A method for preparing a murine model of Staphylococcus aureus pneumonia and model evaluation

Abstract

The present invention discloses a method for preparing a murine model of Staphylococcus aureus pneumonia and model evaluation, and the steps are as follows: Step 1: in vitro labeling of Staphylococcus aureus; Step 2: establishment of a murine model of Staphylococcus aureus pneumonia. By adopting the method of exposing the trachea and quantitatively aerosolizing bacteria through the oral trachea using a murine tracheal quantitative aerosol nebulization device, the amount inhaled by the mice can be controlled at the same level, with smaller errors in each group and higher quality of model establishment; the operation difficulty is small, the success rate of model establishment can be effectively improved, and the damage to animals is small. By observing the lung imaging of mice in vivo through a near-infrared imaging system, if obvious and uniform near-infrared fluorescence images are observed in the lungs of the mice, it indicates that the model establishment is successful. It can know whether the model is successfully established in the first time, avoiding lag. Moreover, this evaluation method does not require a series of invasive operations to extract samples and subsequent series of detections. The method is simple to operate and takes a short time.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing a Staphylococcus aureus pneumonia mouse model and model evaluation. Background Art

[0002] Staphylococcus aureus (S. aureus) is one of the most common bacterial pathogens, causing an untold number of uncomplicated skin infections and potentially more serious invasive infections worldwide each year. It is a major pathogen of pneumonia, other respiratory tract infections, surgical site, prosthetic joint and cardiovascular infections, and intranasal bacteremia. Due to the widespread use of antibiotics in recent decades, S. aureus has rapidly developed resistance to multiple antibiotics, increasing the risk of fatal infections in immunocompromised patients. Therefore, establishing a stable and reliable S. aureus pneumonia model is important for basic research such as studying its resistance mechanism.

[0003] There are many methods for constructing existing pneumonia animal models, mainly including: nasal instillation, oropharyngeal tracheal instillation, atomization inhalation, tracheal intubation, tracheotomy and other methods. However, these methods all have certain disadvantages, mainly reflected in: 1. Nasal instillation, oropharyngeal tracheal instillation, atomization inhalation and other methods cannot achieve quantitative, the amount of mouse inhalation cannot be controlled at the same level, and the modeling quality is low. 2. Tracheal intubation requires certain technical level requirements, the operation is difficult, it is easy to mistakenly enter the esophagus, the modeling success rate cannot be guaranteed, and repeated insertion is easy to cause laryngeal edema to increase the difficulty of insertion, and even cause the death of mice. 3. Tracheotomy, damage to the trachea, easily lead to suffocation and death of mice.

[0004] In addition, the established model lacks an effective evaluation method. Currently, most methods involve measuring body temperature after the model is established, and detecting inflammatory factors in the alveolar lavage fluid. These evaluation indicators are lagging, cumbersome, and have low specificity and sensitivity.

[0005] The shortcomings of existing technologies in model building are as follows:

[0006] 1. The nasal instillation method, the oropharyngeal tracheal instillation method, the atomization inhalation method and other methods cannot achieve quantitative results. The amount of inhalation by mice cannot be controlled at the same level. The errors among the groups are large, and the modeling quality is low.

[0007] 2. Tracheal intubation requires a certain level of technical skills and is difficult to operate. It is easy to enter the esophagus by mistake, and the success rate of modeling cannot be guaranteed. Repeated insertion can easily cause laryngeal edema, aggravating the difficulty of insertion, and even causing the death of mice, increasing animal costs;

[0008] 3. Tracheotomy causes great trauma to mice, especially damage to the trachea, which can easily lead to suffocation and death of mice, increasing animal costs;

[0009] The disadvantages of the prior art in the model evaluation method are as follows:

[0010] 1. Currently, most model evaluation methods measure the body temperature of mice and collect blood samples or perform aseptic thoracotomy to detect the secretion of related inflammatory factors after the model is established. These evaluation methods can only be detected after a certain period of model establishment, and it is impossible to know whether the model is successfully established in a timely manner, resulting in a certain lag.

[0011] 2. The influencing factors of body temperature and the changes in blood or alveolar lavage fluid are complex. Therefore, the specificity and sensitivity of these evaluation methods are not high.

[0012] 3. These evaluation methods require a series of invasive operations to extract samples and subsequent series of detections, with the methods being cumbersome and time-consuming. Summary of the Invention

[0013] In view of the deficiencies of the prior art, the present invention provides a method for preparing a mouse model of Staphylococcus aureus pneumonia and a model evaluation, which solves the problems raised in the background art.

[0014] To achieve the above objectives, the present invention is realized through the following technical solutions: A method for preparing a mouse model of Staphylococcus aureus pneumonia and a model evaluation, the steps are as follows:

[0015] Step 1: In vitro labeling of Staphylococcus aureus;

[0016] Step 2: Establishment of a mouse model of Staphylococcus aureus pneumonia.

[0017] Preferably, the in vitro labeling of Staphylococcus aureus includes the following steps:

[0018] a: Strain and bacterial culture;

[0019] b: Near-infrared fluorescent dye and bacterial labeling.

[0020] Preferably, the strain and bacterial culture step includes the following:

[0021] S1: Activation. After thawing the glycerol strain of Staphylococcus aureus stored in a refrigerator at -80°C to -90°C, inoculate it on an agar culture dish using an inoculation loop and culture it overnight at 37°C to 45°C;

[0022] S2: Pre-culture. Inoculate the bacterial liquid obtained in step S1 at a ratio of 1:100 (v / v) into 30 ml of fresh NB liquid medium and culture it at 37°C to 45°C and 250 rpm for 6 h to 10 h until the OD600 reaches 0.8;

[0023] S3: Formal culture: Inoculate the bacterial liquid obtained in S2 into 30 ml of fresh NB liquid medium at a ratio of 1:100 (v / v), and culture it at 37°C - 45°C and 250 rpm for 4 h until the OD600 reaches 0.6;

[0024] S4: Adjust the OD of the bacterial liquid. Collect 1 mL of the bacterial liquid obtained in S3 into a sterile EP tube, centrifuge at 4000 rpm for 10 min to collect the bacteria, discard the supernatant of the culture medium, wash it 3 - 4 times with PBS at a rotation speed of 4000 rpm, for 10 - 15 minutes, at a temperature of 25°C - 30°C, and then adjust the concentration of the bacterial liquid to about 2×108 CFU / ml of the bacterial solution with PBS.

[0025] Preferably, the near-infrared fluorescent dye is an amino water-soluble quantum dot, and the excitation and emission wavelengths are 650 ± 20 nm.

[0026] Preferably, the method for labeling bacteria is to add 2 μg / ml of quantum dot dye to the bacterial solution with a concentration of 2×108 CFU / ml, ice-bath the bacterial liquid for 10 - 15 minutes, water-bath at 42°C for 90 seconds, then immediately ice-bath for 2 minutes, and then place it in a constant temperature and humidity incubator at 37°C - 45°C for 60 minutes. After the culture is completed, centrifuge at 4000 rpm for 6 minutes, discard the supernatant, wash it 3 - 5 times with PBS at a rotation speed of 4000 rpm, for 10 - 15 minutes, at a temperature of 25°C - 30°C, and then resuspend it with 1 ml of PBS.

[0027] Preferably, the experimental animals for the Staphylococcus aureus pneumonia mouse model are 30 male BALB / c mice with a body weight of 20 - 22 g. The experimental mice are fed in the animal house of the Naval Medical Center of Chinese People's Liberation Army. The indoor relative humidity of the breeding environment is 50% - 60%, and the temperature is controlled at 18 - 22°C; they are fed with conventional feed, given free drinking water, the feces are flushed regularly by ventilation, the environment is kept quiet, and the experiment is started after 1 - 2 weeks of adaptive feeding.

[0028] Preferably, the establishment of the Staphylococcus aureus pneumonia mouse model also includes inoculating bacteria to the experimental animals, and adopting the method of exposing the trachea and quantitatively aerosolizing the bacteria into the trachea of the mouse through the oral trachea with a mouse tracheal quantitative aerosol nebulizer.

[0029] Preferably, the method for inoculating bacteria is as follows:

[0030] a: Anesthetize the mouse with sodium pentobarbital, fix the mouse in a supine position on the operating table, cut the skin of the neck, free the trachea, hang the front teeth of the mouse with a rubber band, and gently pull out the mouse's tongue with forceps;

[0031] b: After pulling out the mouse's tongue, hold the laryngoscope with the left hand, insert it into the oral cavity from the upper lip teeth of the mouse, and adjust the angle until the glottis fissure can be clearly seen when reaching the epiglottis margin of the mouse;

[0032] c: Hold the mouse tracheal quantitative aerosol atomization device pre - filled with a pre - determined amount of near - infrared fluorescently labeled Staphylococcus aureus in the right hand. Insert the aerosol atomization nozzle into the trachea for 15 - 20 mm. Lift the trachea to determine if the needle is accurately inserted into the trachea, and then quickly inject 50 μl of Staphylococcus aureus bacterial solution.

[0033] d: After the injection is completed, suture the wound. The blank group is not treated, and the saline control group is injected with the same amount of sterile saline.

[0034] Preferably, place the modeled mice in a small animal in - vivo imager. Observe the imaging situation of the mice's lungs in - vivo through the near - infrared imaging system. If obvious and uniform near - infrared fluorescence images are observed in the mice's lungs, the modeling is successful.

[0035] The present invention provides a method for preparing a mouse model of Staphylococcus aureus pneumonia and model evaluation. It has the following beneficial effects:

[0036] By adopting the method of exposing the trachea and atomizing and delivering bacteria through the mouse tracheal quantitative aerosol atomization device via oral tracheal atomization, the amount inhaled by the mice can be controlled at the same level, with relatively small errors in each group and high - quality modeling; the operation difficulty is small, the modeling success rate can be effectively improved, the damage to animals is small, and the animal cost is reduced; at the same time, this model evaluation method places the modeled mice in a small animal in - vivo imager and observes the imaging situation of the mice's lungs in - vivo through the near - infrared imaging system. If obvious and uniform near - infrared fluorescence images are observed in the mice's lungs, the modeling is successful. It can know whether the model is successfully established in a timely manner, avoiding lag, and this evaluation method does not require a series of invasive operations to extract samples and subsequent series of detections. The method is simple to operate and takes a short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a schematic diagram of the infrared fluorescence image of the present invention;

[0038] Figure 2 It is a schematic diagram of mice inoculated with bacteria of the present invention;

[0039] Figure 3 It is a schematic diagram of the infection dose of the Staphylococcus aureus pneumonia model of the present invention;

[0040] Figure 4 It is the fluorescence image of Staphylococcus aureus labeled with Cy5.5 in vitro, and the fluorescence microscope images under 20× and 40× magnifications respectively;

[0041] Figure 5The labeled Staphylococcus aureus and the unlabeled control group were continuously cultured in NB medium for 12 h, and samples were taken every 2 h to measure the OD600 value to evaluate the bacterial proliferation. It can be seen from the figure that there is no significant difference in the proliferation of the labeled bacteria compared with the control group. Detailed implementation mode

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0043] Example 1:

[0044] The steps are as follows:

[0045] Step 1: In vitro labeling of Staphylococcus aureus;

[0046] Step 2: Establishment of a mouse model of Staphylococcus aureus pneumonia.

[0047] The in vitro labeling of Staphylococcus aureus includes the following steps:

[0048] a: Strain and bacterial culture;

[0049] b: Near-infrared fluorescent dye and bacterial labeling.

[0050] The steps of the strain and bacterial culture include the following:

[0051] S1: Activation. After thawing the glycerol strain of Staphylococcus aureus stored in a -80 °C refrigerator, it was inoculated onto an agar culture dish using an inoculation loop and cultured overnight at 37 °C.

[0052] S2: Pre-culture. The bacterial solution obtained in step S1 was inoculated into 30 ml of fresh NB liquid medium at a ratio of 1:100 (v / v), and cultured at 37 °C and 250 rpm for 6 h until the OD600 reached 0.8.

[0053] S3: Formal culture: The bacterial solution obtained in S2 was inoculated into 30 ml of fresh NB liquid medium at a ratio of 1:100 (v / v), and cultured at 37 °C and 250 rpm for 4 h until the OD600 reached 0.6.

[0054] S4: Adjust the OD of the bacterial solution. Collect 1 mL of the bacterial solution obtained in S3 into a sterile EP tube, centrifuge at 4000 rpm for 10 min to collect the bacteria, discard the supernatant of the culture medium, wash 3 times with PBS at a rotation speed of 4000 rpm for 10 - 15 minutes at 25°C, and then adjust the concentration of the bacterial solution to approximately 2×108 CFU / ml with PBS to obtain a bacterial solution.

[0055] The near-infrared fluorescent dye is an amino water-soluble quantum dot, and the excitation and emission wavelengths are 650±20 nm.

[0056] The method for labeling bacteria is to add 2 μg / ml of quantum dot dye to the bacterial solution with a concentration of 2×108 CFU / ml, ice-bath the bacterial solution for 10 minutes, water-bath at 42°C for 90 seconds, then immediately ice-bath for 2 minutes, and then place it in a constant temperature and humidity incubator at 37°C for 60 minutes. After the incubation, centrifuge at 4000 rpm for 6 minutes, discard the supernatant, wash 3 times with PBS at a rotation speed of 4000 rpm for 10 minutes at 25°C, and then resuspend with 1 ml of PBS.

[0057] The experimental animals for the mouse model of Staphylococcus aureus pneumonia are 30 male BALB / c mice with a body weight of 20 - 22 g. The experimental mice are fed in the animal house of the Naval Medical Center of Chinese People's Liberation Army. The relative humidity in the indoor breeding environment is 50%, and the temperature is controlled at 18°C; they are fed with conventional feed and free drinking water, and the feces are flushed and the ventilation is carried out on time to keep the environment quiet. The experiment starts after 1 week of adaptive feeding.

[0058] The establishment of the mouse model of Staphylococcus aureus pneumonia also includes inoculating bacteria to the experimental animals by means of exposing the trachea and quantitatively aerosolizing and atomizing the bacteria into the trachea through the oral cavity using a mouse tracheal quantitative aerosol atomizing device.

[0059] The method for inoculating bacteria is as follows:

[0060] a: Anesthetize the mouse with sodium pentobarbital, fix the mouse in a supine position on the operating table, cut the skin of the neck, free the trachea, hang the front teeth of the mouse with a rubber band, and gently pull out the mouse's tongue with forceps;

[0061] b: After pulling out the mouse's tongue, hold the laryngoscope with the left hand, insert it into the oral cavity from the upper lip teeth of the mouse, and adjust the angle until the glottis fissure can be clearly seen when reaching the edge of the mouse's epiglottis;

[0062] c: Hold the mouse tracheal quantitative aerosol atomizing device pre-quantitatively filled with near-infrared fluorescently labeled Staphylococcus aureus with the right hand, insert the aerosol atomizing jet head 15 - 20 mm into the trachea, lift the trachea to determine whether the needle is accurately inserted into the trachea, and then quickly inject 50 μl of Staphylococcus aureus bacterial solution;

[0063] d: After the bolus injection, suture the wound. The blank group is not treated, and the saline control group is injected with the same amount of sterile saline.

[0064] Put the modeled mice into a small animal in vivo imager, and observe the lung imaging of the mice in vivo through the near-infrared imaging system. If obvious and uniform near-infrared fluorescence images are observed in the lungs of the mice, the modeling is successful.

[0065] Example 2:

[0066] The steps are as follows:

[0067] Step 1: In vitro labeling of Staphylococcus aureus;

[0068] Step 2: Establishment of a mouse model of Staphylococcus aureus pneumonia.

[0069] The in vitro labeling of Staphylococcus aureus includes the following steps:

[0070] a: Strain and bacterial culture;

[0071] b: Near-infrared fluorescent dye and bacterial labeling.

[0072] The steps of the strain and bacterial culture include the following:

[0073] S1: Activation. After thawing the glycerol strain of Staphylococcus aureus stored in a -90°C refrigerator, inoculate it on an agar culture dish using an inoculation loop and culture it overnight at 45°C;

[0074] S2: Pre-culture. Inoculate the bacterial solution obtained in step S1 into 30 ml of fresh NB liquid medium at a ratio of 1:100 (v / v), and culture it at 45°C and 250 rpm for 10 h until the OD600 reaches 0.8;

[0075] S3: Formal culture: Inoculate the bacterial solution obtained in S2 into 30 ml of fresh NB liquid medium at a ratio of 1:100 (v / v), and culture it at 45°C and 250 rpm for 4 h until the OD600 reaches 0.6;

[0076] S4: Adjustment of the bacterial solution OD. Collect 1 mL of the bacterial solution obtained in S3 into a sterile EP tube, centrifuge at 4000 rpm for 10 min to collect the bacteria, discard the supernatant of the culture medium, wash it 4 times with PBS at a rotation speed of 4000 rpm, a time of 15 minutes, and a temperature of 30°C, and then adjust the concentration of the bacterial solution to about 2×108 CFU / ml of the bacterial solution with PBS.

[0077] The near-infrared fluorescent dye is amino water-soluble quantum dots, and the excitation and emission wavelengths are 650±20 nm.

[0078] The bacterial labeling method is as follows: Add 2 μg / ml of quantum dot dye to a bacterial solution with a concentration of 2×108 CFU / ml. Ice-bath the bacterial solution for 15 minutes, then water-bath at 42°C for 90 seconds, immediately followed by ice-bathing for 2 minutes. Then place it in a constant temperature and humidity incubator at 45°C for 60 minutes. After the incubation, centrifuge at 4000 rpm for 6 minutes, discard the supernatant, wash 5 times with PBS at a rotation speed of 4000 rpm, for 15 minutes at a temperature of 30°C, and then resuspend with 1 ml of PBS.

[0079] The experimental animals for the established Staphylococcus aureus pneumonia mouse model are 30 male BALB / c mice with a body weight of 20 - 22 g. The experimental mice are fed in the animal house of the Naval Medical Center. The indoor relative humidity of the breeding environment is 60%, and the temperature is controlled at 22°C; they are fed with conventional feed, given free access to drinking water, the feces are flushed regularly by ventilation, the environment is kept quiet, and the experiment starts after 2 weeks of adaptive feeding.

[0080] The establishment of the Staphylococcus aureus pneumonia mouse model also includes inoculating bacteria into the experimental animals, using the method of exposing the trachea and quantitatively aerosolizing and atomizing bacteria into the trachea of the mice through an oral tracheal atomizing device.

[0081] The method for inoculating bacteria is as follows:

[0082] a: Anesthetize the mouse with sodium pentobarbital, fix the mouse in a supine position on the operating table, cut the skin of the neck, free the trachea, hang the front teeth of the mouse with a rubber band, and gently pull out the mouse's tongue with forceps.

[0083] b: After pulling out the mouse's tongue, hold the laryngoscope in the left hand, insert it into the oral cavity from the upper lip teeth of the mouse, and adjust the angle until the glottis fissure can be clearly seen when reaching the epiglottis margin of the mouse.

[0084] c: Hold the mouse tracheal quantitative aerosol atomizing device pre-quantitatively filled with near-infrared fluorescently labeled Staphylococcus aureus in the right hand, insert the aerosol atomizing jet head into the trachea by 15 - 20 mm, lift the trachea to determine whether the needle is accurately inserted into the trachea, and then quickly inject 50 μl of Staphylococcus aureus bacterial solution.

[0085] d: After the injection is completed, suture the wound. The blank group is not treated, and the saline control group is injected with the same amount of sterile normal saline.

[0086] Put the mice with successfully established models into a small animal in vivo imager, and observe the imaging situation of the lungs of the mice in vivo through the near-infrared imaging system. If obvious and uniform near-infrared fluorescence images are observed in the lungs of the mice, the model establishment is successful.

[0087] By adopting the method of exposing the trachea and quantitatively atomizing bacteria through oral tracheal atomization with a mouse tracheal quantitative aerosol atomization device, the amount inhaled by the mouse can be controlled at the same level, with relatively small errors in each group and high modeling quality; the operation difficulty is small, the modeling success rate can be effectively improved, the damage to animals is small, and the animal cost is reduced; at the same time, in this model evaluation method, the modeled mice are placed in a small animal in vivo imager, and the imaging situation of the mouse lungs is observed in vivo through a near-infrared imaging system. If obvious and uniform near-infrared fluorescence images are observed in the mouse lungs, it indicates that the modeling is successful. It is possible to know whether the model is successfully established in a timely manner, avoiding lag, and this evaluation method does not require a series of invasive operations to extract samples and subsequent series of detections. The method is simple to operate and takes a short time.

[0088] The foregoing has shown and described the basic principles, main features and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, in any regard, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to embrace all changes that fall within the meaning and scope of the equivalent elements of the claims in the present invention. Any reference numerals in the claims should not be regarded as limiting the claims involved.

[0089] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative manner of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a murine model of Staphylococcus aureus pneumonia, characterized in that: The steps are as follows: Step 1: In vitro labeling of Staphylococcus aureus; the in vitro labeling of Staphylococcus aureus includes the following steps: a: Strain and bacterial culture; the steps of strain and bacterial culture are as follows: S1: Activation. After thawing the glycerol strain of Staphylococcus aureus stored in a refrigerator at -80°C to -90°C, inoculate it onto an agar culture dish using an inoculation loop and culture it overnight at 37°C - 45°C. S2: Pre-culture. Inoculate the bacterial solution obtained in step S1 into 30 ml of fresh NB liquid medium at a ratio of 1:100 (v / v), and culture it at 37°C - 45°C and 250 rpm for 6 h - 10 h until the OD600 reaches 0.

8. S3: Formal culture. Inoculate the bacterial solution obtained in S2 into 30 ml of fresh NB liquid medium at a ratio of 1:100 (v / v), and culture it at 37°C - 45°C and 250 rpm for 4 h until the OD600 reaches 0.

6. S4: Adjust the OD of the bacterial solution. Collect 1 mL of the bacterial solution obtained in S3 into a sterile EP tube, centrifuge at 4000 rpm for 10 min to collect the bacteria, discard the supernatant of the culture medium, wash 3 - 4 times with PBS at a rotation speed of 4000 rpm for 10 - 15 minutes and at a temperature of 25°C - 30°C. Then adjust the concentration of the bacterial solution to approximately 2×10 8 CFU / ml bacterial solution; b: Near-infrared fluorescent dye and bacterial labeling. The near-infrared fluorescent dye is amino water-soluble quantum dots, with an excitation and emission wavelength of 650 ± 20 nm. The bacterial labeling method is to add 2 μg / ml of quantum dot dye to a bacterial solution with a concentration of 2×108 CFU / ml, ice-bath the bacterial solution for 10 - 15 minutes, water-bath it at 42°C for 90 seconds, then immediately ice-bath it for 2 minutes, and then place it in a constant temperature and humidity incubator at 37°C - 45°C for 60 minutes. After the culture is completed, centrifuge it at 4000 rpm for 6 minutes, discard the supernatant, wash it 3 - 5 times with PBS at a rotation speed of 4000 rpm, for 10 - 15 minutes, at a temperature of 25°C - 30°C, and then resuspend it with 1 ml of PBS. Step 2: Establishment of a mouse model of Staphylococcus aureus pneumonia. The experimental animals for the mouse model of Staphylococcus aureus pneumonia are 30 male BALB / c mice, weighing 20 - 22 g. The experimental mice are fed in the animal house of the Naval Medical Center of the Chinese People's Liberation Army. The indoor relative humidity of the breeding environment is 50% - 60%, and the temperature is controlled at 18 - 22°C. They are fed with conventional feed, given free access to drinking water, and the feces are flushed regularly and the ventilation is carried out on time to keep the environment quiet. After 1 - 2 weeks of adaptive feeding, the experiment is started. The establishment of the mouse model of Staphylococcus aureus pneumonia also includes inoculating bacteria into the experimental animals, using the method of exposing the trachea and quantitatively aerosolizing the bacteria into the trachea of the mouse through an oral tracheal aerosolization device. The method of inoculating bacteria is as follows: a: Anesthetize the mouse with sodium pentobarbital, fix the mouse in a supine position on the operating table, cut the skin of the neck, free the trachea, hang the front teeth of the mouse with a rubber band, and gently pull out the mouse's tongue with forceps. b: After pulling out the mouse's tongue, hold the laryngoscope in the left hand, insert it into the mouse's oral cavity from the upper lip teeth, and adjust the angle until the glottis fissure can be clearly seen when reaching the epiglottis margin of the mouse. c: Hold the mouse tracheal quantitative aerosolization device pre-loaded with near-infrared fluorescently labeled Staphylococcus aureus in the right hand, insert the aerosolization injection head into the trachea for 15 - 20 mm, lift the trachea to determine whether the needle is accurately inserted into the trachea, and then quickly inject 50 μl of Staphylococcus aureus bacterial solution. d: After the bolus injection, suture the wound. The blank group is not treated, and the saline control group is injected with the same amount of sterile normal saline. Put the modeled mice into a small animal in vivo imager, and observe the lung imaging of the mice in vivo through the near-infrared imaging system. If obvious and uniform near-infrared fluorescence images are observed in the lungs of the mice, the modeling is successful.

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