TSPO-PET/CT Imaging to Detect Infarct Formation in a Mouse Model of Ischemic Stroke

Take anesthetized control mice and mice with restricted blood flow to the somatosensory cortex.

This restriction causes neuronal death and infarct formation in the brain, triggering microglial activation and peripheral immune cell infiltration.

The immune cells overexpress the mitochondrial translocator protein (TSPO).

Secure the mice on the PET/CT scanner bed.

Perform a CT scan using a rotating X-ray source that passes X-rays through the mouse's body.

A detector captures the X-rays and processes them into images of major anatomical landmarks.

Inject a TSPO radiotracer via the tail vein.  

The radiotracer travels to the infarct area and binds to TSPO in immune cells.

Perform a PET scan.

The radiotracer undergoes positron decay, emitting positrons that collide with nearby electrons to produce gamma rays.

A detector captures these rays and reconstructs a 3D image of the radiotracer distribution.

Visualize the overlaid PET/CT images to identify infarct areas with higher tracer uptake.

Begin by positioning the anesthetized mice prone on the PET/CT scanner bed, ensuring they are straightened and securely in the nose cones. Tape the head and body of each mouse to the bed with soft surgical tape, ensuring that breathing is not restricted by the placement of the tape. Once animals are secure in the bed and respiration is stable, turn on the laser crosshairs and move the scanning bed so that they align with the brain of all four mice.

Move the scanner bed into the acquisition position with the brains of the mice as close to the center of the field of view as possible. Acquire a scout view image of the mice to verify their position, and adjust the position by dragging the field of view box on the interface if necessary. Finally, click Start Workflow in the scanner software to begin the CT scan, making sure to select Display interactive user prompts, so the PET scan can be manually started prior to tracer injection.

Once the mice automatically advance from CT to PET, set up the back of the scanner for the radiotracer injection. Place protective absorbent padding on a ledge and make sure scissors and lighter are on hand. Snip the sealed catheter tubing with scissors. Check catheter lines are clear of any bubbles and confirm the cannula is still within the vein by performing a 10 to 20 microliter saline flush. Then load previously measured dose syringes into each of the four catheters, keeping track of which dose was given to each mouse.

Click OK when the PET scan is ready to start, while simultaneously starting a 10 second timer. Have two researchers at the back of the scanner, with the dose syringes in hand to inject all four mice simultaneously upon the timer reaching zero. Flush each catheter with 50 to 100 microliters of saline to make sure the full dose enters the tail vein and reseal the tubing once again using a lighter.

Next, measure the dose syringes using a dose calibrator to obtain a residual radioactivity value. Take note of the values and the time they are recorded. Once the scan is complete, return the PET bed to the original position using the horizontal home button within the motion control panel. Remove the mice from the scanner and carefully remove the catheter. Gently apply pressure to the cannulation site to prevent excessive bleeding. Then measure the residual activity in the catheter using a dose calibrator.

Finally, reconstruct the data by opening the post-processing managing software, which will automatically reconstruct each scan using the histogram data that was generated from the first file. For PET analysis, begin by opening image analysis software. Clicking on the Open Data icon to load the CT image. And selecting the append data icon to load the dynamic PET. Perform a visual quality control of the data via the time series operator in the Dropdown menu. Select Reference and Global and apply an appropriate Min and Max for the color scale.

Visualize the dynamic PET data frame by frame, verifying radioactivity uptake and checking for any motion confounds within the scan. Then create an average PET image using the arithmetic function. Choose Average Selected, deselect Reference and ensure input one. Input two and input star are selected to create an average of all PET frames.

Go to the Data Manager tab and drag the Average Image up to the input one position to allow for visualization of the PET signal using the average PET image. Then redistribute the color scale by clicking on the Automatic calculation in the MinMax tool. Next, register the CT to the average PET file using the automatic 3D function. In the Reorientation Registration Dropdown menu, select Ref and input 1 and choose Rigid Fast input one to ref registration.

Visually check the registration in all three dimensions and manually adjust, if necessary in the manual 3D tab using the translation and rotation functions. When satisfied with the registration, select Input 2 and Input Star and apply it to all pet frames by clicking the Check Mark. Right click on the CT and PET files in the DM and save as raw. Next, select Cropping from the Dropdown menu and drag the image boundaries to crop the head of one mouse at a time below the brain stem.

Manually reorient the PET and CT images so that the skull is straight in all dimensions. Load in the MR image for that mouse using the append data button on the top left of the interface. Move the MR using the manual 3D reorientation and fit it to the skull within the CT image. Next, turn off the PET visualization by deselecting it within the visual controller tab, and use only the MR and the CT to draw the Region Of Interest or ROI.

In the 3D ROI tool, click on the Add ROI button to create a new ROI and name it infarct. Select the spline tool, left clicking to draw the ROI border and right click to close it. Following that, create a new ROI and label it contralateral. Right click on the Infarct ROI and select Export. Then move the ROI to position 2, Input one, to allow visualization and manual reorientation of the new ROI.

With only input one selected, tick the ROI box and choose View Only to allow visualization of the infarct ROI without reorienting it. Within the reorientation registration menu, apply a left right flip using the operator function and manually move the new ROI to the identical region on the contralateral side. Then select the arithmetics operator and apply a scalar multiplication of 2 to the new ROI, permitting independent quantification of ROIs.

Return to the 3D ROI tool. Go to the Export And Experimental tab and click on the Import ROI button. Select input one from the dialog box to load the new volume as the contralateral ROI. Finally, right click on the Average PET image and unload it and turn the PET back on. Generate the quantitative uptake results using the export results icon within the 3D ROI tool.