The overall goal of this experiment is to study the immune responses in the lung during bacterial infection. This matter can help in answering some key questions in the field of lung inflammation such as what are the key regulatory mechanisms and genes that inhibit or promote lung inflammation. The major advantage of this technique is that it is very efficient and highly reproducable because of the direct delivery of the bacterial inoculum into the lower respiratory tract.
To begin, plate the NTHi on a chocolate agar plate. Incubate the plate upside down over night, at 37 degrees Celsius with 5%carbon dioxide. The following day, culture the bacteria in a brain-heart infusion broth.
Then, in a microcentrifuge tube, add one milliliter of the broth. Spin the mix down at 4, 000 g's for five minutes at room temperature. Then, discard the supernatant and resuspend the pellet in one milliliter of PBS.
Repeat this wash step one more time. Next, dilute 100 microliters of resuspended solution in 900 microliters of PBS, and measure the culture's absorbance at 600 nanometers. Use this information to adjust the inoculum to 20 million CFUs per milliliter.
Next, determine the viability and CFUs of the inoculum. Culture one to ten serial dilutions of the broth on chocolate agar plates, and incubate the plates overnight. Begin with anesthetizing the mouse, and verifying it is adequately anesthetized using a toe pinch.
Then, position the mouse supine and shave the ventral area of the neck. Disinfect the exposed skin. Now, lift the skin of the upper ventral part of the neck with rat tooth forceps, and using a scalpel, make a one half to one centimeter incision above the thymus.
Then, carefully separate the muscle to expose the trachea. Into the trachea, slowly inject 50 microliters of air, followed by 50 microliters of prepared NTHi, followed by another 50 microliters of air. For a negative control procedure, inject saline instead of NTHi.
After the injection, keep the animal vertical for around a minute. Then, close the incision with tissue adhesive and keep the mouse laterally recumbent and warm for about 10 minutes. 24 hours after infection, euthanize the infected animal and open the abdominal cavity with anatomical scissors.
Then, cut the ventral aorta to bleed the animal. Next, expose the ventral part of the lung to access the trachea. Intubate the trachea with a 20 gauge catheter.
Using the catheter, instill 0.8 milliliters of BALF buffer and collect the lavage by dental aspiration. Repeat this procedure three more times, pooling the four lavage collections into a single suspension. Take a 100 microliter aliquot and spin down for two minutes at 2, 000 RPM.
Remove the supernatant and resuspend in RBC lysis buffer. Spin it down and resuspend the pellet with 1x PBS. Now, count the total number of cells in 100 microliters of the suspension using a hemocytometer under 10x magnification with trypan blue staining.
Then, prepare slides to stain the cells. Aliquot 100 microliters of BALF suspension into the appropriate wells of cytospin slides, and spin the slides at 500 g's for five minutes. Then, dry the slides for 10 minutes at room temperature, and stain the cells using a modified Giemsa stain according to the manufacturer's protocols.
Use the remaining cells for subsequent analyses, such as facts, confocal microscopy, and so forth. After collecting lavage from the infected lung, take a 20-40 milligram piece of lung and incubate it in RNA stabilizing reagent at four degrees Celsius overnight, followed by long term storage at 80 degrees Celsius. When ready, wash the lung tissue samples in cold PBS to remove the RNA stabilization reagent.
Then, place the tissue into a 1.5 milliliter centrifuge tube containing lysis buffer with beta mercaptoethanol. Next, with small scissors, chop the tissue into pieces and homogenize the pieces using a motorized pestle for 20-40 seconds. Keep the lysate on ice for five minutes, then spin them down at 18, 000 g's for three minutes.
Carefully remove the supernatant and transfer it to a new tube. Then add one volume of 70%ethanol and triturate the mixture. Let the mixture rest at 25 degrees Celsius for two to four minutes.
Then, transfer up to 700 microliters to a spin column on a two milliliter collection tube and centrifuge the column at 9600 g's for 15 seconds. Discard the flow-through, add 350 microliters of wash buffer and repeat the spin. Next, incubate the membrane with 80 microliters of DNase I Solution for 15 minutes at room temperature.
Then, add 350 microliters of wash buffer to the column and repeat the spin. Next, add 500 microliters of RNA wash buffer. Repeat the short spin, and discard the flow-through.
Do two washes with RNA wash buffer. Finally, over a new collection tube, add 30-50 microliters of RNase-free water directly to the column and spin it for one minute to collect the RNA sample in the flow-through. The procedure was performed as described with normal, healthy, mice.
Intratracheal installation resulted in a markedly increased number of leukocytes in the bronchoalveolar lavage fluid of mice innoculated with NTHi compared to saline treatment controls. The differential count analysis of the leukocytes clearly shows increased neutrophil infiltration. Facts analysis of the cells in the BALF further confirms the increased number of neutrophils.
HNE stained sections of the lung tissue show increased airway inflammation. Collectively, these data suggest that intratracheal installation induces airway inflammation in mice. In a similar experiment with mutant and control mice, lung tissue RNA was analyzed by whole transcriptome sequencing.
Compared to wild-type controls, Itch deficiency resulted in differential expression of several genes. Itch is thought to be a regulator of inflammatory signalling pathways, so this result can be used to foster new studies. All through its development this technique paved ways for researchers in the field of lung diseases to explore cell signalling in immune cells during lung infection and inflammation.
While attempting this procedure, it is important to remember to take extra precautions during procedures like exposure of trachea and installation of bacteria into trachea. Once mastered, intratracheal installation can be performed within ten minutes and vial fluid can be collected within 10-15 minutes. After this procedure, additional matters such as RNA sequencing, flow cytometry, and immunoblocking can be performed to address additional questions such as, which are the genes that are upregulated or downregulated.
