Hi, I am Jeffrey Young from the division of Neuroradiology of the Department of Radiology of the Brigham Women's Hospital, a teaching hospital of Harvard Medical School. Hi, I'm Brad Quo. I'm from the gastrointestinal unit and Department of Medicine, as well as the Martino Center for Radiology at Massachusetts General Hospital.
Today we'll show you a procedure for bioluminescence imaging of he oxygenase one regulation in a live mouse model. We use this technique in our laboratory to study the systemic upregulation of Hema oxygenation. One with a procedure called guha, a traditional Chinese medicine technique involving skin scraping.
So let's get started. Female HO one luciferase transgenic mice are weighed prior to being anesthetized so that the correct dosage of Lucifer can be calculated later before beginning hair removal. Confirm by the lack of a toe pinch reflex that the mice are fully anesthetized.
Then use a cotton swab to apply a hair remover lotion over the back of the animal. Wait for five to 10 seconds, then use a clean cotton swab to wipe off the hair After that, dip a piece of tissue in distilled water and use the tissue to wipe clean the remaining hair. Apply distilled water to lubricate the skin areas targeted by gua a few times during the procedure.
Scrape the hair-free region of the back of the mouse gently but firmly using a plastic spoon. Continue scraping until the back skin turns red, which is a sign of subcutaneous blood extravasation. This is usually achieved within two to three minutes.
Place the mice in the induction chamber while you prepare the Lucifer solution, which will be shown in the next section. Prepare a 7.5 milligram per milliliter Lucifer solution by dissolving the Lucifer in sterile water. The dose of Lucifer is 65.5 milligrams per kilogram of body weight.
Preload a syringe with a 26 gauge needle with a single aliquot containing the full dose of Lucifer solution. Quickly inject the needle into the abdomen of the mouse and deliver the entire dose in a single injection. Place a piece of non fluorescent black paper on the imaging of an IVUS 100 station.
To reduce background noise during the imaging. To begin imaging, transfer the mouse to the imaging chamber on the IVUS 100 optical imaging station. The imaging chamber is continuously infused with 1.5%of isof fluorine to keep the mouse anesthetized, position the mouse in a supine position.
Abdomen up. Set the image acquisition to medium benning and the exposure time to 30 seconds. Set the machine to repeat the image acquisition every three minutes, either manually or automatically.
Begin acquiring images after acquisition of the first image. Draw a region of interest or ROI to cover the abdominal chest and head areas. Then copy this ROI and paste it to the subsequent images.
Using the living image software, signal intensity is measured in photons per second. Mark the time taken for the signal intensity to reach its peak value and continue imaging the mouse for about five to 10 minutes. After the peak time, when the image acquisition at the supine position is done, turn the mouse over and lay it in the prone position.
Back up this time, draw the ROI to cover the back and the head areas. After acquiring the first image, continue imaging the animal for five to 10 minutes. When the image acquisition at the prone position is complete, turn off the isof fluorine.
Transfer the mouse from the imaging chamber to the induction chamber. For recovery. The induction chamber is now infused with medical air only, and the mouse should wake up in less than one minute.
Save the imaging data for post-processing. After the initial imaging, additional imaging is carried out for several days following the gua procedure. Before imaging, repeat the hair removal procedure on the back of the mice.
If the hair has grown back in vivo, upregulation of HO one in response to guha is observed. The front view of the same mouse is shown before guha at 18 hours, 36 hours, and 120 hours post guha from left to right respectively. As can be seen, the bioluminescence signal intensity progressively increases in multiple organs after gu sha.
This graph shows the quantitative temporal change over 120 hours in optical flux from the whole body of the same mouse. In response to the guha procedure, You have just watched how to image the in vivo upregulation of heme oxygenase one with bioluminescence imaging. When doing this procedure, it's important to keep the area to be imaged clean, to avoid interference from dust and hair.
Careful control of exposure. Time is required to avoid saturation in the imaging results. So that's it.
Thank you very much for watching and good luck with your experiments.
