A method for constructing a chronic thromboembolic pulmonary hypertension rat model

Abstract

The application provides a method for constructing a chronic thromboembolic pulmonary hypertension rat model, and belongs to the technical field of animal models. The method comprises the following steps: selecting polyvinyl alcohol microspheres as embolization material, and preparing a microsphere suspension; fixing the rat after anesthesia, exposing a side of the jugular vein, and establishing a venous injection channel through a cannula needle; preparing the prepared microsphere suspension into a microsphere injection solution, injecting the microsphere injection solution into the rat through the established venous channel, and forming a persistent mechanical obstruction; after the microsphere injection is completed, the animal is treated for hemostasis, suture and recovery, and the rat is normally bred for 7 days after the operation to obtain the chronic thromboembolic pulmonary hypertension rat model. The method overcomes the defects of the traditional autologous thrombus model, such as easy dissolution of the thrombus, unstable embolization degree, and difficulty in maintaining chronic pathological changes, and improves the stability, repeatability and drug efficacy evaluation applicability of the model.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, and in particular to a method for constructing a rat model of chronic thromboembolic pulmonary hypertension. Background Technology

[0002] Chronic thromboembolic pulmonary hypertension (CTEPH) is a type of pulmonary hypertension caused by long-term residual and organized thrombi in the pulmonary arteries, leading to secondary pulmonary vascular remodeling. Establishing stable and reproducible animal models is crucial for studying the pathogenesis of CTEPH and evaluating drug interventions. Existing CTEPH animal models mainly include autologous thrombus injection models, pulmonary artery ligation models, and foreign body microsphere embolization models.

[0003] Autologous thrombus injection is one of the earliest methods used to simulate pulmonary thromboembolism. This method typically involves preparing a blood clot in vitro, then infusing it via the animal's jugular or peripheral vein, allowing the clot to enter the pulmonary artery and cause embolism. This method has been applied in rats, dogs, rabbits, and other animals, and has also been reported in mouse models with smaller blood vessels. Its advantages include a good ability to simulate the process of deep vein thrombosis and pulmonary artery embolism in humans; respiratory distress and hemodynamic changes can be observed in animals shortly after embolism injection; and the operational principle is relatively intuitive.

[0004] However, this method still has significant drawbacks. First, the size, shape, texture, and post-injection distribution of in vitro-prepared autologous thrombi are difficult to maintain consistently, leading to instability in the extent and degree of pulmonary artery obstruction and significant individual variability within the model. Second, the experimental animals have a strong fibrinolytic system, making it easy for the injected autologous thrombi to dissolve, be absorbed, or recanalize, hindering the formation of long-term, stable pulmonary artery obstruction. Therefore, it cannot adequately simulate the pathological processes of long-term thrombus retention, organization, and persistent pulmonary vascular remodeling in CTEPH. These issues directly affect the model's stability, reproducibility, and the reliability of subsequent pharmacodynamic evaluation results.

[0005] Besides autologous thrombus injection, existing techniques also include pulmonary artery ligation and foreign body embolization. For example, some studies have used foreign materials such as polystyrene microspheres and dextran microspheres to embolize the pulmonary artery, in order to prolong the pulmonary artery obstruction time and induce pulmonary hypertension. In large animal models such as pigs, some studies have used fluoroscopic guidance to percutaneously inject dextran microspheres with a diameter of approximately 100-300 μm into the pulmonary artery multiple times, inducing CTEPH-related changes after 1-2 months. In canine models, there are also reports of right ventricular dysfunction observed after repeated infusions of 100-300 μm dextran microspheres every 3-4 days for several months. The above methods can induce persistent pulmonary artery obstruction to a certain extent, but they usually require imaging equipment, have high operational technical requirements, long modeling cycles, and high experimental costs. They are more suitable for large animal studies and are not conducive to large-scale pharmacodynamic evaluation under routine experimental conditions.

[0006] In rat models, studies have also used fibrinogen / collagen-encapsulated polystyrene microspheres combined with thrombin to induce pulmonary embolism through repeated tail vein injections. This method can induce CTEPH-related manifestations to some extent, including increased right ventricular systolic pressure, right ventricular cardiomyocyte hypertrophy, pulmonary artery remodeling, elevated serum brain natriuretic peptide levels, and thrombofibrosis. However, these models typically require multiple injections or complex microsphere surface treatment steps, making the operation relatively cumbersome. Furthermore, some models show limited increases in right ventricular systolic pressure, and the manifestations of pulmonary hypertension and chronic vascular remodeling are not stable enough, making it difficult to fully meet the requirements for model stability, repeatability, operability, and persistent pathological changes in CTEPH efficacy evaluation.

[0007] In summary, while existing animal models of pulmonary embolism (CTEPH) can simulate pulmonary embolism and its subsequent pathological changes to some extent, they still suffer from problems such as easy clearance of autologous thrombi by the fibrinolytic system, unstable embolism severity, insufficient model reproducibility, long modeling cycles, and complex or costly procedures. Therefore, there is an urgent need to establish a small animal CTEPH model that is easy to operate, has stable embolization effects, good reproducibility, and can continuously induce pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular remodeling to meet the needs of CTEPH pathogenesis research and drug efficacy evaluation. Summary of the Invention

[0008] The purpose of this invention is to provide a method for constructing a rat model of chronic thromboembolic pulmonary hypertension (CTEPH), overcoming the problems of easy dissolution of emboli, unstable degree of embolism, and insufficient long-term modeling effect in autologous thrombosis models, and improving the stability, repeatability, and operability of CTEPH rat model construction.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for constructing a rat model of chronic thromboembolic pulmonary hypertension, comprising the following steps: (1) Polyvinyl alcohol microspheres were selected as the embolization material and a microsphere suspension was prepared. (2) After anesthetizing the rats, fix them and expose one jugular vein. Establish an intravenous injection route through a cannula. (3) The microsphere suspension prepared in step (1) is prepared as a microsphere injection solution and injected into rats through the intravenous access established in step (2) to form a persistent mechanical obstruction; (4) After the microspheres are injected, the animals are subjected to hemostasis, suturing and resuscitation to obtain a rat model of chronic thromboembolic pulmonary hypertension.

[0010] Preferably, the microsphere suspension in step (1) is prepared by mixing polyvinyl alcohol microspheres with physiological saline.

[0011] Preferably, the particle size of the polyvinyl alcohol microspheres in step (1) is 450~550 μm.

[0012] Preferably, the concentration of the microsphere suspension in step (1) is 180~220 mg / mL.

[0013] Preferably, the jugular vein mentioned in step (2) is the left jugular vein.

[0014] Preferably, the amount of microsphere injection solution injected into the rat in step (3) is 0.8~1.2 mL.

[0015] Preferably, the preparation method of the microsphere injection solution in step (3) is as follows: take 0.3~0.5 mL of microsphere suspension and mix it with 0.5~0.7 mL of physiological saline.

[0016] The present invention also provides an application of the rat model of chronic thromboembolic pulmonary hypertension constructed by the above-described construction method in drug screening.

[0017] This invention uses polyvinyl alcohol microspheres instead of traditional autologous thrombi as the embolic material, and injects them into the pulmonary artery via intravenous injection. Utilizing the stable particle size, resistance to rapid degradation, and ability to create persistent mechanical obstruction, polyvinyl alcohol microspheres induce chronic pulmonary artery obstruction and secondary pulmonary vascular remodeling in rats. This method overcomes the shortcomings of traditional autologous thrombosis models, such as easy thrombus dissolution, unstable embolism, and difficulty in maintaining chronic pathological changes, thus improving the model's stability, repeatability, and applicability for efficacy evaluation. Attached Figure Description

[0018] Figure 1 The results show the evaluation of right ventricular systolic pressure and right ventricular hypertrophy in the model rats and control rats in Example 1. Detailed Implementation

[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Example 1

[0021] 1. Selection and preparation of laboratory animals

[0022] Eighteen male SD rats weighing 200-230 g were selected as experimental animals and divided into three groups: control group (Control), experimental group 1 (Model 1), and experimental group 2 (Model 2). Before the experiment, the rats were allowed free access to water and food to acclimatize them, maintaining their normal diurnal rhythm and providing ample feed and water. Before modeling, the rats' mental state, activity level, respiratory status, and weight changes were observed, and rats in good general condition were selected for subsequent experiments.

[0023] 2. Preparation of polyvinyl alcohol microsphere suspension

[0024] The microsphere material used in this embodiment is polyvinyl alcohol microspheres, i.e., PVA microspheres. The PVA microspheres are available in two sizes: 500 μm and 180 μm. First, the PVA microspheres are weighed and added to sterile physiological saline to prepare a PVA microsphere stock solution with a concentration of 200 mg / mL. Before use, the solution is thoroughly mixed to ensure uniform dispersion of the PVA microspheres in the physiological saline, preventing uneven injection dosage due to microsphere sedimentation. During modeling, 0.4 mL of the above PVA microsphere stock solution is drawn using a 1 mL syringe, followed by 0.6 mL of sterile physiological saline. The mixture is then stirred to prepare a 1 mL PVA microsphere injection solution. This injection solution contains 0.4 mL of the PVA microsphere stock solution, sufficient for a single rat modeling injection.

[0025] 3. PVA microsphere injection molding

[0026] Experimental Group 1 Model Establishment: After anesthetizing and fixing the rats, the left jugular vein was exposed. Using an 18G cannula, 1 mL of PVA microsphere injection solution (PVA microsphere particle size of 500 μm) was slowly injected into the left jugular vein. During injection, the injection rate should be slow and steady, and the rat's chest rise and fall, respiratory rate, and general condition should be continuously observed to avoid acute pulmonary circulatory overload or sudden death due to excessively rapid injection. The PVA microsphere injection was performed once. After injection, sterile cotton balls were immediately applied to the puncture site to stop bleeding. Once there was no significant bleeding, the rats were returned to their cages for continued rearing. After model establishment, the rats were raised according to their normal diurnal rhythm, and their mental state, activity level, food and water intake, and respiratory status were continuously observed.

[0027] Modeling in Experimental Group 2: The procedure was the same as in Experimental Group 1, except that the injection site was the left femoral vein of the rat, and the PVA microspheres had a particle size of 180 μm.

[0028] 4. Post-modeling observation and material selection

[0029] After PVA microsphere injection, rats were fed normally for 7 days. During the modeling period, rats were observed for signs such as reduced activity, changes in respiratory rate, and decreased mental state. Samples were taken and relevant indicators were measured on day 7 after modeling.

[0030] 5. Evaluation of model success

[0031] The success of the model was evaluated by measuring right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVHI). The results are as follows: Figure 1 As shown in the diagram, right ventricular systolic pressure reflects changes in pulmonary circulation pressure, while right ventricular hypertrophy reflects the increased right ventricular load after prolonged elevation of pulmonary circulation resistance. Specifically, right ventricular systolic pressure can be measured via right heart catheterization, and the right ventricular hypertrophy index (RV / (LV+S)) is calculated by weighing the right ventricle and the left ventricle plus the interventricular septum. If the model rats exhibit elevated right ventricular systolic pressure and increased right ventricular hypertrophy, it suggests that this method can induce pathological changes related to chronic thromboembolic pulmonary hypertension.

[0032] Depend on Figure 1 The results showed that the RVSP of rats in the Control group was (16.8±0.6) mmHg, in the Model1 group it was (28.0±1.6) mmHg, and in the Model2 group it was (18.0±1.4) mmHg. Compared with the Control group, the RVSP of the Model1 group was significantly increased (P<0.0001), while there was no statistically significant difference between the Model2 group and the Control group (P>0.05). The RVHI of the Control group, Model1 group, and Model2 group were 0.30±0.01, 0.66±0.04, and 0.32±0.02, respectively; compared with the Control group, the RVHI of the Model1 group was significantly increased (P<0.0001), while there was no statistically significant difference in RVHI of the Model2 group (P>0.05). The above results indicate that injection of 500 μm diameter, 200 mg / L polyvinyl alcohol microspheres via the left jugular vein significantly increases right ventricular systolic pressure and induces right ventricular hypertrophy in rats, effectively mimicking CTEPH-related pulmonary hypertension and right ventricular remodeling. In contrast, injection of 180 μm polyvinyl alcohol microspheres via the left femoral vein did not cause significant changes in these indicators. Therefore, Model 1 is significantly more effective than Model 2 and is more suitable for constructing a CTEPH rat model.

[0033] Compared with existing methods for constructing CTEPH animal models, this method uses polyvinyl alcohol microspheres with a particle size of approximately 500 μm and a concentration of 200 mg / L, which are injected via the left jugular vein. The microspheres are carried by the blood flow into the pulmonary artery and form a persistent embolism. This method has the advantages of relatively simple operation, controllable embolization material parameters, and high model stability and repeatability.

[0034] The particle size and injection concentration of polyvinyl alcohol (PVA) microspheres can be preset, with minimal batch-to-batch variation. They are not easily degraded by the body's fibrinolytic system and can form relatively persistent and repeatable mechanical obstruction within pulmonary vessels, thus reducing individual variability caused by embolus dissolution and size inhomogeneity. Compared to smaller-diameter microspheres, the 500 μm PVA microspheres used in this study resulted in more significant increases in pulmonary artery pressure and right ventricular hypertrophy, which is beneficial for obtaining a stable CTEPH-related hemodynamic phenotype. Microsphere embolization, which establishes persistent pulmonary vascular obstruction and induces pulmonary vascular remodeling and increased right ventricular load, has been used to construct chronic pulmonary embolism models.

[0035] In summary, this method, through standardized control of polyvinyl alcohol microsphere particle size and injection concentration, can induce continuous and stable pulmonary vascular occlusion without complex open-chest surgery, resulting in a significant increase in RVSP and RVHI. It has the advantages of simple operation, controllable embolus particle size, stable embolism, poor solubility, low equipment dependence, good batch consistency, and easy large-scale modeling. It is suitable for experimental research on the pathogenesis, drug efficacy, and intervention measures of CTEPH.

[0036] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a rat model of chronic thromboembolic pulmonary hypertension, characterized in that, Includes the following steps: (1) Polyvinyl alcohol microspheres were selected as the embolization material and a microsphere suspension was prepared. (2) After anesthetizing the rats, fix them and expose one jugular vein. Establish an intravenous injection route through a cannula. (3) The microsphere suspension prepared in step (1) is prepared as a microsphere injection solution and injected into rats through the intravenous access established in step (2) to form a persistent mechanical obstruction; (4) After the microspheres are injected, the animals are hemostatically stopped, sutured and resuscitated. After being fed normally for 7 days, a rat model of chronic thromboembolic pulmonary hypertension is obtained.

2. The construction method according to claim 1, characterized in that, The method for preparing the microsphere suspension in step (1) is as follows: take polyvinyl alcohol microspheres and mix them with physiological saline.

3. The construction method according to claim 1, characterized in that, The polyvinyl alcohol microspheres mentioned in step (1) have a particle size of 450~550 μm.

4. The construction method according to claim 1, characterized in that, The concentration of the microsphere suspension in step (1) is 180~220 mg / mL.

5. The construction method according to claim 1, characterized in that, The jugular vein mentioned in step (2) is the left jugular vein.

6. The construction method according to claim 1, characterized in that, The preparation method of the microsphere injection solution in step (3) is as follows: take 0.3~0.5 mL of microsphere suspension and mix it with 0.5~0.7 mL of physiological saline.

7. The application of a rat model of chronic thromboembolic pulmonary hypertension constructed by the construction method according to any one of claims 1 to 6 in drug screening.

Images