The overall goal of this imaging protocol is to quantify the in vivo realtime uptake of glucose from the bloodstream into specific tissues in mice. This method can help answer key questions in the diabetes and metabolism field, such as the impact of any diabetic treatments on glucose uptake and metabolism. The main advantage of this technique is that experiments can be performed longitudinally to assess for example the impact of age or diet on glucose metabolism.
Learning this method provides information about glucose metabolism in sclero muscle. It is also useful in other tissues such as liver and brain and is transferrable to rats. Visual demonstration of this method is critical, as the imaging acquisition and our analysis steps must be completed correctly to ensure accurate results.
First, wipe down the induction chamber, the imaging bed, and the heat pad with 80%ethanol. Place the mouse subject in the induction chamber and anesthetize the mouse with 5%isoflurane and oxygen. Secure an electric heating pad and a sensor pad on the imaging bed.
Place the mouse in a prone position and continue delivering one liter per minute of 1.5 to 2%isoflurane by a nose cone. Apply veterinary ointment to the mouse's eyes to prevent dryness. Then, warm the mouse's tail for one to two minutes.
Once the lateral tail vein is dilated, insert a 30 gauge needle into the vein. Fix the needle in place with surgical glue and connect a catheter to the needle. Load the imaging bed into the PET/CT scanner and position the bed so the catheter is accessible from the rear of the machine.
Secure a syringe containing the ten mega back roll F18-FDG dose to the catheter. After a set time following induction of anesthesia, start a one hour PET scan and inject the F18-FDG dose. Upon completion of the PET scan, perform a 10 minute CT scan.
After the CT scan, move the imaging bed to its initial position and remove the mouse from the bed. Either euthanize the mouse by cervical dislocation under anesthesia, or allow the mouse to recover in single housing under observation. To begin the image processing, load the reconstructed PET and CT images.
Designate the CT data as source and the PET data as target. Co-register the data and align the images as needed. Then, select ROI quantification.
Use the panning and zoom tools to locate the desired region of interest in the image. Navigate to the create tab and select the paintbrush tool. Draw an ROI corresponding to the gastrocnemius muscle.
Then, use the save ROI quantification function to extract the tissue time activity data as a CSV file. De-convolve the estimated system point spread function for five iterations with a Van Cittert deconvolution method. Next, draw an ROI corresponding to the vena cava.
Export the blood input function time activity data from the post de-convolution images as a CSV file. Import the CSV files into spreadsheet software. Save the data as new CSV files and convert the uptake values to percent injected dose per centimeter cubed.
Plot the time activity curves with the spreadsheet software. Next, save the processed vena cava data to a new CSV file. Convert the vena cava data to a plasma input function.
Select kinetic in the kinetic modeling tool. Import the tissue and plasma total activity count data files. Choose a two tissue, three compartment model.
Ensure that the F18-FDG dephosphorylation rate, K4, is unchecked and has a value of zero. Set the blood volume fraction, VB, to 2%To add dispersion to the vena cava derived input function, use a program such as disp4dft to create multiple input functions with different dispersion times. Then load the input functions with dispersion one by one and select fit current region.
Repeat to find the input function with dispersion that has the lowest ChiSquared value. Using the optimized input function, set the blood volume fraction to a floating value. Fit the region again to determine the regional rate constants.
Then calculate the regional influx constant. Six week old DBDB mice treated with either insulin or phosphate-buffered saline were imaged with this method. The insulin treated mice showed increased F18-FDG activity in the gastrocnemius muscle compared to the control mice.
No increased activity was observed in the vena cava. A two tissue, three compartment model was used for kinetic modeling. No significant difference from control was observed in the rate of F18-FDG transport from arteriol plasma to the intracellular space or vice versa.
However, the F18-FDG phosphorylation rate was significantly higher in the insulin-treated mice. The influx constant was correspondingly higher for the insulin-treated mice. After watching the video, you should have a good understanding of how to correctly image the mice, draw regions of interest, and calculate kinetic parameters.
Don't forget that working with radiation can be hazardous and precautions such as proper training, use of shielding and monitoring of exposure should always be taken when performing this procedure.
