The overall goal of this conceptual method is to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis demonstrate their central nervous system protection during the onslaught of immune cell infiltration or as a consequence of immune cell infiltration suppression. This method can help answer key questions in the neural immunological field. Such as, how does manipulating EAE treatment times differentiate the effects on immune cell infiltration versus protection.
The implications of this technique also extend toward the therapy of multiple sclerosis, since there are currently no neural protective treatments for this disease. We first had the idea for this method when we discovered that treating EAE mice with an inhibitor of the system Xc-transporter was both neuroprotective and immunomodulatory. Though this method can be used to study drug treatments, it can also be applied to studies using genetic knockout mice subjected to EAE or other manipulations.
Begin by using sterile scissors to mince the brains and spinal cords from individual animals into small pieces. Then use one three milliliter syringe plunger per tissue to mash each sample over individual 70 micron cell strainers into individual 50 milliliter conical tubes while rinsing the strainers with medium. When all the pieces have been macerated, bring the final volume in the tubes up to 50 milliliters with fresh medium, then centrifuge the cell suspensions.
Resuspend each pellet in four milliliters of 40%density gradient solution. Then carefully layer the cell slowly down the walls of individual 15 milliliter conical tubes on top of two milliliters of 70%density gradient solution per tube. Separate the cells by centrifugation and use one milliliter transfer pipettes to carefully discard the upper myelin layers.
Next, transfer the viable cells from the sample interfaces into new 15 milliliter conical tubes and wash the cells in a final volume of 15 milliliters of fresh medium per tube. Resuspend the pellets in 200 microliters of fresh medium and transfer the cell suspensions into individual wells of a 96 well round bottom plate. Centrifuge the plate and flick off the supernatants.
Then, resuspend the pellets in 200 microliters of restimulation medium and incubate the cells at 37 degrees Celsius for four hours. At the end of the incubation, centrifuge the plate again and remove the supernatants. Then wash the cells in 200 microliters of PBS supplemented with 2%FCS and resuspend the cells in 200 microliters of PBS supplemented with 2%FCS and FC-block for 10 to 15 minutes on ice.
At the end of the incubation, wash the cells as just demonstrated and resuspend the pellets in 15 microliters of cell surface stain cocktail for 15 minutes on ice. Then spin down the cells and wash the samples two times in 200 microliters of PBS. Next, resuspend the cells in FOXP3 transcription factor staining reagents according to the manufacturer's instructions for 30 minutes to overnight at 4 degrees Celsius.
At the end of the incubation, wash the cells in 150 microliters of permeabilization buffer. Then centrifuge the cells and resuspend the pellets in the intracellular antibodies of interest in 15 microliters of permeabilization buffer on ice. After 30 minutes, spin down the cells followed by three washes in 200 microliters of permeabilization buffer.
After the last wash, resuspend the pellets in 200 microliters of PBS and analyze the samples by flow cytometry, gating on the live CD4-positive, TCR beta-positive cells. It's also important to assess peripheral T cell proliferation because changes in the periphery can affect immune cell infiltration into the CNS. To quantify the number of astrocytes and glial cells present in the spinal cord tissue samples after EAE induction, obtain images of each section at a 4x magnification and save the images as TIFF files.
Next, in ImageJ, select the image of interest and use the polygon selections tool to trace the entire spinal tissue section. Under the image menu, select type and 16-bit to convert the image to 16-bit. Then, open the process menu and select subject background to set the rolling ball radius to at least the size of the largest object that is not part of the background.
Check the sliding paraboloid and click OK.Then, under image, select adjust and threshold, and use the sliding bars to set the lower threshold level. To obtain the percent of the thresholded area within the selected region, under analyze and set measurements, select area fraction. Ensure that the limit to threshold is unchecked and that display label is checked, and click OK.To obtain the measurements, under analyze, select measure.
A results popup box will appear. Save this data and compare the area fraction values between the treatment groups. To quantify the myelin basic staining by optical density, open the image of interest and use the polygon sections tool to draw a region of interest within the sample.
Under analyze and set measurements, select mean gray value and confirm that limit to threshold is unchecked, and that display label is checked. Click OK.Then, under the analyze menu, select measure. A new results popup box will appear with the myelin basic protein mean gray value data.
Spinal cord removal and flow cytometric analysis allows the assessment of central nervous system immune cell infiltration, where immune cell infiltration is maximum at the peak of disease. Statistical analysis of the CD4-positive, interferon gamma-positive, IL-17-positive, and FOXP3-positive cell numbers can reveal the significance of changes observed in the CD4-positive infiltrating T cell numbers in drug treated animals, versus control animals, with in depth coexpression analysis essential for ruling out skewing as a confounding factor in the overall reduction of CD4-positive infiltrating T cell number. To eliminate the possibility that a reduction in central nervous system infiltrating T cells is a consequence of inhibiting proliferation, activation, and differentiation in the periphery, the number of actively proliferating T cells and the proportion of T cell subtypes can be evaluated.
For quantitative evaluation of the myelin integrity, statistical analysis of the optical density of myelin basic protein staining can be performed, revealing a significant demyelination of the spinal cord tissue in unspecified genetic knockout animals compared to wild type littermates, for example. To further substantiate that neuroinflammation is sustained or decreased by therapeutic interventions, reactive gliosis can also be assessed by measuring the mean fraction area for reactive gliosis as just demonstrated. Once mastered, the flow cytometric technique can be completed in two days if it's performed properly, while quantification of the tissue staining can be performed in about two hours per stain.
The immunomodulation can be assessed by introducing the treatments before the immune cell infiltration to quantify the migratory and proliferative changes in the T cells. The CNS protection can be assessed by introducing the treatments during the peak of disease when the immune cells have already infiltrated into the CNS. While attempting this procedure it's important to remember to evaluate optimum threshold settings in ImageJ for the specific highlighting of cellular staining only.
After watching this video, you should have a good understanding of how to assess the effects of specific treatments on central nervous system, T cell proliferation, and migration responses. By altering the treatment times before and after immune cell infiltration.
