The overall goal of this imaging technique to to visualize and quantify optic neuritis and brain inflammation in the EAE model of multiple sclerosis in living mice. The method can help answer key questions in the neuroimmunology field, such as novel mechanisms which impact the course of EAE and multiple sclerosis. The main advantage of this technique is that EAE evoked optic neuritis and brain inflammation can be observed and quantified in living mice throughout the course of the disease.
The implications of this technique extend toward therapy because we can observe effect of medication, which do not reveal from the EAE scores. In this procedure dissolve PTX at two micrograms per milliliter in 1x PBS and mix well. Then inject 100 microliters of PLP/CFA in the mice subcutaneously twice at the base of the tail during short isoflurane anesthesia.
Do not inject into the back of the neck, because any immune reactions in the skin in the upper back or neck will disturb imaging of the head and spinal cord. One to two hours after immunization, inject 100 microliters of PTX in the mice intraperitoneally. Then 24 hours after immunization, inject the mice with 100 microliters of PTX again.
For the control mice, inject 100 microliters of CFA without PLP twice at the base of the tail. From day seven post immunization, assess the clinical symptoms daily according to the standard scoring systems. When the mice reach a score larger than one, which will occur around day 10 to 12 after immunization, perform the first imaging at the onset of the disease.
Then perform the second imaging at the peak of the disease, which is about one to two days after the initial symptoms develop, and will last for one to three days. Subsequently, the mice will fully recover within seven to 10 days. Imaging during the intervals may still show vascular leaks, but inflammation indicators should be negative.
To perform in vivo imaging keep the mice under 2%to 2.5%isoflurane anesthesia during all imaging procedures. Position two mice next to each other in the apparatus, and position the upper spines of the mice in the center. Next, shield the site of immunization with black cloth.
Open the software for image capture and initialize the camera. Then take a baseline image to assess the correct positioning of the mice. Subsequently use B focus with a 6.5 centimeter distance to the camera for capturing all the images.
Afterward mix the ready-to-use chemiluminescent reagent at 40 milligrams per milliliter before filling the syringe. Then, inject 100 microliters of it in the mice intraperitoneally and capture the bioluminescence images five, 10, and 15 minutes after injection. In this procedure open the bioluminescence analysis software.
In the upper menu bar click on the File Browser icon. Go to the directory of the experiment folder and open the files, and then configure the columns showing the descriptions relevant to the experiment. For quality control click on a file of a non-responder mouse without the symptoms of EAE, or a naive mouse to check the specificity of the EAE signals.
As further control of the specificity of the signal, check the baseline image before the probe injection for each mouse, which should be negative. To select an image click on the first file of the first EAE mouse and check the bioluminescent intensity and localization. Check all images one by one.
For quantification, use the image with the strongest signal for each mouse. To adjust and export the image, double click the first file to be quantified. In a new popped up window under the Options button, customize the labels to display in each screen.
In the right tool palette, go to Image Adjustment. By default minimum and maximum intensities are set to auto and displayed in rainbow pseudocolor. Select Manual to change the settings if necessary.
Next, click in Image Export, select PNG, the directory, and an image name. For quantification of the regions of interest go to the ROI Tool in the tool palette. Select the ROI method and the number of ROIs.
In the ROI window within the image window, adjust the size and position of the ROI. Use identical ROI thresholds for all the images when the auto ROI tool is used. Use identical areas for all the images if the ROI sizes and positions are defined manually.
After that, click on Measure ROI. Customize the columns on the experimental details in a new pop-up window and save them. When ready, select all and copy and paste the table into a spreadsheet.
After that, export the image as a PNG with the ROIs in place. Then, save and close the image file. Here are the examples of two SJL/J mice that were imaged at day 10 and 14 after EAE induction.
The bioluminescent signal around the eyes was highest at day 10, and disappeared when the mice started to recover. The bar charts here show the quantification of the bioluminescent signals at day 10 and 14, whereas the clinical EAE scores are shown here. In this figure the bioluminscent images of five mice in each group show the effect of the medication in which optic neuritis was reduced in mice treated with R-flurbiprofen.
The quantification of the total bioluminescent counts in ROIs confirmed the significant treatment efficacy. Once mastered, this technique can be done in two to three hours, depending on the number of mice to be imaged. While attempted this procedure, it's important to remember to use white mice and to closely observe the clinical EAE course.
Following this procedure other methods like FACS analysis or immunohistochemistry can be performed in order to answer additional questions like immune cell infiltration of the spinal cord. After watching video, you should have a good understanding of how to asses optic neuritis and brain inflammation in the EAE model of multiple sclerosis using bioluminescent imaging.
