Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with ¹⁸F-FDG

The overall goal of this collaborative project is to image changes in brain distribution of a radioactive glucose probe in rats before and after stroke. This is accomplished by initiating cerebral ischemia in rats using a middle cerebral artery occlusion procedure. The next step is to inject the Fluor deoxy glucose radiotracer and collect positron emission tomography images.

The final step is to analyze the data using a cerebral volume of interest template that is anatomically coregistered with the rat brain. Ultimately, pet imaging of rats undergoing cerebral ischemia may be used to measure regional differences in brain uptake of radio probes like Fluor deoxy glucose during longitudinal studies of disease progression. The main advantage of this technique over existing methods like necropsy and histology, is that pet imaging is non-invasive and may be used in a longitudinal fashion and study treatments and linear progression of stroke.

Generally, individuals new to this method will struggle to establish the animal stroke model. The use of anatomical landmarks like the internal and external carotid arteries will greatly ease the procedure. Visual demonstration of this method is critical to perform the VOI analysis that is enabled by merging the PMOD brain template Atlas with the cerebral pet data of the specimen understudy.

The surgeon should change into clean sterile gloves after prepping the area and use sterile instruments. For this study, use male spro dolly rats weighing between 220 and 270 grams and begin by obtaining a pre-surgery PET and CT scan. To provide a baseline for F 18 FDG uptake, anesthetized rats with 2.5%isof fluorine using a nose cone.

Then place the animal in dorsal recumbent on a heating pad, assure that the animal is fully anesthetized with a paw pinch. Then tap down the front legs. Next, shave the dorsal surface of the animal's neck, and prep the shaved area with 70%ethanol, followed by 10%povidone iodine solution.

Now using scissors, make a two to 2.5 centimeter incision 0.5 centimeters to the right of and parallel to the trachea. Then using blunt dissection, locate the carotid artery and use retractors to help visualize the vessel Once identified, place a micro clamp on the common carotid artery. Next, locate the first branching point, which will be the external carotid artery or ECA as well as the internal carotid artery or ICA cauterize smaller branches attached to the external carotid such as the occipital artery.

Then ligate the ECA near the branch to the thyroid artery with a four to zero silk suture, providing extra length to allow hemostats to hold the suture in place. To clamp the sutures with the hemostats, pull the ECA cauley and it will be parallel with the common carotid. Now cauterize the ECA above the suture.

Next, locate the ICA and use another micro clamp to occlude this artery. Then make a small hole in the ECA using small spring scissors. Insert the occlude and tie a suture around it to prevent blood flow.

Now, remove the micro clamp on the internal carotid and advance the occlude until resistance is felt. Make sure the occlude advances into the internal carotid and not the tego palatine artery. Next, remove the micro clamp from the common carotid and cut any excess occluder or suture.

Then use nine millimeter wound clips to close the skin incision. Remove the animal from anesthesia and allow the animal to awaken. Then perform a PET CT Scan 1.5 hours after the stroke procedure after two hours.

Again, fully anesthetize the rat with isof fluorine and remove the wound clips. Then locate the end of the occlude and remove it from the middle cerebral artery by gently pulling on it until the white tip of the occlude comes into contact with the sutures. However, do not pull it all the way out as this will cause bleeding.

Finally, replace the wound clips. Then remove the animal from anesthesia and allow the animal to awaken. Then scan the animal again, 24 hours post reperfusion.

To quantify the brain tissue damage due to stroke injury. As previously mentioned, the animal should be scanned before inducing stroke. Then 1.5 hours after stroke, and finally 26 hours post-stroke.

To quantify brain tissue damage due to stroke injury prior to imaging, anesthetize the rat under 2.5%isof fluorine gas in an anesthesia chamber. Then inject approximately 500 micro curies of fluoro deoxy glucose into the tail vein. Allow the rat to recover after one hour.

Then re anesthetize for imaging. Place the anesthetized rat on a standard rat bed with a nose cone for ISO fluorine anesthesia. Measure the distance in millimeters between the nose of the rat and the edge of the bed and record this as the horizontal offset.

Next, place the animal into the scanner and set up for the study, including the horizontal offset animal weight injection, compound dose time and date. Then click the start study button to begin the scan and reconstruct the data once the scan is acquired. Perform image analysis described here using the PMOD analysis software in conjunction with the W Schiffer brain atlas.

Begin by navigating to the manual co-registration tab at the top of the screen. Then select the slicing tab. Next, use the open white rectangle to rotate the micro PET scans and the filled white rectangle.

To move the micro PET scans, align the two scans by locating landmarks such as the hard glands and the top and rear cerebral features, which can be used to match the micro PET scan with the brain model. Then adjust the micro PET scan until it aligns with the brain atlas. If required, rotate the micro PET scan 180 degrees on the coronal view and raise the scan significantly in the sagittal view along with the other minor orientation changes.

Next, navigate the full screen fusion tab. Select source A at the top right of the screen. Then navigate to template atlas.

At the bottom of the page select rat from the dropdown menu. Optionally return to the manual co-registration tab where the atlas should appear overlaid on a brain atlas. The atlas can be used to help align the micro PET scan and the brain atlas after alignment.

Return to the full screen fusion tab. A template will appear on the brain indicating which sections of the brain will be measured for the VOI statistics. Now select source B at the top right of the screen.

Then select the VOI statistics button. Save the spreadsheet that appears by selecting save and then save to file system. Type the desired file in the file name field.

Next image analysis can be performed using this data to visualize data, create image sequences using vol view imaging software. See the manuscript accompanying this video for details regarding analysis and visualization steps. Representative imaging data for PET and x-ray CT scans are shown here for a rat at the 24 hour pre and 24 hour post reperfusion time points at 24 hours, there was a dramatic decline in glucose uptake to the ipsilateral hemisphere suggesting widespread tissue damage due to the induced ischemic stroke.

White arrows indicate location of decreased FDG uptake due to stroke damage. Here we see the FDG PET data of a rat 24 hours after we profusion fused with the VOI brain template atlas. For analysis, crosshatched colors indicate the separate vois of the brain template atlas.

Finally, here we see ratios of right to left hemisphere FDG pet signal and standard uptake units from each region of the W Schiffer Rat brain Atlas reported for scans taken before ischemic stroke event, 1.5 hours and 24 hours post reperfusion. After watching this video, you should have a solid understanding of how to establish a model cerebral ischemia in rats, as well as perform FDG PET imaging of the animals and utilize a brain template atlas to perform rigorous VOI analysis of the data.