A method for constructing an animal model of chronic obstructive pulmonary disease combined with pulmonary arterial hypertension

Abstract

A mouse air-anesthetized breathing mask for constructing a mouse animal model is provided. A method for constructing an animal model of chronic obstructive pulmonary disease (COPD) complicated with pulmonary hypertension is also provided, comprising: taking 6-week-old male BALB / c mice and allowing them free access to water and food; injecting porcine pancreatic elastase solution into the airways of the mice; starting smoke exposure 1 day later and continuing smoke exposure for 8 weeks to obtain a mouse model of COPD complicated with pulmonary hypertension.

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 obstructive pulmonary disease 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] Chronic obstructive pulmonary disease (COPD) is the most common cause of type III pulmonary embolism (PH), which leads to long-term damage to the lung parenchyma or interstitium, hypoxia, and secondary damage to the pulmonary vascular bed, thereby causing PH. The pathophysiological mechanisms of this type of PH involve multiple aspects, including hypoxia-related pulmonary vasoconstriction / remodeling, vascular endothelial and smooth muscle dysfunction, inflammation, and hypercoagulable state.

[0006] To address this real-world clinical problem, there is an urgent need to study the pathogenesis of COPD complicated with pulmonary hypertension (COPD-PH), which necessitates animal disease models that can reflect the patient's condition.

[0007] Currently, some animal models of COPD-PH differ from the pathogenesis of COPD, making it difficult to simulate the development and progression of COPD and hindering the exploration of disease mechanisms. Other models are time-consuming and costly to establish; furthermore, the disease induced by these models is often mild and progresses inconsistently, making it difficult to stably assess COPD-PH. In addition, the details of existing model establishment are not standardized, and the conditions that lead to pulmonary hypertension are unclear, resulting in inconsistent modeling processes and effects.

[0008] Previous animal models were limited to idiopathic pulmonary hypertension, COPD, or emphysema, which could not accurately simulate the development and progression of the disease in patients. The lack of a stable COPD-PH model has resulted in a lack of research on this disease.

[0009] This application aims to propose a mouse disease model that can simulate COPD complicated with pulmonary hypertension, providing a suitable research tool for studying the occurrence, development, and intervention of this disease. Summary of the Invention

[0010] Existing air anesthesia devices for mice are complex in structure, expensive, and mostly planar in design, making them difficult to infuse into the airway when used in lung models. The applicant provides a simple air anesthesia device for mice that adapts well to the physiological structure of mice. This device allows the mouse to be held on an inclined plane, facilitating surgical procedures and airway infusion, and allowing the medication to flow smoothly into the lungs.

[0011] The mouse anesthesia device of this application includes: a back plate for placing the mouse body, wherein the back plate is connected to a base plate, making the back plate 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. The mouse anesthesia device also includes an anesthesia mask and a mask fixing mechanism, wherein the mask fixing mechanism fixes the anesthesia mask to the back plate.

[0012] One end of the air-assisted anesthesia mask is a sloping funnel, and the other end is an air tube connector that connects to an air source. A wire is installed inside the sloping funnel, passing through the junction of the sloping funnel and the air tube connector, and has a distal end.

[0013] The inclined funnel, when used in conjunction with the line, allows the air-anesthesia mask to completely cover the mouse's head. During use, the mouse's back is first placed against the backplate, and the mouse's incisors are hooked onto the line. Then, the distal end of the line is pulled, causing the mouse's body to move along the line until the mouse's head enters the inclined funnel. After anesthesia, the mouse's own weight is sufficient to maintain its contact with the air-anesthesia mask, eliminating the need for a separate fixation mechanism.

[0014] In some embodiments, the friction between the thread and the air-mask is sufficient to support the mouse's own weight, thus eliminating the need to secure the distal end of the thread. In other embodiments, the backplate is provided with protrusions for securing the distal end, thereby enhancing the fixation of the mouse.

[0015] In some embodiments, the diameter of the endotracheal connector of the air-assisted anesthesia mask is 0.5-0.8 cm. In some embodiments, the angle between the inclined surface of the slanted funnel and the axial direction of the endotracheal connector is 45-60 degrees. In some embodiments, the length of the inclined surface of the slanted funnel is approximately 2 cm.

[0016] In another aspect, this application provides a method for constructing an animal model of chronic obstructive pulmonary disease (COPD) complicated with pulmonary hypertension, which includes: taking 6-week-old male BALB / c mice and allowing them free access to water and food; injecting porcine pancreatic elastase solution into the airways of the mice; starting smoke exposure 1 day later and continuing smoke exposure for 8 weeks to obtain a mouse model of COPD complicated with pulmonary hypertension.

[0017] In some embodiments, the concentration of the porcine pancreatic elastase solution is 2.5 mg / mL, and the volume of the porcine pancreatic elastase solution is 2 μL / g mouse body weight.

[0018] In some embodiments, the step of injecting porcine pancreatic elastase solution into the airway of the mouse uses the mouse air-anesthesia breathing mask as described above in this application.

[0019] In some implementations, the smoke exposure is a full-body smoke exposure for 6 days / week, lasting 2 hours each day.

[0020] In some implementations, cardiovascular ultrasound of the obtained mouse model is also included to make analogies to detection parameters in humans.

[0021] In some implementations, the method also includes measuring the right ventricular pressure of the obtained mouse model using a catheter under anesthesia to make an analogy with the pulmonary artery pressure in humans.

[0022] This application utilizes a dual-pronged approach of porcine pancreatic elastase and smoke exposure to create a low-cost, stable, uniform animal model that conforms to the disease's development and progression. Furthermore, this application designs a mouse air-anesthesia breathing mask to achieve the goal of keeping the mouse's body upright during modeling, enabling efficient surgical modeling at a lower cost. Attached Figure Description

[0023] Figure 1 This is a three-dimensional structural diagram of the mouse air anesthesia device of this application;

[0024] Figure 2 This is a schematic diagram of the structure of the mouse air anesthesia breathing mask of this application;

[0025] Figure 3 This is a schematic diagram illustrating the use of the mouse air anesthesia device of this application;

[0026] Figure 4 For the assessment of pulmonary vascular and right ventricular function in the experimental and control groups of this application;

[0027] Figure 5 For the lung function assessment of the experimental and control groups in this application;

[0028] Figure 6-7 The pathological changes in the experimental and control groups of this application are shown. 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] This application uses BALB / c mice to establish a COPD-PH animal model. BALB / c mice have albino coats and are one of the most widely used inbred mouse models in biomedical research, particularly in immunology and infectious disease studies. They exhibit the highest blood pressure compared to other inbred mouse strains and display spontaneous hypertension.

[0033] To better simulate the pathophysiological state of COPD-PH patients, this application combines airway injection of porcine pancreatic elastase and smoke exposure. Airway infusion of porcine pancreatic elastase alone results in a shorter and faster model establishment time, which differs from the pathogenesis of COPD, while smoke exposure alone is more time-consuming. By combining the two methods, the pathogenesis of COPD can be better simulated, and the desired disease severity can be rapidly achieved, thus realizing a stable COPD-PH animal model.

[0034] Example 1: Mouse Air Anesthesia Device

[0035] Existing air anesthesia devices for mice are complex in structure, expensive, and mostly planar in design, making them difficult to infuse into the airway when used in lung models. The applicant provides a simple air anesthesia device for mice that adapts well to the physiological structure of mice. This device allows the mouse to be held on an inclined plane, facilitating surgical procedures and airway infusion, and allowing the medication to flow smoothly into the lungs.

[0036] See Figure 1-3 The mouse anesthesia device of this application includes: a back plate 10 for placing the mouse body, wherein 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. The mouse anesthesia device also includes an anesthesia mask 20 and a mask fixing mechanism 11, wherein the mask fixing mechanism 11 fixes the anesthesia mask to the back plate 10.

[0037] One end of the air-assisted mask 20 is a sloping funnel 21, and the other end is an air tube connector 22, which is connected to an air source. A wire 23 is provided inside the sloping funnel 21, and the wire 23 passes through the junction of the sloping funnel 21 and the air tube connector 22 and has a distal end 24.

[0038] The inclined funnel 21, when in use, engages with the thread 23 to allow the air anesthesia mask 20 to completely cover the mouse's head. During use, the mouse's back is first placed against the backplate 10, and the mouse's incisors are hooked onto the thread 23. Then, the distal end 24 of the thread 23 is pulled, causing the mouse's body to move along the thread until the mouse's head enters the inclined funnel 21. After anesthesia, the mouse's own weight is sufficient to maintain its contact with the air anesthesia mask 20, eliminating the need for a separate fixation mechanism.

[0039] In some embodiments, the friction between the thread 23 and the air-mask 20 is sufficient to support the mouse's own weight, thus eliminating the need to secure the distal end 24 of the thread 23. In other embodiments, the backplate 10 is provided with protrusions 12 for securing the distal end 24, thereby enhancing the fixation of the mouse.

[0040] In some embodiments, the diameter of the endotracheal connector 22 of the air-assisted anesthesia mask is 0.5-0.8 cm. In some embodiments, the angle between the inclined surface of the inclined funnel 21 and the axial direction of the endotracheal connector 22 is 45-60 degrees. In some embodiments, the length of the inclined surface of the inclined funnel 21 is approximately 2 cm.

[0041] Example 2: Model Building

[0042] I. Experimental Materials

[0043] 1. Laboratory animals

[0044] Commercially available BALB / c mice were used and purchased from Beijing Huafukang Biotechnology 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.

[0045] 2. Reagents

[0046] Porcine pancreatic elastase solution: Dissolve 5 mg of porcine pancreatic elastase (Sigma, E7885) powder in 2 mL of sterile PBS (phosphate buffer) to prepare a 2.5 mg / mL porcine pancreatic elastase solution.

[0047] Marlboro cigarettes (soft red 2.0), 75% medical alcohol, medical iodine, isoflurane.

[0048] 3. Instruments

[0049] 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 mouse anesthesia mask, adhesive tape, several cotton balls, a surgical light, a warm light, a 100 μL microsyringe, a 26G long-tipped adhesive dispensing needle, a 200 μL pipette, 200 μL pipette tips, and 1.5 mL centrifuge tubes. All instruments were autoclaved the day before surgery.

[0050] The structure of the mouse air paralysis mask is as shown in Example 1 and... Figure 1-3 As shown.

[0051] II. Operating Procedures

[0052] Six-week-old male BALB / c mice (weighing 20g-22g) were selected and divided into a model group and a control group, with six mice in each group. A 12 / 12 light / dark cycle was used, and the mice had free access to water and food. The housed mice were kept at a constant temperature of 26℃ and a room humidity of 50%, and each mouse was ear-tagged.

[0053] Before starting the modeling process, place the prepared drug system on ice for later use.

[0054] 1. Intratracheal injection of porcine pancreatic elastase

[0055] 1) Select mice, 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;

[0056] 2) Use a pipette to draw 2.5 mg / mL of porcine pancreatic elastase solution (2 μL of mouse 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.

[0057] 3) After the mouse is fully anesthetized, remove the mouse, fix its incisors, put on an anesthesia mask, and fix it to the operating table with tape. Adjust the isoflurane concentration to 2%.

[0058] 4) Disinfect the center of the mouse's neck with iodine-soaked cotton balls, and then remove the iodine with alcohol-soaked cotton balls, always from the center outwards.

[0059] 5) Use scissors to make a vertical opening in the middle of the mouse's neck, about 3 mm long. 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 thyroid cartilage process of the mouse.

[0060] 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 mouse's respiratory rhythm. The mouse should breathe faster at this time. If the mouse experiences respiratory distress or apnea, immediately stop injecting the drug and continue administering it after the mouse's breathing recovers. The experimental group received a 2.5 mg / mL solution of porcine pancreatic elastase at a concentration of 2 μL (body weight), while the control group received the corresponding volume of PBS.

[0061] 7) After the drug injection is completed, use a needle holder and 6-0 surgical sutures to suture the mouse's neck. Use simple interrupted sutures, and suture 3 to 4 stitches. After suturing, disinfect the mouse's neck with iodine-soaked cotton balls and alcohol-soaked cotton balls;

[0062] 8) Turn off the gas anesthesia machine, remove the unaware mouse from the operating table, place it flat in the mouse cage, turn on the heat lamp to irradiate it, and leave only when the mouse wakes up and its breathing and heartbeat return to normal.

[0063] 2. Smoke exposure

[0064] Starting from the second day after injection, the experimental group mice were exposed to whole-body smoke using Marlboro cigarettes (Soft Red 2.0) and a DSI smoke exposure device, once in the morning and once in the afternoon, for 2 hours each time, for 6 days a week. The mice were weighed weekly to record changes in body weight.

[0065] Eight weeks after injection, a mouse model of chronic obstructive pulmonary disease complicated with pulmonary hypertension was obtained. Clinical indicators were then measured.

[0066] III. Model Results

[0067] 1. Assessment of pulmonary vascular and right ventricular function

[0068] On day 55 after injection, hair was removed from the chest and abdomen of all mice and chest ultrasound was performed to detect indicators such as right ventricular free wall thickness, pulmonary artery blood flow acceleration time, and tricuspid annulus systolic displacement, thereby enabling multi-dimensional assessment of pulmonary vascular and right ventricular function.

[0069] On day 56 after injection, the right ventricular pressure of mice was measured using a catheter under gas anesthesia. This method of measuring the right ventricular pressure of mice is more accurate and easier to compare with the pulmonary artery pressure in humans.

[0070] 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.

[0071] 1) On day 56 after injection, arterial blood was drawn through the apex of the heart under gas anesthesia. The mice were then sacrificed, and 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.

[0072] 2) Separate the heart from the mouse after lung removal, rinse off the blood with physiological saline, and cut off the atria and residual blood vessels;

[0073] 3) The right ventricular wall (RV) is freed from the pulmonary artery outlet, and the rest is left ventricle + interventricular septum tissue (LV + S);

[0074] 4) After the filter paper has absorbed the moisture, weigh each of the filters separately;

[0075] 5) According to the formula RVHI=RV / [LV+S], when the pulmonary artery pressure increases, the right ventricle undergoes compensatory hypertrophy due to the increased afterload.

[0076] Figure 4 The data compare the experimental group (COPD) and the control group (CON). The right ventricular systolic pressure was significantly higher in the experimental group mice. Figure 4 A), the right ventricular hypertrophy index was significantly elevated ( Figure 4 B), the PAT / PET ratio indicated a significantly reduced ratio of lung acceleration time (PAT) to lung ejection time (PET) (Figures C and F), and a significantly reduced systolic displacement of the tricuspid annulus (Figures D and G). In summary, compared with the control group, the experimental group mice showed increased pulmonary artery pressure and significantly decreased right ventricular function, consistent with the clinical characteristics of pulmonary hypertension.

[0077] 2. Lung function assessment

[0078] On day 56 post-injection, lung function changes in mice were assessed using a pulmonary function instrument under tribromoethanol anesthesia.

[0079] Figure 5 The data comparison between the experimental group and the control group is shown. The ratio of forced expiratory volume in 50 ms (FEV50) to forced vital capacity (FVC), FEV50 / FVC, was significantly lower in the experimental group mice. Figure 5 A), Functional Residual Gas (FRC) increased significantly ( Figure 5 B), inspiratory volume (IC) and vital capacity (VC) were significantly increased ( Figure 5 C, D), peak expiratory flow (PEF), flow rate at 50% forced expiratory capacity (FEF50), and maximum mid-expiratory flow rate (MMEF) were significantly reduced. Figure 5 (EG). In summary, compared with the control group, the experimental group mice showed a significant decrease in all lung function indicators, consistent with the clinical characteristics of chronic obstructive pulmonary disease.

[0080] 3. Sliced ​​data

[0081] After the left lung was fully fixed, it was embedded and sectioned, and H&E and α-SMA immunohistochemical staining were performed to calculate the degree of inflammation, emphysema and vascular remodeling.

[0082] Figure 6-7 The data comparison between the experimental group and the control group is shown. Figure 6 The comparison of vascular remodeling levels between the experimental and control groups is shown. Figure 7The changes in lung tissue structure between the experimental and control groups are shown. MLI (mean lining septum) reflects the mean alveolar diameter, which was significantly higher in the experimental group than in the control group. COPD indicates the experimental group, and CON indicates the control group. The degree of emphysema and vascular muscularization in the experimental group were significantly higher than those in the control group, consistent with the clinical characteristics of pulmonary hypertension associated with chronic obstructive pulmonary disease.

[0083] 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.

[0084] 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.

[0085] 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 obstructive pulmonary disease complicated with pulmonary hypertension, comprising: Six-week-old male BALB / c mice were given free access to water and food. The mice were injected via airway with porcine pancreatic elastase solution. Smoke exposure began one day later and continued for eight weeks to establish a mouse model of chronic obstructive pulmonary disease complicated with pulmonary hypertension. The smoke exposure referred to here is whole-body smoke exposure for 2 hours each day for 6 days per week. The concentration of the porcine pancreatic elastase solution was 2.5 mg / mL, and the volume of the porcine pancreatic elastase solution was 2 μL / g mouse body weight. The step of injecting porcine pancreatic elastase solution into the airway of the mouse uses a mouse air-anesthetized breathing mask comprising the following structure: A backplate, which is inclined at an angle to the horizontal plane, is used to place the mouse body; An air-induced anesthesia mask is fixed to the back plate, and a string is provided inside the air-induced anesthesia mask for attaching it to the mouse's incisors to secure the mouse's head. The air-assisted anesthesia mask includes an inclined funnel and an air tube connector. The inclined funnel is connected to the air tube connector. A wire is disposed on the funnel, passing through the position between the inclined funnel and the air tube connector, and has a distal end. The back plate is also provided with a protrusion, and the distal end of the line is hung on the protrusion.

2. The method according to claim 1, characterized in that, The angle between the inclined surface of the inclined funnel and the axial direction of the tracheal connector is 45-60 degrees.

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

4. The method according to claim 1 or 2 further comprises measuring the right ventricular pressure of the obtained mouse model under anesthesia using a catheter to make an analogy with the pulmonary artery pressure in humans.

Images