The overall goal of this procedure is to perform in vivo imaging of mouse neonates to investigate the dissemination of luminescent cytomegalovirus. This is accomplished by first preparing the appropriate dilution of the virus from a stock solution. Next neonates are injected with the luminescent MCMV in the peritoneal cavity.
At time points after injection. The pups are anesthetized and injected with Lucifer before in vivo imaging. Ultimately, in vivo imaging is used to show and quantify the replication and dissemination of the virus in the peritoneal cavity and central nervous system.
The main advantage of this technique over existing methods like viral filtrations in organs after euthanasia of infected animals, is that less time and fewer resources are consumed. Demonstrating the procedure will be Mr.Il Macan, a technician from my laboratory. A strain of MCMV expressing Luciferase obtained from Ulrich Kowski laboratory is used in the following study.
To amplify the strain, add the virus to cultured cells in serum free medium at 37 degrees Celsius. After one hour, remove the supernatant and replace it with DMEM supplemented with 10%fetal calf serum. Five days later, the culture is harvested and aliquots are stored at minus 80 degrees Celsius.
Until further use. After titrating the viral suspension by plaque assay, dilute the sample to 50 pfu in 50 microliters of DMEM and leave on ice until ready to use injections are performed in neonates that are between four and 20 hours old. Before handling the pups, manipulate the litter and feces with gloved hands.
To avoid introducing foreign odors that could stress the mother to perform a practice injection, fill an insulin syringe with 1%methylene blue diluted in DPBS. When ready, hold the neonate by the dorsal skin as seen here with the ventral side up. Introduce the needle under the four leg and gently push it subcutaneously into the peritoneal cavity.
Once the needle tip is in the correct position, inject the methyl in blue. Next, slowly withdraw the needle and wipe away any blood from the injection site. Return the neonate back to the home cage after injection after a successful practice injection, repeat this procedure with the viral suspension prepared earlier.
Images are taken seven days after the injection, handling the neonates cautiously as shown previously. Inject each with a mixture of anesthetics and Lucifer while waiting 12 to 15 minutes For the LUCIFERIAN to reach its maximum emission, make sure that the platform in the acquisition chamber of the imaging device is heated to maintain the correct body temperature. After the pups are anesthetized and tagged for identification, they are ready to be placed in the imaging system.
First, perform acquisition of the light emitted by the whole body. Since the virus has replicated in several target organs, the duration of exposure should be short. Here, acquisition is performed for 10 to 20 seconds.
It is helpful to obtain low resolution images of several animals within one round of acquisition, and then move the platform closer in order to focus on one part of a particular animal or an isolated organ following dissection. To acquire a light signal from only the head, cover the rest of the body with a thick, dark paper that will mask the photons, perform acquisition for five to 10 minutes, and repeat this for the opposed side of the animal. Under the same conditions.
After imaging return the pups to a heated cage to maintain their body temperature while anesthetized. These images were taken at day 7, 9, 11, and 14 after infection, the overall viral titer in the animal appears to decrease over time. In this example, the body is covered, leaving the head exposed.
This reveals a discreet spot at the level of the left ear whose intensity increases between day seven and 14, suggesting that the dissemination of MCMV to the brain can be dynamically observed in vivo. Following this procedure, other methods like immunohistochemistry can be performed in order to answer additional questions about the precise location of the virus in the brain.
