免疫細胞の下流分析のための中枢神経系組織および関連する髄膜の単離

Rapid isolation of CNS tissues including the meninges allows for a comprehensive analysis of immune cell phenotype, function, and localization in homeostatic and pathogenic conditions. The two demonstrated methods allow for the quick extraction of CNS tissues with the meninges, ideal for downstream analysis using single cell techniques and histological methods. Although the representative results focus on the analysis of immune cells, these two methods can be used to analyze CNS resident cells, including microglia, astrocytes, pericytes, endothelial cells, stromal cells, and neurons.

Begin by isolating brain and spinal cord samples from euthanized mice. After removing the head, make a midline incision in the skin and flip it over the eyes to free the skull. Cut at the nasal bone to release the mandible from the skull.

Then remove the mandible, tongue, and eyes. Cut along the lateral aspects of the skull to release the tissue along the external auditory meatus and trim it. To collect the spinal cord sample, separate the rib cage from the spinal column by cutting parallel to the spine with sharp scissors.

Make a small cut at the lower lumbar region to isolate the spinal column, then trim and remove any remaining muscle along the spine to expose the vertebrae. When performing decalcification, check the bone daily to determine if it is soft and pliable. Remove the tissue from the EDTA solution with forceps, place it on a Petri dish, and gently test the bone softness with a 25 gauge needle.

If the needle easily penetrates the bone, the decalcification process is complete. To extract the skull cap in the brain, make a midline incision in the skin and flip the skin over the eyes. Place the scissors within the foramen magna and begin cutting the skull laterally along the cortices towards the olfactory bulb, keeping the incisions above the external auditory meatus and mandible.

Perform the same cuts on the opposite side with cuts meeting at the olfactory bulb to free the skull cap from the brain. Using forceps, peel back the skull cap and place it into a 15 milliliter conical tube containing five milliliters of cold RPMI medium supplemented with 25 millimolar HEPES. Place curved forceps below the base of the brain and lift them to free the brain from the skull cap.

Extract the vertebrae column and spinal cord tissue by cutting parallel to the spine to separate the rib cage from the spinal column. Then make a small cut at the lower lumbar region to isolate the vertebrae column. Trim and remove any remaining muscle along the spine to expose the vertebrae.

Next, place the extra fine surgical scissors within the vertebral column and cut along the lateral edge of the column. Cut the opposite lateral edge completely to divide the vertebral column into an anterior and posterior portion. Use forceps to peel away the spinal cord slowly and carefully from the vertebral column and place it in a 15 milliliter conical tube containing five milliliters of cold RPMI with 25 millimolar HEPES.

Then transfer the anterior and posterior portions of the spinal column to another tube. To remove the meninges from the skull cap, take the skull cap out of the RPMI media and use sharp forceps to score around the outer edge. Peel the meninges away from the edge of the skull cap, scraping to remove the dural and arachnoid meninges and place the meninges on a Petri dish.

To remove the meninges from the vertebral column, take the column out of the media and score around the edges of the vertebral column with sharp forceps to free the meninges. Use curved forceps to peel away the meninges from the edge of the vertebrae and place the meninges on a Petri dish. Place a nylon mesh strainer in a 50 milliliter conical tube.

To create a cell suspension, move the meninges into a strainer and add three milliliters of HEPES supplemented RPMI, then use a plunger from a five milliliter syringe to grind the tissue and media through the strainer. Pour the brain or spinal cord tissue with the media into a 100 millimeter Petri dish and use forceps to move the tissue to the bottom. Mince the brain tissue with a sterile razor blade and gather the tissue at the bottom of the plate.

Add three milliliters of RPMI supplemented with 10%FCS to the Petri dish, then use a 10 milliliter serological pipette to resuspend the tissue in the media and transfer it to a 15 milliliter conical tube. Add collagenase type 1 and DNase 1 to the tissue and incubate the tubes in a 37 degree Celsius water bath for 40 minutes. Invert the tubes every 15 minutes to thoroughly mix the tissue with the enzymes.

After incubation, add EDTA to each tube for a final concentration of 0.01 molar and incubate the tubes for an additional five minutes to inactivate the collagenase. Add nine milliliters of RPMI supplemented with 10%FCS to each tube, then centrifuge the tubes at 450 G for five minutes at four degrees Celsius. Use a Pasteur pipette with a vacuum to aspirate the supernatant, taking care not to touch the cell pellet.

Add three milliliters of 100%stock isotonic density gradient solution to the cell pellet, then add additional RPMI 10%FCS media to bring the final volume to 10 milliliters and resuspend the cell pellet. Invert and mix the tube well, then insert a serological pipette with one milliliter of 70%stock isotonic density gradient solution into the bottom of the tube. Slowly underlay the solution, being careful not to make bubbles.

Remove the serological pipette from the tube, making sure not to disturb the gradient. Centrifuge the tube at 800 G for 30 minutes at four degrees Celsius, then aspirate the supernatant, including the myelin debris layer, until two to three milliliters remain in the tube. Use a one milliliter pipette to harvest the cell layer between the 30%and 70%density gradient and transfer it to a new 15 milliliter tube.

Add RPMI 10%FCS media to bring the final volume to 15 milliliters, then centrifuge the cells at 450 G for five minutes at four degrees Celsius. This protocol was used to assess B and T cells in the meninges, brain, and spinal cord during progressive multiple sclerosis. Gating was conducted to distinguish CD45 high peripherally derived infiltrating immune cells from CD45 low microglia and CD45 negative neurons, astrocytes, and oligodendrocytes.

Few CD45 high cells were present in the spinal cord and brain tissue of sham-treated mice. The same gating cutoff for CD45 high expression was used in the meninges to identify CD45 high immune cells and exclude non-immune cells. In all TMEV-IDD CNS tissues, increased percentages of CD45 high immune cells were observed compared to sham treated mice.

During chronic TMEV-IDD, the percentage of B cells among CD45 high immune cells in the brain and spinal cord was higher compared to the meningeal compartment. Decalcified brains and spinal cords were imaged to identify the localization of B and T cells within the CNS compartment. Conventional extraction of the brain from the skull cap resulted in an intact pia layer, but the remaining meningeal layers were excluded.

In both decalcified brains and spinal cords, all meningeal layers were intact. Isotype switched B cells and T cells were localized with IgG and CD3 expression, respectively. Costaining IgG with ER-TR7 revealed that B cells were present in the CNS parenchyma and the meninges, while cellular aggregates within ER-TR7 positive meninges contained multiple IgG positive B cells and CD3 positive T cells.

A critical step in these protocols is the careful extraction of the CNS tissues, essential for improving the purity of single cell suspensions and for obtaining histology tissue with high quality morphology. The described techniques allow for the comprehensive analysis of cells occupying the CNS compartment, ideal for examining cell phenotype, function, and localization under homeostasis and during disease pathogenesis.