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

Protokol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. PET/CT Calibrations and Workflow Setup (6 Days Post-dMCAO Surgery)

1. Create an imaging workflow in the scanner-operating software to include a CT attenuation acquisition, 60-minute C-11 dynamic PET acquisition (350 - 650 keV level discrimination, 3.438 ns coincidence window), histogram (20 frames: 5 x 15 sec, 4 x 1 min, 11 x 5 min; with dead time correction) and a 3DOSEM-OP reconstruction (2 iterations, 18 subsets) to create 128 x 128 x 159 images with 0.776 x 0.776 x 0.96 mm voxel size.
2. Perform x-ray source conditioning via the CT calibration panel located at the top left corner of the interface. This calibration must be carried out weekly or before the scan if the system has not been used in the past 48 h.
3. Perform dark/light (D/L) and center offset (C/O) calibrations.
   1. Press the CT calibration button (X) in the top left of the interface.
   2. Select D/L and C/O for the CT file that you will be running, remove the bed from the gantry and run the D/L calibration.
   3. Insert the calibration tool bed into the scanner and run the C/O calibration, making sure to switch the selection on the interface to "calibration tool" instead of "70 mm palette".
4. Remove the calibration tool and return the standard PET bed, making sure to change the selection on the interface back to "70 mm palette".
5. Secure a 4-mouse imaging bed onto the scanner platform using tape and attach the anesthesia tubing (Figure 1A). Ensure that isoflurane is flowing through the tubes and that there are no kinks.
6. Push the bed forward so it is in the center of the field of view (FOV), close the CT door and obtain a scout view of the CT to ensure the bed is in the correct position.
7. Perform a "standard" calibration of the PET/CT scanner using an in-house manufactured phantom containing a known dose of C-11 solution as a radiation source.
   1. Prepare a 20 mL syringe filled with the tracer dose equivalent to that administered to one mouse (~250 - 350 µCi/9 - 13 MBq of C-11 tracer diluted in saline).
   2. Record the activity in the standard using a dose calibrator and note the time of measurement.
   3. Conduct a PET/CT scan of the standard using the exact same parameters that will be used to image mice (as described above). Do this weekly to create a correction factor for the PET scanner to apply to the imaging data.

2. Workspace Setup for PET/CT Imaging

1. Create a sterile environment by using a disinfectant with virucide (see Table of Materials) and placing protective absorbent padding on all surfaces.
2. Ensure isoflurane and oxygen tanks are adequately filled.
3. Prepare tail vein catheters by filling a 1 mL syringe (fitted with a 27.5 G needle tip) with 0.9% sodium chloride (sterile saline) and flushing through a 27.5 G, 24 cm butterfly catheter. Cut off the wings of the catheter before cannulating to ensure they do not block the view of the tail vein and to help with ease of moving the mice into the scanner without displacing the catheter.
4. Ensure all essential equipment is laid out at the workstation including spare "flush" syringes (filled with sterile saline), eye lubricant, ethanol swabs, heat lamps, prepared catheters (pre-filled with saline), surgical tape, tissue glue, 0.5 mL dose syringes, scissors, and a lighter to seal catheter after successful placement in tail vein (Figure 1B).

3. Animal Preparation and Cannulation

1. Weigh mice to determine the maximum volume allowed to be injected into each mouse (i.e., volume of tracer and any saline administered must not exceed 10% of body weight).
2. Anesthetize mice in an induction chamber using 3% Isoflurane and maintain at 1-2% (2 L/min 100% O₂).
3. Apply eye lubricant to each mouse and confirm anesthetization via pedal reflex (toe pinch). Adjust anesthesia levels if necessary.
4. Place the mouse onto a heated bed fitted with a nose cone to deliver isoflurane at 1-2% (2 L/min 100% O₂).
5. While the mouse is anesthetized, perform tail vein cannulation using the following protocol:
   1. Place mouse on its side to expose one of the lateral veins, while head remains in the nose cone.
   2. Warm the tail using a heat lamp, being careful not to overheat or burn the tail, and swab with an alcohol wipe to dilate the vein and sterilize the injection site.
   3. Hold the needle with the bevel up and align it with the vein at an acute angle.
   4. Lightly apply pressure to puncture the skin and level out the needle so it is in line with the vein.
   5. Gently push forward a few millimeters past the bevel so the needle enters the vein.
   6. Confirm the catheter is in by administering a small (10-20 µL) flush of saline. The saline should leave the syringe smoothly and the vein should clear. If any resistance or back pressure is observed, it is likely the catheter is not in the vein and re-attempting cannulation is advisable. If clotting is observed, use heparin (1,000 units heparin per mL saline) for cannulation setup and flushing.
      NOTE: We have assessed cannulation with and without heparin in the mouse strain of interest, and since no clotting was observed, saline alone was used for cannulations.
   7. Secure the catheter to the tail using a small drop of tissue glue, followed by surgical tape, to ensure that the catheter remains immobile when transferring the mice to the scanner.
   8. Remove the flush syringe from the end of catheter and seal the end with lighter, ensuring the researcher is not near any isoflurane or ethanol.
   9. Repeat for 3 additional mice so that all 4 mice to be scanned are cannulated and prepared.
6. Turn on the anesthesia flow (2.5% Isoflurane, 2 L/minute 100% O₂) connected to the PET/CT and carefully position the mice prone in the scanner bed, ensuring catheters remain in place and each mouse’s head is straight and secure within the nose cone. Tape the head and the body of each mouse to the bed with soft surgical tape, ensuring breathing is not restricted by the placement of the tape. Record the position of each mouse to allow for correct location and group allocation for image analysis.
7. Keep mice heated throughout the procedure (e.g., using a heat lamp or hot air pump system to ensure mice are kept warm without overheating). Monitor respiratory rate of all mice, either visually if using an open gantry or through a remote monitoring system using respiratory pads, and alter the anesthesia levels as necessary.

4. CT Acquisition

1. Once animals are secure in the bed and respiration is stable, turn on the laser cross hairs and move the scanning bed so that they align with the brain of all four mice. Move the scanner bed in to the acquisition position (position 3) with the brains of the mice as close to the center of the FOV as possible.
2. Acquire a scout view image of the mice to verify their position (use a 200 mm FOV), and adjust the position by dragging the FOV box on the interface if necessary. 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.

5. [¹¹C]DPA-713 Dose Preparation

1. Synthesize [¹¹C]DPA-713, ensuring you are wearing appropriate PPE (personal protective equipment) for handling radioactivity, including a lab coat, gloves, and personal finger and body dosimeters. Ensure you change gloves regularly to prevent radioactive contamination, and increase your distance from the radioactive source when possible.
2. Use forceps to carefully transfer the radiotracer vial behind a lead shield.
3. Prepare 0.5 mL dose syringes for each mouse containing approximately 250-350 µCi/9-13 MBq in 100-200 µL volume to ensure a dose adequate for a 60-minute dynamic PET scan (dose administered should be determined considering the half-life of the isotope and time-line of the study design, with the volume depending on the mouse weight).
4. Measure the activity using a dose calibrator set to C-11, located in close proximity to the cannulation site, and record the times of measurement and injection to enable decay correction. Draw up the doses just before the CT ends to limit decay and ensure the desired level of radioactivity will be injected in each mouse.
5. Verify that there are no air bubbles in the dose syringe before measuring the activity and injecting into each mouse.

6. PET Acquisition

1. Once the mice automatically advance from CT to PET, set up the back of the scanner for [¹¹C]DPA-713 injection (Figure 1C). Place protective absorbent padding on a ledge and make sure scissors and lighter are on hand.
2. 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 10-20 µL saline flush. Load the measured dose syringes from step 5.4 into each of the 4 catheters, keeping track of which dose was given to each mouse.
3. 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 4 mice simultaneously upon the timer reaching zero. Flush each catheter with 50-100 µL of saline (depending on the length of catheter tubing — i.e., the dead volume) to make sure the full dose enters the tail vein, and re-seal the tubing once again using a lighter.
4. Measure the dose syringes using a dose calibrator to obtain a residual radioactivity value (any tracer left in the syringe). Take note of the values and the time they are recorded.
5. Once the scan is complete, home 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.
6. Measure the residual activity in the catheter using a dose calibrator.
7. If mice are to be recovered ensure this is done in a warm environment (e.g., in a box with a heated pad underneath or containing a glove filled with warm water) to ease recovery. If planning to euthanize the mice, place the mice into an induction chamber containing isoflurane so that they remain anaesthetized prior to euthanasia via perfusion.
8. To reconstruct the data, open the post-processing managing software (see Table of Materials), which will automatically reconstruct each scan using the histogram data that was generated from the lst file.

7. Dynamic PET Image Analysis

1. Open image analysis software (see Table of Materials) and click on the "open data" icon to load the CT image (as the source) and the "append data" icon to load the dynamic PET (as the reference).

2. Perform a visual quality control of the data via the time-series operator in the drop-down menu: Select reference ("ref") 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.

3. Create an average PET image using the "arithmetic operator".

1. Choose "average selected", unselect "ref", and ensure input 1 ("Inp1"), input 2 ("Inp2") and input star ("Inp*"-includes the rest of the PET frames in the scan) are selected to create an average of all the PET frames.

2. Go to the "data manager" tab (DM) and drag the average image up to the "input1" position for visualization purposes. Redistribute the color scale by clicking on the automatic calculation in the "min-max" tool.

4. Register the CT to the average PET file using the "automatic 3D" function in the "re-orientation/registration" drop-down menu.

1. Select "ref" and "Inp1", and choose "rigid", "fast", "Inp1 to Ref" registration. Visually check the registration in all 3 dimensions and manually adjust if necessary in the "manual 3D" tab using the "translation" and "rotation" functions.

2. When satisfied with the registration, select "Inp2" and "Inp*", and apply to all PET frames by clicking the checkmark. Right click on the CT and PET files in the DM and save as raw.

5. Crop the brain of one mouse at a time for brain analysis using the CT as a guide: Select "cropping" from the drop-down menu and drag the image boundaries to crop the head of the mouse below the brainstem. Re-orientate the PET and CT images using the "manual 3D reorientation" function as described above so that the skull is straight in all dimensions.

6. Load in the MR image for that mouse (in DICOM format) using the "append data" button on the top left of the interface. Move the MR using the "manual 3D reorientation" and fit to the skull within the CT image (make sure all modalities are in the same orientation).

7. Draw the stroke ROI on the MR image using the "3D ROI tool".

1. Turn off the PET visualization by deselecting it within the visual controller tab (VC) and use only the MR and the CT to draw the ROI.

2. 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.

3. Repeat through all slices encompassing the stroke, making sure not to capture any of the skull in the ROI, with best practice being to leave a voxel gap between the skull border and stroke ROI.

8. Generate a contralateral ROI using the infarct volume.

1. Create a new ROI and label it "contralateral". Right click on the Infarct ROI and select "export". Drag the ROI to position 2 ("Inp1").

2. With only "Inp1" selected, apply a left right flip using the "operator" function within the "reorientation/registration" menu. Tick the "ROI" box, choose "view only", and manually move the new ROI to the identical region on the contralateral side. Select the "arithmetic's" operator and apply a scalar multiplication of 2 to the new ROI, permitting independent quantification of ROIs.

3. Return to the 3D ROI tool. Go to the "expert and experimental" tab and click on the "import ROI" button. Select Inp1 from the dialog box to load the new volume as the contralateral ROI.

9. 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.

10. Perform additional split brain analysis if desired (i.e., automated ROI generation of right versus left brain hemisphere regions using a 3D mouse brain atlas plugin module for Vivoquant software).

1. Re-load the registered PET/CT images.

2. Import the mouse brain atlas by clicking on the "advanced modules" menu and selecting the 3D brain atlas tool. Select "all regions left/ right" in the "advanced settings" and click "run" to import the 3D atlas.

3. Manually fit the atlas within the brain using the skull as a border.

4. Re-run the atlas making sure that "import 3D ROI" is checked to generate a spreadsheet of results for all 14 left and right hemisphere ROIs (medulla, cerebellum, midbrain, pons, cortex, hippocampus, thalamus, hypothalamus, striatum, pallidum, olfactory bulbs, corpus callosum and white matter).

11. Quantify tracer uptake in the spleen using the scanner operating software (see Table of Materials).

1. Load PET and CT image files by highlighting them in the database and clicking on "general analysis".

2. Click on the registration tab and co-register PET and CT images clicking on the "rigid registration" icon.

3. Click on the ROI quantification tab, click on the "create ROI" icon and name it spleen.

4. Choose the "sphere" tool to draw spleen ROIs using the CT file for reference, ensuring there is no overlap with kidney uptake (using the PET image and signal to avoid spillover from kidneys).

5. Edit the ROIs to maintain consistent ROI volumes between animals.

12. Calculate a standard correction value for uptake normalization.

1. Load the PET/CT data from the standard scan and create a cylinder ROI encompassing the 20 mL syringe using the "manual 3D ROI" tool.

2. Obtain the level of radioactivity contained within the standard using the spreadsheet icon.

3. Use this nCi/cc result and the original recorded radioactivity for the standard (i.e., the dose calibrator measurement of the standard in nCi/cc) to create a correction factor for PET uptake values. That is, divide the radioactivity of the standard recorded by the dose calibrator by the radioactivity calculated from the PET image of the standard.

13. Use the dose activities and time of measurements to decay correct to the time of PET acquisition for all mice (i.e calculate the dose activity at the start of the PET scan).

14. Repeat for the residual values and subtract from the decay corrected dose to calculate the exact activity each animal received.

15. After applying this decay correction, also apply the standard correction to make sure the data are at the right activity level. Ensure these corrections are applied to the manually drawn ROI results, and brain atlas ROI data relevant brain regions for dMCAO location (i.e., cortex, hippocampus and striatum).

16. Calculate the %ID/g for all ROIs using the following equation: %ID/g = (ROI radioactivity in nCi/cc / decay corrected dose received in nCi/cc) x100. Plot %ID/g as a function of time using graphing software to generate time activity curves for each ROI.

17. Use scanner software for final image visualization and figure generation. Normalize images according to the decay corrected dose received by each mouse at the time of scanning, ensuring all images are on the same %ID/g scale.

NOTE: This is necessary to enable accurate comparison of images from different mice and/or images from studies performed on different days.

Sonuçlar

Figure 1: PET Scanner and Workspace Set-up. All workspaces were covered in protective absorbent padding to create a sterile environment. (A) After calibrations, a 3D-printed mouse bed, equipped for imaging 4 mice simultaneously was secured in the scanner and nose cones for all 4 mice attached to the anesthesia. (B) Necessary equipment for PET imaging were prepared in advance, including saline-filled 27.5 G catheters, eye lubricant, ethanol swabs, heat lamps, surgical tape, tissue glue, 0.5 mL dose syringes, scissors and a lighter. (C) For radiotracer injection, place saline-flush syringes and scissors at the back of the scanner.

Malzemeler