The overall goal of the Experimental Autoimmune Encephalomyelitis or EAE model, is to facilitate the study of the potential molecular mechanisms underlying the development of multiple sclerosis. This method can help answer key questions in the multiple sclerosis field such as, at which stage of the disease do immune cells infiltrate the central nervous system? The main advantage of this technique is that the EAE model mimics many of the main characteristics of multiple sclerosis, such as demyelination and immune cell infiltration.
The implications of this technique extend towards therapy of multiple sclerosis because drugs tested with this method have already been approved for this disease. Generally, individuals new to this method would struggle because there are thorough conditions which have to be taken into consideration when inducing EAE. Mice must be kept in a stress-free environment, and a pertussis toxin should be freshly prepared.
Official demonstration of this method is critical as the isolation of the lymph nodes and the extraction of the spinal cord are difficult to learn by description alone. Begin by subcutaneously injecting 10 to 13 week-old female mice with 200 micrograms of myelin oligodendrocyte glycoprotein. emulsified in 200 microliters of complete Freund's adjuvant, containing 400 micrograms of mycobacterium tuberculosis.
Immediately and 24 hours later, intraperitoneally inject the mice with 0.2 micrograms of pertussis toxin in 200 microliters of PBS. It is essential that mice have been adapted both to handling and the experimental environment before EAE induction toward inducing stress which can prevent the development of clinical symptoms. One week after the injection, examine the mice daily for clinical symptoms as described in the table.
To evaluate the EAE induced lymph node immune cell population, at the appropriate time point post-induction, wet the incision area with 80%isopropanol, and carefully remove the skin over the hip region. Using forceps, carefully remove the inguinal lymph node from the fat tissue. Then weigh the node, and place the sample in PBS on ice.
To analyze the spinal cord cells, wet the back of the animal with isopropanol, and use a scalpel to make a longitudinal cut down the spine. Then remove the skin, and dissect out the lumbar portion of the spine, innervating the hind limbs. Flush the spine with a PBS-filled syringe to extract the spinal cord.
After removing approximately 1/3 of the lumbar cord, weigh the spinal fragment, and store the tissue in PBS on ice. To analyze the splenic immune cell population, open the tissue to get access to the lower part of the abdomen. Next, remove the spleen, and cut off approximately 1/8 of the tissue.
Weigh the spleen fragment, and store the tissue in PBS on ice. Then use the plunger from a two mL syringe to macerate the rest of the tissue through a 70 micron mesh sieve on a 50 milliliter tube, followed by a five milliliter PBS wash of the strainer. Centrifuge the filtered cells.
Resuspend the pellet in 500 microliters of cell lysis buffer, and transfer the cells into a 1.5 milliliter tube. After 10 minutes at room temperature, wash the cells two times in 500 microliters of PBS. After the second wash, resuspend the pellet in 100 microliters of 0.2%BSA in PBS, and add two microliters of Fc receptor one blocking buffer to the cells.
After 15 minutes in the dark at room temperature, label the cells with 13 microliters of antibody cocktail for another 15 minutes at room temperature in the dark. At the end of the incubation, wash the cells at least two times in 500 microliters of PBS, and resuspend the final pellet in 500 microliters of fresh PBS. Then transfer the cells into a flow cytometry tube on ice.
Finally, to analyze the samples by flow cytometry, directly before the flow cytometry measurement, add 30 microliters of flow cytometric absolute count standard to the cells for determining the absolute cell counts. Then analyze the samples on the flow cytometer. As this figure illustrates, a transient increase in all of the immune cell populations within the lymph nodes is observed during the acute phase of EAE induction.
In the spleen however, only macrophages, B cells, T cells, and neutrophils demonstrate an increased infiltration. In the blood, all of the immune cell populations demonstrate a transient increase in their peripheral circulation during the pre-clinical phase that corresponds to a similar disease-dependent increase in the lumbar spinal cord during the acute phase of the disease, excluding the monocyte population, which presumably differentiates into macrophages after entrance into the spinal cord. Here, the gating strategy for the evaluation of the different cell populations is shown.
The numbers of cells are related to the amount of tissue or blood volume analyzed, reflecting the absolute cell count. In the lymph nodes, IL-23 mRNA expression is increased during the pre-clinical phase, while the expression of IL-6 increases in a disease-dependent manner. In the spleen, IL-6 and IL-23 expression increase in the pre-clinical phase, whereas IL-1 beta mRNA expression is not affected until the onset of the disease.
In the blood, the expression of only TNF-alpha mRNA is upregulated and in a disease-dependent manner. In the lumbar spinal cord, TNF-alpha, IL-1 beta and interferon gamma are induced during the acute phase, whereas IL-6 is upregulated during the onset phase of the disease only. Once mastered, the EAE model, subsequent cell isolation, and effects analysis can be completed in eight hours for ten mice if they are performed properly.
Following this procedure, other methods like Western Blot or ELISA can be performed to confirm the results of the mRNA expression analysis. After its development, this technique paved the way for researchers in the field of multiple sclerosis to explore the mechanism of demyelination in the central nervous system. After watching this video, you should have a good understanding of how to induce the EAE model and how to isolate immune cells to obtain reproducible flow cytometry results.
Don't forget that working with complete Freund's adjuvant can be extremely hazardous, and add precautions such as lifting the skin of the mouse and ensuring complete skin penetration by the syringe should be taken while performing this procedure.
