The overall goal of this study on an IPF-like mouse model is to longitudinally monitor lung fibrosis with non-invasive imaging techniques such as FMT and micro-CT. This method can advance what sequence in regarding this specific molecular events occurring in lung fibrosis. The main advantage of this techniques is that anatomical changes of specific molecular events can be monitored noninvasively.
The applications of this technologies extend towards therapy of IPF pathology because the fusion of FMT and micro-CT represents the possibility to study disease progression and new therapy evaluation. Start by positioning an anesthetized mouse on the intubation platform hanging it by its incisors placed on the wire. Turn on the laryngoscope, then take a pair of blunt-ended forceps and gently open the mouth.
Place the laryngoscope blade towards the back of the mouth until the opening of the trachea is visualized. Use the other hand to insert the delivery tube connected to the end of the PE tubing into the trachea. Rotate the three-way valve to deliver 50 microliters of bleomycin.
After installation quickly remove the tube from the trachea to prevent suffocation. Hold the mice upright for a few seconds. For probe injection take the mouse by the tail and allow it to grip the cage lid.
Then grasp across the shoulders and hold the mouse by the scruff. Immerse the tail in a beaker of warm water to cause the veins to swell for easier visualization and needle insertion. Find the two caudal veins on either side of the tail, not the artery on the underside of the tail.
Insert the 26 gauged needle of the MMP loaded 1 milliliter syringe into the vein and inject the MMP probe solution at 10 milliliters per kilogram of body weight. To begin initialize the date acquisition software and open a new study in the database for the experiment. Before starting the imaging, transfer the shaved and anesthetized mouse to the middle of imaging cassette.
Keep the mouse flat, secure, and gently compressed against both windows of the imaging cassette. Then in the scanning window of the software, click on Subject"and then preview to see the live image. Draw the scan region sufficiently large so that the tissues surrounding the anticipated area of fluorescence is captured.
Once the ROY is drawn, click Add to Reconstruction Queue"and then click Scan"to image the mouse. When the image acquisition is complete, remove the mouse from the imaging cassette and place back into the home cage. Quantify the picomoles of fluorescence using the ROY tool and reduce the ROY around 700 to 800 cubed millimeters on the signal coming from lung region.
Repeat imaging every seven days or as required. To image by micro-CT, first turn on the micro-CT by pressing the green power button and launch the software to warm up the x-ray source. Create the database by clicking on New Database"and build the browser based on the number of mice in the experiment.
Before starting the scan, select acquisition parameters in the software control window as follows. Set x-ray tube voltage to 90 kilovolts, the CT x-ray tube current to 160 micro angstroms, live x-ray tube current to 80 micro angstroms, and the field of view to 36 millimeters. Select no Gating Technique"and High Resolution"and four minutes for scan technique.
After anesthetizing the mouse by inhalation of 3%isoflurane, place it onto the animal bed inserted into the small bore with a nose cone providing a constant supply of anesthetic. Immobilize the paws with tape on the bed to allow the chest to be exposed. Next side the instrument door closed and turn on live mode to see the mouse position in real time by pressing the capture button.
Move the animal bed to align the chest with field of view using the buttons on the CT instrument. Once the mouse is centered, rotate the gantry to confirm optimal bed position by selecting 90 degrees and clicking Set"Then click the CT scan button. Click Yes"to the message that informs that the x-ray source will be turned on.
After imaging place the animals back into the cage and monitor until fully recovered from anesthesia. Repeat imaging every seven days or as required. Staining with Masson's trichrome reveals the morphology in the lung tissue.
Pictures of the bleomycin treated group showed a pronounced pattern of fibrosis starting from day seven mainly as single fibrotic masses and progressed at day 14 to confluent conglomerates of substituted collagen and remained unaltered until day 21. Repeated micro-CT imaging allowed for quantification and segmentation of airways in fibrosis lung lobes. The airways were divided into a central and distal part.
The distal part of the airways is the intrapulmonary tract and splits into the lung lobes as the right cranial lobe, right middle lobe, right caudal lobe, right accessory lobe, and left lung. Quantification of airway radius showed increased radius in the bleomycin treated mice compared to saline controls. Each point represents the mean standard error of the mean of five animals for a total of 30 mice.
Total lung fibrosis quantification of vehicle or bleomycin-treated mice at different time points demonstrates that these micro-CT parameters agree with histological findings. Once mastered this technique can be performed for drug screening routinely. After its development this technique paved the way for researcher in the field of lung fibrosis disease to explore efficacies of the repeating treatment in IPF-like animal models.
