Experimental Model to Evaluate Resolution of Pneumonia

This protocol provides a reproducible model of pneumonia that generates a robust, acute-lung injury. Other advantages of this model is can be used to reproduce infectious ARDS or acute respiratory distress syndrome in mice. As I mentioned, this model is highly reproducible where the mortality and injury level can be easily titrated to evaluate early and late phases of acute lung injury and resolution, which serves as a platform to evaluate therapeutic strategies.

Although this protocol does not provide with a novel approach for the diagnosis or treatment of lung injury, it is a reliable model where new therapies can be tested, which is particularly useful for areas related to immunology, pulmonary diseases and infectious diseases. One of the advantages of this technique is its reproducibility. However, people performing this technique for the first time may experience difficulties inserting the catheter intratracheally.

That is why ideally, people learning this technique should start practicing in euthanized animals. To begin, warm a blood agar plate for 10 minutes at 37 degrees Celsius in a heated shaker or incubator. Take a new bacterial stock vial from the freezer and thaw it down by gentle agitation in a 37-degrees-Celsius water bath until the vial is completely thawed.

Avoid touching the O-ring or cap with warm water. To manually count the bacterial colonies on a plate, perform a 1-times-10-to-the-sixth dilution and plate 200 microliters from the last dilution on a pre-warmed blood agar plate. Incubate the plate overnight at 37 degrees Celsius.

The next day, determine the new bacterial concentration by applying the given formula. Place the anesthetized mouse on a clean and sterilized surface. Hang the mouse by the incisors and gently tape the forelimbs.

Shave the neck region and disinfect the area with chlorhexidine and 70%alcohol. Then using surgical scissors, make a one-centimeter, superficial-midline neck incision to visualize the trachea. If an abundance of fat tissue is seen, carefully dissect the adipose tissue vertically to visualize the trachea.

Pull the tongue outward gently and introduce a 20-gauge angio-catheter through the mouth, advancing the catheter into the trachea. Apply gentle pressure over the trachea to facilitate intubation. After intubation, connect the mouse to a respirator to confirm intubation.

Set the ventilator parameters to 200 microliters of tidal volume and 200 strokes per minute. After confirming intubation, disconnect the mouse from the respirator and carefully instill 50 microliters of the bacterial agent through the angio catheter using a 200-microliter pipette, gel-loading tip. After installation, connect the mouse to the respirator again to help restart breathing.

Leave the mouse on the respirator for 30 to 60 seconds to monitor breathing. If a slow-breathing pattern is observed, connect the mouse to the respirator again. Close the incision by adding one drop of glue to the skin.

Bring together the skin folds and apply gentle pressure until the glue dries. Lay the euthanized mouse supine on a clean surgical board and hang it by the incisors. After spraying the mouse skin with 70%ethanol, using scissors, make a small, superficial, midline neck excision to visualize the trachea.

Cannulate the trachea with a 20-gauge catheter and carefully add one milliliter of calcium free PBS intratracheally using a one-milliliter syringe. Allow full lung expansion, then aspirate the fluid using the same syringe. Repeat the procedure two times for a total of two milliliters.

Transfer the bronchoalveolar lavage, or BAL, into a two-milliliter aliquot. Using scissors, open the thoracic cavity to expose the lung, heart and trachea. Carefully dissect the diaphragm and remove the rib cage ensuring not to pinch the lung tissue.

Transect the abdominal aorta to allow exsanguination. Perfuse the lung tissue by making a small incision of approximately one to two millimeters in the right ventricle using scissors and injecting five milliliters of cold PBS, using a 20 gauge catheter. Upon successful perfusion, the lung tissue turns white and pale, and the PBS leaves the intravascular compartment through the abdominal aorta.

Then carefully extract the lung and dissect it from the trachea. Alternatively, if the lung is being used for histology, carefully insert a 20-gauge catheter to inflate the lungs up to 25 centimeters with formalin solution. Once the lungs are insufflated, pass a 3/O suture string about five centimeters long underneath the trachea and tie it firmly twice to ensure the formalin stays in the lung tissue.

Gently dissect the lung from the rest of the tissues and place it in a 15-milliliter conical tube containing 10 milliliters of formalin solution. Centrifuge the BAL at 500 G at four degrees Celsius for five minutes and remove the cell-free supernatant in a separate tube. Lyse the red blood cells by adding 100 microliters of lysing buffer for one minute.

Neutralize the lysing reaction by adding one milliliter of PBS. Centrifuge the BAL, and remove the supernatant before re-suspending the pellet in 100 to 300 microliters of PBS. Next, perform a cell count with 0.4%trypan blue stain by automated cell counting.

Gently dissect the lung from the tissues in a 15 milliliter conical tube containing five milliliters of cold PBS. After dissection, remove the lung from the PBS and dry it using a paper towel. Dissect the right and left lung from other tissues that were also extracted such as the trachea.

Prepare a digestion cocktail as described in the text manuscript and transfer the lung to a C-tube containing one milliliter of digestion cocktail. Transfer the tube to the tissue dissociator and follow the standardized protocol for processing lung tissue. After dissociation, add 10 milliliters of cold PBS into the tube and mix properly.

Filter the single-cell suspension using a 70 micron cell strainer on top of a 50 milliliter conical tube on ice. Centrifuge the suspension and remove the supernatant. Add one millimeter of lysing buffer for one minute at room temperature.

Then add 10 milliliters of cold PBS to stop the lysing reaction and remove the supernatant. Re-suspend the cells in PBS and perform a cell count with trypan blue stain by automated cell counting. After bacterial-pneumonia-induced lung injury in mice, the infected groups displayed lower body weight compared to the uninfected control.

The streptococcus pneumoniae or Spn group recovered their body weight toward baseline while Klebsiella pneumoniae, or Kpn-infected mice displayed slow recovery after six days of infection. BAL-protein concentration and the total cell count for both the BAL and lungs were markedly higher in the infected groups. Representative histological sections displaying the inflammatory process in both models were obtained at day two, four, and six post-inoculations showing evidence of persistent alveolar inflammation in the Kpn-infected mice, even at day 10.

Kpn-infected mice continued the injury by day 10 while Spn-infected mice resolved the lung inflammation by day six. Immune-cell landscape by multicolor flow cytometry after six days of Spn infection showed an increased number of granulocytes, interstitial macrophages, monocytes, B cells, and T Cells, including natural killer cells. While attempting this method, insertion of the catheter is the key to have replicable infections.

This is a reliable method that serves as a platform for the identification of targets for the study of acute lung injury and resolution.