerable effort to establishing reliable animal models, but a stable model is still lacking. Many CTEPH animal models have achieved varying degrees of success, but no mature animal model can replicate the natural progression and main characteristics of human CTEPH, especially the transformation from acute pulmonary thromboembolism to chronic pulmonary thromboembolism.

[0010] Autologous thrombus injection was one of the earlier methods attempted for research. Specifically, it involves infusing a pre-prepared blood clot into the jugular vein of an animal to directly induce pulmonary embolism. This method simulates pulmonary embolism similar to the process of deep vein thrombosis (DVT) embolism 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. Discomfort symptoms or hemodynamic changes can be observed immediately after embolism injection, but the size, shape, and distribution of the embolus are unclear. The advantage of this method is its ability to simulate the process of pulmonary embolism caused by venous thrombosis in humans. However, due to the strong fibrinolytic system in animals, autologous thrombi tend to dissolve easily and cannot stably embolize the pulmonary artery long-term.

[0011] Other studies on pulmonary embolism have employed ligation of the pulmonary artery or embolization with foreign bodies, such as polystyrene microspheres. One study in a pig model used percutaneous puncture under fluoroscopic guidance to embolize the pulmonary artery four times over 1-2 months to establish a CTEPH model. In a canine model, right ventricular dysfunction was observed after several months of infusion of 100-300 μm dextran microspheres every 3-4 days. In a rat model, one study used fibrinogen / collagen-encapsulated polystyrene microspheres and thrombin via three consecutive tail vein injections; after the third embolization, rats exhibited characteristic changes of CTEPH, including a slight increase in right ventricular systolic pressure (<33 mmHg), right ventricular cardiomyocyte hypertrophy, pulmonary artery remodeling, elevated serum brain natriuretic peptide levels, and thrombus fibrosis.

[0012] Foreign body embolization models are more stable and easier to perform than autologous thromboembolization models. However, their drawbacks include the fact that existing foreign body embolization methods all use insoluble substances, making it difficult to simulate the thrombus formation characteristics in the pathophysiological changes of CTEPH, failing to simulate the mechanism of pulmonary thrombosis in humans, and thus limiting the exploration of the occurrence and development mechanism of thrombus insolubility.

[0013] The lack of animal models hinders the progress of translational medicine research. Therefore, a widely applicable animal model is needed to accelerate research into the pathogenesis of CTEPH, fully understand its natural course, and develop new treatment strategies and intervention targets. This application aims to address the key technical challenge of maximally simulating pulmonary artery obstruction and in situ thrombosis in the pathophysiological changes of CTEPH. Summary of the Invention

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

[0015] Adult healthy male SD rats weighing 180-200 grams were used and allowed free access to water and food;

[0016] A soluble gelatin sponge solution was injected into the jugular vein of the rats.

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

[0018] The rats were then allowed free access to 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 Sugen5416 stock solution with an equal volume of physiological saline and then sonicating to dissolve it before use, and 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 a 1 mL 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 physiological saline before use, wherein the gelatin sponge stock solution is a 5 mg / mL gelatin sponge physiological saline solution.

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

[0024] In some implementations, the method of this application further includes measuring right ventricular systolic pressure and right ventricular hypertrophy index in the obtained rat model.

[0025] In another aspect, this application provides the use of the aforementioned model animals in drug screening.

[0026] This application utilizes a single injection of soluble gelatin sponge, which is simple and easy to operate. Combined with the endothelial injury drug Sugen5416 to induce endothelial damage, it can establish a severe CTEPH animal model. The method of this application is simple to operate, low in cost, suitable for large-scale sample experiments, and the experimental animals are small and easy to obtain, requiring no multiple people to cooperate. The experimental procedure does not require repeated injections, resulting in minimal surgical trauma. The experimental animals have strong tolerance to acute ischemia and hypoxia, exhibiting model stability, ease of replication, and cost-effectiveness. This facilitates widespread application and provides a foundation for the construction of CTEPH animal models and subsequent drug and device development. Attached Figure Description

[0027] Figure 1 The graph shows the right ventricular systolic pressure test results for the experimental group and the control group in an embodiment of this application.

[0028] Figure 2 The image shows the right ventricular hypertrophy index detection results for the experimental and control groups in an embodiment of this application. Detailed Implementation

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Among rodent experimental animals, rats have systemic blood pressure similar to humans, are more tolerant of acute ischemia and hypoxia, and exhibit stronger adaptability, survival ability, and resistance to infection than mice. They are also readily available, require no multiple personnel, and are reasonably priced compared to other medium to large animals. Compared to mice, rats are larger, making jugular vein thrombectomy relatively easier. However, like mice, rats exhibit excessive fibrinolysis, making injected thrombi prone to dissolution and regression. Despite these limitations, considering cost-effectiveness and efficiency, rats are currently the ideal animal model for constructing small CTEPH models. Current rat models primarily utilize insoluble foreign body thrombi, thrombus additives, or a combination of factors to enhance thrombus stability. Rat models can, to some extent, explain some of the molecular mechanisms underlying CTEPH, such as inflammation and endothelial damage.

[0033] This application uses the SD rat (Sprague-Dawley rat) as the basic experimental animal to develop an animal model. The SD rat is named after its creators, two American biologists, Sprague and Dawley, who bred it in the 1920s. This rat is characterized by its medium size, docile temperament, and long lifespan; it is typically white. The SD rat's gentle nature and relatively long lifespan make it an ideal subject for research experiments, widely used in various biomedical studies, including toxicology, pharmacology, genetics, and immunology. SD rats have strong resistance to diseases, especially respiratory diseases.

[0034] To better simulate the pathophysiological state of CTEPH patients, this application combines soluble gelatin sponge and the endothelial injury drug Sugen5416. Gelatin sponge is a novel biomaterial composed of gelatin and sponge. It possesses excellent biocompatibility, biodegradability, plasticity, and transparency. Soluble gelatin sponge is not easily cleared by the fibrinolytic system like autologous thrombi, and it overcomes the shortcomings of insoluble emboli such as polystyrene microspheres, which are unable to simulate the thrombus formation characteristics 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 various different impacts. This application uses soluble gelatin sponge to increase the pulmonary circulation load, and uses the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor Sugen5416 to block the VEGF signaling pathway, causing damage to the vascular endothelium. This establishes a rat animal model that can relatively objectively simulate the pathophysiological process of CTEPH caused by pulmonary thromboembolism in humans.

[0035] This application utilizes a single injection of soluble gelatin sponge, which is simple and easy to operate. Combined with the endothelial injury drug Sugen5416 to induce endothelial damage, it can establish a severe CTEPH animal model. The method of this application is simple to operate, low in cost, suitable for large-scale sample experiments, and the experimental animals are small and easy to obtain, requiring no multiple people to cooperate. The experimental procedure does not require repeated injections, resulting in minimal surgical trauma. The experimental animals have strong tolerance to acute ischemia and hypoxia, exhibiting model stability, ease of replication, and cost-effectiveness. This facilitates widespread application and provides a foundation for the construction of CTEPH animal models and subsequent drug and device development.

[0036] I. Experimental Materials

[0037] 1. Laboratory animals

[0038] SD rats were used and purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experiments were conducted in accordance with the relevant provisions of the "Regulations on the Administration of Laboratory Animals of the People's Republic of China" and have been reviewed and approved by the Animal Ethics Committee of the Clinical Research Institute of 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 DMSO solution, vortex continuously, and sonicate to aid dissolution. When needed, dilute with physiological saline to a working solution of 10 mg / mL, sonicate to dissolve, and obtain a Sugen5416 solution for use.

[0043] Prepare 25U / mL heparinized saline solution on the day of pressure measurement: dissolve 12500U heparin sodium injection in 500mL of saline solution.

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

[0045] 3. Instruments

[0046]

[0047]

[0048] II. Operating Procedures

[0049] Adult healthy male SD rats weighing 180-200 grams were used. The animals were housed according to a normal diurnal rhythm, with relative humidity of 60-85% and indoor temperature controlled at 20-25℃. The cages were cleaned regularly, and all rats had free access to water and food.

[0050] The experimental animals were divided into four groups: sham control group, gelatin sponge group, 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 rats with isoflurane gas;

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

[0054] 3) Carefully shave off the fur around the neck and disinfect the surgical site;

[0055] 4) Make a longitudinal incision of about 0.5 cm in length on the skin of the left side of the neck. Use tissue forceps to bluntly dissect layer by layer until the left jugular vein is exposed. At the same time, 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) Use an 18G cannula to puncture the left jugular vein, with the bevel of the needle facing upwards and the cannula running parallel to the jugular vein. Withdraw the needle and connect the syringe.

[0057] 6) If the syringe can be injected smoothly and there is no extravasation of liquid, the puncture is successful. After aspirating the air, blood return can be observed. Then inject the gelatin sponge solution and observe the chest rise and fall and respiratory rate of the rat. Avoid injecting too quickly to avoid sudden death (stop the injection if rapid breathing or distress occurs).

[0058] 7) After the gelatin sponge is completely injected, withdraw the needle, apply pressure with a sterile cotton ball to stop the bleeding, rinse the incision with a small amount of normal saline to prevent tissue adhesion, then suture and disinfect, and apply povidone-iodine to the incision to prevent infection.

[0059] 8) Observe the rat's condition, and after it recovers, put it back into the feeding cage.

[0060] 2. Sugen5416 injection

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

[0062] A rat model of chronic thromboembolic pulmonary hypertension was obtained by allowing rats free access to water and food and raising them for 28 days. Clinical indicators were then measured in the rats.

[0063] III. Model Results

[0064] The obtained rat model of chronic thromboembolic pulmonary hypertension (CTEPH) was tested and found to reproduce the elevated right ventricular systolic pressure (RVSP) in CTEPH patients, while the right ventricular hypertrophy index (RVHI) indicated right ventricular remodeling.

[0065] 1. Right ventricular systolic pressure measurement

[0066] 1) Right ventricular systolic pressure was measured using a Millar catheter connected to a pressure transducer;

[0067] 2) Anesthetize rats with isoflurane gas, fix the rats in a supine position, prepare the skin, and disinfect the surgical site;

[0068] 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.

[0069] 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.

[0070] 1 cm, then continue insertion; when a faint right atrial waveform is displayed, continue to advance the catheter 1-2 cm 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 had a significantly increased right ventricular systolic pressure compared with the sham-operated control group, the gelatin sponge group, and the Sugen5416 group.

[0072] 2. Evaluation of the degree of right ventricular hypertrophy

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

[0074] 1) Separate the heart from the rat after lung removal, rinse off the blood with physiological saline, and cut off the atria and residual blood vessels;

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

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

[0077] 3) After the filter paper has absorbed the moisture, weigh each of the filters separately;

[0078] 4) Calculate RVHI according to the formula RV / [LV+S]. When the pulmonary artery pressure increases, the right ventricle undergoes compensatory hypertrophy due to the 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 surgery control group, the gelatin sponge group, and the Sugen5416 group, which represents right ventricular wall hypertrophy, leading to a reduction in right ventricular function, similar to the indications for patients with clinical pulmonary hypertension.

[0080] 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.

[0081] 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.

[0082] 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 chronic thromboembolic pulmonary hypertension, comprising: Adult healthy male SD rats weighing 180-200 grams were used and allowed free access to water and food; 1 mL of a 2 mg / mL soluble gelatin sponge solution was injected into the left jugular vein of the rat. One day later, a single subcutaneous injection of 10 mg / mL Sugen5416 solution was administered at a dose of 10 mg / kg rat body weight. The rats were then allowed free access to water and food for 28 days to obtain a rat model of chronic thromboembolic pulmonary hypertension.

2. The method of claim 1, wherein the Sugen5416 solution is a Sugen5416 suspension obtained by mixing Sugen5416 stock solution with an equal volume of physiological saline and dissolving by sonication before use, and the Sugen5416 stock solution is a solution of Sugen5416 dissolved in DMSO at a concentration of 20 mg / mL.

3. The method according to claim 1, wherein the soluble gelatin sponge solution is prepared by mixing 0.4 mL of gelatin sponge stock solution with 0.6 mL of physiological saline before use, wherein the gelatin sponge stock solution is a 5 mg / mL gelatin sponge physiological saline aqueous solution.

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