A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

The overall goal of this procedure is to generate consistent and reproducible airway neutrophilia by exposure to aerosolized LPS. This is accomplished by first loading LPS suspended in saline into a nebulizer connected to an exposure chamber. Next mice are exposed to the aerosolized LPS and then bronchoalveolar lavage is collected and inflammatory cells in the lavage are enumerated.

Finally, the lung tissue is fixed in, formally in for embedding and sectioning and immunohistochemical analysis. Ultimately, the total and differential cell counts of the bronchoalveolar lavage can be used to assess neutrophil recruitment to the lungs. The main advantages of this technique over existing methods like intratracheal LPS administration are the even distribution of aerosolized LPS throughout the lungs, the ease of performance and the minimal training required for successful application To generate an LPS aerosol using proper PPE and within a ventilated level two biohazard hood begin by inserting a red inlet into a nebulizer and then connecting the nebulizer to pressurized room air by the tubing provided by the manufacturer.

Next, use an air filter to connect the outlet of the nebulizer to a mass flow meter, and then connect the mass flow meter to an electrical supply. Adjust the air flow to five liters per minute, keeping the pressure at one to two bar, and then remove the mass flow meter and disconnect the airflow. Next, connect the outlet of the nebulizer to a 15.9 millimeter bifurcating tube connected to 2 1 50 by 1 63 by 2 0 5 millimeter plexiglass boxes fitted with removable lids.

Each box should have a five millimeter hole in the side opposing the inlet to prevent pressure buildup. Now place up to five mice into each box and close the lids. Then open the nebulizer.

Fill the insert with four to eight milliliters of LPS dissolved in saline or saline vehicle alone, and reconnect the inlet to the air supply. Allow the aerosol to flow into the closed plexiglass boxes with continuous monitoring of the animals and making sure the air supply remains tightly secured to the inlet of the nebulizer during the aerosolization. After 10 minutes, disconnect the air supply and leave the animals in the plexiglass boxes for another two minutes.

Then open the lids. Allow the aerosol to disperse and return the mice to their cages at the experimental endpoint For each animal in turn, first use scissors to make a single anterior posterior cut to expose the thorax. Next, lift the ribcage by the anterior tip of the sternum and puncture the diaphragm at the most ventral point without cutting into any lung tissue.

Then open the ribcage with two cuts that meet below the jaw in the anterior posterior direction. Once the ribcage has been opened, use forceps to gently pull it apart and cut the trachea below the larynx. Now lift the trachea and cut the ligaments connecting the lung lobes to the thoracic cavity.

Then gently pull the lungs and trachea up and away from the adipose and cardiac tissue and place them on a piece of syringe package paper. Next, insert a 0.965 millimeter polyethylene tube into the trachea and secure it with a string of silk thread. Tie off the multi lobe with silk thread as well, and then insert a 23 gauge needle into the polyethylene tube.

Then to collect the bronchoalveolar lavage, use a one milliliter syringe to slowly inject 250 microliters of ice called sterile PBS into the single lobe, and to carefully tap the lungs and against the surface of the work bench 30 times slowly draw the liquid back into the syringe and collect it in a tube after repeating the procedure with 200 microliters of fresh PBS, place the bronchoalveolar lavage on ice for later analysis. To fix the lung tissue for histological analysis, remove the multi lobe and snap. Freeze it on dry ice.

Store the lobe at minus 80 degrees Celsius. To fix the tissue for histological analysis, mount a 60 milliliter syringe without a plunger onto a metal support stand insufflate the single lobe with formalin for five minutes. Then disconnect the needle and pull on the silk thread to remove it together with the polyethylene tube from the trachea.

Simultaneously closing the trachea and retaining the pressure in the lung. Next, submerge the lobe in Formin for 24 hours at four degrees Celsius. The next day, wash the fixed tissue three times for at least 20 minutes each time in 70%ethanol.

After embedding the dehydrated tissue in para film, cut the lobe into four to five micron sections and stain the sections with hematin and eoin for histological assessment. The total cell number in the bronchoalveolar lavage fluid of mice exposed to an aerosol generated with vehicle only is typically around or below 200, 000 cells. The cells consist of 95 to 100%mononuclear cells with only a few lymphocytes and no neutrophils.

Mice challenged with one milligram per milliliter of aerosolized.LPS. However, exhibit an increased total cell number in the bronchoalveolar lavage fluid, typically greater than 500, 000 cells after six hours with cell infiltrates remaining high after 24 hours, and with a cell profile shifted towards a predominance of neutrophils. A similar inflammatory cell profile and pulmonary neutrophilia is observed with nebulization of five milligrams per milliliter LPS as well.

Neutrophils were observed in the epithelial submucosa as well as in the spaces surrounding the conducting airways and blood vessels after six and 24 hours of LPS challenge, but not in control. Saline treated mice. The total protein content in the bronchoalveolar lavage fluid of LPS challenge mice is increased compared to mice exposed to saline and the expression of the neutrophil chemo attractant chemokines c, XCL one and C XCL two are increased in LPS challenged mice as well.

Though this method can provide insights into LPs induced lung inflammation. It can also be applied to other disease models such as bacterial or viral infections, as the aerosolized vapor can be adapted to deliver many different challenges.