This protocol is significant because we are learning that glia are regionally heterogeneous, morphologically, functionally, and genetically. The primary advantage of this technique is that you can study three separate glia subsets from four distinct regions of the central nervous system. Demonstrating this procedure will be myself and Rachel Tinky, a graduate student in my lab.
Use fine scissors to cut the cutaneous layer along the midline of the pup's head, starting caudally and moving rostral until reaching the snout. Avoid cutting deeply into the skull to prevent any tissue damage. Angling the head down, pull the cutaneous layer to each side of the skull and use spring scissors to make an incision along the skull midline, starting at the foramen magnum.
Pull the skull halves apart with fine tipped forceps exposing the cortex, cerebellum, and brainstem. Once exposed, gently lift the brain out of the skull and place it into a 10 centimeter petri dish with 10 milliliters of PBS and antibiotic solution. Ensure that the brain remains undamaged with the hind-brain attached.
Pinch off the cerebellum with fine curved tipped forceps. Place into a designated 15 milliliter conical tube on ice. Separate the mid-brain from the cortex.
Remove the cortical meninges and place it in a designated conical tube. The tissue must be carefully examined to ensure complete removal of the meninges. If meningeal cells remain in the culture they can be detrimental to the growth of glial cells.
To dissect the spinal cord, place it ventral side up and use fine spring scissors to cut the vertebrae, alternating between the left and right sides until the spinal cord tissue is exposed. Gently remove the spinal cord and the meninges and place them in the designated conical tube on ice. Add one milliliter of 0.05%trypsin with 0.53 millimolar EDTA to each tube with tissue.
Triturate the mixture with a 10 milliliter pipette approximately 20 times. Then transfer the cell suspension to an empty 50 milliliter conical tube. Incubate the solution at 37 degrees Celsius and 5%carbon dioxide for 15 minutes.
Gently agitating the lysates after eight minutes. After the incubation, add five milliliters of MGM and 200 microliters of DNase I one to each tube for a final concentration of 50 micrograms per milliliter. Triturate each lysate 10 times with a 10 milliliter pipette.
Then let the cell suspension sit for three minutes at room temperature to allow nondissociated tissue to settle at the bottom of the tube. Transfer the cell suspensions to new 50 milliliter conical tubes leaving behind the nondissociated tissue. After centrifugation, resuspend the cell pellet in five milliliters of MGM and triturate it 20 times Plate the cell suspensions on coated T25 flasks and incubate the cells at 37 degrees Celsius and 5%carbon dioxide.
Change the media after 24 hours to remove cell debris. To perform oligodendrocyte precursor cell isolation, shake the cells for 15 hours, then remove the supernatant from the flask and plate it on a sterile petri dish. Incubate the supernatant at 37 degrees Celsius and 5%carbon dioxide for 30 minutes.
Swirling after 15 minutes to remove remaining microglia. Remove non-adherent cell supernatant, count the cells and plate them on a Poly-D-Lysine coated surface at the desired concentration. Return the cells to the incubator for at least one hour.
Then gently aspirate 95%of media and slowly add warm OPC media, pipetteing it against the wall of the well to minimize disruption of OPCs. This method was used to demonstrate that interferon gamma signaling influences OPC differentiation and maturation. Without the interferon gamma receptor, cortical OPCs do not differentiate into mature myelinating oligodendrocytes says readily, which is evidenced by the absence of MBP staining.
Expression of GFAP was also analyzed. Interferon gamma deficient cells strongly express GFAP, suggesting that they might be adopting an astrocytic phenotype. Regional heterogeneity in CNS cells is evidenced by varying astrocyte morphology in the cortex, cerebellum, brainstem and spinal cord.
Astrocytes from the same region may also exhibit morphological heterogeneity, supporting the notion that this glial subtype is highly dynamic. Regional responses of OPCs to differential cytokine stimulation were also explored. Cytokine signaling significantly influences stem and immune cell behavior and may impact how the OPCs differentiate into mature myelinating oligodendrocytes.
Following regional glial cell isolation cells can be used in a variety of experimental conditions and acid using several techniques including immunohistochemistry, quantitative real-time PCR and Western blotting.
