Construction method of idiopathic pulmonary fibrosis and pulmonary arterial hypertension combined animal model

Abstract

The rat numbness device comprises a back plate, the back plate is connected with a bottom plate or a supporting plate, an inclined angle is formed between the back plate and the horizontal plane, and the back plate is used for containing the body of a rat; the metal wire is fixed on the back plate and is hung on the incisor of the rat during use; the main body of the gas anesthesia mask is a hollow cylinder, one end of the gas anesthesia mask is connected with a gas source, the other end of the gas anesthesia mask is an inclined plane, a v-shaped groove is formed in the side, opposite to the inclined plane end, of the main body, and the v-shaped groove is matched with the metal wire when the gas anesthesia mask is used, so that the head of the rat can be completely covered with the gas anesthesia mask. The invention also provides a construction method of the idiopathic pulmonary fibrosis combined pulmonary hypertension animal model, which comprises the following steps: taking a 6-week SD male rat, and freely taking water and food; the method comprises the following steps: anesthetizing a rat by using a rat gas anesthesia device, and injecting a bleomycin sulfate solution into the airway of the rat; and enabling the rat to freely ingest water and food for 4 weeks to obtain the idiopathic pulmonary fibrosis combined pulmonary arterial 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 idiopathic pulmonary fibrosis combined with pulmonary hypertension. Background Art

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

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

[0004] Clinically, PH is divided into five categories: (1) PH due to 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] 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 gradually worsening dyspnea without any new shadows on imaging.

[0006] Previous animal models only had idiopathic pulmonary hypertension or idiopathic pulmonary fibrosis animal models, which could not accurately simulate the occurrence, development and changes of the disease in patients. Existing animal models of idiopathic pulmonary fibrosis have different administration methods, and the number of administrations, frequency, dosage and modeling time are not uniform. Even with the same administration method, the experimental schemes are not exactly the same, the experimental results vary greatly, and the results are often unable to be reproduced. Therefore, the existing modeling methods have the disadvantages of many modeling steps, cumbersome procedures, unstable modeling, difficult to reproduce, and mild disease severity. At the same time, their evaluation methods are also relatively simple and cannot be standardized.

[0007] This application hopes to propose a rat disease model that can simulate idiopathic pulmonary fibrosis combined with pulmonary hypertension, providing a suitable research tool for studying the occurrence, development and intervention of such diseases. Summary of the invention

[0008] In another aspect, the present application provides a rat gas anesthesia device, comprising:

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

[0010] In some embodiments, the body diameter of the gas anesthetic mask is about 2.5 centimeters.

[0011] In some embodiments, the angle between the inclined surface 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-shaped groove is located in the lower middle portion of the slope, and the depth of the V-shaped groove is about 2 cm.

[0013] On the other hand, the present application provides a method for constructing an animal model of idiopathic pulmonary fibrosis combined with pulmonary hypertension, which comprises: taking 6-week-old SD male rats and allowing them to freely consume water and food; anesthetizing the rats using a rat gas anesthesia device and injecting bleomycin sulfate solution into the airways of the rats; allowing the rats to freely consume water and food for 4 weeks to obtain a rat model of idiopathic pulmonary fibrosis combined 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 embodiments, the method further comprises performing cardiovascular ultrasound on the obtained rat model to compare with human detection indicators.

[0016] In some embodiments, the method further comprises measuring the right ventricular pressure of the rat under gas anesthesia using a catheter to compare it with the pulmonary artery pressure of humans.

[0017] In yet another aspect, the present application provides use of the above-mentioned model animals in screening drugs.

[0018] The animal model of idiopathic pulmonary fibrosis combined with pulmonary hypertension obtained by the method of the present application is low-cost, and the modeling results are stable and uniform, which conforms to the laws of disease occurrence and development, and provides a suitable research tool for studying the occurrence, development and intervention of such diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the three-dimensional structure of the rat gas anesthesia device of the present application;

[0020] Figure 2 A schematic diagram of a rat gas anesthesia mask of the present application;

[0021] Figure 3-4 A schematic diagram of the use of the rat gas anesthesia device of the present application;

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

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

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

[0025] Figure 8 This is a comparison chart of the vascular remodeling degree data between the experimental group and the control group of this application. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] Among rodent experimental animals, rats have 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.

[0030] 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.

[0031] In the present application, an animal model of pulmonary fibrosis combined with pulmonary hypertension is established by injecting bleomycin into the airways of SD rats. The obtained animal model can quickly reach the desired disease severity, and is similar to pulmonary hypertension caused by human idiopathic pulmonary fibrosis as evaluated by multiple indicators.

[0032] Example 1: Rat gas anesthesia device

[0033] Existing rat gas anesthesia devices have complex structures, high costs, and are mostly flat, which makes it difficult to perfuse the airway when used in lung models. The applicant provides a rat gas anesthesia device with a simple structure that can well adapt to the physiological structure of rats, which can keep the rat on an inclined plane, facilitate surgical operations and airway instillation operations, and allow the drug solution to flow smoothly into the lungs.

[0034] See also Figure 1-4The rat gas anesthesia device of the present application includes: a backboard 10, which is used to place the rat body. In the figure, the backboard is connected to a bottom plate, so that the backboard 10 is inclined at an angle to the horizontal plane. A support plate or a support column can also be used to make the backboard inclined to the horizontal plane. A metal wire 11 is arranged on the backboard 10, which is hung on the rat's incisors when in use. The rat gas anesthesia device also includes a gas anesthesia mask 20, the main body 21 of the gas anesthesia mask 20 is a hollow cylinder, one end 22 is connected to the gas source, and the other end is an inclined surface 23. A V-shaped groove 24 is arranged on the opposite side of the main body at the inclined surface end. The V-shaped groove 24 cooperates with the metal wire 11 when in use, so that the gas anesthesia mask 20 can completely cover the rat's head.

[0035] During use, the rat's back is first attached to the back plate 10, and the rat's front teeth are hung on the metal wire 11. Then, the gas anesthesia mask 20 is moved from the top of the rat's head along the extension direction of the rat's body, so that the metal wire 11 enters the V-shaped groove 24. The movement is maintained until the metal wire 11 reaches the bottom of the V-shaped groove 24. After anesthesia, the rat's own weight and the gas anesthesia mask 20 can maintain the two in coordination, without the need for a separate fixing mechanism.

[0036] In some embodiments, the body 21 of the gas anesthesia mask has a diameter of about 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-shaped groove 24 is located in the middle and lower part of the inclined surface and has a depth of about 2 cm.

[0037] Example 2: Model construction

[0038] 1. Experimental Materials

[0039] 1. Experimental Animals

[0040] Commercially available 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 reviewed and approved by the Animal Ethics Committee of the Clinical Research Institute of the 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 buffered saline), transfer to a 15 mL centrifuge tube after complete dissolution, use 1 mL of sterile PBS to wash the reagent tube and transfer the liquid to a 15 mL centrifuge tube, then use sterile PBS to make up the total volume of the drug solution to 6000 mL to prepare a 0.5 mg / mL bleomycin sulfate solution. Place the prepared drug system on ice for later use.

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

[0044] 3. Equipment

[0045] Surgical instruments and experimental tools include: scissors, curved forceps, needle holder, 6-0 surgical suture, sterile surgical drape, adjustable angle small animal operating table, gas anesthesia machine, homemade rat gas anesthesia mask, tape, cotton balls, surgical lamp, warm lamp, 100μL micro syringe, 26G long-head dispensing needle, 200μL pipette, 200μL pipette tip, 1.5mL centrifuge tube. The above instruments were steam sterilized one day before the operation.

[0046] 2. Operation steps

[0047] Six-week-old SD male rats were selected and divided into a modeling group and a control group, with 6 rats in each group. A 12 / 12 light and night cycle was used, and the rats were free to drink water and eat. The breeding temperature was constant at 26°C, the room humidity was 50%, and each rat was ear-tagged.

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

[0049] 1. Airway injection of bleomycin

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

[0051] 2) Use a pipette to draw 0.5 mg / mL bleomycin sulfate solution (weight of rat × 4 (μL)) into a 1.5 mL centrifuge tube, use a 100 μL microsyringe with a bent 26G long-tip dispensing needle to draw the solution, and place 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, and adjust the isoflurane concentration to 2%;

[0053] 4) Use iodine cotton balls to disinfect the center of the rat's neck, and then use alcohol cotton balls to deiodine, in the order of from the center to both sides;

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

[0055] 6) Adjust the isoflurane concentration to 1.5%, then insert the dispensing needle into the trachea through the small hole, slowly push the drug solution, and observe the rat's breathing rhythm at the same time. At this time, the rat's breathing should accelerate. If the rat has difficulty breathing or respiratory arrest, stop pushing the drug immediately, and continue to administer the drug after the rat's breathing recovers. The drug solution for the experimental group is 0.5 of body weight × 4 (μL)

[0056] mg / mL bleomycin sulfate solution, and the control group rats received the corresponding volume of PBS;

[0057] 7) After the drug injection is completed, the rat's neck is sutured with 4-0 surgical sutures using a needle holder, and simple interrupted sutures are used for 3 to 4 stitches. After the suturing is completed, the rat's neck is disinfected with iodine cotton balls and alcohol cotton balls;

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

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

[0060] The rats were allowed to freely take in water and food, and after 4 weeks of breeding, a rat model of idiopathic pulmonary fibrosis combined with pulmonary hypertension was obtained, and clinical indicators were tested.

[0061] 3. Model Results

[0062] 1. Physiological index data

[0063] On the 27th day after injection, the chest and abdomen of all rats were depilated and chest ultrasound was performed, and the pulmonary artery acceleration time and ejection time were measured to evaluate the severity of pulmonary hypertension.

[0064] On the 28th day after injection, dynamic compliance, static compliance, vital capacity, forced vital capacity and 100ms forced expiratory volume were measured using a pulmonary function meter under anesthesia to determine changes in the rats' respiratory function for comparison with the patients' lung function.

[0065] The right ventricular pressure of rats was measured using a catheter under gas anesthesia. This method is more accurate and convenient for comparison with human pulmonary artery pressure. Figure 5 The comparison of physiological index data between the experimental group and the control group is shown. 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 100ms, and RVSP indicates right ventricular systolic pressure.

[0066] 2. Cardiac hypertrophy index

[0067] Right ventricular hypertrophy index (RVHI) is a commonly used hemodynamic index 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 from the abdominal aorta under gas anesthesia, and then the rats were killed. The pulmonary circulation was lavaged with heparinized saline, and then the lungs were taken out. The right lung was quickly frozen with liquid nitrogen, and the left lung was perfused with formalin.

[0069] 2) The heart was separated from the rat after the lungs were removed, the blood was flushed out with saline, and the atrium and remaining blood vessels were cut off;

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

[0071] ventricular septum tissue (LV+S);

[0072] 4) After the filter paper absorbs the water, weigh each weight;

[0073] 5) According to the formula RVHI = RV / [LV+S], when the pulmonary artery pressure increases, the right ventricle undergoes compensatory hypertrophy due to increased afterload.

[0074] In pulmonary hypertension, the right side of the heart has to work harder to push blood through the pulmonary arteries, and over time, the right ventricle becomes thicker and larger. Figure 6 The data of right ventricular hypertrophy index between the experimental group and the control group are shown, indicating that pulmonary hypertension occurred in the experimental group and persisted for a long time.

[0075] 3. Slice data

[0076] After the left lung was fully fixed, it was embedded and sectioned, and Masson and α-SMA immunohistochemical staining were performed, and the degree of pulmonary fibrosis and vascular remodeling were calculated, respectively. Figure 7 The data of the degree of pulmonary fibrosis between the experimental group and the control group are shown. Figure 8 The data of vascular remodeling degree between the experimental group and the control group are shown. Bleomycin or BLM indicates the experimental group, and PBS or CON indicates the control group. The degree of pulmonary fibrosis in the experimental group was significantly higher than that in the control group, and the degree of vascular muscularization was also significantly higher than that in the control group.

[0077] 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.

[0078] 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.

[0079] 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 rat gas anesthesia device, comprising: A back plate, the back plate is connected to a bottom plate or a support plate, the back plate is inclined at an angle to a horizontal plane, and the back plate is used to place the body of the rat; A metal wire fixed to the back plate for hanging rat incisors when in use; The gas anesthesia mask has a main body which is a hollow cylinder, one end of which is connected to the gas source and the other end of which is an inclined surface. A V-shaped groove is arranged on the opposite side of the main body at the inclined surface end. The V-shaped groove cooperates with the metal wire when in use, so that the gas anesthesia mask can completely cover the head of a rat.

2. The rat anesthesia device of claim 1, wherein the body diameter of the anesthesia mask is about 2.5 centimeters.

3. The rat gas anesthesia device as claimed 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.

4. The rat gas anesthesia device as claimed in claim 1, wherein the V-shaped groove is located in the lower middle part of the inclined surface, and the depth of the V-shaped groove is about 2 cm.

5. A method for constructing an animal model of idiopathic pulmonary fibrosis combined with pulmonary hypertension, comprising: Six-week-old SD male rats were given free access to water and food; Anesthetize a rat using the rat gas anesthesia device according to claims 1 to 4, and inject bleomycin sulfate solution into the airway of the rat; The rats were allowed to freely take in water and food for 4 weeks to obtain a rat model of idiopathic pulmonary fibrosis combined with pulmonary hypertension. 6 . The method according to claim 5 , wherein 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.

7. The method of claim 5, further comprising performing cardiovascular ultrasound on the obtained rat model to compare with human detection indicators.

8. The method according to claim 5, further comprising measuring the right ventricular pressure of the rat using a metal catheter under gas anesthesia in the obtained rat model to make an analogy with the pulmonary artery pressure of a human.

9. Use of the rat model obtained by the method of claims 5-8 in screening drugs for treating idiopathic pulmonary fibrosis combined with pulmonary hypertension.

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