虚血性脳卒中のマウス モデルで [¹¹C] DPA-713 Neuroinflammation のイメージングをペットします。

The overall goal of this experiment is to accurately quantify the spatial distribution and extent of neuroinflammation in a mouse model of ischemic stroke using TSPO-PET and MR imaging and to validate these in vivo findings using ex vivo autoradiography. I'm really excited to share with you this step-by-step guide for how to use PET imaging to visualize neuroinflammation in a living subject. Here we're using a mouse model of stroke as an example.

Now, why might you want to use this technique? There are a couple of reasons. The first is that neuroinflammation, which is just inflammation that occurs in the central nervous system, is thought to be intimately associated and really something that underlies many, many brain diseases, including stroke but also Alzheimer's disease, multiple sclerosis, Parkinson's, and the list goes on.

So it's incredibly important that we have these techniques that really allow us to study these biological phenomena. Secondly, PET imaging offers a number of advantages over some of the more traditional techniques. For example, most of these traditional techniques rely heavily upon using both human and mouse post-mortem brain tissue, and while they have afforded a number of important insights, these techniques are static by nature, meaning that they can only really tell us information about one moment in time.

Since we know that both the brain and the immune system are highly dynamic, it makes sense that we'd want to have a technique that allows us to really capture these molecular processes in living, intact systems, so in their native environment in real time, and that's exactly what PET imaging does. Here we'll show you how we use a specific PET radiotracer, DPA-713, which is known to bind to the TSPO or translocator protein. We'll show you specifically how we inject an image not only in one mouse but four mice at the same time dynamically, and why that's such a big deal is that it really helps to increase the number of mice you can image on any given day with the same batch of tracer, thus really streamlining your experiments.

While this is technically challenging, really hoping that this video can help to guide you so that you can perform this in your own lab. 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 the 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 Okay 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 1, Input 2, and Input 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 1 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 Min/Max 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 1 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 and apply 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 1, to allow visualization and manual reorientation of the new ROI. With only Input 1 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 two to the new ROI, permitting independent quantification of ROIs. Return to the 3D ROI tool.

Go to the Expert and Experimental tab, and click on the Import ROI button. Select Input 1 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. The resulting PET/CT images and time activity curves display increased radiotracer uptake in the ipsilateral versus contralateral hemispheres. Quantification of dynamic PET brain images using summed data from 50 to 60 minutes revealed significant increase in tracer uptake in the ipsilateral compared to the contralateral hemisphere in dMCAO but not in sham mice using the manually-drawn ROI approach.

After watching this technique, you should have a good understanding of how to accurately and efficiently quantify neuroinflammation in a mouse model of ischemic stroke via TSPO-PET, using both automatic and manual image analysis methods. Importantly, you should also be able to confirm these in vivo findings using ex vivo digital autoradiography. Since the Carbon 11 half-life is so short, it is critical to map out a clear experimental plan and timeline prior to attempting this procedure in order to maximize the quantity and quality of your data.

Following this procedure, other methods such as immunohistochemistry can be performed to answer additional questions such as the correlation between glial activation and TSPO expression. Keep in mind that using radioactivity can be extremely hazardous. To minimize your exposure, utilize protective clothing, lead shields, and increasing your distance when performing this procedure.