Rapid and Efficient Enrichment of Mouse Spinal Cord Microglia
September 22nd, 2023
Microglia are regarded as some of the most versatile cells in the body, capable of morphological and functional adaptation. Their heterogeneity and multifunctionality enable the maintenance of brain homeostasis, while also being linked to various neurological pathologies. Here, a technique for purifying spinal cord microglia is described.
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.
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Overview
This study focuses on the heterogeneity and multifunctionality of microglia, particularly in the spinal cord. It introduces a method for purifying spinal cord microglia from animal models of neurological pathologies, enhancing experimental accuracy.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Neuroinflammation
Background
- Microglia exhibit diverse phenotypes and functions.
- They contribute to brain homeostasis and are implicated in neurological disorders.
- Traditional classifications are often insufficient for categorizing microglial behavior.
- Achieving high-purity isolation of microglia remains a significant challenge.
Purpose of Study
- To enhance the understanding of spinal cord microglia under various conditions.
- To provide a robust protocol for purifying microglia with high purity.
- To explore cytokine signaling pathways involved in chronic pain.
Methods Used
- Isolation of spinal cord microglia from euthanized mice using a surgical approach.
- The protocol involves somatic digestion followed by density gradient centrifugation.
- Key operational details include incubation, vortexing, and multiple centrifuge steps.
- Extensive purification yielded up to 99% pure microglial cells.
Main Results
- A novel purification method significantly decreases contamination from astrocytes.
- Microglia showed an average size of 10 micrometers, consistent with nonactivated cells.
- Specific cytokine pathways were identified during chronic pain scenarios.
- The protocol supports higher cellular yield for downstream applications and experiments.
Conclusions
- This study demonstrates an effective method for isolating spinal cord microglia.
- The findings have implications for understanding microglial roles in neurological conditions.
- Insights gained may enhance the study of neuroinflammatory responses and therapeutic strategies.
