This model provides experimental opportunities to study various aspects of myelination of the peripheral nervous system in vitro. It enables myelin quantification and the examination of Axon-Schwann cell interactions. The co-culture develops a robust myelination from day 14 onward.
The DRG explant cultures provide the advantage of intact structural architecture in comparison to dissociated neuronal cultures. This method is available to screen for tropotic compounds for inflammatory and neurodegenerative diseases of the peripheral nerve system. To begin, sterilize the work area in the euthanized rat's torso with 70%ethanol.
Open the rat's dorsal lower left limb with scissors, and carefully remove the bicep's femorous muscle. Loosen the sciatic nerve by smooth elevation with curved forceps, ensuring not to bruise the nerve. Using forceps, Transfer the clipped nerves to a tissue culture dish with ice cold Leibovitz's L-15 medium with 50 micrograms per milliliter of gentamycin.
Then, using a stereo microscope, remove the fat, muscle, and blood vessels from the nerves with two pairs of fine forceps. Identify the proximal and distal ends of the sciatic nerve and remove the epineurium with one pair of fine forceps in proximal to distal direction while holding the proximal nerve end with the second pair of fine forceps. After transferring the purified nerves to another tissue culture dish with ice cold Leibovitz's L-15 medium with Gentamycin, tease the isolated nerve fascicles to separate and isolate single nerve fibers using two pairs of fine forceps.
Transfer the nerve fibers to a 50 milliliter tube using a 10 milliliter serological pipette, and take up as little medium as possible. Then add 50 milliliters of leibovitz's L 15 medium with gentamycin to the nerve fibers in slew a few times. Centrifuge the tube containing the nerve fibers at 188 G for five minutes at four degrees Celsius.
After removing the supernatant, transfer the pellet with the remaining Leibovitz's L-15 medium into a 60 millimeter tissue culture dish. Rinse the 50 milliliter tube with 10 milliliters of the enzymatic digestion solution and add it to the nerve fibers dish. Distribute the nerve fibers in the dish carefully with the tip of a pipette.
Incubate at 37 degrees Celsius and 5%carbon dioxide for 18 hours. And stop the enzymatic digestion by adding 10 milliliters at 40%FCS in HBSS. Transfer the digested nerves into a 50 milliliter tube to centrifuge at 188 G for 10 minutes at four degrees Celsius.
After discarding the supernatant, re suspend the pellet in 10 milliliters of DMEM containing 10%FCS and 50 micrograms per milliliter of gentamycin. Next, filter the cell suspension through a 100 micrometer cell strainer. after centrifuging at 188 G for 10 minutes at four degrees Celsius, re suspend the pellet with four milliliters of DMEM containing FCS and gentamycin.
Add two milliliters of the cell suspension to each of the two polylysine and laminin coated 60 millimeter tissue culture dishes. Incubate at 37 degrees Celsius and 5%carbon dioxide, leaving the plates untouched for two days in the incubator. Centrifuge the Schwann cell suspension at 188 G for 10 minutes at four degrees Celsius, and re suspend the cell pellet in 90 microliters of magnetic cell separation buffer per one times 10 to the seventh cells.
Then add 10 microliters of ti1 micro beads per one times 10 to the seventh cells. Incubate the re suspended solution for 15 minutes in the dark at eight degrees Celsius. Next, add two milliliters in the magnetic cell separation buffer to the cell suspension.
After centrifuging at 300 G for 10 minutes at four degrees Celsius, re suspend the pellet in 500 microliters of magnetic cell separation buffer. Place the magnetic cell separation column in the cell separator, and moisten the column with one milliliter of the magnetic cell separation buffer. Then apply the cells to it to collect the flow through for centrifugation.
Carefully remove the uterus from a euthanized pregnant rat and place it into a 100 millimeter tissue culture dish with ice cold HBSS. Holding the uterus using curved forceps, open the uterus wall with fine forceps. Remove one amniotic sack and open it carefully by pinching a hole with fine forceps.
Quickly remove all the embryos from the uterus and transfer them into a dish filled with HBSS. Gently, place one embryo torso into a 35 millimeter dish filled with HBSS. under a stereo microscope, Open the dorsal part of the torso to divide the embryo into two halves.
Turn one half to the side and identify the strand of dorsal root ganglia, or DRG, located in the line at the dorsal part of the embryo. Cut out the DRG as a whole strand using fine forceps and micro scissors. After placing the DRG in a fresh dish filled with two milliliters of HBSS, Separate a single DRG from the remaining tissue using fine forceps and micro scissors.
Take a 4 well plate containing 190 microliters of DRG growth medium in each well, and carefully transfer a single DRG into the center of each well using fine forceps and a spatula. Then place the DRG explant cultures in the incubator at 37 degrees Celsius and 5%carbon dioxide. The next day, carefully add 50 microliters of the DRG growth medium to each well and observe DRG explant adherence and axon outgrowth using a microscope daily.
For co-culturing on day three of the DRG explant culture, carefully replace the DRG growth medium with 250 microliters of the co-culture medium containing 30, 000 schwann cells per well. For performing immunocytochemical staining, fix the cells on the cover slips by incubating the DPBS washed cells in 4%paraformaldehyde for 10 minutes. Take pictures of the immunocytochemically stained samples at eight defined regions surrounding the DRG explant in the center using an inverted microscope.
The appearance of schwann cells with an elongated and spindle shaped morphology and the thin DRG axons in a co-culture is depicted here. Myelination in the co-culture was assessed on different days by staining the DRG explants and Schwann cells for myelin basic protein or MBP, beta three tubulin, and DAPI. The axonal network in the co-culture was dense, and did not change visibly in the time course of the observation.
First signs of myelination were visible on days 10 and 12 of co-culture. From day 14, the MVP signal was more pronounced, and myelin-enwrapped axons were detected. The myelination increased with the co-culture time until day 20.
The myelination was quantified as a ratio of the MVP and beta three tubulin positive areas. A significant increase in myelination was observed on days 18 and 20 compared to day 10. DRG explant cultures are fragile, and need to be handled carefully.
For example, when taken out of the incubator or during medium change and fixation. Gene expression analysis of co-culture samples can be formed to investigate myelin in greater detail. Here, we detected the myelin markers PMP22, MAG, Oct6, Egr2, Olig1 on day 22.
