This technique uses PET imaging to elucidate the in vivo distribution and dynamics of B cells in the central nervous system. Our approach is beneficial for studying neurological diseases, where the affected spinal cord makes analysis more challenging. So there are a couple of main advantages to our technique.
First, we have created a new tool to track a pan B cell biomarker that allows us to capture a range of B cell subsets and visualize them in vivo. Secondly, our spinal cord analysis method enables highly accurate and reproducible quantification of PET signals in this region. The great thing about our technique is that it is disease-agnostic.
It can be used from preclinical to clinical PET imaging in any scenario, where B cells, and/or the spinal cord are of interest. 18 to 24 hours after radiolabeling and injecting the antibody into the mouse, prepare the mouse for scanning by applying eye gel to the eyes. Ensure the four-mouse scanning bed is equipped with a heating pad with isoflurane set from 1.5 to 2%Place the mouse in a supine position on the scanning bed, and gently pull the mouse tail to straighten the spine.
Once the mouse is in a supine position, securely tape it with soft microscope tape over the head and belly to minimize the motion from breathing. Make a record of the scanning position for each mouse in a lab notebook. After securing the first group, close the bed, and check the bed placement by running a CT scan.
Click on CT Center Field of View, and once the bed is in position, run the CT test scan to ensure the placement is correct. Repeat until the bed position is satisfactory. Place a small white tape on the scanner bed to mark the correct bed placement for the remainder of the study.
Open the Motion Controller menu, and click on PET Center Field of View to move the mice into the PET ring. Once the bed is in the PET ring, initiate the scanning sequence by clicking on Run, and wait for the scanner to automatically complete the PET scan, and move from the PET ring to the CT for CT acquisition. Since experimental autoimmune encephalomyelitis, or EAE, mice have pronounced curvature of the spine due to disease progression, scanning them while on their backs helps to straighten the spine.
Make an incision down the dorsal side of the animal, and remove the skin and fur to expose the spinal column. Cut along three transverse planes through the spinal column at the neck, directly under the rib cage, and at the top of the pelvis to separate the lumbar from the cervical and thoracic regions. Carefully remove the segmented spinal column to get two pieces, lumbar and cervical thoracic.
Then isolate the lumbar spinal column by carefully trimming the spinal column from the pelvic end until the lumbar spinal cord is visible. To expel the lumbar spinal cord, use a slip-tip syringe filled with PBS and create a seal between the syringe and the spinal column using the thumb and forefinger. Gently push the PBS through the syringe to expel the spinal cord onto an absorbent pad, and repeat for cervical thoracic spinal cord by inserting the syringe from the cervical side.
Place the spinal cord tissues in a gamma counting tube. Record the dry weight, and add PBS to ensure the tissue is at the bottom of the tube to avoid drying. Place the tube on ice until ready for counting.
To begin the analysis of regions of interest in the spinal cord, open the 3D Region of Interest Tool from the Navigation menu. Under the Regions of Interest header, use the plus sign at the bottom of the menu to create six regions of interest, lumbar region of interest, cervical thoracic region of interest, lumbar skeleton, thoracic skeleton, lumbar spinal cord, thoracic spinal cord. To avoid visual interference from the PET signal, click on F3 to turn off the PET.
Go to the top of the 3D Region of Interest Tool Operator, and click on the solid dot at the right of the cursor symbol to open the 3D Paint Mode, and Erode/Dilate menu. Select Sphere, and change the size to 20 pixels. Likewise, set dilate to plus five.
Before proceeding further, go to the bottom of the menu, and ensure the lumbar region of interest is selected. On the CT, find the L6 vertebra of the spinal column. Starting with one vertebra above L6, draw a rough lumbar region of interest over the five vertebrae above the hips.
Then switch to cervical thoracic region of interest, and trace the remainder of the spine to the base of the skull. After drawing the generalized regions of interest, go to the top of the operator, and select the Segmentation Algorithms menu. From the dropdown menu, select Otsu Thresholding, then select lumbar region of interest for the input, and ensure lumbar skeleton is chosen at the bottom of the menu.
In the dropdown menu next to Image, ensure the CT scan is selected, indicated here by the number zero. Click on Apply, and repeat the procedure for cervical thoracic region of interest and thoracic skeleton. After using Otsu Thresholding to create the skeleton regions of interest, return to the Navigation menu, and delete the region of interest, or check mark the H column for both the rough lumbar and cervical thoracic regions of interest to hide them.
Check mark the I column for both skeletal regions of interest, so they cannot be edited. Finally, return to the top of the 3D Region of Interest Tool Operator, and go to the 3D Paint menu to draw the spinal cord regions of interest. Select the Sphere tool again, and trace the spinal cord within the skeleton for both the lumbar and thoracic, ensuring the correct region of interest is selected at the bottom of the menu.
To erase any region of interest, click on Command/Control and draw over the part to be erased. Check the spinal cord region of interest from all three planes to ensure no region of interest is drawn outside the spinal column. If the PET signal was turned off, press F3 after the spinal cord regions of interest are drawn to turn the PET back on, or select the Visual Controller, and click on the PET bar.
Go back to the Navigation menu. Click on the Grid icon to show table. Copy the table into spreadsheet software, and Save the file.
PET imaging revealed elevated radiotracer binding in the brain and thoracic spinal cord of EAE mice compared to the naive mice. Ex vivo gamma counting showed increased binding in both lumbar and cervical thoracic spinal segments, and the brain of EAE mice compared to naive. Ex vivo autoradiography images showed increased radiotracer binding in sagittal brain sections, specifically in the brainstem, cerebellum, and ventricles of EAE mice compared to naive mice.
Similarly, an increased radiotracer binding was observed in both the cervical thoracic and lumbar spinal cord segments of EAE mice compared to naive spinal cords. After PET imaging and gamma counting, we can investigate the relationship between PET signal and the target of interest through molecular biology techniques, such as flow cytometry and immunohistochemistry. Our technique has paved the way for researchers to ask questions about the in vivo role of B cells in multiple disease areas, including stroke, multiple sclerosis, other autoimmune diseases, and cancer.
