Our research focuses on the heterogeneity of microglia, which can exhibit both pro and anti-inflammatory behaviors across different pathologies and region like the brain and spinal cord. We aim to enable a deeper understanding of spinal cord microglia behavior in diverse scenarios. The heterogeneity of microglia has made it difficult to classify them.
This year, a new and broad classification has been proposed that goes beyond just a pro or anti-inflammatory status. Currently, achieving high purity angio in isolating spinal cord microglia from animal models of neurological pathologies is a significant experimental challenge. We identified a novel cytokine signaling pathway in the spinal cord microglia during chronic pain.
Our protocol allows for a more controlled and somatic digestion, followed by a density gradient. Both result and less contamination by astrocytes compared to currently employed techniques for the purification of microglia from the spinal cord, brain, or embryos. To begin, with a scalpel, make an incision along the dorsal midline of a euthanized mouse from the occipital to the sacral region.
Using a stereoscopic surgical microscope, carefully dissect through layers until the lumbar spine. Identify the last costal arc. Identify the last cervical vertebra and the sacrum, then make a cut to separate the spine.
With a pair of dissecting forceps, perform a dorsal laminectomy of the vertebra to extract the spinal cord. Remove the nerves emerging through the foramina, which constitute the peripheral nervous system. Now place the extracted spinal cord on a dissecting board.
With a pair of Vannas scissors, cut it into two millimeter sized pieces, then transfer the pieces to a two milliliter flat bottomed microtube. Add one milliliter of HBSS-LD working solution to the microtube. Incubate the mixture at 37 degrees Celsius for 25 minutes.
Make sure to vigorously vortex the mixture every five minutes. Next, pass the contents of each digestion through a 40 micron sized strainer. Then add seven milliliters of HBSS with 2%FBS to neutralize the digestion, and crush the remaining tissue.
Transfer the suspension to a 50 milliliter conical tube before centrifuging it at 220 G for five minutes at four degrees Celsius. Now use a one milliliter micropipette to discard the supernatant, then re-suspend the pellet in two milliliters of HBSS FBS in a new 15 milliliter conical tube. Add two milliliters of 90%isotonic density gradient medium to the tube, and centrifuge the mixture at 160 G for 25 minutes at 18 degrees Celsius.
Finally, with a transfer pipette, retrieve the interface and transfer it to a 15 milliliter conical tube. Wash the cells with 10 milliliters of PBS, then centrifuge the cells at 220 G for five minutes at five degrees Celsius before further experimentation. The purification of the extracted microglial cells yielded up to 99%cell yield, as confirmed by fact staining.
Double positive cells with an average size of 10 micrometers consistent with nonactivated microglia were observed.
