Method for establishing acute plateau low-pressure low-oxygen lung injury mouse model

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to an acute plateau low-pressure low-oxygen lung injury mouse model building method which comprises the following steps: selecting SPF-level male SD (Sprague Dawley) rats for adaptive feeding; performing low-oxygen sensitivity pretreatment on the rats after the adaptive feeding is finished; debugging the closed low-pressure oxygen cabin to an initial standard state, putting the pretreated rat into the closed low-pressure oxygen cabin, and closing the cabin door; performing staged low-pressure and low-oxygen synergistic exposure on the rats in the cabin, and obtaining an acute plateau low-pressure and low-oxygen lung injury rat model after the exposure is finished; according to the method, a staged low-pressure low-oxygen collaborative exposure design of pressure dominated low oxygen and oxygen concentration collaborative low oxygen is adopted, so that the core characteristic that the natural plateau oxygen partial pressure is gradually reduced along with the altitude is completely reproduced; the periodic fluctuation mode of the oxygen concentration cooperation stage further fits the oxygen concentration change of the real plateau environment, and it is ensured that the model can truly reproduce the progressive pathological process from the initial hypoxia stress to the acute inflammation injury.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedicine, and particularly relates to a method for establishing an acute high-altitude low-pressure and low-oxygen lung injury mouse model. BACKGROUND

[0002] High-altitude pulmonary edema is one of the most severe types of acute high-altitude disease, and its pathological mechanism is closely related to the destruction of alveolar-capillary barrier and the activation of inflammatory response, which poses a significant threat to the health of people in high-altitude areas. Due to the complex environmental conditions and difficulties in sample acquisition, the establishment of an animal model that can accurately simulate the pathological process of high-altitude low-pressure and low-oxygen has become a key basis for revealing the pathogenesis of high-altitude pulmonary edema and developing prevention and treatment drugs.

[0003] The prominent feature of the high-altitude environment is that the oxygen partial pressure gradually decreases with the increase of altitude. This stepwise decrease will trigger the progressive pathological changes from initial hypoxic stress to acute inflammatory injury, which is the core driving factor for the occurrence and development of high-altitude pulmonary edema. However, existing animal models generally do not follow the gradual rule of oxygen partial pressure with the increase of altitude in the natural high-altitude environment, but use a one-time rapid pressure reduction method to simulate the high-altitude environment, ignoring the real process of gradually reducing pressure and slowly decreasing oxygen concentration when the human body enters the high-altitude. This leads to mechanical damage to the lung tissue of experimental animals due to sudden pressure changes, and cannot reproduce the stepwise pathological process of high-altitude pulmonary edema from vasoconstriction, increased permeability to inflammatory cell infiltration. The dynamic correlation between hypoxia and lung injury is simplified, making it difficult to accurately reflect the core mechanism of hypoxia-induced lung injury in the real high-altitude environment, which seriously restricts the supporting value of the model for the study of high-altitude pulmonary edema. SUMMARY

[0004] In view of the above problems in the prior art, the application provides a method for establishing an acute high-altitude low-pressure and low-oxygen lung injury mouse model to solve the problems in the background art.

[0005] In order to solve the above technical problems, the application adopts the following technical scheme: A method for establishing an acute high-altitude low-pressure and low-oxygen lung injury mouse model, characterized in that it comprises the following steps: Step 1: Select SPF male SD rats, adaptively feed, and exclude individuals with respiratory tract infection and cardiopulmonary basic lesions; Step 2: Pre-treat the rats after adaptive feeding with low oxygen sensitivity; Step 3: After adjusting the closed low-pressure oxygen cabin to the initial standard state, put the rats pre-treated with low oxygen sensitivity into the closed low-pressure oxygen cabin, and close the cabin door; Step 4: Perform a low-pressure and low-oxygen exposure on the rats in the closed low-pressure oxygen cabin in stages, and obtain an acute high-altitude low-pressure and low-oxygen lung injury mouse model after the exposure is completed. Step five: multi-dimensional verification of acute high-altitude low-pressure and low-oxygen lung injury mouse model is carried out to determine whether the model is successfully established.

[0006] Further, the specific steps of step one are as follows: SPF male SD rats weighing 220-250 g are selected, adaptive feeding is carried out for 7 days, and during the feeding period, the rats are free to eat and drink water, and the respiratory rate and activity state of the rats are observed daily to exclude respiratory tract infection and cardiopulmonary basic lesion individuals.

[0007] Further, the low-oxygen sensitivity pretreatment in step two refers to placing the rats after adaptive feeding for 1 h in a normal-pressure low-oxygen environment for 3 consecutive days, and the oxygen concentration of the normal-pressure low-oxygen environment is decreased by 0.5% per day from 16% at the beginning to 15%.

[0008] Further, the initial standard state of step three is that the pressure in the closed low-pressure oxygen cabin is 101 kPa, the oxygen concentration is 21%, the temperature in the cabin is controlled at 22±2℃, the humidity is 35-40%, the CO2concentration is ≤0.5%, and the air flow velocity is ≤0.3 m / s, and the nitrogen proportion is maintained stable by gas control means.

[0009] Further, the step four includes: The pressure-dominated hypoxia stage is to reduce the pressure in the closed low-pressure oxygen cabin from 101 kPa to 40-45 kPa at a rate of 0.3-0.7 kPa / min, maintain the oxygen concentration at 21% during the period, and reduce the oxygen partial pressure from 21.2 kPa to 8.5-9.5 kPa in a stepwise manner, and the whole process lasts for 1-2 h to induce initial hypoxic stress; The oxygen concentration coordinated hypoxia stage: the cabin pressure is kept at 40-45 kPa, the 21% oxygen concentration of the pressure-dominated hypoxia stage is maintained, the oxygen concentration is reduced from 21% to 14-16% at a rate of 0.3-0.7% / min, the oxygen partial pressure is further reduced from 8.5-9.5 kPa to 6-7 kPa, and then enters the periodic hypoxia fluctuation mode, and the whole process lasts for 6-8 h to induce acute inflammatory injury of lung tissue, and during the period, nitrogen is supplemented to maintain the stability of the total amount of gas in the cabin to avoid pressure fluctuations caused by oxygen concentration adjustment.

[0010] Further, the periodic hypoxia fluctuation mode includes: maintaining the oxygen concentration at 14-16% for 1.5-2.5 h, then increasing the oxygen concentration to 17-19% and maintaining for 8-12 min, and then reducing the oxygen concentration to 14-16%.

[0011] Further, the multi-dimensional verification in step five includes the following indicators: The lung tissue W / D is ≥5.5; The white blood cell count in the bronchoalveolar lavage fluid is ≥1×106 0.5-1.0 x 106cells / mL; The lung tissue pathological section shows that the alveolar septum thickening and inflammatory cell infiltration; The relative expression of hypoxia inducible factor HIF-1 alpha mRNA in lung tissue is increased by more than 3 times compared with the control group, and the protein content of inflammatory factor IL-6 is more than 80 pg / mg.

[0012] Further, the control group is the same batch as the acute high altitude low pressure and low oxygen lung injury model group, and the rest of the operation steps are consistent except that the staged low pressure and low oxygen exposure is not performed.

[0013] Further, the lung tissue pathological section is observed by HE staining, and when red blood cell exudation appears in the alveolar cavity, it is determined as a severe acute high altitude low pressure and low oxygen lung injury model.

[0014] Compared with the prior art, the present application has the following beneficial effects: 1. The present application adopts a staged low pressure and low oxygen exposure design, first simulates the oxygen partial pressure drop at a medium-high altitude of about 5500 meters through a pressure-dominated hypoxia stage, and then simulates a hypoxic environment at an altitude of about 8000 meters through an oxygen concentration coordinated hypoxia stage, which completely reproduces the core feature of the gradual decrease of oxygen partial pressure with altitude on a natural plateau, and the "total pressure constant + oxygen concentration regulation" in the oxygen concentration coordinated hypoxia stage replaces the physical path of "total pressure decrease + oxygen concentration constant" on a natural plateau, which not only avoids mechanical damage to the lung tissue of experimental animals caused by sudden pressure drop, but also accurately reproduces the hypoxic intensity at a very high altitude, achieving a perfect balance between "simulating real plateau low pressure and low oxygen" and "ensuring the physiological safety of experimental animals", which is an equivalent simulation of the essence of the natural plateau low pressure and low oxygen environment rather than a simple replication, and the present application firmly grasps the essential feature of the gradual decrease of oxygen partial pressure on a natural plateau, and ensures that the model can truly reproduce the progressive pathological process from initial hypoxic stress to acute inflammatory damage in a more scientific way, which is highly consistent with the clinical pathogenesis of human high altitude pulmonary edema; 2. The periodic fluctuation mode of the oxygen concentration coordinated stage of the present application accurately simulates the natural fluctuation characteristics of oxygen concentration caused by cloud cover and terrain changes on a real high altitude, which is consistent with the oxygen concentration change of the real high altitude environment, and the oxygen concentration of 14-16% corresponds to the hypoxic environment at an altitude of about 8000 meters, which is the core hypoxic intensity for inducing acute inflammatory damage to the lung tissue, and the temporary increase of the oxygen concentration to 17-19% accurately simulates the possible temporary hypoxic relief scene on a real high altitude, which not only improves the natural simulation of the model and the consistency of the model with the pathological and physiological process of the real high altitude lung injury, but also makes the pathological changes of the model more clinically valuable, and reduces the probability of non-specific death of rats caused by constant extreme hypoxia; 3. The present application from the precise screening of experimental rats, adaptive breeding to exclude respiratory tract infection and cardiopulmonary basic lesion individuals, hypoxia sensitivity pretreatment to reduce individual hypoxia tolerance difference, to the initial state debugging of the closed low-pressure oxygen cabin, forming a whole-process standardized operation closed loop, the above steps are progressive, the physiological differences of the rats before the core low-pressure low-oxygen operation are minimized, and the interference of individual differences on the modeling results is avoided. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The flowchart of the embodiment of the method for establishing an acute high-altitude low-pressure and low-oxygen lung injury rat model of the present application. DETAILED DESCRIPTION

[0016] In order for those skilled in the art to better understand the present application, the technical solutions of the present application will be further described below in combination with the drawings and examples.

[0017] Among them, the drawings are only used for exemplary illustration, and the representation is only a schematic diagram, not a physical diagram, and cannot be understood as a limitation on the present application; in order to better illustrate the embodiments of the present application, some components in the drawings will be omitted, enlarged or reduced, and do not represent the size of the actual product; for those skilled in the art, it is understandable that some known structures and their descriptions in the drawings can be omitted.

[0018] The same or similar reference numerals in the drawings of the embodiments of the present application correspond to the same or similar components; in the description of the present application, it should be understood that if the terms "upper", "lower", "left", "right", "inner", "outer" and the like indicating the orientation or positional relationship are based on the orientation or positional relationship shown in the drawings, only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, therefore the positional relationship described in the drawings is only used for exemplary illustration, and cannot be understood as a limitation on the present application, for those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0019] In the description of the present application, unless otherwise explicitly specified and limited, if the term "connection" and the like indicating the connection relationship between components appears, the term should be broadly understood, for example, it can be fixedly connected, or it can be detachably connected, or it can be integrated; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication or interaction relationship between two components. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0020] Example 1 As Figure 1As shown, the present application, in particular, a method for establishing a mouse model of acute high-altitude low-pressure and low-oxygen lung injury, characterized in that it comprises the following steps: Step one: select SPF male SD rats, adaptively feed, exclude respiratory tract infection and cardiopulmonary basic pathology individuals; Step two: the rats after adaptive feeding are pretreated for low oxygen sensitivity; Step three: after adjusting the closed low-pressure oxygen cabin to the initial standard state, the rats pretreated for low oxygen sensitivity are put into the closed low-pressure oxygen cabin, and the cabin door is closed; Step four: the rats in the closed low-pressure oxygen cabin are subjected to phased low-pressure and low-oxygen exposure, and the mouse model of acute high-altitude low-pressure and low-oxygen lung injury is obtained after the exposure is completed; Step five: the mouse model of acute high-altitude low-pressure and low-oxygen lung injury is subjected to multi-dimensional verification to determine whether the model is successfully established.

[0021] In an embodiment, the present application establishes a model establishment method framework of screening, pretreatment, cabin preparation, phased modeling and verification, ensures that each step serves the goal of "establishing a stable and repeatable mouse model of high-altitude low-pressure and low-oxygen lung injury", and breaks through the limitations of the existing technology of one-time rapid decompression simulation of high-altitude environment, simulates the natural high-altitude oxygen partial pressure change law through phased step-by-step regulation, lays a foundation for the pathological relevance of the model, makes the model more suitable for the pathological and physiological process of lung injury in the real high-altitude environment, and provides a stable and reliable experimental carrier for the mechanism research, drug screening and the like of acute high-altitude lung injury.

[0022] Among them, the specific steps of step one are: select SPF male SD rats, weighing 220-250g, adaptively feed for 7 days, feed freely during the feeding period, observe the respiratory rate and activity state of the rats every day to exclude respiratory tract infection and cardiopulmonary basic pathology individuals.

[0023] In the embodiments, in the animal selection, the SPF male SD rats with a body weight of 220-250 g are selected, the SPF rats ensure no specific pathogen infection, and the influence of infection factors on the subsequent lung injury judgment is excluded, the male rats avoid the physiological state fluctuation caused by the sex hormone cycle change of female rats, and the body weight of 220-250 g is the stable body weight interval of the adult SD rats, the heart and lung functions of the rats at this stage are mature and stable, the consistency of the experimental individuals on the physiological basis is ensured, the difference in the hypoxia tolerance caused by the age and weight difference is reduced, and the reliability of the experimental results is ensured, in the adaptive feeding link, the environmental temperature is controlled to be 22±1 ℃, the humidity is controlled to be 50±5%, and the 12-hour light-dark cycle is maintained, the 7-day feeding cycle allows the rats to have enough time to adapt to the new feeding environment and relieve the stress caused by transportation, the respiratory rate and activity state are observed every day, the individuals with respiratory tract infection and heart and lung basic diseases are excluded, the superposition of the basic diseases and the lung injury induced by the subsequent low pressure and low oxygen is avoided, the probability of experimental failure caused by the basic health problems of the experimental animals is reduced, the experimental results are more persuasive, and the success rate of the model establishment is improved.

[0024] In the step two, the low oxygen sensitivity pretreatment refers to that the rats after the adaptive feeding are exposed to a normal pressure and low oxygen environment for 1 hour every day for 3 days, and the oxygen concentration of the normal pressure and low oxygen environment is decreased by 0.5% per day from 16% to 15%.

[0025] In the embodiments, the low oxygen sensitivity pretreatment is a key innovative point for improving the model quality and experimental stability, can effectively reduce the low oxygen stress difference between the rats, and if the high-intensity low pressure and low oxygen exposure is directly performed, some low oxygen sensitive individuals may appear acute shock or even death due to the incapability of rapid adaptation, the experimental sample loss is caused, and the data deviation is large, the mild low oxygen pretreatment for 3 days: the oxygen concentration is 16% on the first day, the oxygen concentration is 15.5% on the second day, and the oxygen concentration is 15% on the first day, can gradually activate the basic low oxygen adaptation mechanism in the rats, so that all the experimental rats reach a similar low oxygen tolerance baseline before entering the core modeling stage, the mortality of the rats in the subsequent low pressure and low oxygen exposure process is greatly reduced, the sufficiency of the experimental sample amount is ensured, and the pretreatment mode is in line with the adaptation law in the natural plateau environment, in the real plateau environment, the human or animal entering the high altitude area usually experiences a step-by-step adaptation process, the embodiment simulates the natural adaptation characteristics, avoids the non-physiological damage caused by the one-time sudden increase in the altitude, further improves the relevance between the model and the pathological process of the real plateau lung injury, and makes the research results based on the model more reflect the actual situation, and provide a more reliable basis for the clinical transformation of subsequent research.

[0026] The initial standard state of step three is that the internal pressure of the closed low-pressure oxygen cabin is 101 kPa, the oxygen concentration is 21%, the internal temperature is controlled at 22±2℃, the humidity is 35-40%, the CO2concentration is ≤0.5%, the airflow velocity is ≤0.3 m / s, and the proportion of nitrogen is maintained stable by the gas control means.

[0027] In the embodiment, the initial state is set to be the pressure of 101 kPa and the oxygen concentration of 21%, which replicates the standard atmospheric environment parameters in the plain area, so that the rats enter the cabin from the normal physiological environment starting point, and the environment of the temperature of 22±2℃, the humidity of 35-40%, the CO2concentration of ≤0.5% and the airflow velocity of ≤0.3 m / s is the more suitable living environment for the SD rats, which avoids the false hypoxia state or the early stress reaction caused by abnormal initial environment parameters, ensures that the low-pressure and low-oxygen exposure in the subsequent stage is the only core variable, and lays a foundation for accurately studying the damage effect of low-pressure and low-oxygen on the lung tissue; in addition, the proportion of nitrogen as the main filling gas in the cabin is stable, which provides convenience for the parameter adjustment in the subsequent stage of low-pressure and low-oxygen exposure.

[0028] The step four of the staged low-pressure and low-oxygen exposure includes: In the pressure-dominated hypoxia stage, the internal pressure of the closed low-pressure oxygen cabin is reduced from 101 kPa to 40-45 kPa at a rate of 0.3-0.7 kPa / min, the oxygen concentration is maintained at 21% during the period, the oxygen partial pressure is reduced from 21.2 kPa to 8.5-9.5 kPa in a stepwise manner, and the whole process lasts for 1-2 h to induce initial hypoxic stress; In the oxygen concentration coordinated hypoxia stage, the internal pressure of the cabin in the pressure-dominated hypoxia stage is kept unchanged at 40-45 kPa, the oxygen concentration is reduced from 21% to 14-16% at a rate of 0.3-0.7% / min based on the 21% oxygen concentration in the pressure-dominated hypoxia stage, the oxygen partial pressure is further reduced from 8.5-9.5 kPa to 6-7 kPa, and then enters the periodic hypoxia fluctuation mode, and the whole process lasts for 6-8 h to induce acute inflammatory damage of the lung tissue, during which the total amount of gas in the cabin is maintained stable by supplementing nitrogen to avoid the pressure fluctuation caused by the adjustment of the oxygen concentration.

[0029] In embodiments, the staged low-pressure and low-oxygen exposure design achieves a breakthrough innovation in terms of simulating natural plateau oxygen partial pressure variation. The pressure-dominated hypoxia stage completely follows the core mechanism of "altitude increase-atmospheric pressure decrease-oxygen partial pressure decrease" in natural plateau environments. By reducing the cabin pressure from 101 kPa to 40-45 kPa while maintaining the oxygen concentration at 21%, the oxygen partial pressure is accurately reduced from 21.2 kPa to 8.5-9.5 kPa. The oxygen partial pressure is calculated as: total pressure x oxygen concentration. This oxygen partial pressure range is highly consistent with the oxygen partial pressure at an altitude of about 5500 meters on a real plateau, accurately simulating the hypoxic state of a medium-high altitude plateau environment and providing a low-pressure and low-oxygen stimulus that conforms to natural laws for inducing initial hypoxic stress in rats. The oxygen concentration-coordinated hypoxia stage accurately simulates the extremely high-altitude hypoxic environment while avoiding non-specific damage. In this stage, the cabin pressure is kept constant at 40-45 kPa to avoid further reducing the pressure to about 33 kPa, which is the pressure corresponding to an altitude of 8000 meters, to cause lung tissue decompression injury in rats. Instead, the oxygen concentration is reduced from 21% to 14-16%, further reducing the oxygen partial pressure to 6-7 kPa. This oxygen partial pressure level is relatively close to the oxygen partial pressure at an altitude of about 8000 meters on a real plateau. This design essentially achieves the same goal of gradually reducing oxygen partial pressure by a different means. The "constant total pressure + oxygen concentration regulation" replaces the natural plateau "total pressure reduction + constant oxygen concentration" physical path, which not only avoids mechanical damage to the lung tissue of experimental animals due to sudden pressure drop, but also accurately reproduces the hypoxic intensity at extremely high altitudes, achieving a perfect balance between "simulating real plateau low-pressure and low-oxygen" and "ensuring the physiological safety of experimental animals". This is an equivalent simulation of the essence of the natural plateau low-pressure and low-oxygen environment rather than a simple replication, which significantly improves the pathological relevance of the model.

[0030] The periodic hypoxic fluctuation mode includes maintaining an oxygen concentration of 14-16% for 1.5-2.5 hours, then increasing the oxygen concentration to 17-19% for 8-12 minutes, and then reducing the oxygen concentration to 14-16%.

[0031] In this implementation, the periodic hypoxia fluctuation pattern closely matches the oxygen concentration variation characteristics of the real high-altitude environment. An oxygen concentration of 14-16% corresponds to the extremely high altitude hypoxic environment at around 8000 meters, which is the core hypoxic intensity that induces acute inflammatory damage in lung tissue. Briefly raising the oxygen concentration to 17-19% accurately simulates the brief hypoxic relief scenarios that may occur in real high-altitude environments. This upgrades the model from constant hypoxic exposure to more natural fluctuating hypoxic exposure, improving the model's natural simulation and consistency with the pathophysiological processes of lung injury in real high-altitude environments. Secondly, this model can effectively reduce the risk of non-specific mortality in rats, ensuring sufficient experimental sample size. The oxygen partial pressure of 6-7 kPa at an altitude of around 8000 meters is relatively low for rats. In extreme hypoxic environments, if constant hypoxia exposure is maintained for 6–8 hours, some rats may die nonspecifically due to their inability to withstand prolonged extreme hypoxic stress. The cause of death may be systemic multi-organ failure rather than specific lung injury, interfering with experimental results. A hypobaric hypoxia duration of 1.5–2.5 hours can ensure sufficient hypobaric hypoxia stimulation intensity to induce lung tissue inflammation and damage. A brief oxygen concentration recovery of 8–12 minutes can provide a respiratory buffer period for rats, temporarily relieving the excessive pressure of extreme hypobaric hypoxia on the respiratory and circulatory systems, allowing the rats to recover their physiological state temporarily. At the same time, because the recovery time is short, it will not break the overall hypobaric hypoxia stress and will not affect the hypobaric hypoxia-induced lung injury effect.

[0032] The multi-dimensional verification in step five includes the following indicators: Lung tissue W / D (wet / dry weight ratio) ≥ 5.5; White blood cell count ≥1×10 in bronchoalveolar lavage fluid 6 cells / mL; Lung tissue pathological sections showed thickening of alveolar septa and infiltration of inflammatory cells; The relative expression level of hypoxia-inducible factor HIF-1α mRNA in lung tissue was ≥3 times higher than that in the control group, and the protein content of inflammatory factor IL-6 was ≥80 pg / mg.

[0033] The implementation method covers multi-dimensional verification including physiological function, cellular level, pathological morphology, and molecular mechanisms. A lung tissue wet / dry weight ratio (W / D) ≥ 5.5 is a classic physiological indicator reflecting the degree of lung edema. One of the core pathological features of acute high-altitude hypobaric hypoxic lung injury is alveolar fluid exudation caused by damage to the alveolar-capillary barrier. This indicator directly quantifies the degree of lung edema and can quickly determine whether the model shows typical physiological changes of lung injury. A white blood cell count ≥ 1 × 10⁻⁶ in the bronchoalveolar lavage fluid is also included. 6The concentration of HIF-1α cells / mL at the cellular level demonstrated significant inflammatory cell infiltration in the lung tissue, distinguishing "hypobaric hypoxia-induced inflammatory lung injury" from non-inflammatory mechanical injury, further clarifying the specificity of the injury type. Pathological sections of the lung tissue showed alveolar septal thickening and inflammatory cell infiltration, providing direct morphological evidence to confirm the occurrence of lung injury at the tissue structure level, which is one of the criteria for judging the success of the model, allowing direct observation of organic changes in the lung tissue. The expression indicators of HIF-1α mRNA and IL-6 protein in the lung tissue revealed the nature and pathway of the injury at the molecular mechanism level. HIF-1α is a specific transcription factor in response to hypobaric hypoxia stress, and it is significantly activated and highly expressed only under hypobaric hypoxia conditions. Its relative mRNA expression level was ≥3 times higher than that of the control group, directly proving that the lung injury in the model was induced by hypobaric hypoxia stimulation, ruling out the possibility of false positive models caused by other non-hypobaric hypoxia factors such as infection and stress. IL-6... It is a core regulatory factor of acute inflammatory response, with a protein content ≥80pg / mg, proving that hypobaric hypoxia has successfully induced a significant inflammatory response in lung tissue, and the intensity of inflammation has reached the level that causes substantial damage. It forms a complete molecular mechanism loop of "hypobaric hypoxia stress → HIF-1α activation → inflammatory response (increased IL-6) → lung tissue damage (edema, pathological changes)," making the determination of the model's effectiveness more scientific and convincing.

[0034] The control group consisted of rats from the same batch as the acute high-altitude hypobaric hypoxic lung injury model group, and all operating procedures were identical except for the absence of staged hypobaric hypoxic synergistic exposure.

[0035] In the implementation method, a control group is clearly defined, and the principle of single variable in animal experiments is strictly followed. Only the core difference of whether or not staged hypoxia exposure is performed is retained. The interference factors of batch differences and differences in feeding management, pretreatment and cabin environment are excluded to ensure that the changes in the relative expression of hypoxia-inducible factor HIF-1α mRNA in lung injury and lung tissue in the model group can be accurately attributed to hypoxia stimulation, thus avoiding the distortion of experimental results.

[0036] The lung tissue pathological sections were stained with hematoxylin and eosin (HE). When red blood cell exudation was observed in the alveolar spaces, it was identified as a severe acute high-altitude hypobaric hypoxic lung injury model. In the embodiment, one of the typical pathological manifestations of patients with severe high altitude pulmonary edema is that the alveolar capillary barrier is severely damaged, leading to leakage of red blood cells in the blood vessels into the alveolar cavity, and the patients often have symptoms such as hemoptysis and severe hypoxemia, which is one of the core diagnostic bases for patients with severe high altitude pulmonary edema. In the embodiment, the appearance of red blood cell exudation in the alveolar cavity is used as the judgment standard of the severe model, which directly corresponds to the key clinical characteristics, so that the acute high altitude low pressure and hypoxia lung injury model established becomes an experimental carrier for studying the pathological mechanism of patients with severe high altitude pulmonary edema, developing first-aid drugs, and exploring treatment schemes, which can more accurately simulate the pathological state of clinical severe patients, builds a more reliable bridge for basic research to clinical application, solves the problem that the existing model focuses on mild or moderate injury and is difficult to meet the research needs of severe patients, expands the application scenario of the model, and the lung tissue pathological section adopts HE staining, which is the most common and mature staining method in pathological research, has a very high laboratory popularization rate, is simple to operate and has low cost, and also reduces the technical difficulty and cost of experimental operation.

[0037] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

[0038] The above is only an embodiment of the present application, and the circuit and electronic components and modules involved are all prior art, which can be implemented by those skilled in the art without further description. The content protected by the present application does not involve the improvement of software and methods. The specific structures and characteristics of the scheme known in the art are not described in detail here. Those skilled in the art know all the ordinary technical knowledge in the field of the application before the filing date or the priority date, can obtain all the prior art in the field, and have the ability to apply conventional experimental means before that date. Those skilled in the art can perfect and implement the present scheme based on their own ability under the guidance of the present application. Some typical known structures or known methods should not be an obstacle for those skilled in the art to implement the present application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present application, which should be considered as the protection scope of the present application. These will not affect the effect and practicality of the patent.

Claims

1. A method for establishing a mouse model of acute high-altitude hypobaric hypoxia lung injury, characterized in that, Includes the following steps: Step 1: Select SPF-grade male SD rats, acclimatize them, and exclude individuals with respiratory infections and underlying cardiopulmonary diseases; Step 2: After the acclimatization period, the rats were subjected to hypoxia-sensitive pretreatment. Step 3: After adjusting the closed hypobaric chamber to its initial standard state, place the rats that have undergone hypoxia sensitivity pretreatment into the closed hypobaric chamber and close the chamber door; Step 4: Rats were subjected to staged low-pressure and low-oxygen synergistic exposure in a closed hypobaric chamber. After the exposure, an acute high-altitude hypobaric and hypoxic lung injury rat model was obtained. Step 5: Perform multi-dimensional validation on the mouse model of acute high-altitude hypobaric hypoxia lung injury to determine whether the model has been successfully established.

2. The method for establishing an acute high-altitude hypobaric hypoxic lung injury mouse model as described in claim 1, characterized in that, The specific steps of step one are as follows: Select SPF-grade male SD rats weighing 220-250g and acclimatize them for 7 days. During the feeding period, the rats have free access to food and water. Observe the respiratory rate and activity status of the rats every day to exclude individuals with respiratory tract infections and underlying cardiopulmonary diseases.

3. The method for establishing a mouse model of acute high-altitude hypobaric hypoxia lung injury as described in claim 1, characterized in that, The hypoxia sensitivity pretreatment in step two refers to exposing rats to a normobaric hypoxia environment for 1 hour each day for 3 consecutive days after the completion of acclimatization feeding. The oxygen concentration of the normobaric hypoxia environment is reduced by 0.5% to 15% each day from the initial 16%.

4. The method for establishing an acute high-altitude hypobaric hypoxic lung injury mouse model as described in claim 1, characterized in that, The initial standard state of step three is a closed low-pressure oxygen chamber with an internal pressure of 101 kPa, an oxygen concentration of 21%, a chamber temperature of 22±2℃, a humidity of 35~40%, a CO2 concentration of ≤0.5%, and an airflow velocity of ≤0.3m / s. The nitrogen ratio is maintained stable through gas regulation.

5. The method for establishing an acute high-altitude hypobaric hypoxic lung injury mouse model as described in claim 4, characterized in that, The phased low-pressure, low-oxygen synergistic exposure in step four includes: During the pressure-dominated hypoxia phase, the pressure inside the closed hypobaric chamber was reduced from 101 kPa to 40–45 kPa at a rate of 0.3–0.7 kPa / min, while maintaining an oxygen concentration of 21%. The oxygen partial pressure was gradually reduced from 21.2 kPa to 8.5–9.5 kPa, and this process lasted for 1–2 hours to induce initial hypoxic stress. Oxygen concentration-coordinated hypoxia phase: Maintain the chamber pressure at 40-45 kPa, continue the pressure-dominated hypoxia phase with a baseline oxygen concentration of 21%, and reduce the oxygen concentration from 21% to 14-16% at a rate of 0.3-0.7% / min, further reducing the oxygen partial pressure from 8.5-9.5 kPa to 6-7 kPa. Then, enter a periodic hypoxia fluctuation mode, which lasts for 6-8 hours to induce acute inflammatory damage to lung tissue. During this period, nitrogen is supplemented to maintain a stable total gas volume in the chamber and avoid pressure fluctuations caused by oxygen concentration adjustments.

6. The method for establishing an acute high-altitude hypobaric hypoxic lung injury mouse model as described in claim 5, characterized in that, The periodic hypoxia fluctuation pattern includes maintaining an oxygen concentration of 14–16% for 1.5–2.5 hours, then raising the oxygen concentration to 17–19% and holding it for 8–12 minutes, and then lowering the oxygen concentration to 14–16%.

7. The method for establishing an acute high-altitude hypobaric hypoxic lung injury mouse model as described in claim 1, characterized in that, The multi-dimensional verification in step five includes the following metrics: Lung tissue W / D ≥ 5.5; White blood cell count ≥1×10 in bronchoalveolar lavage fluid 6 cells / mL; Lung tissue pathological sections showed thickening of alveolar septa and infiltration of inflammatory cells; The relative expression level of hypoxia-inducible factor HIF-1α mRNA in lung tissue was ≥3 times higher than that in the control group, and the protein content of inflammatory factor IL-6 was ≥80 pg / mg.

8. The method for establishing a mouse model of acute high-altitude hypobaric hypoxia lung injury as described in claim 7, characterized in that, The control group consisted of rats from the same batch as the acute high-altitude hypobaric hypoxic lung injury model group, and all operating procedures were identical except for the absence of staged hypobaric hypoxic synergistic exposure.

9. The method for establishing a mouse model of acute high-altitude hypobaric hypoxia lung injury as described in claim 7, characterized in that, The lung tissue pathological sections were stained with hematoxylin and eosin (HE). When red blood cell exudation was observed in the alveolar cavity, it was determined to be a severe acute high-altitude hypobaric hypoxic lung injury model.

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