Construction method of plateau pulmonary edema animal model

Abstract

The invention provides a construction method of a plateau pulmonary edema animal model. The construction method comprises the following steps: S1, establishing a simulation environment; s2, adjusting an initial environment state; s3, experimental animals are put in; s4, setting an environment variable; s5, preparation before detection; s6, detecting the BALF protein concentration and the W / D value of the lung tissue; s7, observing pathological changes; by accurately simulating factors such as sunlight, sunshine, rain, temperature difference change and carbon dioxide concentration of the plateau environment, a simulation space close to the real plateau environment is created for an experimental animal, and the animal is experimented in the simulation environment, so that the influence of the plateau environment change on the animal is more accurately reflected, and the experimental accuracy is improved. Finally, a more accurate plateau pulmonary edema animal model is established, the adaptability and stress response of organisms in the plateau environment can be simulated by elaborately simulating the plateau environment and accurately controlling the plateau environment, and important experimental data is provided for deeply knowing the action mechanism of the plateau environment on the organisms.

Description

Technical Field

[0001] The invention discloses a method for constructing an animal model of high altitude pulmonary edema, belonging to the technical field of animal model construction. Background Art

[0002] Pulmonary edema refers to a pathological state in which the amount of extrapulmonary fluid increases due to the accumulation of body fluid in the pulmonary interstitium or alveoli. It is mostly caused by increased permeability of the pulmonary capillary wall or increased intracapillary pressure. The clinical manifestations of acute pulmonary edema are sudden onset, dyspnea, cyanosis, cough, coughing up colorless or pink foam, diffuse moist rales in the lungs, and death from respiratory failure in severe cases. It is one of the clinical emergencies. Many factors can cause pulmonary edema, but its mechanism is still unclear. Therefore, experimental studies on the pathogenesis and related treatments are often carried out with the help of some animal models. Existing animal models of HAPE mainly use low-pressure and low-oxygen environments for simulation, attempting to induce HAPE by changing atmospheric pressure and oxygen concentration. However, plateau environmental changes are far more than these, including multiple factors such as changes in air pressure, low temperatures, and low-intensity ultraviolet radiation at plateaus. Existing models are limited to simulating low-oxygen environments and only provide low-pressure and low-oxygen environments for experiments. The environmental simulation is incomplete and cannot restore the actual changes in the plateau environment, resulting in low accuracy of the obtained animal models. In order to solve the above technical problems, a method for constructing an animal model of high altitude pulmonary edema was proposed. Summary of the invention

[0003] In view of this, the present invention provides a method for constructing an animal model of high altitude pulmonary edema to solve or alleviate the technical problems existing in the prior art and at least provide a beneficial option.

[0004] The technical solution of the present invention is achieved as follows: A method for constructing an animal model of high altitude pulmonary edema comprises the following steps: S1. Establish a simulation environment; S2, adjust the initial environment state; S3, input of experimental animals; S4. Set environment variables; S5. Preparation before testing; S6. Detect BALF protein concentration and lung tissue W / D value; S7. Observe pathological changes.

[0005] Further preferably, in said S1, when establishing the simulation environment, the simulation environment is constructed with actual plateau environmental information and environmental change cases as parameters, and the simulation of environmental information such as sunshine, rain, alternating cold and hot changes and carbon dioxide adsorption is completed in a closed environment.

[0006] Further preferably, in S2, the simulated environment is divided into a plateau cold group and a plateau normal temperature group, the parameters of the two groups of simulated environments are initialized, and the environmental parameters are set to the average state of the corresponding environment to avoid extreme environmental parameters at the beginning of the experiment.

[0007] Further preferably, in S3, 100 healthy rats are randomly divided into two groups, each with 50 rats. The two groups of rats are respectively put into the simulated environment, and each group is divided into five subgroups according to the length of time the rats stay in the environment, with 10 rats in each subgroup.

[0008] Further preferably, in S4, environmental changes are simulated, environmental parameters are changed, and extreme environments are randomly inserted to simulate abnormal environmental conditions.

[0009] Further preferably, in S5, the experimental animal is taken out at a corresponding time point after the start of the experiment, and alveolar lavage fluid is injected, and the alveolar lavage fluid and lung tissue are taken out after anesthesia.

[0010] Further preferably, in S6, the protein concentration of the alveolar lavage fluid and the W / D value of the lung tissue are tested for each group of experimental animals, and the data of each experimental animal in each group are entered into a chart for statistical analysis.

[0011] Further preferably, in S7, lung tissue slices are prepared and HE-stained to observe pathological changes, and lung tissue slices of normal experimental animals are obtained for comparison and reference.

[0012] The embodiment of the present invention has the following advantages due to the adoption of the above technical solution: 1. The present invention creates a simulation space close to the real plateau environment for experimental animals by accurately simulating multiple factors such as sunshine, sunlight, rain, temperature difference and carbon dioxide concentration in the plateau environment, allowing animals to experiment in the simulated environment, thereby more accurately reflecting the impact of plateau environmental changes on animals, and finally establishing a more accurate animal model of high-altitude pulmonary edema, which helps the study of the effects of plateau environmental factors on diseases, and observes the pathological changes of lung tissue through HE-stained pathological sections to compare the lung reactions and damage degrees of animals under different environmental conditions, so as to study the mechanism of the influence of plateau environment on organisms. By carefully simulating the plateau environment and precisely controlling it, the adaptability and stress response of organisms in the plateau environment can be simulated, providing important experimental data for a deep understanding of the mechanism of action of the plateau environment on organisms; 2. The present invention selects male animals as modeling animals. In altitude sickness, the symptoms of male animals are more obvious than those of female animals, and the size of the animal will also affect the animal's oxygen demand and oxygen consumption. Animals with larger volumes require more oxygen. The present invention targets modeling animals of specific weight to make them more susceptible to inducing the production of pulmonary edema.

[0013] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 is a flow chart of the method of the present invention; Figure 2 This is a flow chart of the method of Embodiment 4 of the present invention. DETAILED DESCRIPTION

[0016] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0017] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0018] Embodiment 1 like Figure 1 As shown, the embodiment of the present invention provides a method for constructing an animal model of high altitude pulmonary edema, comprising the following steps: S1. Establish a simulation environment, using actual plateau environmental information and environmental change cases as parameters to construct a simulation environment, and complete the simulation of environmental information such as sunshine, rain, alternating cold and hot changes, and carbon dioxide adsorption in a closed environment; S2. Adjust the initial environmental state, divide the simulated environment into a plateau cold group and a plateau normal temperature group, initialize the parameters of the two groups of simulated environments, and set the environmental parameters to the average state of the corresponding environment to avoid extreme environmental parameters at the beginning of the experiment; S3. Experimental animals were selected and 100 healthy rats were randomly divided into two groups, 50 rats in each group. The two groups of rats were placed in the simulated environment respectively, and each group was divided into five subgroups according to the length of stay in the environment. The five subgroups corresponded to stay for 8h, 24h, 48h, 72h and 120h, and there were 10 rats in each subgroup. S4, setting environmental variables, changing environmental parameters, and randomly inserting extreme environments to simulate abnormal environmental conditions; S5. Preparation before testing: remove the experimental animals at the corresponding time point after the start of the experiment, inject alveolar lavage fluid, and remove the alveolar lavage fluid and lung tissue after anesthesia; S6. Detecting the BALF protein concentration and the lung tissue W / D value, detecting the alveolar lavage fluid protein concentration and the lung tissue W / D value of each group of experimental animals, and entering the data of each experimental animal in each group into a chart for statistical analysis; S7. Observe the pathological changes. Prepare lung tissue slices and perform HE staining to observe the pathological changes. Obtain lung tissue slices from normal experimental animals for reference.

[0019] Embodiment 2 like Figure 1 As shown, the embodiment of the present invention also provides a method for constructing an animal model of high altitude pulmonary edema, comprising the following steps: S1. Establish a simulation environment, using actual plateau environmental information and environmental change cases as parameters to construct a simulation environment, and complete the simulation of environmental information such as sunshine, rain, alternating cold and hot changes, and carbon dioxide adsorption in a closed environment; S2. Adjust the initial environmental state, divide the simulated environment into a plateau cold group and a plateau normal temperature group, initialize the parameters of the two groups of simulated environments, and set the environmental parameters to the average state of the corresponding environment to avoid extreme environmental parameters at the beginning of the experiment; S3. Experimental animals were selected and 150 healthy rats were randomly divided into three groups, 50 rats in each group. Two groups of rats were placed in the simulated environment respectively. Each group was divided into five subgroups according to the length of stay in the environment, with 10 rats in each subgroup. The remaining group of rats was induced with pulmonary edema by intravenous infusion as the control experimental group; S4, setting environmental variables, changing environmental parameters, and randomly inserting extreme environments to simulate abnormal environmental conditions; S5. Preparation before testing: remove the experimental animals at the corresponding time point after the start of the experiment, inject alveolar lavage fluid, and remove the alveolar lavage fluid and lung tissue after anesthesia; S6. Detecting the BALF protein concentration and the lung tissue W / D value, detecting the alveolar lavage fluid protein concentration and the lung tissue W / D value of each group of experimental animals, and entering the data of each experimental animal in each group into a chart for statistical analysis; S7. Observe the pathological changes. Prepare lung tissue slices and perform HE staining to observe the pathological changes. Obtain lung tissue slices from normal experimental animals for reference.

[0020] Embodiment 3 The embodiment of the present invention also provides a method for constructing an animal model of high altitude pulmonary edema, comprising the following steps: S1. Establish a simulation environment, using actual plateau environmental information and environmental change cases as parameters to construct a simulation environment, and complete the simulation of environmental information such as sunshine, rain, alternating cold and hot changes, and carbon dioxide adsorption in a closed environment; S2. Obtaining plateau environmental parameters of a designated area, using a drone equipped with a sensor to enter the designated environmental area, and obtaining the environmental status of the area; S3, adjusting the initial environmental state, adjusting the parameters of the simulated environment to the obtained environmental parameters of the designated area, and adjusting the subsequent environmental parameters based on the changes in the environment; S4. Experimental animals were selected and 100 healthy rats were randomly divided into two groups, 50 rats in each group. The two groups of rats were placed in the simulated environment respectively, and each group was divided into five subgroups according to the length of time they stayed in the environment, with 10 rats in each subgroup; S5. Preparation before testing: remove the experimental animals at the corresponding time point after the start of the experiment, inject alveolar lavage fluid, and remove the alveolar lavage fluid and lung tissue after anesthesia; S6. Detecting the BALF protein concentration and the lung tissue W / D value, detecting the alveolar lavage fluid protein concentration and the lung tissue W / D value of each group of experimental animals, and entering the data of each experimental animal in each group into a chart for statistical analysis; S7. Observe the pathological changes, prepare lung tissue slices, and perform HE staining to observe the pathological changes and obtain the animal model of high altitude pulmonary edema in the designated plateau area.

[0021] Embodiment 4 like Figure 2 As shown, the embodiment of the present invention also provides a method for constructing an animal model of high altitude pulmonary edema, comprising the following steps: S1. Select 14 clean-grade SD male rats, with the weight of each rat controlled at 190g-210g, and place the rats in a low-pressure and low-oxygen environment to move freely for 1 day. The conditions of the low-pressure and low-oxygen environment are: temperature of 4-10 degrees Celsius, oxygen concentration of 0.020mol / L-0.027mol / L, and air pressure of 50KPa. In this environment, the rats are given a diet equivalent to that of normal times; S2, the rats in S1 were raised for 1, 3, and 7 days respectively, with normal diet, to establish the animal model of high altitude cerebral edema; S3. After one day of the experiment, the rats were identified and monitored for pulmonary edema symptoms using cardiac ultrasound, dry-wet ratio measurement, and pathological section methods.

[0022] In one embodiment, a dry-wet ratio determination method is used. A rat is placed on an operating table, anesthetized to death, and the chest is opened to remove both lungs. The left lung is taken out, and the water on the surface of the lung is absorbed with absorbent paper and weighed with an electronic balance, which is the wet weight of the lung. The lung tissue is then placed in a constant temperature drying box, and the drying temperature is set to 70 degrees Celsius. After 72 hours, the dried rat brain is taken out and weighed.

[0023] In one embodiment, the stained sections are subjected to pathological analysis, and the analysis process is as follows: A. Baking: Place the slices in a 60℃ constant temperature drying oven for 2 hours; B. Conventional dewaxing and hydration: After dewaxing the sections in xylene for 5 min*2 times, place them in 100%, 100%, 95%, 95%, and 90% ethanol for 5 min each to remove the xylene; C. Rinse with distilled water for 5 minutes*3 times to wash away the ethanol; D. Hematoxylin staining for 15 minutes, rinse with running water for 15 seconds; E. Decolorize with 1% hydrochloric acid alcohol for 3 seconds, and rinse with running water for 15 minutes; F. Stain with eosin for 2 minutes, rinse with running water for 15 seconds to remove floating color; G. Dehydration: Place the sections in 90%, 95%, 95%, 100%, and 100% ethanol for 5 minutes each, and xylene for 5 minutes*2 to make the specimens transparent; H. Take out the transparent slide from xylene, add an appropriate amount of neutral gum, cover the specimen with a coverslip, and observe the pathological changes of lung tissue under an optical microscope.

[0024] In one embodiment, a depilatory cream is applied to the chest and upper abdomen of a rat, and the area to be depilated is wiped with a cotton swab, the rat is anesthetized in an anesthesia induction box, the anesthetized rat is fixed in a supine position on an animal operating platform, the mouth and nose of the rat are stuffed with an anesthesia mask, anesthesia is maintained with isoflurane at a concentration of 1-2% and a flow rate of 0.6-1 L / min, a conductive paste or coupling agent is applied to the electrodes of the animal platform, and the rat's paw is fixed to the electrode pad with tape, a correct electrocardiogram is collected, the mouse body temperature is maintained, the respiratory rate is checked, the pulmonary artery blood flow acceleration time (PAT) and the pulmonary artery ejection time (PET) are measured, and the breathing is avoided and measured three times in different cardiac cycles.

[0025] When the present invention is working, it constructs a simulated environment with actual plateau environmental information and environmental change cases as parameters, completes the simulation of environmental information such as sunshine, rain, alternating cold and hot changes and carbon dioxide adsorption in a closed environment, divides the simulated environment into a plateau cold group and a plateau normal temperature group, initializes the parameters of the two groups of simulated environments, sets the environmental parameters to the average state of the corresponding environment, avoids the occurrence of extreme environmental parameters at the beginning of the experiment, selects 100 healthy rats and randomly divides them into two groups, each group has 50 rats, and the two groups of rats are respectively put into the simulated environment, and each group is divided into five subgroups according to the length of stay in the environment, and the five subgroups correspond to stay for 8h, 24h, 48h, At 72h and 120h, 10 animals were put into each subgroup and the experiment started. The environmental parameters were changed and extreme environments were randomly inserted to simulate abnormal environmental conditions. The experimental animals were taken out at the corresponding time points after the start of the experiment, and alveolar lavage fluid was injected. The alveolar lavage fluid and lung tissue were taken out after anesthesia, and the BALF protein concentration and the W / D value of the lung tissue were detected. The protein concentration of the alveolar lavage fluid and the W / D value of the lung tissue were detected for each group of experimental animals. The data of each experimental animal in each group were entered into charts for statistics, and the lung tissues were prepared and stained with HE to observe pathological changes, and lung tissue sections of normal experimental animals were obtained for reference.

[0026] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of various changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for constructing an animal model of high altitude pulmonary edema, characterized in that: The following steps are involved: S1. Establish a simulation environment; S2, adjust the initial environment state; S3, input of experimental animals; S4. Set environment variables; S5. Preparation before testing; S6. Detect BALF protein concentration and lung tissue W / D value; S7. Observe pathological changes.

2. The method for constructing an animal model of high altitude pulmonary edema according to claim 1, characterized in that: In said S1, when establishing the simulation environment, the simulation environment is constructed with actual plateau environmental information and environmental change cases as parameters, and the simulation of environmental information such as sunshine, rain, alternating cold and hot changes and carbon dioxide adsorption is completed in a closed environment.

3. The method for constructing an animal model of high altitude pulmonary edema according to claim 1, characterized in that: In S2, the simulated environment is divided into a plateau cold group and a plateau normal temperature group, the parameters of the two groups of simulated environments are initialized, and the environmental parameters are set to the average state of the corresponding environment to avoid extreme environmental parameters at the beginning of the experiment.

4. The method for constructing an animal model of high altitude pulmonary edema according to claim 1, characterized in that: In S3, 100 healthy rats were randomly divided into two groups, each with 50 rats. The two groups of rats were placed in a simulated environment respectively, and each group was divided into five subgroups according to the length of time the rats stayed in the environment, each with 10 rats.

5. The method for constructing an animal model of high altitude pulmonary edema according to claim 1, characterized in that: In S4, environmental changes are simulated, environmental parameters are changed, and extreme environments are randomly inserted to simulate abnormal environmental conditions.

6. The method for constructing an animal model of high altitude pulmonary edema according to claim 1, characterized in that: In S5, the experimental animal is taken out at a corresponding time point after the start of the experiment, and alveolar lavage fluid is injected, and the alveolar lavage fluid and lung tissue are taken out after anesthesia.

7. The method for constructing an animal model of high altitude pulmonary edema according to claim 1, characterized in that: In S6, the protein concentration of the alveolar lavage fluid and the W / D value of the lung tissue are detected for each group of experimental animals, and the data of each experimental animal in each group are entered into a chart for statistical analysis.

8. The method for constructing an animal model of high altitude pulmonary edema according to claim 1, characterized in that: In S7, lung tissue sections are prepared and HE-stained to observe pathological changes, and lung tissue sections of normal experimental animals are obtained for reference.

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