The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.
The overall goal of this procedure is to real-time monitor lung inflammation in mice after a noninvasive challenge with a pro-inflammatory stimulus such as a bacterial culture supernatant. This method can help to answer specific questions regarding host-pathogen interactions such as which bacterial products are responsible for respiratory infection diseases. The main advantages of this technique are that it is not invasive and that it allows the same animals to be monitored repeatedly overtime.
Visual demonstration of this method is important because installation steps are difficult to learn by text alone. Demonstrating the procedure will be Francesca Ruscitti, Angela Sandri, Federico Boschi, all external collaborators of my research group. For in vivo gene delivery, first warm the nucleic acid complex to room temperature.
Next, warm the tail of the experimental animal in 50 to 53 degrees Centigrade water for 30 seconds. When the tail veins have dilated, place the mouse in an appropriate restraining device and insert a 27 to 30 gauge needle into a dilated tail vein at a 20 to 30 degree angle to inject 200 microliters of the complex. When the entire volume has been administered, remove the needle and apply pressure to the injection site.
Twenty-four to 48 hours later, intraperitoneally inject 10 milliliters per kilogram of freshly prepared D-Luciferin into up to five fully anesthetized complex injected animals 15 minutes before the bioluminescent imaging. Open the in vivo imaging system and place a piece of black card stock into the imaging chamber and then place the mice into the imaging chamber. Open the in vivo imaging system software and initialize the system.
Add a check next to luminescent and confirm that the excitation filter is set to block and the emission filter is set to open. For the luminescent imaging mode, select a five-minute exposure time, a binning of eight, and an F/Stop of one. For the photograph imaging mode, select a binning of four and an F/Stop of eight.
Under the field of view menu, select the D field of view with a 19 by 19 centimeter area and a subject height of 1.5 centimeters. When all of the parameters have been set, click Acquire to image the animals. Once the image has been acquired, return the animals to their cages with monitoring until full recovery.
To quantify photons emitted from specific regions of interest, open the imaging system software and select the region of interest tool. In the type menu, select Measurement Region of Interest and use the square tool to draw a square region of interest around the thorax of one animal. Copy and paste this region of interest onto each of the other animals to generate regions of interest with the same dimensions and click Measure Region of Interest to measure the total intensity of all the regions of interest.
Then click Export and select the folder where the regions of interest measurement data will be saved. At least seven days after the in vivo gene delivery, connect the five milliliter syringes with a spring, a 100 microliter Hamilton syringe, and a disposable gauge to a three-way stop cock. Connect a piece of PE 190 micro medical tubing to the disposable gauge and to the pen sentry needle.
Fill the five milliliter syringe with 800 microliters of air. Turn the stop cock and draw up the supernatant in the tube by filling the 100 microliter syringe and then place the anesthetized experimental mouse into the plexiglass intubation platform and secure the animal by its incisors. Turn on the laryngoscope with the nondominant hand and use blunt-ended forceps in the other hand and the tip of the laryngoscope to gently pry open the mouth.
Use the forceps to pull the tongue to the side and guide the laryngoscope blade towards the back of the mouth. Press the laryngoscope very gently at a 90 degree downward angle until the opening of the trachea is visible. Using the other hand, insert the delivery tube connected to the end of the PE tubing into the trachea and rotate the three-way valve to deliver the Pseudomonas aeruginosa inoculum, quickly removing the tube as soon as all of the inoculum has been delivered.
Hold the mouse upright for a few seconds to allow the inoculum to be inhaled into the lungs. Finally, intraperitoneally inject 150 milligrams per kilogram of D-Luciferin into the inoculated animals and image the lungs in the in vivo imaging system four, 24, and 48 hours after the intratracheal stimulation installation, followed by photon quantification as just demonstrated. Here, bioluminescent imaging was used for the real-time in vivo monitoring of lung inflammation in bovine interleukin-8 luciferase transiently transgenic BALB/c mice challenged with concentrated bacterial supernatant containing secreted virulence factors as just demonstrated.
The induced inflammatory response was detected as an increase in the bioluminescence signal as early as 2.5 hours post-installation with the photons emission peaking between five and 24 hours and remaining detectable until at least 48 hours after stimulation. Following this procedure, other methods like bronchoalveolar lavage can be performed to answer additional questions about the number and types of immune cells created upon treatment. After watching this video, you should have a good understanding of how to use lung inflammation in mice and to monitor it in vivo by using bioluminescence imaging.
