Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

All procedures were conducted in accordance with the guidelines of the National Council for the control of Animal Experimentation (CONCEA, Brazil) and were approved by the Ethics Committee for Animal Research of the Medical School of the University of Sao Paulo (CEUA-FMUSP, Brazil – protocol number: 25/15).

NOTE: In this protocol, we show how to induce a lysolecithin rat model of multiple sclerosis and how to acquire and analyze the myelin PET images.

1. Lysolecithin solution preparation

1. Weigh lysolecithin (L-α-Lysophosphatidylcholine from egg yolk) on an analytical scale using a conic plastic tube (1.5 mL).
2. Add sterile saline to the tube to make 1% solution (for example: weigh 1 mg of lysolecithin and dissolve it with 100 µL of saline) and dissolve the lysolecithin by shaking the tube (shaking from side to side and not turning from top to bottom).
3. Prepare the solution just before starting the animal model induction (do not stock the final ready solution).

2. Lysolecithin rat model – Stereotaxic surgery

1. Use male Wistar rats weighing between 220 – 270 g. Use gloves and mask during all procedures.
2. Induce anesthesia with 5% isoflurane mixed in 100% O₂ (1 L/min) using an induction box. Check if the animal is anesthetized by observing absence of movement (only breathing should be observed).
3. Place the animal in stereotaxic apparatus on a heating pad. Fix the nose and ears of the animal to the equipment.
   ​NOTE: Details of how to perform stereotaxic surgery can be found in JoVE Science Education Database. Neuroscience. Rodent Stereotaxic Surgery. JoVE, Cambridge, MA, 2021.
   1. Monitor the anesthesia for the whole procedure (including surgery and image acquisition). To adjust the isoflurane concentration, observe the animal breathing rate. A fast breathing rate requires higher concentration and a slow breathing rate requires lower anesthetic concentration.
4. Inject the analgesic (ketoprofen – 5 mg/kg) subcutaneously (dilute the medication to 1 mL in saline, this will help to hydrate the animal).
5. Put eye cream in the animal's eyes to protect against dehydration.
6. Use a 0.5% chlorhexidine solution to clean the incision area.
7. Inject 100 µL of lidocaine hydrochloride 2% subcutaneously in the region of incision.
8. Shave the skull area.
9. Use sterile instruments for this procedure.
10. Using a scalpel, make an incision of about 2 cm in the skin over the skull.
11. Expose the skull with Bulldog clamps.
12. Use a swab to clean the skull area with 1% hydrogen peroxidase.
13. Locate and mark the bregma (a stereotaxic rat brain atlas can help to identity the Bregma).
14. Position the Hamilton syringe (µL neuros syringes) at the bregma.
15. Using the bregma and a stereotaxic atlas as reference, position the Hamilton syringe at the following coordinates: Antero-posterior: -0.30 mm, latero-lateral: -3.0 mm, and ventral until it touches skull bone, and mark the skull and note the coordinates orientation on paper.
16. Drill the skull at the marked coordinate. Take care with dura mater (damaging dura mater can cause a lot of bleeding).
17. Fill the Hamilton syringe with 7 µL of lysolecithin solution (1% in saline). A greater volume can be placed in the syringe, but only 7 µL will be injected (take the bubbles out of the syringe to measure the volume correctly).
18. Position the Hamilton syringe at the previous coordinates to start the drug injection (total of 7 µL into 3 different ventral stereotactic coordinates). Considering the previously noted coordinates, lower the Hamilton syringe to ventral coordinate -5.0 mm.
19. Very slowly inject 2 µL of lysolecithin solution (1 µL/10 min). Wait 3 min.
20. Take the needle up to the next ventral stereotaxic coordinate (-4.2 mm) and slowly inject 2µL of lysolecithin solution (1 µL/ 10 min). Wait 3 min.
21. Inject the third ventral coordinate -3.0 mm (3 µL of lysolecithin solution – 1 µL/ 10 min).
22. Wait 5 min and then remove the Hamilton syringe from the brain.
23. Suture the skin.
24. Remove the animal from the stereotactic apparatus and allow the animal to awaken.
25. Return the animal to the home cage, keep the animal alone and under supervision to check for signs of discomfort.
26. Inject subcutaneous analgesic (ketoprofen – 5 mg/kg) diluted in saline, at 24 h and 48 h after the surgery.

3. PET acquisition

1. Take 7 to 20 MBq of ¹¹C-PIB radioactivity in a syringe (1 mL is the maximum volume allowed for intravenous injection in rats).
2. Anesthetize the animal with 5% isoflurane mixed in 100% O₂ (1 L/min) using the induction box.
3. Inject ¹¹C-PIB (radioactivity defined in item 4.1 above) into the penile vein, or tail vein of the rat (both administration sites are fine, the decision is a personal choice. We only advice not to use a retro-orbital injection since the location is too close to the region of interest (the brain) and can impair image quality.
   NOTE: Image acquisition of baseline time point is performed before stereotaxic injection and the other 2 time points at 1 week and 4 weeks after surgery.
4. Remove the animal from anesthesia to allow the animal to awaken (leave the animal on a warm pad until it is completely awake).
5. Return the animal to the home cage.
6. Wait 30 min for the next step.
7. Anesthetize the rats with 5% isoflurane mixed in 100% O₂ (1 L/min) using an induction box.
8. Open PET scanner software.
9. Select Scan > Pet Ready.
10. Complete principal investigator details, Study ID, Series ID, Animal ID, Animal weight (g), and Additional notes. Select Next.
    NOTE: Use dot (.) for decimal number in the animal weight.
11. Edit ROI area for scanning (usually between 3 and 8). This is the area that will be included in the image (areas between the numbers 3 and 8 of the bed cover will appear in the final image).
12. Position the anaesthetized rat on a standard rat bed of the PET scanner and turn the anesthesia on (3% isoflurane in 100% O₂ is a good start, and then the anesthesia rate should be adjusted as necessary). To adjust the anesthesia, check the monitoring data of the scanner software to verify that the animal is breathing in a constant and slow rhythm.
    NOTE: Turn on the vacuum pump coupled to the activated carbon filter to collect isoflurane gas excess (if the pump is available).
13. Apply eye cream to protect the animal's eyes.
14. Push the animal bed inside the PET scanner.
15. Complete Activity details, Activity Calibration Time, Isotope (C-11), and Scan duration (20 minutes). Select Start Scan.
    NOTE: A Pet Moving Bed message will appear, and the animal bed will move into the equipment and stop at the previously selected ROI position. Time of acquisition will start count down and number of counts per seconds will appear (CPS).
16. In the scanner software, navigate to the Monitor tab on the left of the screen, check Change Threshold to Respiratory parameters change (BPM).
    NOTE: A window will pop up, complete with threshold parameter (500). Select OK.
17. Navigate to 0% POWER to change temperature parameters to heat the animal during scan. A window will pop up; complete the parameter (80 – 100). Select OK.
    NOTE: Choose between 80 and 100% power based on room temperature conditions (the more power, the warmer it will get).
18. Navigate back to SCAN tab.
19. Navigate to configuration tool (gear icon) and complete Injection Time, Remaining Activity, Remaining Activity Calibration Time. Select Save.
    NOTE: A window will pop up "Are you sure you want to modify the protocol metadata?"> YES
20. Navigate back to the Monitor tab (check the animal's respiratory parameters, and if necessary, increase or decrease isoflurane concentration – usually 3% in 100% O₂ is enough).
21. When PET acquisition is finished, Pet Ready message will appear on the Scan tab, check Arrow Out Icon (left of configuration tool) to move out the animal bed.
22. Remove the animal from anesthesia and allow to awaken on a warm pad.
23. For reconstructing the image, navigate to Reconstruct tab.
24. Check plus icon at bottom left of the screen, and select the study file which will be reconstructed. Select Next.
25. Navigate to Energy Resolution tool. A window will pop up. Configure the energy peak (keV) to the equipment parameter (based on monthly quality control). Select Close.
    NOTE: Energy peak can change every month when performing monthly equipment calibration.
26. Keep all other parameters as the default (Isometric voxel size: 400 mm; number of iterations: 30; Energy resolution: 30%; Save last iteration only; uncheck Keep binary data) .
27. Check Add.
    ​NOTE: A window will pop up "The reconstruction was added to the queue and will start once the others have finished". Select OK. A file will appear in the reconstruction list on the Reconstruct tab and the status will appear as waiting or % (progress) or finished.

4. Image Analysis

NOTE: Perform image analysis using dedicated image analysis software. In the current protocol the demonstration uses a specific software program, but if it is not available, other options can be used.

1. Open PMOD > Fuse it.
2. Navigate to the Matching tab at the top of the screen.
3. Open the Load Input menu in the right middle of the screen and select Autodetect files, select PET file (DICOM, Interfile or NifTI), add to selected, click with Operations, Reorient to Standard Orientation, click Load and Close.
4. Verify if the animal Species is correct at the bottom of the screen (RAT).
5. Check Crop box at the bottom right of the screen.
6. Adjust the yellow box size in the PET image to take the whole brain.
7. Click the Rigid button on the right of the screen. At confirmation window click Yes.
8. Click on the brain tool at the right middle of the screen to open Reference Atlas. Select Px Rat (W.Schiffer) – T2.
9. Select Match Results from the top right tab (Select processing stage tab).
10. Click Data reslicing (4^thtab at top right menu).
11. Align the PET image with reference template using the rotate tool (white icon at center of PET image). Check alignment for 3 anatomical planes.
12. Save the co-registration file (right side menu of the screen).
13. Select output format (DICOMor NifTI), directory and file prefix. Click Save.
    NOTE: If co-registered output is saved as NifTi the header image information will be lost.
14. Click VOIs menu at the bottom of the screen.
15. Navigate to Template > Atlas at the bottom of the screen (below VOIs window).
16. Select Px Rat(W.Schiffer) from the drop-down menu.
    NOTE: If you need to analyze only specific VOIs you can select only the brain areas of interest. If you want all brain areas of the brain atlas, no action is needed.
17. Click Outline at the bottom of the screen.
    NOTE: VOIs of brain areas will appear in VOIs window.
18. Manually draw the VOI in the lesion site and contralateral brain hemisphere area (¹¹C-PIB low uptake area and contralateral brain hemisphere, respectively).
19. Click in the area of PET image with ¹¹C-PIB low uptake.
20. Select New VOI > OK.
    NOTE: A spreadsheet will appear
21. Navigate to SPHERE icon in the middle part of the screen (to the left of the VOIs window)
    NOTE: A spreadsheet will appear.
22. Choose the VOI name: lesion and select Apply.
    NOTE: A sphere will appear in the co-registered image.
23. Align the sphere in the lesion area and adjust the VOI for all anatomical planes.
24. Click Close (at the bottom part of the spreadsheet).
25. Select the lesion VOI (from VOIs list).
26. Navigate to VOI mirroring operations icon (at the top right of the VOIs window) and select Clone and mirror left/right.
27. Navigate to Calculate Selected VOI Statistics at the top of the VOIs list window. Navigate to Select statistics to be calculated icon to choose the output data (to the right side of the VOI statistics icon). Usually for myelin content check Statistics for group of VOIs, Average, SD, Min, Max).
    NOTE: A spreadsheet will appear. The default setting Data Unit is kBq/cc. On the left top of the spreadsheet (VOI Statistics) you can check the Data Unit as SUV (Standardized Uptake Value). If you completed the radiotracer administration and image acquisition details correctly in the scanner software, as described previously, the SUV data will be calculated automatically by PMOD software, if not, you can edit the details.
28. Navigate to Copy using system locale number format.
    NOTE: Verify if all statistics are selected to copy as output (Check icon at the right top of the screen). The data that will be copied depends on the Data Unit selected.
29. Paste the output data in a notepad or spreadsheet.
    NOTE: Take care with software settings for dot and comma symbols between numbers. This can be different between language configurations.
30. Save the file with study name and details.