A method for modeling acute respiratory failure by non-invasive selective unilateral pulmonary drug administration in rats

Abstract

The application provides a method for modeling acute respiratory failure of a rat by non-invasive selective unilateral pulmonary drug administration, and relates to the technical field of biomedical experiments. First, anesthetized rats are fixed on a rotatable platform, a special curved thin catheter is inserted into and oriented to a main bronchus on one side through the mouth, and a predetermined dose of damage-causing reagent solution is slowly injected into the lung through the catheter. During the administration process, the body position of the rat is adjusted so that the lung on the administration side is in a low position, and short-time high-frequency oscillation is used to make the solution uniformly distributed in the lung. After injection, the catheter is kept for a certain period of time to avoid liquid extravasation, and then the catheter is pulled out to let the rat wake up and breathe independently for about 5 hours, and then the rat is subjected to 4 hours of mechanical ventilation support. The method does not require tracheostomy and other invasive operations, and can make the reagent act on the unilateral lung and induce the lung to appear typical lesions of ARDS, including pulmonary edema, severe hypoxemia, pathological changes, inflammatory infiltration, alveolar capillary barrier changes and the like, while the contralateral lung not directly contacting the damage-causing reagent also appears damage.

Description

Technical Field

[0001] This invention relates to the field of biomedical experimental technology, specifically to a method for inducing acute respiratory failure in rats through non-invasive selective unilateral lung administration. Background Technology

[0002] Acute respiratory distress syndrome (ARDS) is an acute, diffuse inflammatory lung injury syndrome caused by multiple risk factors. It has a high mortality rate and lacks specific treatments. Significant heterogeneity exists among ARDS patients; patients with different etiologies and extent of injury respond differently to the same treatment. Current clinical guidelines tend to classify and intervene based on the type of lung injury. For example, the latest ARDS definition (2023) clearly distinguishes between unilateral and bilateral lung injury for the first time, emphasizing the necessity of differentiated intervention studies for different types. Approximately 22.7% of patients with acute respiratory failure present with unilateral lung injury, and nearly 20% of these will progress to bilateral infiltrative ARDS. Therefore, unilateral lung injury accounts for a significant proportion of ARDS and has unique clinical significance.

[0003] Because previous diagnostic criteria for ARDS required simultaneous damage to both lungs, research on unilateral lung injury has lagged behind. However, the characteristics of unilateral lung injury induced by mechanical ventilation (VILI) are more pronounced: the injured lung is prone to atelectasis, while the healthy lung may overinflate due to compensatory ventilation; these two injury mechanisms are difficult to distinguish in bilateral lung injury. Research on unilateral lung injury will contribute to a deeper understanding of the pathogenesis of ARDS and provide a basis for personalized ventilation strategies. However, before clinical trials, reliable animal models are needed to validate the effectiveness and safety of interventions for unilateral lung injury.

[0004] Currently, there are relatively few studies on animal models of unilateral lung injury ARDS. Reported methods include inducing unilateral lung injury in large mammals (such as pigs and dogs) through intrabronchial injection of saline or acidic solutions. These methods are costly, complex, and the unclear genetic background of large animals limits subsequent mechanistic studies and intervention trials. Furthermore, small rodents (such as mice) are unsuitable as ideal subjects for unilateral ARDS models due to their small lung capacity, the high precision required for mechanical ventilation, and the difficulty in obtaining arterial blood. In contrast, rats have a suitable body size: ensuring sufficient lung volume for precise mechanical ventilation and repeated blood gas analysis, while maintaining the advantages of rodents in terms of genetic manipulation and antibody detection. Therefore, developing a rat-based unilateral lung injury ARDS model is of great significance.

[0005] Currently, no sufficiently stable and reproducible rodent unilateral ARDS model has been reported. Existing methods for attempting unilateral lung administration have significant shortcomings: for example, invasively inserting a needle into the bronchus to infuse the drug, or adjusting the animal's position to allow the animal to inhale the drug via the nose. These methods make it difficult to ensure that the drug is uniformly distributed within only one lung lobe, often resulting in excessive accumulation of drug at the hilum or leakage into the contralateral lung, leading to poor model stability and reproducibility. Furthermore, previous unilateral lung injury models often used corrosive reagents such as hydrochloric acid to simulate aspiration lung injury. The proportion of ARDS caused by aspiration lung injury is far lower than that caused by pulmonary infection and sepsis, and the model's conformity to the pathophysiological characteristics of ARDS lacks verification. Statistically, lung inflammation caused by Gram-negative bacterial infection is one of the most common causes of clinical ARDS. Therefore, it is necessary to establish a unilateral lung injury ARDS model based on bacterial endotoxin-induced ARDS and evaluate and validate it using authoritative standards. Based on the above background, this invention provides a non-invasive, stable, and reproducible method for preparing a rat unilateral lung injury ARDS model to fill the gap in the existing technology. Summary of the Invention

[0006] A method for establishing a rat model of acute respiratory failure induced by non-invasive selective unilateral lung administration includes the following steps: anesthetizing rats, inserting a specially designed curved endotracheal tube orally into their trachea and directing it into one main bronchus; slowly injecting a predetermined volume of pathogenic reagent solution into the lung on the affected side through the tube; adjusting the rat's position during injection so that the lung on the treated side is in a lower position, and applying mechanical vibration to the rat to promote uniform distribution of the solution in the lung; keeping the tube in place for a period of time and then removing the tube; subsequently allowing the rat to breathe spontaneously for a certain period of time to induce unilateral lung injury to develop into an acute respiratory distress syndrome state, and, if necessary, providing mechanical ventilation support to the rat for a period of time; if mechanical ventilation can be performed and the injured state can still be maintained, finally verifying through pathological and physiological indicators that the unilateral lung injury has reached the criteria for an acute respiratory distress syndrome model.

[0007] Furthermore, the endotracheal tube has an approximately 160° bend at the tip and is marked with an orientation. The outer diameter of the tube is approximately 0.85 mm and the length is approximately 12 cm. During intubation, the tip of the tube is advanced toward the target lung until it is approximately 8.5 cm away from the rat's incisors, so that the tip of the tube enters the main bronchus on the target side.

[0008] Furthermore, the rat's body position was adjusted by tilting the body of the administration side downwards at approximately 70 degrees and fixing it in place. After the liquid was injected through the catheter, the rat was shaken for 1 minute at a frequency of approximately 1000 times / minute using an oscillator to promote the uniform distribution of the liquid in the lung on that side.

[0009] Furthermore, the resting time after the injection of liquid is approximately 30 minutes, during which the catheter is kept inserted to block the bronchial opening on the administration side, and the rat is kept in an inclined position to retain the liquid in the lung on the administration side; after the resting period, the catheter is removed and the rat's position is returned to horizontal.

[0010] Furthermore, the pathogenic agent is Gram-negative bacterial endotoxin lipopolysaccharide (LPS) or its functionally equivalent acute lung injury factor.

[0011] Furthermore, the dosage of the LPS solution is approximately 2.5 mg per rat, and the injection volume of the solution is 0.5–0.6 mL.

[0012] Furthermore, after extubation, the rats were allowed to breathe spontaneously for 4–6 hours to induce acute damage to one lung. Subsequently, the rats were given mechanical ventilation support for 3–5 hours to simulate the clinical mechanical ventilation treatment process, in which a lung-protective ventilation strategy with a tidal volume of 6–8 mL / kg was adopted.

[0013] Furthermore, the following indicators were used to verify whether the rat unilateral lung injury model reached the state of acute respiratory distress syndrome (2022 American Thoracic Society publication): significantly decreased arterial blood oxygen partial pressure and oxygenation index, extensive inflammatory cell infiltration and pulmonary edema in the injured lung, and significantly increased protein content and inflammatory factor levels in bronchoalveolar lavage fluid. Compared with the prior art, the present invention has the following advantages: Non-invasive targeted drug delivery, stable and reproducible model. This invention develops a method for inducing acute respiratory failure in rats through non-invasive selective unilateral lung administration. By using a rotating platform and a curved endotracheal tube, the drug solution is delivered almost 100% targeted to the target area in the left lung of the rat, avoiding the uneven distribution of drug solution that may be caused by traditional invasive intubation or postural inhalation methods. Compared with existing methods, this invention can ensure that the liquid is confined to a single lung lobe and evenly distributed.

[0014] Using the steps of this invention, a damaging treatment (such as administration of LPS or other reagents) can be performed on the left lung of the same experimental rat, while the right lung remains undamaged as a control. This allows for the acquisition of paired samples of the treated and control lungs in a single animal, resulting in higher statistical efficiency than traditional methods that require separate experimental and control groups.

[0015] This invention successfully established an animal model meeting the definition criteria for ARDS by injecting bacterial endotoxin LPS into a single lung of rats to induce lung injury. By optimizing the LPS dosage, fluid volume, and observation time, this model achieved a stable ARDS injury state in one lung after 5 hours of spontaneous breathing, exhibiting typical characteristics such as deteriorating oxygenation and decreased lung compliance during subsequent mechanical ventilation. This model, validated by multiple indicators, conforms to the pathophysiological characteristics of ARDS and can serve as a reliable platform for studying ARDS mechanisms and interventions. Furthermore, this method is flexible and scalable: by changing the inducing factor (such as Staphylococcus aureus Gram-positive bacterial toxin-hemolysin, Streptococcus pneumoniae toxin PLY protein, etc.), unilateral ARDS models with different etiologies can be constructed, providing new methods and perspectives for basic research related to ARDS. Attached Figure Description

[0016] Figure 1 A schematic diagram of the procedure for non-invasive selective administration of drugs to one lung of a rat; Figure 2 The results validate the operational stability of the unilateral lung drug delivery technique (Blue staining tracing in the field of Evans Biomedical Laboratory Technology). Figure 3 The results validate the stability of the fluid delivery position in a unilateral lung over 24 hours. Figure 4 Immunohistochemical staining results of caspase-11 expression in rat lung tissue after LPS administration; Figure 5 A schematic diagram of the experimental procedure for a unilateral ARDS model (including the timeline of the spontaneous breathing phase and the mechanical ventilation phase). Figure 6 Comparison of the gross appearance of lung tissue in rats from different groups 5 hours after intratracheal injection of LPS; Figure 7 Comparison of the gross appearance of lung tissue in rats from different groups 9 hours after intratracheal injection of LPS; Figure 8 The graph shows the change of oxygenation index (PaO2 / FiO2) over time for each experimental group. Figure 9 Comparison of HE staining images of rat lung tissue from different time points (magnified 200 times). Figure 10 Immunohistochemical results of myeloperoxidase (MPO), a marker of neutrophils, in each experimental group; Figure 11 The concentrations of tumor necrosis factor (TNF-α) in plasma and BALF; Figure 12Results for indicators related to alveolar-capillary barrier damage, including total protein concentration in BALF, wet-to-dry weight ratio, alveolar epithelial cell damage marker (SP-D), and endothelial cell damage marker (Ang-2). Detailed Implementation

[0017] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed merely to enable those skilled in the art to better understand and implement the subject matter described herein, and are not intended to limit the scope, applicability, or examples set forth in the claims. The function and arrangement of the elements discussed may be changed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the various examples. For example, the described methods may be performed in a different order than described, and steps may be added, omitted, or combined. Furthermore, features described in some examples may be combined in other examples.

[0018] As used herein, the term "including" and its variations represent open terms, meaning including but not limited to. The term "based on" indicates at least partially based on. The terms "one embodiment" and "an embodiment" refer to at least one embodiment. The term "another embodiment" refers to at least one other embodiment. The terms "first," "second," etc., may refer to different or the same objects. Other definitions, whether explicit or implicit, may be included below. Unless explicitly indicated by the context in the field of biomedical experimental technology, the definition of a term remains consistent throughout the specification.

[0019] Example A method for establishing a rat model of acute respiratory failure induced by non-invasive selective unilateral lung administration includes the following steps: anesthetizing rats, inserting a specially designed curved endotracheal tube orally into their trachea and directing it into one main bronchus; slowly injecting a predetermined volume of pathogenic reagent solution into the lung on the affected side through the tube; adjusting the rat's position during injection so that the lung on the treated side is in a lower position, and applying mechanical vibration to the rat to promote uniform distribution of the solution in the lung; keeping the tube in place for a period of time and then removing the tube; subsequently allowing the rat to breathe spontaneously for a certain period of time to induce unilateral lung injury to develop into an acute respiratory distress syndrome state, and, if necessary, providing mechanical ventilation support to the rat for a period of time; finally, verifying through pathological and physiological indicators that the unilateral lung injury has met the criteria for an acute respiratory distress syndrome model.

[0020] Furthermore, the endotracheal tube has an approximately 160° bend at the tip and is marked with an orientation. The outer diameter of the tube is approximately 0.85 mm and the length is approximately 12 cm. During intubation, the tip of the tube is advanced toward the target lung until it is approximately 8.5 cm away from the rat's incisors, so that the tip of the tube enters the main bronchus on the target side.

[0021] Furthermore, the rat's body position was adjusted by tilting the body of the administration side downwards at approximately 70 degrees and fixing it in place. After the liquid was injected through the catheter, the rat was shaken for 1 minute at a frequency of approximately 1000 times / minute using an oscillator to promote the uniform distribution of the liquid in the lung on that side.

[0022] Furthermore, the resting time after the injection of liquid is approximately 30 minutes, during which the catheter is kept inserted to block the bronchial opening on the administration side, and the rat is kept in an inclined position to retain the liquid in the lung on the administration side; after the resting period, the catheter is removed and the rat's position is returned to horizontal.

[0023] Furthermore, the pathogenic agent is Gram-negative bacterial endotoxin lipopolysaccharide (LPS) or its functionally equivalent acute lung injury factor.

[0024] Furthermore, the dosage of the LPS solution is approximately 2.5 mg per rat, and the injection volume of the solution is 0.5–0.6 mL.

[0025] Furthermore, after extubation, the rats were allowed to breathe spontaneously for 4–6 hours to induce acute damage to one lung. Subsequently, the rats were given mechanical ventilation support for 3–5 hours to simulate the clinical mechanical ventilation treatment process, in which a lung-protective ventilation strategy with a tidal volume of 6–8 mL / kg was adopted.

[0026] Furthermore, the following indicators were used to verify whether the rat unilateral lung injury model reached the state of acute respiratory distress syndrome: a significant decrease in arterial blood oxygen partial pressure and oxygenation index, extensive inflammatory cell infiltration and pulmonary edema in the injured lung, a significant increase in protein content and inflammatory factor levels in bronchoalveolar lavage fluid, and secondary injury signs in the contralateral lung that was not directly damaged.

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are for illustrating the present invention and not for limiting the scope of the present invention. Without departing from the spirit of the present invention, those skilled in the art can make various substitutions and modifications based on the ideas proposed in the present invention.

[0028] I. Non-invasive selective unilateral lung targeted drug delivery procedure First, the test rats were anesthetized. Healthy adult SD rats (weighing approximately 300–350 g) were selected and anesthesia was induced by intraperitoneal injection of sodium pentobarbital at a dose of 50 mg / kg. Subsequent procedures were performed after the rats lost consciousness. The anesthetized rats were then fixed onto a specially designed unilateral lung drug delivery platform. This platform consists of a lower base and a rotatable upper fixation plate. The lower base is placed on a horizontal experimental table, while the upper plate can rotate freely around its central axis and lock into position at any angle. The rats were fixed supine on the upper platform, initially in a horizontal position.

[0029] Next, directional endotracheal intubation was performed. With the rat's mouth open, a specially designed narrow-diameter endotracheal tube was slowly inserted into the trachea through the mouth. The endotracheal tube was approximately 12 cm in total length and 0.85 mm in outer diameter. The tip was shaped to an angle of approximately 160 degrees and marked with orientation. The tube was positioned to the left of the rat during insertion (approximately at the 9 o'clock position from a frontal view). During intubation, the tube was guided under direct vision, ensuring it passed the glottis and was gradually inserted into the trachea. When the tip of the tube reached a depth of approximately 8.5 cm from the rat's incisors, further insertion was stopped and the tube was secured. At this point, the tip of the tube was essentially inside the lumen of the left main bronchus. This depth was determined through extensive preliminary experiments based on rat airway anatomy. Maintaining this intubation depth in rats weighing between 300 and 360 g ensured stable entry of the tube into the left lung without excessive insertion into the alveoli.

[0030] To verify that the catheter is correctly located in the left bronchus, the following auxiliary judgment measures can be used: First, combining the insertion depth and catheter orientation markings mentioned above, it can be roughly inferred that the catheter is in the left bronchus; Second, place a thin thread or cotton swab at the opening of the catheter and observe whether the thread swings regularly during the rat's spontaneous breathing to indicate that airflow is passing through the catheter; Third, connect the end of the catheter to a syringe containing a small amount of sterile saline (e.g., 100L) and observe whether the droplet fluctuates up and down with respiration. These methods can further confirm that the catheter has not mistakenly entered the right bronchus or stomach, but is located in the left lung airway.

[0031] After successful intubation and positioning, the drug can be administered to the left lung via the catheter. In this embodiment, we selected lipopolysaccharide (LPS) solution as the drug delivery fluid to induce lung injury (the specific modeling dose is described later). Before administration, the platform was rotated and tilted so that the rat was in a low-lying position with its left side facing down. Preferably, the angle between the rat's body and the horizontal plane was adjusted to approximately 70 degrees, and the platform position was locked to maintain this position. This helps to prevent the fluid from flowing back into the right bronchus by utilizing gravity. Then, a predetermined volume of fluid (e.g., 0.6 mL) was slowly injected into the left lung through the inserted endotracheal tube. After the fluid injection was completed, the rat and platform were immediately lifted and gently placed on a micro-oscillator, which was oscillated at a frequency of approximately 1000 times / minute for 1 minute. This oscillation step further dispersed the injected fluid evenly within the left lung, helping to cover the entire left lobe. After oscillation, the rat was kept in the original low-lying left position for approximately 30 minutes. During this process, the endotracheal tube remains in the left main bronchus to act as an embolism, and gravity helps to keep the fluid mostly in the left lung, preventing it from flowing into the right lung due to respiratory movements or slight changes in body position. After resting for 30 minutes, the endotracheal tube is carefully removed, and the rat is then returned to a horizontal left lateral decubitus position to allow it to breathe freely and awaken.

[0032] In the above-mentioned non-invasive directional intubation drug delivery process, three key points ensured that the fluid was limited to the left lung and evenly distributed: (1) Precise catheter positioning in the field of biomedical experimental technology: Through the design of the curved catheter and the control of the insertion depth, the tip of the catheter accurately entered the left main bronchus, providing a pathway for directional fluid infusion; (2) Body position and occlusion in the field of biomedical experimental technology: The tilted left low body position combined with the occlusion effect of the intubation itself prevented the fluid from escaping into the healthy lung; (3) Oscillation to aid distribution in the field of biomedical experimental technology: Short-term high-frequency oscillation caused the fluid to diffuse from the hilum to the distal bronchus, avoiding confinement to the vicinity of the hilum. These measures worked together to ensure that the left lung was evenly infiltrated by the fluid, while the right lung remained uncontaminated.

[0033] Figure 1 This diagram illustrates the non-invasive selective unilateral lung drug delivery procedure for rats as described in this invention. The diagram shows the rat being fixed to a rotatable platform, with a specially designed curved catheter inserted orally into the left main bronchus. The platform rotates to tilt the rat downwards on its left side, allowing the reagent solution to be injected into the left lung through the catheter. After injection, the entire unit is placed on a shaking device and vibrated to ensure even distribution of the drug solution within the left lung. This schematic diagram visually demonstrates the key steps and device configuration for achieving targeted unilateral lung drug delivery in rats.

[0034] To demonstrate the effectiveness and reproducibility of the unilateral lung drug delivery procedure described above, we conducted a dye visualization experiment. A visual dye (such as Evans Blue, a biomedical experimental technology dye) without pathogenic factors was selected and injected into the left lung via catheter following the same procedure. The rats were sacrificed 24 hours later, and their lungs were harvested for dye distribution observation. The results showed that in each experiment, the dye stained only the left lung; the right lung showed no visible staining, and the dye distribution within the left lung remained stable. Repeated experiments yielded consistent results, indicating that the unilateral lung targeted drug delivery technology provided by this invention has good stability and reproducibility.

[0035] Figure 2 The results demonstrate the validation of the operational stability of the unilateral lung drug delivery technique. Figure 2 This is a typical rat lung photograph. It shows that after injection of Evans' Blue dye (from the biomedical experimental technology field), the left lung lobe exhibits uniform blue staining, while the right lung tissue remains unstained and retains its normal color. Repeated experiments yielded similar results, demonstrating that the method of this invention can reliably confine liquid to the left lung without contaminating the contralateral lung.

[0036] Furthermore, we verified the positional stability of the fluid injected into the left lung using this method. Even one day after administration, the fluid remained largely within the left lung without leaking into the right lung or systemic circulation. Lungs of rats injected with the dye using the above method were harvested several hours and 24 hours after administration to compare the dye coverage. The results showed that even after 24 hours, the dye coverage in the left lung remained essentially the same as several hours prior, while the right lung remained unstained. This phenomenon demonstrates that single-lung administration of the present invention is stable over a longer period and does not cause significant changes in fluid position due to respiration or changes in body position.

[0037] Figure 3 The results demonstrate that the fluid administered to one lung remained stable over 24 hours. The figures show a comparison of the rat lung appearance at 5 and 24 hours post-administration: the dye-marked areas in the left lung remained similar across different time points, while no staining was observed in the right lung. This demonstrates that, using the procedure of this invention, the fluid injected into the left lung will not migrate to the healthy lung or other parts of the body over a prolonged period.

[0038] The aforementioned verification experiments also included an examination of biological effects. It is known that Gram-negative bacterial endotoxin LPS can induce inflammatory responses in lung tissue, such as activating the expression of inflammatory proteases like caspase-11. We injected a certain dose of LPS solution into the left lung of rats according to the method of this invention to simulate a focal infectious ARDS model. Twenty-four hours after administration, lung tissues from both sides were harvested for immunohistochemical staining of caspase-11 protein. The results showed that compared to control rats that only received an equal volume of saline, the LPS-injected group showed significantly enhanced caspase-11 positive staining in the left lung tissue, while the right lung tissue of the same rat showed no significant difference in caspase-11 expression compared to the control group. This further demonstrates at the molecular level that the unilateral administration method of this invention can stably deliver the damaging factor to the target lung, inducing a local inflammatory response, while the contralateral lung, which did not directly contact the damaging factor, remains essentially normal.

[0039] Figure 4 Images showing the immunohistochemical staining results of caspase-11 protein after LPS injection into the left lung of rats. In the image, A, B, and C are tissue sections of the left lung of the control group (left lung injected with saline), the left lung of the LPS group, and the right lung of the LPS group, respectively. D is a statistical graph of the average optical density to grayscale value of the corresponding caspase-11 positive expression. Figure 4 As can be seen, after LPS injection into the left lung, a large area of ​​brown positive staining appeared in the lung tissue (indicated by arrows), indicating a significant upregulation of caspase-11 expression. In contrast, the right lung sections from the same rat and the control group showed only sporadic background staining with no obvious positive signal. Quantitative bar chart analysis further confirmed that the caspase-11 expression level in the left lung of the LPS-injected group was significantly higher than that in the control group (P < 0.05), while there was no statistically significant difference between the right lung of the LPS-injected group and the right lung of the control group. These results, at the microscopic level, validate the effectiveness of the non-invasive selective unilateral lung drug delivery method of this invention. The damaging factor only exerts its effect in the target lung, successfully inducing a focal inflammatory response, while the contralateral lung remains unaffected.

[0040] II. Construction and Validation of a Unilateral Lung Injury ARDS Model Building upon the successful establishment of unilateral lung-targeted drug delivery technology, this invention further provides a method for constructing a unilateral lung injury ARDS model using this technology. The specific steps for constructing the ARDS model and the resulting outcomes are described in detail below.

[0041] First, the selection of the damaging factor and the dosage: In this embodiment, Gram-negative bacterial endotoxin lipopolysaccharide (LPS) was selected as the damaging factor for inducing ARDS. This is because infectious ARDS caused by bacterial endotoxins is relatively common in clinical practice, and using LPS can improve the correlation between the model and clinical findings. The LPS reagent was derived from Escherichia coli strain O55:B5. Through preliminary exploration, we determined the optimized dosage to be approximately 2.5 mg / kg (equivalent to 2.5 mg LPS per approximately 300g rat), dissolved in sterile saline to prepare a solution with a total volume of 600 L. The actual volume of fluid injected into the left lung of each experimental rat via catheter was approximately 0.6 mL, containing approximately 2.5 mg LPS. An equal volume of sterile saline was selected as the control solution for single-lung administration to the control group rats.

[0042] The following is the modeling schedule and treatment plan: After successfully injecting LPS solution into the left lung, immediately shake for 1 minute and let stand for 30 minutes as described above, then place the rats on their left side on the experimental table. For the next 5 hours, allow the rats to maintain free and spontaneous breathing. This spontaneous breathing phase was used to observe the development of unilateral lung injury without mechanical ventilation intervention. Based on our findings, rats showed typical signs of ARDS (such as a significant decrease in oxygenation index and extensive inflammatory infiltration in the lungs) approximately 4–5 hours after LPS administration to one lung. Therefore, we considered the ARDS model to have been successfully established 5 hours after administration. Subsequently, to simulate the scenario of severe ARDS patients receiving mechanical ventilation support in clinical settings, we implemented mechanical ventilation on some rats at the 5-hour time point. Mechanical ventilation was performed using a small animal ventilator, with ventilation parameters set according to a protective ventilation strategy: tidal volume 6–8 mL / kg, respiratory rate approximately 80 breaths / min, and appropriate positive end-expiratory pressure (PEEP) to maintain oxygenation. Mechanical ventilation continued for 4 hours, ending the experiment 9 hours after administration. Throughout the process, deep anesthesia was maintained in rats as needed, and vital signs were monitored. Arterial blood was collected via a catheter in the right femoral artery at different time points (e.g., baseline condition before drug administration and various time points after drug administration) for blood gas analysis, monitoring indicators such as arterial oxygen partial pressure (PaO2), carbon dioxide partial pressure (PaCO2), and pH value; mean arterial pressure (MAP) and other circulatory indicators were continuously monitored via femoral artery catheterization to assess physiological changes during model development.

[0043] In the experimental design, we set up several control and experimental groups to verify the specific role of the unilateral lung injury ARDS model. Specific groups included: a left lung LPS group (FL group, LPS injected into the left lung unilaterally), a left lung saline group (FS group, an equal volume of saline injected into the left lung unilaterally), and corresponding bilateral LPS groups (UL group, half the dose of LPS injected into each lung, with the total dose the same as the FL group) and bilateral saline groups (US group). A normal control group (NC group) was also included, which did not receive any fluid injection. The FL and FS groups were used to compare the effects of different injected substances (LPS vs. saline) on the occurrence of ARDS, while the UL and US groups were used to compare the differences between bilateral and unilateral lung injury. The NC group provided a baseline reference. The number of rats in each group was selected according to statistical requirements (e.g., n=8–12 per group). Some rats were sacrificed at the end of spontaneous respiration 5 hours after administration to assess early ARDS damage; the remaining rats were mechanically ventilated until the end of 9 hours to assess late ARDS changes.

[0044] Figure 5 This is a schematic diagram of the experimental procedure for a unilateral lung injury ARDS model. The flowchart visually illustrates the treatment process for different experimental groups along the timeline: all rats first received intrapulmonary fluid injection (LPS or saline) into the left lung via endotracheal intubation at time 0, subsequently entering the spontaneous breathing phase. Blood gas analysis was performed on the rats at multiple time points during this period (as shown by the multiple sampling points marked in the figure). Five hours after drug administration, some rats in the FL and UL groups, as well as the NC group, were sacrificed under deep anesthesia for sampling (indicating the end point of the spontaneous breathing phase in the model). The remaining rats received mechanical ventilation support starting at 5 hours (as shown in the 5-9 hour segment in the figure). At the 9-hour endpoint, the remaining rats in all groups were sacrificed under deep anesthesia for sample collection. Figure 5 The flowchart shown clearly illustrates the time progression of model induction and the arrangement of each observation node.

[0045] (I) Gross observation and oxygenation function assessment of the ARDS model: With the passage of time following unilateral intrapulmonary injection of LPS, the morphology and function of the rat lungs changed significantly. Five hours after administration (at the end of spontaneous respiration), the gross appearance of the lungs in each group was observed by thoracotomy: the lung tissue of the normal control group (NC) rats was pinkish-white, smooth, and without local congestion; while the left lung of the rats that underwent LPS injection showed obvious dark red consolidation, with a solid texture, indicating severe alveolar exudative consolidation. In contrast, the lungs of the rats that underwent simultaneous bilateral LPS injection (UL group) also showed diffuse red consolidation, but the consolidation of the left lung of the rats that underwent unilateral LPS injection (FL group) was more concentrated and obvious; the right lung of the rats that did not receive LPS injection showed less overall appearance change because it was not directly damaged, with only a few signs of congestion visible in the area in contact with the diseased left lung.

[0046] Figure 6 These are comparative images of the lung appearance of rats in different groups 5 hours after intratracheal injection of LPS. Image A shows the lung of the normal control group (NC), which is uniformly pinkish-white. Image B shows the left lung of the LPS group (FL), where a large area of ​​dark red consolidation is visible in the left lung, while the right lung appears nearly normal. Image C shows the lungs of the bilateral LPS group (UL), where diffuse red consolidation is observed in both lungs. Figure 6 As can be clearly seen, the unilateral lung injury induced by this invention results in significant solid exudative lesions in the left lung, while the gross appearance of the healthy lung remains largely normal. This phenomenon is similar to the imaging manifestations of clinical unilateral ARDS patients, demonstrating that the model successfully simulates the lung injury characteristics of unilateral ARDS on a macroscopic level.

[0047] When the experiment continued for 9 hours (including 4 hours after mechanical ventilation), the gross changes in the lungs of rats in all groups became more significant. Especially in the LPS injection group that underwent mechanical ventilation, the consolidation of the injured lung worsened, becoming harder and exhibiting widespread atelectasis; the contralateral lung, having received more ventilation during mechanical ventilation, showed overinflation with pink hemorrhagic spots in some areas. In the bilateral LPS group, both lungs of rats showed severe consolidation and scattered hemorrhages after mechanical ventilation. In contrast, the lungs of rats in the saline control groups (FS and US groups) did not show significant abnormal changes after mechanical ventilation; the lung tissue maintained a relatively soft and pinkish normal appearance. This indicates that unilateral LPS-induced ARDS, after mechanical ventilation, did not reverse the damage and even worsened, which is consistent with the potential for worsening lung injury in clinically critically ill ARDS patients under mechanical ventilation.

[0048] Figure 7 The images show a comparison of the lung appearance of rats in different groups 9 hours after intratracheal administration of LPS. Image A shows the lung of the saline control group rats after mechanical ventilation; the lung tissue color and texture are close to normal. Image B shows the lung of the unilateral LPS group (FL) rats after mechanical ventilation; the left lung shows extensive gray-red consolidation and collapse, and slight patchy hemorrhages are also observed in the right lung. The lower left image shows the lung of the bilateral LPS group (UL) rats after mechanical ventilation; both lungs show diffuse consolidation with hemorrhages. The lower right image shows the control group (saved without ventilation) (and...). Figure 6 (Similar to NC in the field of biomedical experimental technology). Figure 7 It was found that the left lung lesion was more severe in the unilateral lung injury model after mechanical ventilation, while the healthy lung was also affected to some extent, but to a lesser degree. This further confirms that the unilateral ARDS model exhibits the characteristics of simultaneous collapse and atelectasis and hyperventilation injury under ventilatory support, providing an intuitive model for the study of VILI mechanisms.

[0049] Besides the gross morphology of the lungs, one of the most important features of the model in this invention is the significant impairment of oxygenation function. We calculated the change in oxygenation index (OI, i.e., PaO2 / FiO2) over time in each group of rats using arterial blood gas analysis to quantify the degree of ARDS. The results showed that before intratracheal infusion of fluid (whether LPS or saline), the oxygenation index of all groups was above 400 mmHg, with no significant difference, falling within the normal range. Within 5 hours after infusion and the onset of spontaneous respiration, the oxygenation index of all groups (FL and UL groups injected with LPS, and FS and US groups injected with saline) rapidly decreased: reaching its lowest value at 1 hour, approximately half of the baseline (significantly lower than 300 mmHg); although it subsequently recovered slightly, it remained significantly lower than the control group (P < 0.05). This may be related to the larger volume of fluid injected. Notably, at the end of the 5-hour spontaneous respiration period, the oxygenation index levels of the FL and UL groups were similar, indicating that both unilateral and bilateral LPS infusion led to severe hypoxia.

[0050] Upon entering the mechanical ventilation phase, significant differentiation in oxygenation occurred among the different groups: the oxygenation index of the control saline injection groups (FS, US) gradually recovered to near-normal levels (above 400 mmHg) after the start of mechanical ventilation, indicating that unilateral or bilateral saline injection did not cause persistent lung function damage, and mechanical ventilation actually improved the mild hypoxia previously caused by transient atelectasis; while the LPS injection groups (FL, UL) showed a decrease in oxygenation index during mechanical ventilation, from approximately 250 mmHg at 5 hours to approximately 200 mmHg at the end of 9 hours. Four hours after mechanical ventilation, there were statistically significant differences in oxygenation between the FL and UL groups and their respective saline control groups (P < 0.05), but no significant difference between the FL and UL groups. This means that once ARDS develops after 5 hours, regardless of whether it is unilateral or bilateral lung injury, subsequent mechanical ventilation support will not improve oxygenation, and may even lead to further deterioration of oxygenation due to ventilation-related damage. The above results are consistent with clinical observations of severe ARDS patients who still have poor oxygenation even with mechanical ventilation, proving that the model of this invention has clinical relevance.

[0051] Figure 8The graphs show the changes in the oxygenation index (PaO2 / FiO2) over time for each group of rats. Different colored curves represent the FL (left lung LPS), UL (bilateral lung LPS), FS (left lung saline), and US (bilateral lung saline) groups, respectively. It can be seen that during the 0-5 hour spontaneous respiration phase, the oxygenation index of the two LPS-injected groups (OI curves are represented by red and purple) decreased significantly and remained at a low level. The vertical dashed line indicates the start of mechanical ventilation. It can be seen that during the 5-9 hour period, the oxygenation in the saline group improved rapidly, while the oxygenation in the LPS group decreased instead of increasing. Finally, at 9 hours, the oxygenation index of the FL and UL groups was approximately 200 mmHg, significantly lower than the corresponding control group curves. Figure 8 It can be seen that the ARDS model constructed in this invention exhibits typical hypoxemia characteristics, and its condition does not improve under mechanical ventilation support, reflecting the refractory hypoxic characteristics of ARDS.

[0052] In addition to oxygenation function, we also monitored other physiological indicators in the model rats. For example, arterial carbon dioxide partial pressure (PaCO2) initially increased during the initial LPS injection (indicating decreased pulmonary ventilation function), and then partially recovered during spontaneous respiration. However, after mechanical ventilation, PaCO2 in the saline group significantly decreased (indicating a certain degree of hyperventilation), while PaCO2 in the LPS group remained near normal levels (around 40 mmHg), indicating that the damaged lungs still suffered from ventilation dysfunction under ventilation support. The changes in blood pH were also consistent: the saline group showed a tendency towards respiratory alkalosis after mechanical ventilation (pH increased to around 7.5), while the LPS group maintained the pre-injury acid-neutral level. The mean arterial pressure (MAP) showed a generally consistent trend across the groups, suggesting that this model mainly affects pulmonary oxygenation and ventilation function, with a smaller and similar impact on systemic circulation. Details of these data are not elaborated here.

[0053] (II) Assessment of Lung Histology and Inflammatory Response in the ARDS Model: To verify the reproduction of ARDS pathological features by the model of this invention at the tissue and molecular levels, we performed histological examination and measured inflammatory markers on lung tissues from rats in different groups at different time points. This mainly included HE staining of lung tissue pathological sections, immunohistochemical staining and quantification of myeloperoxidase (MPO) in lung tissue, detection of inflammatory factor levels in bronchoalveolar lavage fluid (BALF) and plasma, and detection of alveolar-capillary barrier permeability indicators.

[0054] In terms of pathological histological examination, lung tissues from the FL group (left lung LPS), UL group (bilateral lung LPS), and control group were collected at 5 and 9 hours after drug administration. Paraffin sections were prepared for hematoxylin-eosin (HE) staining to observe changes in alveolar structure. The results showed that lung tissues directly exposed to LPS (such as the left lung of FL and the lung of UL at 5 h, and the left lung of FL and the lung of UL at 9 h) all showed typical pathological damage changes of ARDS, including extensive infiltration of alveolar cavities and pulmonary interstitium by neutrophils, edema and thickening of alveolar septa, hemorrhage, and hyaline membrane changes. The damage was most severe in the left lung of FL and the lung of UL at 9 h. In contrast, lung tissues not directly exposed to LPS (such as the right lung of the FL group) also showed mild inflammatory cell infiltration and septal thickening at 5 h, but the degree was significantly lower than that of the directly damaged lungs, and it worsened at 9 h but was still weaker than the damage in the left lung. This indicates that unilateral lung injury not only causes a severe inflammatory response in the damaged lung, but also has a certain impact on the healthy lung (possibly due to the release of systemic inflammatory mediators), although the damage to the healthy lung is relatively mild.

[0055] Figure 9 HE-stained pathological images of rat lung tissue at different time points (200x magnification). Each small image represents a lung tissue section from one rat: A is the lung tissue of the normal control group (NC), showing intact alveolar structure without obvious inflammation; B is the left lung of the unilateral LPS group at 5 hours, with neutrophil infiltration in the alveolar cavity (black arrow) and slight thickening of the alveolar septa; C is the right lung of the same rat, with only a few scattered inflammatory cells; D is the lung tissue of the bilateral LPS group at 5 hours, with diffuse neutrophil infiltration and alveolar structural destruction; E is the left lung of the unilateral LPS group at 9 hours, with a large number of neutrophils filling the alveoli with hemorrhage and extensive consolidation of the lung tissue; F is the lung of the bilateral LPS group at 9 hours, with a similar degree of damage to E. Figure 9 It is evident that, with prolonged time, LPS-induced lung injury significantly worsened in the directly damaged lung, while secondary damage gradually appeared in the undamaged lung (the right lung in the unilateral model). These pathological results further confirm that the model of this invention successfully induced the pathological changes of acute inflammatory lung injury specific to ARDS.

[0056] To quantify the degree of inflammation, we performed MPO immunohistochemical staining and image analysis. MPO is a characteristic enzyme of neutrophils and can reflect the degree of neutrophil infiltration in lung tissue. By staining lung tissue sections from different groups with MPO and calculating the average optical density of the positive area per high-power field, we found a trend consistent with HE staining observations: at 5 hours after drug administration, both the injured side (left lung, UL lung) and the healthy side (right lung) showed significant MPO positivity enhancement compared to the normal control (P < 0.05), with the injured side showing higher positivity than the healthy side (P < 0.05); and at 9 hours, MPO positivity further increased in all sites, with the highest positivity in the unilateral injured left lung. This indicates that LPS-induced inflammation accumulates and worsens over time and is directly proportional to the degree of damage to the affected lung.

[0057] In addition to tissue sections, we examined the levels of inflammatory factors to evaluate systemic and local pulmonary inflammatory responses. Taking tumor necrosis factor (TNF-α) as a representative, 5 hours after LPS injection, the concentration of TNF-α in rat plasma significantly increased: both the left lung LPS group and the bilateral LPS group were significantly higher than the control group without fluid injection (P < 0.05), with similar values ​​between the two groups. After mechanical ventilation (9 hours), plasma TNF-α in the LPS-injected groups remained significantly higher than their respective saline control groups (P < 0.05), while there was no significant difference between the left and bilateral LPS groups. TNF-α in BALF was also significantly higher in the left lung LPS group than in the saline control group at 9 hours (P < 0.05), but although TNF-α was elevated in the bilateral LPS group, the difference did not reach statistical significance. This suggests that both the single-lung and bilateral ARDS models show significant responses at the systemic inflammatory level, but the local pulmonary inflammatory response is more concentrated in the single-lung model.

[0058] Typical indicators included total protein content in BALF, lung tissue wet-to-dry weight ratio, pulmonary surfactant-related protein D (SP-D) in BALF, and angiopoietin-2 (Ang-2) levels in plasma. Results showed that within 9 hours of LPS injection, compared to the corresponding saline control, the total protein concentration in BALF was significantly increased in both the single-lung and double-lung LPS groups (P < 0.05), with a more significant increase in the single-lung LPS group, but the difference was not statistically significant compared to the double-lung group. This indicates that both unilateral and bilateral ARDS resulted in increased alveolar membrane permeability and protein leakage into the alveoli, with a slightly higher value in unilateral damage due to higher local concentrations. Regarding lung tissue water content (wet / dry weight ratio), at 5 hours, the left lung affected by LPS was significantly higher than the control lung, and at 9 hours, the lobes directly exposed to LPS (left FL, left UL, right UL) were significantly higher than the right FL without LPS exposure (P < 0.05), indicating that pulmonary edema worsened over time and with the expansion of the injury area. In BALF, SP-D concentration served as an indicator of alveolar epithelial cell damage. At 9 hours, the left lung affected by LPS was significantly higher than the control lung, while the UL group showed a slight increase with no statistically significant difference (unilateral injury can damage alveolar epithelial cells). Plasma Ang-2, as an indicator of endothelial permeability damage, showed higher levels in the bilateral LPS group than in the control and unilateral groups at 5 hours (suggesting that simultaneous bilateral lung injury has a greater impact on systemic vascular endothelium), but at 9 hours, Ang-2 levels in both models were elevated and similar. Overall, these results demonstrate that the unilateral ARDS model established by this invention reproduces the key pathological processes of ARDS in terms of pulmonary inflammatory mediator release and changes in pulmonary vascular permeability.

[0059] Based on the verification of the above physiological, tissue, and molecular indicators, the rat unilateral lung injury ARDS model of this invention successfully meets the ARDS model evaluation criteria: significant abnormal changes were observed in four aspects: physiological function (severe hypoxemia, etc.), lung histology (acute inflammatory injury), inflammatory response (elevated inflammatory factors), and alveolar-capillary barrier (protein leakage, edema, cell damage markers). The 2022 version of the ARDS model criteria stipulates that any three aspects must be significant for the model to be considered valid; this model demonstrates significance in all four aspects, indicating that its simulation of ARDS is comprehensive and typical.

[0060] Those skilled in the art will understand that the various embodiments disclosed above can be modified and altered in various ways without departing from the spirit of the invention. Therefore, the scope of protection of this invention should be defined by the appended claims.

[0061] It should be noted that not all steps and units in the above processes are necessary; some steps or units can be omitted as needed. The execution order of each step is not fixed and can be determined as required. The device structure described in the above embodiments can be a physical structure or a logical structure. That is, some units may be implemented by the same physical entity, or some units may be implemented by multiple physical entities, or they may be jointly implemented by certain components in multiple independent devices.

[0062] The specific embodiments described above are exemplary embodiments, but do not represent all embodiments that can be implemented or fall within the scope of the claims. The term "exemplary" as used throughout this specification means used as an example, instance, or illustration, and does not imply preference or advantage over other embodiments. Specific details are included to provide an understanding of the described techniques. However, these techniques can be practiced without these specific details. In some instances, well-known structures and apparatuses are shown in block diagram form to avoid obscuring the concepts of the described embodiments.

[0063] The foregoing description of this disclosure is provided to enable any person skilled in the art to implement or use this disclosure. Various modifications to this disclosure will be apparent to those skilled in the art, and the general principles defined herein can be applied to other variations without departing from the scope of this disclosure. Therefore, this disclosure is not limited to the examples and designs described herein, but is consistent with the widest scope of the principles and novel features disclosed herein.

Claims

1. A method for establishing a rat model of acute respiratory failure induced by non-invasive selective unilateral lung administration, characterized in that, The procedure includes the following steps: anesthetizing rats, inserting a specially designed curved endotracheal tube orally into their trachea and directing it into one main bronchus; slowly injecting a predetermined volume of pathogenic reagent solution into the lung on that side through the tube; adjusting the rat's position during injection so that the lung on the receiving side is in a lower position, and applying mechanical vibration to the rat to promote uniform distribution of the solution in the lung; keeping the tube in place for a period of time before removing it; subsequently allowing the rat to breathe spontaneously for a certain period of time to induce unilateral lung injury to develop into an acute respiratory distress syndrome (ARDS) state; if necessary, providing mechanical ventilation support to the rat for a period of time, and maintaining the ARDS state even after mechanical ventilation; finally, verifying through pathological and physiological indicators that the unilateral lung injury has met the ARDS model criteria.

2. The method for establishing a rat model of acute respiratory failure induced by non-invasive selective unilateral lung administration according to claim 1, characterized in that: The endotracheal tube has an approximately 160° bend at the tip and is marked with an orientation. The outer diameter of the tube is approximately 0.85 mm and the length is approximately 12 cm. During intubation, the tip of the tube is advanced toward the target lung until it is approximately 8.5 cm away from the rat's incisor, so that the tip of the tube enters the main bronchus on the target side.

3. The method for establishing a rat model of acute respiratory failure induced by non-invasive selective unilateral lung administration according to claim 1, characterized in that: The rat's body position was adjusted by tilting the body of the administration side downwards at about 70 degrees and fixing it in place. After the liquid was injected through the catheter, the rat was shaken for 1 minute at a frequency of 1000 times / minute using an oscillator to promote the uniform distribution of the liquid in the lung on that side.

4. The method for establishing a rat model of acute respiratory failure induced by non-invasive selective unilateral lung administration according to claim 1, characterized in that: The incubation period after the injection of the liquid is 30 minutes. During this period, the catheter is kept inserted to block the bronchial opening on the administration side, and the rat is kept in a tilted position to retain the liquid in the lung on the administration side. After the incubation period, the catheter is removed and the rat is returned to a horizontal position.

5. The method for establishing a rat model of acute respiratory failure induced by non-invasive selective unilateral lung administration according to claim 1, characterized in that: The pathogenic agent is Gram-negative bacterial endotoxin lipopolysaccharide (LPS) or its functionally equivalent acute lung injury factor.

6. The method for establishing a rat model of acute respiratory failure induced by non-invasive selective unilateral lung administration according to claim 5, characterized in that: The dosage of the LPS solution was 2.5 mg per rat, 0.6 ml of liquid, and the injection volume of the solution was 0.5–0.6 mL.

7. The method for establishing a rat model of acute respiratory failure induced by non-invasive selective unilateral lung administration according to claim 1, characterized in that: After extubation, rats were allowed to breathe spontaneously for 4–6 hours to induce acute injury to one lung. Subsequently, rats were given mechanical ventilation support for 3–5 hours to simulate the clinical mechanical ventilation treatment process, in which a lung-protective ventilation strategy with a tidal volume of 6–8 mL / kg was adopted.

8. The method for establishing a rat model of acute respiratory failure induced by non-invasive selective unilateral lung administration according to claim 1, characterized in that: The following indicators were used to verify whether the rat unilateral lung injury model reached the state of acute respiratory distress syndrome: a significant decrease in arterial blood oxygen partial pressure and oxygenation index, typical pathological changes, extensive inflammatory cell infiltration and pulmonary edema in the injured lung, a significant increase in protein content in bronchoalveolar lavage fluid (BALF), a significant increase in the levels of inflammatory factors in plasma and BALF, significant changes in alveolar-capillary barrier specific markers, and the observation of secondary injury signs in the uninjured contralateral lung.

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