Pulmonary aspergillosis rat model construction method based on tracheal inoculation under laryngoscope direct view and application thereof

Abstract

The invention belongs to the technical field of model animal preparation, and particularly relates to a pulmonary aspergillosis rat model construction method based on tracheal inoculation under laryngoscope direct view and application thereof. According to the invention, standardized immunosuppression is carried out on rats by using cyclophosphamide, and an immunodeficiency state conforming to a disease basis can be stably and repeatedly induced. Based on direct sight of the laryngoscope, the glottis structure of the rat is clear and visible, and the intubation process is visual and controllable. The technical threshold is obviously reduced, and the success rate and the operation repeatability of a beginner are improved; the method has the advantages that laryngeal injury and mistaken inoculation are avoided to the greatest extent, it is guaranteed that bacterial liquid is accurately fed into the trachea, neck operation wounds are avoided, non-specific inflammation interference is reduced, the uniformity, stability and repeatability of a model are improved, and the modeling success rate reaches 100%. The invention provides a disease research platform which is standardized, quantifiable, high in quality and highly associated with clinic, and can be used for screening the pulmonary aspergillosis treatment medicines and evaluating the effectiveness of the pulmonary aspergillosis treatment medicines.

Description

Technical Field

[0001] This invention belongs to the field of model animal preparation technology, specifically relating to a method for constructing a rat model of pulmonary aspergillosis based on tracheal inoculation under direct laryngoscope vision and its application. Background Technology

[0002] Invasive pulmonary aspergillosis (IPA) is a fungal infection caused by Aspergillus invading the bronchi and lung tissue, leading to localized granulomas or widespread purulent histopathological damage. Its occurrence and manifestations largely depend on the host's immune status, typically occurring in immunocompromised patients. It has a mortality rate as high as 90%, with over 300,000 cases worldwide annually. Clinical diagnostic indicators for IPA include fungal antigen detection, histopathological examination, and etiological examination. Invasive pulmonary aspergillosis (IPA) is a fatal fungal pneumonia primarily affecting immunocompromised individuals, with its incidence increasing annually and exhibiting a high disease burden and mortality rate. Therefore, establishing animal models that accurately simulate the pathophysiological processes of human diseases is of irreplaceable value for a deeper understanding of pathogenesis, screening for novel antifungal targets, and evaluating drug efficacy. The appropriateness of model selection and the reliability of the modeling method directly affect the clinical translational value of experimental results.

[0003] Currently, there is no unified standard for constructing animal models of invasive pulmonary aspergillosis (IPA). In China, the commonly used methods are nasal instillation in mice, non-direct-vision tracheal instillation in rats, or transtracheal puncture in rats and rabbits. However, nasal instillation is extremely imprecise in localization and has poor uniformity. Large amounts of bacterial fluid remain in the upper respiratory tract or enter the digestive tract, making the dose and distribution of infection in the lungs uncontrollable and resulting in significant individual variability. Non-direct-vision tracheal instillation requires a high level of operator skill, and its success rate and uniformity are highly dependent on the operator's experience. Accidental esophageal intubation can lead to model failure, the catheter may damage the airway, and inconsistent intubation depth can result in uneven distribution of pulmonary infection foci. Transtracheal puncture is an invasive procedure with significant interference. The surgical trauma itself can trigger non-infectious inflammation, severely interfering with the interpretation of infection-specific pathology and immune responses. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing a rat model of pulmonary aspergillosis based on tracheal inoculation under direct laryngoscopy and its application, thereby improving the success rate of constructing a rat model of pulmonary aspergillosis, enhancing the model's uniformity, stability, and reproducibility, accurately screening drugs for the treatment of pulmonary aspergillosis, and evaluating the efficacy of drugs for the treatment of pulmonary aspergillosis.

[0005] This invention provides a method for constructing a rat model of pulmonary aspergillosis, comprising the following steps: Standardized immunodeficient rats were obtained by immunosuppression of rats using cyclophosphamide; The immunodeficient rats were anesthetized and fixed using a fixation device. Under direct visualization with a cold light source using a laryngoscope, Aspergillus fumigatus solution was administered into the lungs of the rats via endotracheal intubation through the glottis. This confirmed the establishment of a rat model of pulmonary aspergillosis. The verification included serological immunological verification, specifically the detection of serum galactomannan content. The serum galactomannan content was significantly increased by more than 10% compared to the negative control group, indicating successful model establishment.

[0006] Preferably, the dosage of cyclophosphamide is 30-40 mg / kg / day, administered continuously for 3-5 days.

[0007] Preferably, the cyclophosphamide is administered via intraperitoneal injection.

[0008] Preferably, the dosage of the Aspergillus fumigatus solution is 0.1~0.5 mL / animal, and the concentration of the Aspergillus fumigatus solution is 1×10⁻⁶. 7 ~1×10 9 CFU / mL.

[0009] Preferably, the step of administering Aspergillus fumigatus solution to the lungs of rats includes: making two symmetrical holes in the tube wall 2-3 mm from the blunt tip of the flexible gavage needle or catheter, with the needle tip angled towards the tail end, using a sterile syringe needle at a 15° angle to the tube wall, to obtain the modified flexible gavage needle or catheter. Gently pull the rat's tongue to one side with small tweezers, insert the cold light source of the laryngoscope into the oral cavity, locate the glottis in the pharynx, insert the syringe containing Aspergillus fumigatus liquid into the modified flexible gavage needle tubing or catheter, and slowly insert the rat laryngoscope through the glottis in the pharynx into the trachea. Press the syringe plunger all the way down to inject the Aspergillus fumigatus liquid into the lungs.

[0010] Preferably, the modified flexible gavage needle or catheter has graduation lines on the tube wall at 3cm and 4cm from the blunt tip.

[0011] Preferably, the anesthesia and fixation using a fixation device are performed 24 hours after obtaining the immunodeficient rats; The anesthesia was administered using isoflurane. The method of fixing the rat with the fixation device includes: fixing the anesthetized rat on the endotracheal intubation platform, making the rat supine, fixing the upper incisors to the edge of the operating board, raising the operating board at a 45° angle, and keeping the rat's head and neck back at the same level.

[0012] Preferably, the verification also includes etiological verification, other immunological verification, and pathological verification.

[0013] Preferably, the etiological verification, other immunological verification, and pathological verification include one or more of the following: rat physical examination, routine blood test, cellular immune status test, lung coefficient test, lung tissue fungal colony load, and lung tissue pathological examination.

[0014] This invention provides the application of the rat model of pulmonary aspergillosis constructed by the construction method described above in screening drugs for the treatment of pulmonary aspergillosis and / or evaluating the efficacy of drugs for the treatment of pulmonary aspergillosis.

[0015] Beneficial effects: This invention utilizes cyclophosphamide to induce immunosuppression in rats, resulting in immunodeficient rats. These rats are anesthetized and immobilized using a fixation device. Under direct laryngoscopy, *Aspergillus fumigatus* solution is administered into the rat lungs via endotracheal intubation through the pharyngeal glottis. The presence of serum galactomannan content confirms the establishment of a rat model of pulmonary aspergillosis. This invention utilizes cyclophosphamide to induce immunosuppression in rats, stably and reproducibly inducing an immunodeficient state consistent with the disease basal state. Direct laryngoscopy provides clear visualization of the rat's glottic structure, and the intubation process is intuitive and controllable. This significantly lowers the technical barrier, improves the success rate and reproducibility for beginners, and makes precise inoculation "visual" and "standardized." It minimizes laryngeal injury and mis-inoculation, ensures accurate delivery of the bacterial solution into the trachea, avoids neck surgical trauma, reduces non-specific inflammatory interference, and improves the model's uniformity, stability, and reproducibility, achieving a 100% success rate in animal model establishment. This invention provides a standardized, quantifiable, high-quality, and clinically relevant disease research platform for screening and evaluating the efficacy of drugs for pulmonary aspergillosis. It will significantly enhance the reliability, sensitivity, and scientific value of antifungal drug efficacy evaluation, pathogenic mechanism exploration, and host-fungal interaction studies. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 HE staining results of lung tissue from rats in cyclophosphamide group A in Example 1; Figure 2 HE staining results of lung tissue from rats in the low-dose cyclophosphamide + fungal infection group B in Example 1; Figure 3 HE staining results of lung tissue from rat C in the medium-dose cyclophosphamide + fungal infection group of Example 1; Figure 4 HE staining results of lung tissue from rat D in the high-dose cyclophosphamide + fungal infection group of Example 1; Figure 5HE staining results of lung tissue from rats in the negative control group A of Example 2; Figure 6 HE staining results of lung tissue from rat control group B in Example 2; Figure 7 HE staining results of lung tissue from rat C in the itraconazole capsule group of Example 2; Figure 8 HE staining results of lung tissue from rats in the nebulized formulation group H in Example 2; Detailed Implementation

[0018] This invention provides a method for constructing a rat model of pulmonary aspergillosis, comprising the following steps: Standardized immunodeficient rats were obtained by immunosuppression of rats using cyclophosphamide; The immunodeficient rats were anesthetized and fixed using a fixation device. Under direct visualization with a cold light source using a laryngoscope, Aspergillus fumigatus solution was administered into the lungs of the rats via endotracheal intubation through the glottis. This confirmed the establishment of a rat model of pulmonary aspergillosis. The verification included serological immunological verification, specifically the detection of serum galactomannan content. The serum galactomannan content was significantly increased by more than 10% compared to the negative control group, indicating successful model establishment.

[0019] This invention utilizes cyclophosphamide to suppress the immune system in rats, resulting in standardized immunodeficient rats.

[0020] In one embodiment, the rats described in this invention are SD rats, aged 6-10 weeks. In another embodiment, when the rats described in this invention are female rats, their weight is 193-260 g, and when they are male rats, their weight is 250-335 g.

[0021] In one embodiment, the dosage of cyclophosphamide described in this invention is 30-40 mg / kg / day, administered continuously for 3-5 days; in another embodiment, the dosage of cyclophosphamide described in this invention is 40 mg / kg / day, administered continuously for 3 days. In one embodiment, the method of administration of cyclophosphamide described in this invention is intraperitoneal injection. This invention utilizes a specific concentration of cyclophosphamide to quantitatively suppress the immune system in rats; this method can stably and reproducibly induce an immunodeficiency state consistent with the underlying disease.

[0022] After obtaining the immunodeficient rats, the present invention anesthetizes and fixes the immunodeficient rats using a fixation device. Under direct vision with a cold light source in a laryngoscope, the Aspergillus fumigatus liquid is administered into the lungs of the rats via endotracheal intubation through the pharyngeal glottis. Through verification, a rat model of pulmonary aspergillosis is obtained.

[0023] The validation described in this invention includes serological immunological validation; the serological immunological validation includes the detection of serum galactomannan content, where the serum galactomannan content is significantly increased by more than 10% compared to the negative control group, indicating successful model construction. As one implementation, the validation described in this invention also includes etiological validation, other immunological validations, and pathological validation. As one implementation, the etiological validation, other immunological validations, and pathological validations described in this invention include one or more of the following: rat physical examination, complete blood count, cellular immune status detection, lung coefficient detection, lung tissue fungal colony load, and lung tissue pathological examination. As one implementation, the rat physical examination described in this invention includes tachypnea detection. As one implementation, the cellular immune status detection described in this invention includes the detection of the peripheral blood CD4+ / CD8+ T cell ratio. As one implementation, the lung tissue pathological examination described in this invention includes the detection of hyphal infiltration and the degree of inflammatory destruction. After confirming the positivity of serum galactomannan, this invention further systematically evaluated the rats from multiple dimensions, including physical signs, routine blood tests, immune status, lung coefficient, fungal load in lung tissue, and histopathology. This ensured that the model successfully simulated invasive infection in terms of pathogeny, immunology, and pathology, and laid a solid foundation for evaluating drug efficacy from multiple perspectives in subsequent pharmacodynamic studies.

[0024] In one embodiment, the immunodeficient rats of the present invention are anesthetized 24 hours after birth and then immobilized using a fixation device. In one embodiment, isoflurane is used for anesthesia. In another embodiment, the fixation method described in the present invention includes: immobilizing the anesthetized rat on an endotracheal intubation platform, positioning the rat in a supine position, fixing the upper incisors to the edge of the operating board, raising the operating board at a 45° angle, and keeping the rat's head and neck at the same horizontal level. Using the fixation method described in the present invention facilitates exposure of the glottis and intubation procedures.

[0025] In one embodiment, the present invention provides two symmetrical holes made on the wall of the flexible gavage needle or catheter at a 15° angle to the wall, with the needle tip angled towards the tail end, 2-3 mm from the blunt tip of the flexible gavage needle or catheter. In another embodiment, the specifications of the flexible gavage needle or catheter are: TFEP-X-03, 2.25 mm × 40 mm, or TFEP-001, 2.25 mm × 50 mm. The symmetrical opening of two side holes allows for uniform lateral dispersion of the bacterial solution, solving the problem of uneven distribution during airway injection. In yet another embodiment, the modified flexible gavage needle or catheter has graduation lines at 3 cm and 4 cm from the blunt tip of the blunt tip, allowing for precise control of the insertion depth under direct laryngoscopic visualization, preventing reflux due to shallow insertion or unilateral lung infection due to excessive depth.

[0026] In one implementation method, after obtaining the modified flexible gavage syringe or catheter, the present invention uses small forceps to gently pull the rat's tongue to one side, inserts a laryngoscope with a cold light source into the oral cavity, locates the glottis in the pharynx, inserts a syringe containing Aspergillus fumigatus solution into the blunt tip of the modified flexible gavage syringe or catheter, and slowly inserts it into the trachea through the glottis using the rat laryngoscope. The syringe plunger is then pressed all the way down to inject the Aspergillus fumigatus solution into the lungs. In another implementation method, the modified gavage syringe of the present invention is sterilized by Co60 irradiation.

[0027] This invention utilizes a endotracheal intubation technique via the glottis under direct laryngoscopy to administer Aspergillus fumigatus solution into the lungs of rats. Compared to traditional intranasal or non-direct tracheal instillation, this technique achieves precise and quantitative delivery of Aspergillus fumigatus, ensuring direct and uniform entry of the bacterial solution into the lungs. This significantly reduces variability caused by the inoculation procedure, fundamentally improving the model's uniformity, stability, and reproducibility, resulting in a 100% success rate in animal model establishment. Tracheal puncture is an invasive procedure that may cause complications; nasal instillation cannot precisely control the inoculation volume and distribution. This invention, using glottis intubation, achieves significant advantages in both precision and safety.

[0028] In one embodiment, the dosage of the Aspergillus fumigatus solution of the present invention is 0.1~0.5 mL / animal; in another embodiment, the dosage of the Aspergillus fumigatus solution of the present invention is 0.2~0.4 mL / animal; in yet another embodiment, the dosage of the Aspergillus fumigatus solution of the present invention is 0.3 mL / animal. In one embodiment, the concentration of the Aspergillus fumigatus solution of the present invention is 1×10⁻⁶. 7 ~1×10 9 CFU / mL; as another embodiment, the concentration of the Aspergillus fumigatus solution of the present invention is 2×10⁻⁶ CFU / mL. 7 ~5×10 8 CFU / mL; as another embodiment, the concentration of the Aspergillus fumigatus solution of the present invention is 5 × 10⁻⁶ CFU / mL. 7 ~2×10 8 CFU / mL; as another embodiment, the concentration of the Aspergillus fumigatus solution of the present invention is 1×10⁻⁶. 8 CFU / mL.

[0029] This invention provides the application of the rat model of pulmonary aspergillosis constructed by the construction method described above in screening drugs for the treatment of pulmonary aspergillosis and / or evaluating the efficacy of drugs for the treatment of pulmonary aspergillosis.

[0030] As one implementation method, the pulmonary aspergillosis rat model described in this invention can be used to evaluate the efficacy of inhaled formulations. This invention involves administering the drug to be evaluated to a pulmonary aspergillosis rat model and observing the pathological changes in the lungs of the rat model. If the degree of improvement in the pathological changes in the lungs of the pulmonary aspergillosis rat model is observed, then the drug to be evaluated is effective. This invention provides a reliable, stable, and clinically valuable advanced preclinical research tool, which is expected to significantly improve the efficiency and success rate of new drug development.

[0031] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a method for constructing a rat model of pulmonary aspergillosis based on tracheal inoculation under direct laryngoscope visualization and its application. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0032] The experimental materials used in this invention are as follows: 1. Experimental animals: SD rats; age: 6-10 weeks; weight range: female: 193-260 g; male: 250-335 g.

[0033] 2. Experimental strain: Aspergillus fumigatus ( Aspergillus fumigatus GDMCC No. 3.627.

[0034] 3. Main testing instruments: anesthesia machine, flow cytometer, video laryngoscope, low-speed refrigerated centrifuge, electronic balance, fully automated biochemical analyzer, and quantitative nebulizer for lung fluid.

[0035] 4. Main reagents and positive control: Reagents: PDA culture medium, cyclophosphamide for injection, rat galactomannan (GM) ELISA kit, APC anti-rat CD4, PE anti-rat CD8a; Anesthetic: isoflurane; Positive control: itraconazole capsules and nebulized preparation.

[0036] Example 1 Construction of a rat model of pulmonary aspergillosis 1. Construction of a cyclophosphamide immunosuppression model (1) Eighteen SD rats that passed the adaptability observation were taken, and anticoagulated whole blood was prepared by collecting blood from the anterior vena cava. The blood volume was about 0.25 mL. The blood routine was measured. The rats were randomly divided into three groups according to the total white blood cell count and sex, with 6 rats in each group and half male and half female. The following operations were performed: Treatment Group 1 (40 mg / kg (3 d) group): Cyclophosphamide was administered intraperitoneally once a day for 3 consecutive days at a dose of 40 mg / kg / d, with an injection volume of 4 mL / kg / time. Treatment group 2 (30 mg / kg (5 d) group): Cyclophosphamide was administered intraperitoneally once a day for 5 consecutive days at a dose of 30 mg / kg / d, with an injection volume of 4 mL / kg / time; Treatment group 3 (40 mg / kg (5 d) group): Cyclophosphamide was administered intraperitoneally once a day for 5 consecutive days at a dose of 40 mg / kg / d, with an injection volume of 4 mL / kg / time.

[0037] (2) Blood and bone marrow hematological tests Anticoagulated whole blood was prepared by collecting blood from the anterior vena cava before drug administration and at 4, 6, 8, 10, and 12 days after the first injection. Approximately 0.25 ml of blood was collected. Hematological parameters at these time points were continuously monitored using a fully automated biochemical analyzer, and hemoglobin (HGB) and white blood cell (WBC) counts were analyzed. Twelve days after the first injection, rats were euthanized by exsanguination via the abdominal aorta under isoflurane inhalation anesthesia. The thymus and spleen were harvested, weighed wet, and organ indices were calculated. Bone marrow samples were collected to detect the counts of red blood cells (RBC), hemoglobin (HGB), white blood cells (WBC), neutrophils (NEUT), lymphocytes (LYMPH), monocytes (MONO), eosinophils (EO), and basophils (BASO). Specific test results are shown in Tables 1-4.

[0038] Table 1. Effects of cyclophosphamide on blood leukocytes in SD rats ( ±S, n=6)

[0039] Note: Compared with the 40 mg / kg (3 d) group, # indicates P <0.05, ## indicates P <0.01; compared with the 30 mg / kg (5 d) group, express P <0.05, express P <0.01.

[0040] Table 2 Effects of cyclophosphamide on hemoglobin in SD rats ( ±S, n=6)

[0041] Note: Compared with the 40 mg / kg (3 d) group, # indicates P <0.05, ## indicates P <0.01.

[0042] Table 3. Effects of cyclophosphamide on bone marrow leukocyte classification in SD rats ( ±S, n=6)

[0043] Note: Compared with the 40 mg / kg (3 d) group, # indicates P <0.05, ## indicates P <0.01; compared with the 30 mg / kg (5 d) group, express P <0.05, express P <0.01.

[0044] Table 4. Effects of cyclophosphamide on organ coefficients of thymus and spleen in SD rats ( ±S, n=6)

[0045] Note: Compared with the 40 mg / kg (3 d) group, # indicates P <0.05, ## indicates P <0.01; compared with the 30 mg / kg (5 d) group, express P <0.05, express P <0.01.

[0046] As shown in Tables 1-4, the white blood cell counts in the cyclophosphamide 40 mg / kg (3d), 30 mg / kg (5d), and 40 mg / kg (5d) groups were significantly lower than the pre-injection levels on days 4-12 after the first injection, and the decrease in white blood cells was dose-related. A trend of white blood cell recovery was observed on day 12. After injection, the hemoglobin levels in the cyclophosphamide 40 mg / kg (3d), 30 mg / kg (5d), and 40 mg / kg (5d) groups showed a gradual decreasing trend, and the decrease in hemoglobin was dose-related. The decrease in bone marrow white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, and basophils induced by cyclophosphamide was dose-related. The organ coefficients of the thymus and spleen in rats were significantly lower in the cyclophosphamide 40 mg / kg (5d) group compared to the 40 mg / kg (3d) group. Cyclophosphamide can induce a rat leukopenia model by injecting 40 mg / kg for 3 days, 30 mg / kg for 5 days, or 40 mg / kg for 5 days, thus obtaining standardized immunodeficient rats.

[0047] 2. Strain culture and inoculum preparation Under aseptic conditions, the *Aspergillus fumigatus* suspension is streaked or plated onto PDA medium using an inoculation loop. It is then incubated at 25°C-28°C for 3-7 days, until the *Aspergillus fumigatus* hyphae completely cover the medium. The inoculated *Aspergillus fumigatus* medium is washed with physiological saline or sterile water, the bacterial suspension is collected and mixed, counted using a hemocytometer, and the density of the *Aspergillus fumigatus* suspension is adjusted. It is then stored at 2-8°C for later use.

[0048] 3. Exploratory study of a rat model of invasive Aspergillus fumigatus lung disease (1) Twenty-four SD rats that passed the adaptability observation were selected, with half males and half females, and were randomly divided into four groups according to body weight: cyclophosphamide group (uninfected) (A), cyclophosphamide + Aspergillus fumigatus infection low-dose group (B), cyclophosphamide + Aspergillus fumigatus infection medium-dose group (C), and cyclophosphamide + Aspergillus fumigatus infection high-dose group (D), with six rats in each group, half males and half females. Before infection, each group was given an intraperitoneal injection of cyclophosphamide once a day for 3 consecutive days at a dose of 40 mg / kg / day, with an injection volume of 4 mL / kg / time. Twenty-four hours after the last injection, rats in each group were anesthetized and fixed on the endotracheal intubation platform. The tongue was gently pulled to one side with small forceps, and a laryngoscope was inserted into the oral cavity to locate the glottis in the pharynx. A modified gavage syringe was slowly inserted into the trachea through the glottis via the laryngoscope. The syringe plunger was quickly pressed all the way down, and 0.5 mL of Aspergillus fumigatus solution was injected into the lungs. This model was established once a day to replicate the invasive Aspergillus fumigatus lung disease model. Group A received saline injection, while Group B received an Aspergillus fumigatus solution concentration of 1×10⁻⁶. 7 CFU / mL, the injection concentration of Aspergillus fumigatus solution in group C was 1×10⁻⁶. 8 CFU / mL, the injection concentration of Aspergillus fumigatus solution in group D was 1×10⁻⁶. 9 CFU / mL.

[0049] (2) Serum galactomannan content (GM) determination and white blood cell count On days 3 and 7 of modeling, three animals from each group were selected to collect blood from the anterior vena cava or tail vein for serum galactomannan content (GM) and white blood cell count. The results are shown in Tables 5 and 6.

[0050] Table 5. Serum GM levels in rats with invasive Aspergillus fumigatus lung disease model ( - (x±s, n=3)

[0051] Note: Compared with the negative control group (A), express P <0.05, express P <0.01.

[0052] Table 6. Blood leukocyte count in rats with invasive Aspergillus fumigatus lung disease model ( - (x±s, n=3)

[0053] Note: Compared with the negative control group (A), express P <0.05, express P <0.01.

[0054] (3) Pathological observation of lung tissue On days 3 and 7 of modeling, three animals from each group were anesthetized, euthanized by exsanguination of the abdominal aorta, and lung tissue was dissected for pathological observation. The results were as follows: Figures 1-4 As shown.

[0055] According to Tables 5-6 and Figures 1-4 It can be seen that, compared with the cyclophosphamide control group, the total white blood cell count was significantly reduced in the low, medium and high dose groups of cyclophosphamide + Aspergillus fumigatus infection on day 3, and the total white blood cell count was significantly reduced in the low, medium and high dose groups of cyclophosphamide + Aspergillus fumigatus infection on day 7. The lungs of animals in each infection group showed a small to a large number of hyphae and varying degrees of alveolar epithelial destruction, vascular hemorrhage, increased goblet cells in the bronchial epithelium of the alveoli, and inflammatory cell infiltration.

[0056] Example 2 Effects of drugs on a rat model of invasive Aspergillus fumigatus lung disease mediated by cyclophosphamide immunization 1. Forty SD rats that met the adaptability criteria were selected. Before infection, they were randomly divided into a negative control group (A) and a negative control group (A) with equal numbers of males and females (10 rats per group). The remaining animals were included in the model group. Except for the negative control group, cyclophosphamide was administered intraperitoneally once daily for 3 consecutive days at a dose of 40 mg / kg / day, with an injection volume of 4 mL / kg / injection. 24 hours after the last injection, all model animals were anesthetized, and the rats were fixed on the tracheal intubation platform. The tongue of the animal was gently pulled to one side with small forceps, and a laryngoscope was inserted into the oral cavity to locate the glottis. The modified gavage syringe tubing / catheter was slowly inserted into the trachea through the glottis via the laryngoscope. The syringe plunger was quickly pressed to the bottom, and Aspergillus fumigatus solution (0.1 mL / rat, 2×10⁻⁶) was injected. 8 A CFU / mL solution was injected into the lungs, and the model was established once on the same day (recorded as D1) to replicate the invasive Aspergillus fumigatus lung disease model. On the third day of modeling (D3), blood was collected from all infected rats, and the galactomannan (GM) content in the serum was measured. When the GM value in the serum of the modeling animal increased significantly by more than 10% compared with the GM value in the negative control group, the model was considered to have been successfully established.

[0057] 2. Animals that successfully established the model were selected and randomly divided into three groups based on GM (gross gestational age), body weight, and sex: a model control group (B), an itraconazole capsule group (C), and a nebulized formulation administration group (H), with 10 animals per group (half male and half female). After grouping, animals in the model control group (B) and the nebulized formulation administration group (H) were given nebulized inhalation at a volume of 0.1 mL per animal. The itraconazole capsule group was given itraconazole via gavage (10 mL / kg). All treatments were administered once daily for 4 consecutive days (D4-D7). The negative control group and the model control group received a blank formulation.

[0058] 3. Serum galactomannan (GM) content detection On days 3 and 8, approximately 0.1 mL of blood was collected from each group of animals via the anterior vena cava or abdominal aorta. Serum was separated, and the serum galactomannan content was detected using an ELISA kit. The results are shown in Table 7.

[0059] Table 7. Serum GM levels in rats with invasive Aspergillus fumigatus lung disease model ( - (x±s, n=10)

[0060] Note: D3: Compared with the negative control group (A), express P <0.01; D8: Compared with the negative control group (A), express P <0.01; compared with the model control group (B), ## indicates P <0.01.

[0061] 4. Complete blood count (CBC) On day 3, approximately 0.2 mL of blood was collected from each group of animals, treated with EDTA anticoagulation, and the total white blood cell count was measured. The results are shown in Table 8.

[0062] Table 8. Blood leukocyte count in rats with invasive Aspergillus fumigatus lung disease model ( - (x±s, n=10)

[0063] Note: D3: Compared with the negative control group (A), express P <0.05, express P <0.01.

[0064] 5. Cellular immune markers On day 8, approximately 0.2 mL of blood was collected from each group of animals and treated with EDTA anticoagulation. The percentages of CD4+ and CD8+ lymphocyte subsets were detected by flow cytometry. The results are shown in Table 9.

[0065] Table 9. Proportions of lymphocyte subsets (CD4+ / CD8+) in rats with invasive Aspergillus fumigatus lung disease model (CD4+ / CD8+) - (x±s, n=10)

[0066] Note: Compared with the negative control group (A), express P <0.05, express P <0.01; compared with the model control group (B), ## indicates P <0.01.

[0067] 6. Lung coefficient On day 8, after the animals were euthanized, their lungs were quickly dissected, weighed, and the lung coefficient was calculated. The results are shown in Table 10.

[0068] Table 10 Lung coefficient in rats with invasive Aspergillus fumigatus lung disease model ( - (x±s, n=10)

[0069] Note: Compared with the negative control group (A), express P <0.05.

[0070] 7. Fungal colony load in lung tissue On day 8, after euthanasia, the lungs were dissected and harvested. Fresh right lung tissue from rats was taken under aseptic conditions and homogenized. After weighing with an electronic balance, the tissue was diluted at a ratio of 1g wet lung tissue to 10ml physiological saline. An appropriate amount of the diluted solution was inoculated into a fungal culture medium and placed in a fungal incubator for culture. After 1-4 days, the number of colonies on the plate was read. The inhibition rate was calculated according to the formula: inhibition rate (%) = (1 - (number of colonies in the treatment group - number of colonies in the negative control group) / (number of colonies in the model control group - number of colonies in the negative control group)) × 100%. The results are shown in Table 11.

[0071] Table 11 Fungal colony load in lung tissue of rats with invasive Aspergillus fumigatus lung disease model ( - (x±s, n=10)

[0072] Note: Compared with the negative control group (A), express P <0.05, express P <0.01; D8: Compared with the model control group (B), # indicates P <0.05, ## indicates P<0.01; compared with the itraconazole capsule group (C), □ represents P <0.05, □□ represents P <0.01.

[0073] 8. Histopathological examination On the last treatment day (D8), left lung tissue from each group of animals was collected, fixed in 10% neutral formalin, stained with hematoxylin and eosin (HE), and scored according to the criteria in Table 12. The results are shown in Table 13 and... Figures 5-8 As shown.

[0074] Table 12. Histopathological Diagnosis: Interpretation and Scoring Criteria for Lesion Degree

[0075] Table 13. Lung tissue histological examination scores of rats with invasive Aspergillus fumigatus lung disease model ( - (x±s, n=10)

[0076] Note: Compared with the negative control group (A), express P <0.05, express P <0.01; D8: Compared with the model control group (B), # indicates P <0.05, ## indicates P <0.01.

[0077] According to Tables 7-13 and Figures 5-8As can be seen, compared with the negative control group, the rats in the model control group had significantly decreased body weight, increased GM level, significantly increased proportion of CD8+ cells, significantly decreased CD4+ / CD8+ ratio, and significantly increased lung coefficient and fungal colony load in lung tissue. Combined with the histopathological examination results (degeneration and necrosis of bronchial mucosal epithelial cells, goblet cell proliferation of bronchial mucosal epithelial cells, alveolar edema, perivascular inflammatory cell infiltration, and bacterial clusters / hyphae visible in bronchioles / alveolar cavities / blood vessels), it indicates that the rat model of invasive Aspergillus fumigatus lung disease induced by Aspergillus fumigatus infection was successfully replicated. Compared with the negative control group, on day 3, the GM level in the animals included in the experimental group was significantly increased, and the total white blood cell count in each group was significantly decreased. On day 8, the GM level, lung organ coefficient, and fungal colony load in the lung tissue of the model control group were significantly increased, the proportion of CD8+ cells was significantly increased, and the CD4+ / CD8+ ratio was significantly decreased. Pathological examination of the lung tissue in the model control group showed severe degeneration and necrosis of bronchiolar mucosal epithelial cells, goblet cell proliferation of bronchiolar mucosal epithelial cells, alveolar edema, and perivascular inflammatory cell infiltration, with bacterial clusters / hyphae visible in the bronchioles / alveolar cavities / blood vessels. Compared with the model control group, the serum galactomannan content and fungal colony load in the itraconazole capsule group and the nebulized preparation group were significantly decreased, the lung organ coefficient was decreased, the proportion of CD4+ cells was increased, the proportion of CD8+ cells was decreased, and the CD4+ / CD8+ ratio was significantly increased and returned to near the level of the negative control group, with a reduction in the severity of the aforementioned pathological changes. Compared with the itraconazole capsule group, the nebulized formulation group showed better improvement in lung pathological changes in the model rats. The nebulized formulation group also showed a significantly increased drug concentration in lung tissue compared with the itraconazole capsule group. This invention, based on a rat model of invasive Aspergillus fumigatus lung disease constructed through tracheal inoculation under direct laryngoscopy, can be used to evaluate the efficacy of drugs treating pulmonary aspergillosis.

[0078] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing a rat model of pulmonary aspergillosis, characterized by, Comprise the following steps: Immunosuppression is performed on rats by using cyclophosphamide to obtain standardized immunodeficient rats; Anesthesia and fixation of the immunodeficient rats are performed, and under direct vision of a laryngoscope cold light source, a tracheal intubation inoculation technique is used to administer Aspergillus fumigatus liquid to the lungs of the rats to verify and obtain a pulmonary aspergillosis rat model; the verification comprises serological verification; the serological verification comprises serum galactomannan content detection, and the serum galactomannan content is significantly increased by more than 10% compared with a negative control group, and the model construction is successful.

2. The construction method according to claim 1, characterized in that, The administration dose of the cyclophosphamide is 30-40 mg / kg / d, and the administration is continuously performed for 3-5 d.

3. The construction method of claim 2, wherein, The administration mode of the cyclophosphamide is intraperitoneal injection.

4. The construction method of claim 1, wherein, The administration dose of the Aspergillus fumigatus liquid is 0.1-0.5 mL per one, and the concentration of the Aspergillus fumigatus liquid is 1x10 7 ~1x10 9 CFU / mL.

5. The construction method of claim 1, wherein, The step of administering the Aspergillus fumigatus liquid to the lungs of the rats comprises: A two-symmetry hole is opened at a tube wall 2-3 mm from a blunt tip of a flexible gavage needle hose or catheter at an angle of 15° with the tube wall, with the needle tip inclined surface facing the tail end direction, to obtain a modified flexible gavage needle hose or catheter; A laryngoscope cold light source is gently inserted into the oral cavity to find a pharyngeal glottis, and a syringe containing the Aspergillus fumigatus liquid is inserted into the modified flexible gavage needle hose or catheter, and slowly inserted into the trachea through the pharyngeal glottis under the laryngoscope, and the syringe plunger is pressed to the bottom, and the Aspergillus fumigatus liquid is injected into the lungs.

6. The construction method of claim 5, wherein, The tube wall at the blunt tip of the modified flexible gavage needle hose or catheter 3 cm and 4 cm away is provided with a scale line.

7. The construction method of claim 1, wherein, The anesthesia and fixation of the immunodeficient rats are performed 24 h after the immunodeficient rats are obtained; The anesthesia uses isoflurane; The fixation mode comprises fixing the anesthetized rats on a tracheal intubation platform, making the rats in a supine position, fixing the upper incisors on the edge of an operation board, and raising the operation board to an inclination of 45°, so that the head and the neck back of the rats are kept in the same horizontal position.

8. The construction method of claim 1, wherein, The verification further comprises etiology verification, other immunological verification, and pathological verification.

9. The construction method according to claim 8, wherein, The etiology verification, other immunological verification, and pathological verification comprise one or more of rat sign detection, blood routine detection, cellular immune state detection, lung coefficient detection, lung tissue fungal colony load, and lung tissue pathological examination.

10. Application of the pulmonary aspergillosis rat model constructed by the construction method of any one of claims 1-9 in screening of a pulmonary aspergillosis treatment drug and / or evaluation of the efficacy of a pulmonary aspergillosis treatment drug.

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