Reliable animal models are crucial components of basic research. Our murine approach allows answering immunological questions on the mechanistics and kinetics of acute lung injury development in mammals. Minimal invasiveness, simple handling, as well as good reproducibility are the main advantages of this technique.
In addition, with dose titration, we can modulate the clinical effect. This technique mirrors the clinical situation of patients with severe lung injury and allows to investigate the underlying mechanisms. Store lipopolysaccharide, or LPS, in aliquots at a concentration of five milligrams per milliliter at minus 20 degrees Celsius.
For intratracheal instillation, dilute the thawed LPS in sterile phosphate buffered saline to a final concentration of 2, 000 micrograms per milliliter. Cut a 22-gauge venous catheter to a length of 20 millimeters. Place the mouse in the prone position on a temperature-controlled table to maintain a body temperature of 37 degrees Celsius.
Following anesthesia as described in the text protocol, apply sterile ophthalmic lubricant to prevent desiccation of corneas under anesthesia. Lift the head and hook incisors on a horizontal bar positioned approximately five centimeters above the table while the forepaws remain in close contact with the table. Super-extend the neck in a 90 degree angle relative to the table.
Place a cold light source on the skin above the larynx to help visualize the vocal cords and aim for the trachea. Hold the tongue with forceps to straighten the throat for easier intubation conditions. Proper visualization and identification of the larynx are critical to assure correct intratracheal placement of the catheter.
An external cold light source helps in this step. Insert the catheter approximately 10 millimeters into the trachea. Ensure that the insertion is not too deep, as this will result in unilateral instillation of fluid into the right or left main bronchus.
If resistance of the larynx occurs, retract the catheter a few millimeters before advancing again. Now, inject the LPS-diluted MPBS using a pipette. The injected volume depends on the mouse body weight.
Connect a syringe and add a bolus of 50 microliters of air to ensure the complete liquid volume is distributed in the lungs. Slowly remove the catheter. Keep the mouse's upper body in an upright position for 30 seconds to avoid leakage of the fluid from the trachea.
Fix the euthanized mouse with tape on an operation table and shortly disinfect the fur over the abdomen with 70%ethanol. Open the abdominal cavity carefully in the median line with scissors and tweezers. Remove parts of the intestine to achieve access to the vena cava inferior right to the vertebral column in the abdominal aorta.
Locate the kidney veins and insert a bent, 23-gauge cannula connected to a one milliliter syringe into the vena cava inferior, directly below the confluence of the veins. Aspirate 250 microliters of blood and transfer into a 1.5 milliliter tube filled with 20 microliters of 0.5 molar EDTA solution. Shake gently to facilitate EDTA mixing and put the tube on ice.
For bronchoalveolar lavage, prepare three one milliliter syringes, each with 0.5 milliliters of sterile PBS and 0.1 milliliter of air. Disinfect the fur of the throat with 70%ethanol and carefully expose the trachea with scissors and tweezers. Mobilize the trachea and wrap a suture around it.
Puncture the trachea using micro-scissors and insert a 22-gauge venous catheter, cut to a length of 20 millimeters. Fix the catheter with a suture and instillate 0.5 milliliters of sterile PBS and 0.1 milliliter of air. Aspirate the fluid after 60 seconds.
Repeat the procedure with the additional two syringes and collect the whole aspirate in a 15 milliliter tube on ice. Carefully open the thorax with scissors and tweezers to harvest the lungs. Cut the diaphragm along the costal margin and cut through the ribs with two lateral incisions.
Carefully avoid puncturing the lungs. Lift the sternum cranially and fix or remove it. Prepare two 10 milliliter syringes with warm, 37 degrees Celsius PBS.
Make a small incision into the left ventricle. Puncture the right ventricle with a 26-gauge cannula. Then, flush the pulmonary circulation with a pre-warmed PBS.
Be aware of the lungs turning pale during the procedure. Remove the right lobe of the lungs and cut it into halves. Snap freeze them in liquid nitrogen followed by long-term storage at minus 80 degrees Celsius for further gene expression and protein analysis.
Remove the whole left lung and homogenize it in a 48-well plate by mincing the tissue with scissors and tweezers. Incubate the tissue in two milliliters of digestion buffer at 37 degrees Celsius for 60 minutes. Perform further homogenization by carefully pipetting the lung tissue pieces up and down.
Transfer the blood samples into five milliliter FACS tubes and gently mix the blood with two milliliters of red blood cell lysis buffer. Put the tubes on ice and terminate the reaction after two minutes by adding two milliliters of ice-cold PBS. Centrifuge the sample for five minutes at 400 times g and discard the supernatant.
Resuspend the cell pellet with 60 microliters of FACS buffer and process for subsequent FACS staining according to previously described protocols. Centrifuge BAL fluid for five minutes at 400 times g. Aspirate the supernatant and freeze it in liquid nitrogen followed by long-term storage at minus 80 degrees Celsius for further protein analysis.
After resuspending the BAL cell pellet with two milliliters of cold FACS buffer, transfer the suspension into a five milliliter FACS tube using a 100 micron mesh filter to restrain hairs. After centrifuging the sample again, resuspend the pellet with 60 microliters of FACS buffer and process for subsequent FACS staining according to previously described protocols. Now, transfer the digested left lung tissue into a five milliliter FACS tube using a 100 micron mesh filter to extract clumps.
Terminate the digestion process by adding two milliliters of ice-cold FACS buffer before centrifuging the sample for five minutes at 400 times g. Discard the supernatant and resuspend the pellet with 60 microliters of FACS buffer and process for subsequent FACS staining according to previously described protocols. For a FACS analysis, add 20 microliters of CD16/CD32 antibody blocking solution to 60 microliters of cells in a five milliliter tube.
Incubate the cells at four degrees Celsius for 15 minutes to block nonspecific binding of immunoglobulin to the Fc receptors. Meanwhile, prepare a master mix with FACS buffer and antibodies, as described in the text protocol. After blocking, do not wash the cells.
Add 20 microliters of antibody master mix per sample to obtain a final volume of 100 microliters. Incubate the samples for 20 minutes, in the dark, at four degrees Celsius. Wash each sample with one milliliter of FACS buffer and centrifuge as before.
Discard the supernatant and resuspend the pellet with FACS buffer to the appropriate cell concentration for FACS measurements. Finally, add fixed numbers of commercially available fluorochrome-coupled calibration beads to each sample to determine absolute cell numbers. Perform flow cytometry using the gating strategy shown in the text protocol for blood, BAL, and tissue cells.
24 as well as 72 hours following intratracheal instillation, expression of TNF-alpha in lung tissue was significantly upregulated, reaching a sustained and more than 50-fold increase compared to the control animals. Leucocyte invasion into tissue and alveolar space is a hallmark and characteristic for the development of acute lung injury. FACS analysis revealed a significant infiltration of neutrophile granulocytes into the lung interstitium, with absolute cell count having increased almost nine-fold compared to the controls after 24 hours.
Absolute neutrophile granulocyte count slightly decreased after 72 hours, however, the factor increase compared to the controls remained. Consistent with interstitial neutrophile granulocyte infiltration, MMP-9 expression in whole lung tissue was likewise significantly increased over the total observation period. Neutrophile granulocytes were not only increased in the lung tissue, but also in the BAL fluid.
The fold increase compared to control animals was more pronounced than in the lung tissue. Lung edema due to severe impairment of the alveolocapillary barrier is pathognomonic for the development of acute lung injury. Analysis of albumen content in BAL fluid by ELISA revealed a significant loss of barrier function at 24 hours and 72 hours after LPS instillation.
Proper catheter placement is crucial for bilateral instillation of LPS. Often, changes in respiratory patterns, such as coughing or gasping, indicate correct intratracheal instillation of the fluid. Following induction of acute lung injury, a number of subsequent steps, such as FACS analysis, PCR, a protein detection, may be performed to answer the respective research questions.
