Assessing Microglial Phagocytosis of Myelin Debris in vitro Under Repeated Magnetic Stimulation

[Instructor] The scope of our study focus on research in neuro-rehabilitation and magnetic simulation therapy. This protocol can provide new ideas for magnetic stimulation in exploring how it affects a clear function. We will focus on neuro-rehabilitation and magnetic stimulation therapy in the future.

[Instructor] To begin, obtain a decapitated head of a female rat. Dissect the head on ice. With a pair of scissors, bisect the skull to fully expose the brain and facilitate its complete removal. Use scissors and forceps to meticulously and aseptically excise the membranes, cerebellum, and hippocampus. Clean the brain three times with PBS to remove any residual blood or tissue. Transfer the cleaned brain to 10 milliliters of 0.32 molar sterile sucrose solution. With a pair of microsurgical scissors, cut the brain tissue into pieces to obtain a brain tissue-sucrose mixture. Next, transfer the brain tissue-sucrose mixture to a 50 milliliter sterile homogenizer. Add 30 milliliters of 0.32 molar sterile sucrose solution. Use a 50 milliliter glass homogenizer to grind the tissue for two minutes. To obtain a smooth brain tissue homogenate. Dilute the brain tissue homogenate to 90 milliliters with 0.32 molar sterile sucrose solution, and mix thoroughly. Add 20 milliliters of 0.83 molar sterile sucrose solution into six sterile, thin-walled polypropylene ultracentrifuge tubes. Then slowly dispense 15 milliliters of the brain mixture into the upper part of the tubes. Level the volumes with 0.32 molar sterile sucrose solution. To collect the crude myelin debris, first pre-cool and ultra centrifuge rotor at four degrees Celsius. Centrifuge the sample at 75,000 G for 45 minutes, then collect the myelin debris from the interface between the two sucrose densities using a sterile pasture pipette. For the first isotonic separation and purification, transfer the collected myelin debris solution to a 50 milliliter centrifuge tube. Adjust the volume to 35 milliliters with pre-cooled sterile PBS, and transfer to a new homogenizer tube. After homogenizing for three minutes, distribute the myelin debris homogenate evenly in six 38.5 milliliter sterile, thin-walled, polypropylene ultracentrifuge tubes. Make up the volume with sterile PBS, and then centrifuge. For the second isotonic separation and purification, resuspend the solid white pellet in 10 milliliters of pre-cooled sterile PBS to obtain a myelin debris suspension. Distribute the suspension into ultracentrifuge tubes as before, and centrifuge. After centrifugation, discard the supernatant. Resuspend the solid white pellet in six milliliters of sterile PBS. Divide the myelin debris suspension into six 1.5 centrifuge tubes, and centrifuge again. Finally, resuspend the pellet in 100 microliters of pre-cooled sterile PBS after discarding the supernatant. Thaw the requisite myelin debris at four degrees celsius, or in ice water, and centrifuge as before for 10 minutes. Resuspend the myelin debris in 200 microliters of 50 micromolar CFSE solution. Incubate the suspension at room temperature in the dark for 30 minutes, then centrifuge again. Discard the supernatant, then wash the samples three times with 500 microliters of sterile PBS. Resuspend the myelin debris in 100 microliters of pre-cooled sterile PBS to obtain a suspension of fluorescently labeled myelin debris. Store the sample at -80 degrees Celsius until further use. For in vitro repeated magnetic stimulation, set the treatment frequency to 20 hertz, treatment intensity to 1% of the maximum output intensity, and stimulation time to five seconds. Include a rest period of 20 seconds, and administer 600 pulses over a single treatment time of 2.5 minutes. After predetermining the magnetic stimulation parameters, fix the coil direction perpendicular to the ground and orient it upwards. Sterilize the magnetic stimulation coil with alcohol to prevent cell contamination. After adding 50 micromolars of CFSE, plate 1,000 BV-2 cells in 24 well plates overnight, then aspirate the old medium with a pipette, and immediately add fresh serum-free medium. Change the medium to serum-free medium, and treat cells with one microgram per milliliter lipopolysaccharide for 12 hours. After 12 hours of lipopolysaccharide intervention, add 100 micrograms per milliliter of myelin debris to the medium for co-culture in the dark. Subject the cells to repeated magnetic stimulation, as demonstrated earlier. Spray alcohol over the plate to sterilize it before returning it to the incubator. After a period of co-culture with myelin fragments, wash non-absorbed myelin debris gently with PBS. Fix the cells with 4% paraformaldehyde for 15 minutes. Next, add 150 microliters PBS containing 10% donkey serum albumin and 0.3% Triton X-100 to the wells and incubate. After washing the wells with PBS, add a primary antibody solution at a ratio of 100 to 200 into the wells, and incubate in the cold. Then, incubate the cells in a secondary antibody at a ratio of 100 to 300 for two hours at room temperature before imaging with a confocal microscope. BV-2 microglia cells exhibited a significant reduction in myelin debris phagocytosis following lipopolysaccharide treatment. which was reversed upon repeated magnetic stimulation intervention. Quantitative analysis confirmed a significant decrease in myelin debris area within BV-2 cells in the lipopolysaccharide group compared to the control, with a notable increase in the lipopolysaccharide-treated magnetically-stimulated group,. The percentage of IBA-1 positive microglia co-localized with myelin debris was significantly lower in the LPS group compared to control, while magnetic stimulation significantly increased this percentage. A time-dependent increase in microglial phagocytosis was observed with the lipopolysaccharide-treated magnetically stimulated group for our group showing significantly higher myelin debris uptake.