A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

The overall goal of the following experiment is to quantitatively compare the regional uptake of the brain's two key energy substrates, glucose, and ketones using PET and MRI in rodents. This is achieved by first obtaining a magnetic resonance imaging acquisition of the brain to localize anatomical brain regions As a second step, carbon 11 acetoacetate and flu ROIC glucose or FDG PET acquisitions are performed, which provide images of brain ketone and glucose uptake. Next PET and MR images are coregistered.

In order to measure the regional cerebral uptake of both pet tracers results are obtained that show the regional cerebral metabolic rate of glucose and ketones based on time activity curves of the brain and plasma. This technique can help answer key questions in the field of brain energy metabolism during aging, such as whether deteriorating glucose metabolism with age also occurs with the brain's major alternative fuel ketones. The actual field of view of the scanner will be an important factor and constraint in order to acquire images of both the brain and the heart that are scanned correctly.

The current animal model will require field of view of 7.5 centimeters to accommodate the distribution of the targeted organs. Test scans will locate the final position of the scanner table. We first add the idea for this method when we realize that ketones were important for brain energy metabolism, yet almost nothing is known.

How the brain uses them Demonstrating the procedure will be looked calmly. A research professional and Michelle ett, a grad student from the Sherbrooke Molecular Imaging Center, Begin by allowing rats to acclimatize in the animal facility for a minimum of seven days prior to beginning this protocol then perform Brain MRI scans one to two weeks prior to dual tracer PET to allow the animal's full recovery from anesthesia. Position the rat on the MRI examination table in the headfirst prone position and place a nose cone for isof fluorine.

Also position respiration and rectal probes so that respiration rate can be monitored and body temperature maintained at 37 degrees Celsius with an automated air warming system. Acquire T two weighted MR images of the brain using a fast spin echo pulse sequence. Once imaging is completed, place the rat on a heated mat and monitor recovery from anesthesia one to two weeks after the MR.Imaging fast the rat for 18 hours prior to pet scanning.

Use a small animal pet scanner equipped with avalanche photo diode detectors and axial field of view of approximately 7.5 centimeters and an isotropic spatial resolution of 1.2 millimeters. Begin by placing the rat in an induction chamber. Then transfer the rat onto a heating mat with a nose cone for isof fluorine and assure that the animal is fully anesthetized prior to imaging.

Start carbon 11 acetoacetate synthesis at this time since the synthesis takes 18 minutes from the end of bombardment. Also prepare a PE 50 polyethylene catheter filled with heparinized 0.9%sodium chloride solution. Position the rat on its side and place the catheter in the tail vein for tracer injection.

Install a second catheter in the mid ventral tail artery for blood sampling throughout the acquisitions. Now rapidly transfer the rat to the scanner table in the headfirst prone position with the nose cone in place. Then move the scanner table into the scanner to ensure the appropriate imaging of the brain and the heart simultaneously as an anatomical landmark position the edge of the field of view on the rat's eyes.

Monitor the respiration rate and maintain body temperature at 37 degrees Celsius throughout the experiment. Next, using a concentrated carbon 11 acetoacetate solution of approximately one giga ERL per milliliter, prepare a syringe of about 50 megal of radioactivity. A range of 45 to 55 megal is acceptable.

Then adjust the volume to 300 microliters with saline. Install the syringe in an injection pump and set the bolus injection of radioactive tracer at a rate of one milliliter per minute for an injection duration of about 19 seconds. Then set up a second pump to immediately deliver 300 microliters of saline at a rate of one milliliter per minute to assure optimal injection of tracer into the blood circulation.

Set the regular sampling mode and the energy window at 250 to 650 kilo electron volts and start dynamic pet data acquisition 30 seconds before starting the bolus injection. The total scan duration should be 20 minutes and 30 seconds. The 32nd acquisition prior to tracer injection provides a measure of the ambient background to be subsequently subtracted from the pet data.

Collect two 200 microliter blood samples approximately 15 and 18 minutes after starting the Carbon 11 acetoacetate injection and inject heparinized saline into the catheter after each sampling to avoid blood clotting. Note the time at the beginning and at the end of blood sampling and calculate the meantime. Samples should be centrifuge at 6, 000 RPM for five minutes to collect plasma.

Then radioactivity counts measured using a gamma counter calibrated with the PET scanner. After the carbon 11 acetoacetate scan, allow a waiting period of 20 minutes to ensure that most of the radioactivity has a decayed. During this time, using a concentrated FDG solution, prepare a syringe of about 50 megal.

Again, adjust the volume with saline and set up the infusion pump as previously described. Now start a dynamic acquisition of a total duration of 40.5 minutes, again, including 30 seconds before the injection. Collect two 200 microliter blood samples at approximately 30 and 35 minutes after starting the FDG injection.

Then at the end of the FDG acquisition, take one final 200 microliter blood sample. Once scanning is complete, reconstruct the pet images according to the following timeframe, sequences one by 30, 12 by five, eight by 30, and n by 300 seconds where n equals three for carbon 11 ACETOACETATE acquisition and n equals seven for FDG acquisition. To begin analysis, use P mode software or an equivalent system for small animal pet image analysis to generate a plasma time activity curve.

First load the FDG data then some image frames for the first 60 seconds following injection when the tracer is mainly in the blood. Next, using a manual drawing tool, draw a of interest or VOI on the left ventricular cavity blood pool, approximately one millimeter inside of the edge of the blood pool. This ensures no inclusion of the tissue and avoids tissue radioactivity spill into the blood pool.

Then copy the VOI into the entire dynamic image series and generate a curve of radioactivity as a function of time. Next, in a spreadsheet, use radioactivity counts of the two plasma samples taken during acquisition to correct the plasma time activity curve. Use the equation seen here.

Also verify if residual radioactivity from carbon 11 acetoacetate is present in the first 30 seconds of the FDG scan prior to injection. If so, subtract the following factor from all the subsequent timeframes of the time activity curve. Next, load all 28 FDG timeframes in the same orientation as the MR images and compute the FDG summed image.

Then perform automatic co-registration of the FDG summed image onto the MR images. Apply the transformation to all individual FDG image frames and save the transformation which will later be applied to the carbon 11 acetoacetate images. This is necessary since carbon 11.

Acetoacetate images have a low signal to noise ratio and automatic co-registration is difficult. Next, using segmentation software, select the MRI data and choose the preferred plane for manual drawing. The manual or the semi-automatic drawing tool can be used depending on brain, region size and image contrast.

Locate brain structures according to a standard rat brain atlas segment, the whole brain cortex, hippocampus, satu, and cerebellum as seen here or other regions as necessary. Save the vois, which will be applied to the coregistered FDG and carbon 11 acetoacetate images. First, apply the vois to the FDG coregistered images.

Select the VO i statistics to visualize the brain time activity curve if needed. Correct the curve by subtracting the decay corrected mean radioactivity in the first 30 seconds from all the subsequent timeframes to perform a cerebral metabolic rate calculation, load brain and corresponding plasma time activity curves. Then select the LAC plot kinetic model and set the lumped constant to 0.48 and the corresponding plasma concentration.

Set the maximal relative deviation from the plac plot to 5%and fit the data to the model. Next for carbon 11 acetoacetate to MRI co-registration. First compute the FDG summed image of the 24 first timeframes to have the same number of frames as the Carbon 11 acetoacetate scan.

Then use the FDG two MRI transformation process for carbon 11 acetoacetate image co-registration. Repeat the plasma time activity curve calculations and co-registration steps for the carbon 11 acetoacetate images. Apply the FDG transformation to all individual carbon 11 acetoacetate image frames.

Finally, use the FDG saved vois and again, calculate the cerebral metabolic rate, setting the lumped constant to one. Here we can see that during the co-registration process, Mr.Images are fixed and pet images move due to an alignment in the axial plane. This graph shows kinetic modeling of whole brain FDG uptake.

Using P mode software after brain uptake, steady state is achieved. The curve results in a straight line with the slope representing brain in flux. Typical values of brain influx and distribution volume in the whole brain for FDG are about 0.0165 per minute to the negative one and approximately 0.6425 milliliters of blood per milliliter of tissue respectively.

The expected cerebral metabolic rate of glucose in the whole brain is approximately 25 micromoles per minute, per 100 grams Following this procedure. Other methods such as western blood can be performed in order to answer additional questions. For example, is glucose and ketone brain transporters expression modified in a specific condition.

With the development of this technique, we've opened a new window for researchers interested in brain antigen metabolism to study physiological and pathological processes involved in aging, neurodegenerative disease and tumor development and treatment in rodents and other animal models. Working with radiation requires additional precautions. Handling the radioactivity tracer behind the lead shield.

Wearing a body and ring doza meter are required for this experiment. Conducting personnel and area surveys using a a Geiger is vital to maintaining a contamination free workspace.