A method for constructing an idiopathic pulmonary fibrosis combined with pulmonary arterial hypertension animal model

Abstract

Provided are a rat anesthesia device, which comprises a back plate connected with a bottom plate or a support plate, the back plate is at an inclined angle with the horizontal plane, and the back plate is used for placing a rat body; a metal wire is fixed on the back plate and is used for hanging rat incisors in use; and an anesthesia mask, a main body of the anesthesia mask is a hollow cylinder, one end of the main body is connected with a gas source, the other end is an inclined surface, a v-shaped groove is arranged on the opposite side of the main body at the inclined surface end, and the v-shaped groove is matched with the metal wire in use, so that the anesthesia mask can completely cover a rat head. Also provided is a construction method of an idiopathic pulmonary fibrosis combined with pulmonary arterial hypertension animal model, which comprises the following steps: taking 6-week-old SD male rats, and allowing the rats to freely take water and food; using a rat anesthesia device to anesthetize the rats, and injecting a bleomycin sulfate solution into the airway of the rats; allowing the rats to freely take water and food for 4 weeks, and obtaining an idiopathic pulmonary fibrosis combined with pulmonary arterial 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 idiopathic pulmonary fibrosis complicated with pulmonary hypertension. Background Technology

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

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

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

[0005] Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic lung disease of unknown etiology. Pulmonary hypertension is one of the complications of IPF, which manifests as progressively worsening dyspnea without the presence of new radiographic findings.

[0006] Previous animal models were limited to idiopathic pulmonary hypertension or idiopathic pulmonary fibrosis, which could not accurately simulate the development and progression of the disease in patients. Existing idiopathic pulmonary fibrosis animal models use different administration methods, with inconsistent dosing frequency, dosage, and modeling time. Even with the same administration method, experimental protocols vary, leading to significant differences in experimental results and frequent inability to reproduce outcomes. Therefore, existing modeling methods suffer from drawbacks such as numerous steps, cumbersome procedures, unstable modeling, difficulty in reproducing results, and simplistic assessment methods that fail to achieve standardization.

[0007] This application aims to propose a rat disease model that can simulate idiopathic pulmonary fibrosis complicated with pulmonary hypertension, providing a suitable research tool for studying the occurrence, development, and intervention of this disease. Summary of the Invention

[0008] On the other hand, this application provides a rat gas anesthesia device, which includes:

[0009] A backplate, connected to a base plate or support plate, is inclined at an angle to the horizontal plane and is used to place the rat's body; a metal wire is fixed to the backplate and is used to hang the rat's incisors during use; an air-numbing mask, the main body of which is a hollow cylinder, one end of which is connected to an air source, and the other end is an inclined surface. A V-shaped groove is provided on the opposite side of the inclined surface end of the main body. The V-shaped groove cooperates with the metal wire during use so that the air-numbing mask can completely cover the rat's head.

[0010] In some implementations, the main body diameter of the air-assisted mask is approximately 2.5 centimeters.

[0011] In some implementations, the angle between the inclined plane and the main body is 45-60 degrees, and the angle between the back plate and the horizontal plane is 60-75 degrees.

[0012] In some embodiments, the V-groove is located in the lower middle part of the inclined surface, and the depth of the V-groove is about 2 cm.

[0013] On the other hand, this application provides a method for constructing an animal model of idiopathic pulmonary fibrosis complicated with pulmonary hypertension, which includes: taking 6-week-old male SD rats and allowing them to freely ingest water and food; anesthetizing the rats using a rat anesthesia device and injecting bleomycin sulfate solution into the airway of the rats; allowing the rats to freely ingest water and food for 4 weeks to obtain a rat model of idiopathic pulmonary fibrosis complicated with pulmonary hypertension.

[0014] In some embodiments, the concentration of the bleomycin sulfate solution is 0.5 mg / mL, and the volume of the bleomycin sulfate solution is 4 μL / g rat body weight.

[0015] In some implementations, cardiovascular ultrasound of the obtained rat model is also included to make analogies to human detection parameters.

[0016] In some implementations, the method also includes measuring the right ventricular pressure of the obtained rat model under anesthesia using a catheter to make an analogy to the pulmonary artery pressure in humans.

[0017] In another aspect, this application provides the use of the aforementioned model animals in drug screening.

[0018] The animal model of idiopathic pulmonary fibrosis complicated with pulmonary hypertension obtained by the method of this application is low in cost, and the modeling results are stable and uniform, consistent with the disease occurrence and development pattern, providing a suitable research tool for studying the occurrence, development and intervention of this type of disease. Attached Figure Description

[0019] Figure 1 This is a three-dimensional structural diagram of the rat air anesthesia device of this application;

[0020] Figure 2 This is a schematic diagram of the rat air anesthesia mask of this application;

[0021] Figure 3-4 This is a schematic diagram illustrating the use of the rat air anesthesia device of this application;

[0022] Figure 5 This is a comparison chart of physiological indicators between the experimental group and the control group in this application;

[0023] Figure 6 This is a comparison chart of right ventricular hypertrophy index data between the experimental group and the control group in this application;

[0024] Figure 7 This is a comparison chart of the degree of pulmonary fibrosis between the experimental group and the control group in this application;

[0025] Figure 8 This is a comparison chart of the degree of vascular remodeling between the experimental group and the control group in this application. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] 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.

[0029] Among rodent laboratory animals, rats have similar systemic blood pressure to humans, are more tolerant of acute ischemia and hypoxia, and have stronger adaptability, survival ability and anti-infection ability than mice. They are also easier to obtain, do not require multiple people to cooperate, and are reasonably priced compared to other medium and large animals.

[0030] 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.

[0031] This application uses SD rats to establish an animal model of pulmonary fibrosis combined with pulmonary hypertension by intratracheal injection of bleomycin. The obtained animal model can rapidly reach the desired disease level, and its similarity to pulmonary hypertension caused by human idiopathic pulmonary fibrosis is evaluated by multiple indicators.

[0032] Example 1: Rat anesthesia device

[0033] Existing air anesthesia devices for rats are complex in structure, expensive, and mostly planar in design, making them difficult to administer via the airway when used in lung models. The applicant provides a simple air anesthesia device for rats that is well-suited to the rat's physiological structure. This device allows the rat to be held on an inclined plane, facilitating surgical procedures and airway infusion, while ensuring smooth flow of medication into the lungs.

[0034] See Figure 1-4The rat anesthesia device of this application includes: a back plate 10 for placing the rat's body. The back plate is connected to a base plate, making the back plate 10 inclined at an angle to the horizontal plane. A support plate or support column can also be used to make the back plate inclined to the horizontal plane. A metal wire 11 is provided on the back plate 10 to hold the rat's incisors during use. The rat anesthesia device also includes an anesthesia mask 20. The main body 21 of the anesthesia mask 20 is a hollow cylinder, with one end 22 connected to an air source and the other end being an inclined surface 23. A V-groove 24 is provided on the opposite side of the inclined surface end of the main body. The V-groove 24 cooperates with the metal wire 11 during use, allowing the anesthesia mask 20 to completely cover the rat's head.

[0035] During use, the rat's back is first placed against the backplate 10, and the rat's incisors are hooked onto the metal wire 11. Then, the air anesthesia mask 20 is moved from the top of the rat's head along the direction of the rat's body, allowing the metal wire 11 to enter the V-groove 24. This movement continues until the metal wire 11 reaches the bottom of the V-groove 24. After anesthesia, the rat's own weight and the weight of the air anesthesia mask 20 are sufficient to maintain their fit, eliminating the need for a separate fixation mechanism.

[0036] In some embodiments, the body 21 of the air-assisted mask has a diameter of approximately 2.5 cm. In some embodiments, the angle between the inclined surface 23 and the body 21 is 45-60 degrees. In some embodiments, the V-groove 24 is located in the lower middle part of the inclined surface and has a depth of approximately 2 cm.

[0037] Example 2: Model Building

[0038] I. Experimental Materials

[0039] 1. Laboratory animals

[0040] Commercially purchased SD rats were used, sourced 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 were reviewed and approved by the Animal Ethics Committee of the Clinical Research Institute of China-Japan Friendship Hospital.

[0041] 2. Reagents

[0042] Bleomycin sulfate solution: Dissolve 3 mg of bleomycin sulfate (Selleck, S1214) powder in 1 mL of sterile PBS (phosphate buffer). After complete dissolution, transfer to a 15 mL centrifuge tube. Rinse the test tube with 1 mL of sterile PBS and transfer the liquid to the 15 mL centrifuge tube. Then, use sterile PBS to bring the total drug volume to 6000 mL to prepare a 0.5 mg / mL bleomycin sulfate solution. Keep the prepared drug system on ice for later use.

[0043] Medical 75% alcohol, medical iodine tincture, isoflurane.

[0044] 3. Instruments

[0045] Surgical instruments and laboratory equipment included: scissors, curved forceps, needle holder, 6-0 surgical sutures, sterile surgical drapes, an adjustable-angle small animal operating table, an anesthesia machine, a homemade rat anesthesia mask, adhesive tape, several cotton balls, a surgical light, a warming lamp, a 100μL microsyringe, a 26G long-tipped adhesive dispensing needle, a 200μL pipette, 200μL pipette tips, and 1.5mL centrifuge tubes. All instruments were autoclaved the day before surgery.

[0046] II. Operating Procedures

[0047] Six-week-old male SD rats were selected and divided into a model group and a control group, with six rats in each group. A 12 / 12 light / dark cycle was used, and the rats had free access to water and food. The housed temperature was constant at 26℃, and the room humidity was 50%. Each rat was ear-tagged.

[0048] Before starting the modeling process, place the prepared drug system on ice for later use.

[0049] 1. Bleomycin administered via airway

[0050] 1) Select rats, weigh and record their weight and ear tags. Place them in the induction chamber of an air anesthesia machine and induce anesthesia using 3% isoflurane at a flow rate of 0.3 L / min. Keep the prepared drug system on ice for later use;

[0051] 2) Use a pipette to draw 0.5 mg / mL bleomycin sulfate solution (4 μL of rat body weight) into a 1.5 mL centrifuge tube, and use a 100 μL microsyringe with a bent 26G long-tipped dispensing needle to draw up the solution and place it on ice for later use.

[0052] 3) After the rat is fully anesthetized, remove the rat, fix its incisors, put on an anesthesia mask, and fix it to the operating table with tape. Adjust the isoflurane concentration to 2%.

[0053] 4) Disinfect the center of the rat's neck with iodine-soaked cotton balls, and then remove the iodine with alcohol-soaked cotton balls, always from the center outwards.

[0054] 5) Use scissors to make a vertical opening about 1 cm long in the middle of the rat's neck. Use forceps to bluntly separate the subcutaneous connective tissue to both sides to expose the muscle covering the trachea. Use forceps to bluntly separate the muscle tissue to both sides to expose the trachea. Use a 1 mL syringe needle to make a small hole obliquely above the rat's thyroid cartilage process.

[0055] 6) Adjust the isoflurane concentration to 1.5%, then insert the dispensing needle into the trachea through the small hole and slowly inject the drug solution while observing the rat's respiratory rhythm. The rat should breathe faster at this time. If the rat experiences respiratory distress or apnea, immediately stop injecting the drug and continue administering it after the rat's breathing recovers. The drug solution for the experimental group was 0.5 times its body weight × 4 (μL).

[0056] The control group rats received a bleomycin sulfate solution at a concentration of mg / mL, while the control group rats received the corresponding volume of PBS.

[0057] 7) After the drug injection is completed, use a needle holder and 4-0 surgical sutures to suture the rat's neck. Use simple interrupted sutures, and suture 3 to 4 times. After suturing, disinfect the rat's neck with iodine-soaked cotton balls and alcohol-soaked cotton balls;

[0058] 8) Turn off the gas anesthesia machine, remove the still unconscious rat from the operating table, and lay it flat in the rat cage.

[0059] Turn on the warm light and wait for the rat to wake up and its breathing and heartbeat to return to normal before leaving.

[0060] A rat model of idiopathic pulmonary fibrosis complicated with pulmonary hypertension was obtained after four weeks of free access to water and food. Clinical indicators were then assessed in the rats.

[0061] III. Model Results

[0062] 1. Physiological indicator data

[0063] On day 27 post-injection, all rats underwent hair removal of the chest and abdomen and chest ultrasound to assess the severity of pulmonary hypertension by measuring pulmonary artery acceleration time to ejection time.

[0064] On day 28 after injection, under anesthesia, dynamic compliance, static compliance, vital capacity, forced vital capacity, and forced expiratory volume in 100 ms were measured using a pulmonary function instrument to assess changes in respiratory function in rats and to compare them with the lung function of patients.

[0065] Measuring right ventricular pressure in rats under gas anesthesia via catheterization provides a more accurate measurement and facilitates comparison with human pulmonary artery pressure. Figure 5 The physiological indicators of the experimental group and the control group are compared. Among them, PAT / PET indicates the ratio of lung acceleration time (PAT) to lung ejection time (PET), CDYN indicates dynamic lung compliance, CCHORD indicates static lung compliance, VC indicates vital capacity, FVC indicates forced vital capacity, FEV100 indicates forced expiratory volume in 100 ms, and RVSP indicates right ventricular systolic pressure.

[0066] 2. Cardiac hypertrophy index

[0067] The right ventricular hypertrophy index (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.

[0068] 1) On the 28th day after injection, arterial blood was drawn through the abdominal aorta under gas anesthesia, the rats were sacrificed, the pulmonary circulation was lavaged with heparinized saline, the lungs were then removed, the right lung was flash-frozen with liquid nitrogen, and the left lung was perfused with formalin.

[0069] 2) 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;

[0070] 3) The right ventricular wall (RV) is freed from the pulmonary artery outlet; the remainder is the left ventricle +

[0071] Ventricular septal tissue (LV+S);

[0072] 4) After the filter paper has absorbed the moisture, weigh each of the filters separately.

[0073] 5) 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.

[0074] When pulmonary hypertension occurs, the right side of the heart must forcefully push blood through the pulmonary artery, and over time, the right ventricle thickens and enlarges. Figure 6 The comparison of right ventricular hypertrophy index data between the experimental group and the control group shows that the experimental group developed pulmonary hypertension, which persisted for a long period of time.

[0075] 3. Sliced ​​data

[0076] After the left lung was fully fixed, it was embedded and sectioned, and Masson and α-SMA immunohistochemical staining were performed to calculate the degree of pulmonary fibrosis and vascular remodeling. Figure 7 The data show a comparison of the degree of pulmonary fibrosis between the experimental group and the control group. Figure 8 The data compare the degree of vascular remodeling between the experimental and control groups. Bleomycin or BLM indicates the experimental group, and PBS or CON indicates the control group. The degree of pulmonary fibrosis and vascular muscularization was significantly higher in the experimental group than in the control group.

[0077] 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.

[0078] 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.

[0079] 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 idiopathic pulmonary fibrosis complicated with pulmonary hypertension, comprising: Six-week-old male SD rats were given free access to water and food. The rat was anesthetized using a rat air anesthesia device, and bleomycin sulfate solution was injected into the rat's airway. The concentration of the bleomycin sulfate solution was 0.5 mg / mL, and the volume of the bleomycin sulfate solution was 4 μL / g of rat body weight. The rats were allowed free access to water and food for 4 weeks to obtain a rat model of idiopathic pulmonary fibrosis complicated with pulmonary hypertension. The rat gas anesthesia device includes: A backboard, which is connected to a base plate or a support plate, is inclined at an angle to the horizontal plane and is used to place the rat's body. Metal wires are fixed to the back plate and used to hang rat incisors during use; The air-induced anesthesia mask has a hollow cylindrical body with a diameter of 2.5 cm. One end is connected to an air source, and the other end is a sloping surface. A V-shaped groove is provided on the opposite side of the sloping surface of the main body. The V-shaped groove is located in the lower middle part of the sloping surface and has a depth of 2 cm. When in use, the V-shaped groove cooperates with the metal wire so that the air-induced anesthesia mask can completely cover the rat's head.

2. The method as described in claim 1, wherein the angle between the inclined plane and the main body is 45-60 degrees, and the angle between the back plate and the horizontal plane is 60-75 degrees.

3. The method of claim 1 or 2 further includes performing cardiovascular ultrasound on the obtained rat model to compare with human detection indicators.

4. The method of claim 1 or 2, further comprising measuring the right ventricular pressure of the obtained rat model under anesthesia using a metal catheter to make an analogy to the pulmonary artery pressure in humans.

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