Functional Imaging of Brown Fat in Mice with ¹⁸F-FDG micro-PET/CT

The overall goal of this procedure is to measure brown adipose tissue activity non-invasively in live mice with a standardized micro PET CT protocol. This is accomplished by first placing the animal in cold treatment at four degrees Celsius for four hours to induce brown adipose tissue activity. Next fluorine, 18 FTG is injected and the animal is incubated at four degrees Celsius for one more hour to achieve activity uptake.

Then a sequence of micro CT and micro PET imaging is performed using a small animal dedicated imaging system. Finally, using post imaging processing software, the acquired pet images are coregistered with the CT images and then analyzed for FDG uptake in the interscapular bat area to present bat activity. Brown adipose tissue plays a major role in energy expenditure and the thermogenesis in bone mammals as well as adults.

Although clinical capacity has been used for detecting coding induced bat in humans, this video demonstrates a standardized protocol for imaging and the quantification of bat in small animals Prior to beginning this experiment. First pre chill empty animal cages overnight in a four degree Celsius cold room that has been approved for accommodating laboratory mice. Then on the morning of the experiment, place mice one by one into each of the pre chilled cages at 30 minute intervals each single caged mouse should stay in the cold room for nearly four hours before it is transported to the imaging lab.

Ensure the mice are fasting, but with access to water four hours post cold treatment, begin transport of one animal every 30 minutes to the imaging lab. This can be achieved by filling a styrofoam container with ice and placing a pre chilled housing cage on top of the ice inside the box. Before the beginning of an imaging study order a 10 Milli Curie clinical package of fluorine 18 FTG from a regional vendor.

For arrival to the imaging lab, be sure to follow the institute safety procedures to receive and survey the package containing radioactive materials. With the protection provided by an L block tabletop shield aliquot the FTG and make dilution with sterilized saline. The diluted activity concentration of FDG should be available at 200 to 300 micro curry per 100 microliter for each injection.

Now draw the FDG solution into a one milliliter syringe with a 26 and a half gauge needle and measure the radioactivity of the whole syringe with a dose calibrator. Remove the cage from the cold and place it behind the L block. Then make an intraperitoneal injection of 100 microliters of FDG solution.

Record the injection time and measure the residual radioactivity of the syringe again, with the dose calibrator, place the animal back into the cold cage inside a styrofoam cooler maintained with ice and incubate at four degrees Celsius. For one hour of FDG uptake, calculate the injected FTG activity for each mouse using the formula displayed here. The micro PET CT imaging is acquired with the Siemens Enion dedicated PET system and in beyond multimodality CT SPECT system.

In the DOC mode, start the micro PET and CT imaging. Five hours after the cold treatment and one hour after the FDG injection, anesthetize the animal with 3%isof fluorine in oxygen. Once anesthesia is fully induced, move the animal onto a micro CT animal bed.

With the head resting within a C cone face mask that continuously delivers 2%ISO fluorine at a flow rate of two liters per minute. Place an electric heating pad under the animal to help maintain body temperature. Now slide the animal toward the entrance of the multimodality scanner and position the bed so that the chest of the animal is centered in the horizontal and vertical laser sites.

Then in the laser align window set the first scan type SCT scan and select the PET acquisition included in workflow option at the computer workstation, open the scout view window and acquire a scout view X-ray radiograph. Use the software to adjust the position of the animal bed so that the center field of view of CT is located in the center of the mouse body. And repeat this step if necessary.

Now click start on a pre composed workflow that consists of the following, CT acquisition, pet emission acquisition, pet emission histogram, and PET reconstruction. When prompted, select an appropriate 3D PET CT transformation matrix file to be used in CT reconstruction and choose a recently created normalization file for PET reconstruction with no attenuation. The software will then start CT and PET scan sequentially as programmed.

First, acquire a whole body CT scan with the settings displayed here. This step is linked to a real-time reconstruction option using the common cone beam reconstruction method. Then once the CT is completed, the animal will be automatically slid to the center of the system for a static PET scan.

With the settings seen here in 20 to 25 minutes when the whole workflow is completed, briefly evaluate the quality of the acquired CT and PET images with the A SI Pro analysis software. Archive the imaging data and transfer the data through the network to a post imaging analysis workstation for further analysis. Remove the animal from the imaging systems and place it in a clean housing cage With normal food and water supply for recovery at room temperature, the systems are now ready for the next animal in the queue.

Note the care and handling of post imaging animals should follow the institute's regulations regarding handling of laboratory animals injected with radioactive materials and the relevant waste disposal regulations. To begin analysis, open the Inion research workplace software and manually import both the CT and PET data sets using tools with the general analysis function, coregistered the CT and PET images and assure there is perfect alignment between images in all three dimensions. Next, within the region of interest, quantification window with the anatomic information provided by the coregistered CT images, identify brown adipose tissue at the interscapular region of the neck, the most predominant cold inducible brown adipose tissue in adult mice, and draw a volume of interest over the PET dataset.

Now select voxel intensity as the quantification type and record the mean radioactivity within the VOI as Becca L per liter. A calibration factor, which converts counts per second to bere per milliliter should be previously established by scanning a phantom with known radioactivity. Quantify FDG uptake in brown adipose tissue as percentage injected dose per gram of tissue with decay correction.

The injected dose is the result of the previously calculated injected FDG activity converted to BECCA units. Here we see an example of pet imaging of mouse brown adipose tissue. While the PET imaging encodes the distribution and quantity of FTG uptake throughout the whole body, the CT imaging provides vital anatomical information.

These imaging data can be viewed separately, fused, or demonstrated with a 3D feature, such as maximal intensity projection with the help of a 3D imaging tool. A volume of interest Here, the interscapular BAT region can be drawn over the PET images and the total signals within the VOI can be converted into the percentage injected dose per gram of tissue. Here we see results from an experiment in which pharmacological interventions were applied during the cold treatment precisely 30 minutes before the injection of FTG PET CT imaging and the analysis indicate that the pretreatment of propanolol a beta antagonist significantly reduced the FDG uptake in bat, whereas that of BRL 3 7 3 44 a beta three agonist markedly elevated the uptake as compared with the vehicle control.

A critical step of this protocol is to optimize the duration of the co treatment. A four plus one hour scheme of co treatment is sensitive and consistent in detecting alterations of bad activity in mice. Applications of this method will include various basic studies using mouse models towards a better understanding of bad differentiation and regulation, as well as preclinical research aiming to the discovery of safe, bad stimulating drugs that may benefit the treatment of obesity and diabetes.