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

Summary

The present protocol has been devised to assess the effect of repetitive magnetic stimulation on microglia's ability to phagocytose myelin debris. An in vitro microglia and myelin debris co-culture system has been established to do so.

Abstract

Microglia, the resident phagocytes of the central nervous system (CNS), play a pivotal role in maintaining CNS integrity and homeostasis by removing damaged cells, cellular debris, and myelin remnants. The accumulation of myelin debris is implicated in a range of CNS disorders, including multiple sclerosis, Alzheimer's disease, traumatic brain injury, and spinal cord injury. The presence of myelin debris not only exacerbates neuroinflammation but also hampers the regenerative potential of myelin. Therefore, enhancing the ability of microglia to clear myelin debris through phagocytosis represents a promising therapeutic strategy. Magnetic stimulation has emerged as an innovative treatment modality for CNS diseases, with growing evidence suggesting its potential to promote microglial phagocytosis and support CNS recovery. To further elucidate the effects of magnetic stimulation on microglial clearance of myelin debris, we designed an in vitro experiment involving the co-culture of microglia and myelin debris. The co-culture was subjected to repetitive magnetic stimulation to assess its impact on microglial phagocytic activity in the context of CNS pathology.

Introduction

Myelin is a stable, tubular membrane structure surrounding axons produced by oligodendrocytes in the central nervous system (CNS). This myelin sheath plays a critical role in facilitating the rapid and efficient propagation of action potentials¹. However, various neurological disorders, such as multiple sclerosis (MS), stroke, and traumatic injuries, including spinal cord injury (SCI) and traumatic brain injury (TBI), can lead to the loss of myelin integrity. This results in the generation of myelin debris due to the disruption of the myelin sheath and direct damage to myelin structures^2,3,4. The accumulation of myelin debris not only impedes remyelination and functional recovery but also exacerbates neuroinflammation^5,6. Therefore, effective clearance of myelin debris is essential for resolving neuroinflammation, promoting axonal regeneration, and restoring homeostasis in the nervous system following neurological diseases.

Myelin debris is primarily cleared by phagocytes, such as macrophages and microglia in the central nervous system (CNS). Microglia, the resident immune cells of the CNS, constantly survey their environment for potential threats and protect the CNS by removing damaged myelin and myelin debris. Furthermore, microglial phagocytosis plays a crucial role in promoting neurogenesis through debris clearance^7,8. Increasing evidence suggests that enhancing microglial phagocytosis of myelin debris can mitigate the harmful effects of inflammation, reduce CNS damage, and promote axonal regeneration following nerve injury.^4,9,10.

Magnetic stimulation therapy, a non-invasive neuromodulation technique, has gained widespread use in the treatment of various neurological disorders¹¹. Notably, repeated transcranial magnetic stimulation (rTMS) has shown promise as a therapeutic approach for Alzheimer's disease (AD), brain injury, and stroke^12,13,14. A recent study has demonstrated that rTMS can enhance microglial phagocytosis of amyloid-beta (Aβ) and apolipoprotein E (ApoE), thereby inhibiting the pathological progression of AD in animal models¹⁵. Similarly, our recent work found that repeated trans-spinal magnetic stimulation (rTSMS) promotes microglial phagocytosis of myelin debris following spinal cord injury in rats¹⁶.

Nevertheless, the impact of repetitive magnetic stimulation on microglia has been predominantly substantiated through experimental models in animals^17,18,19. Indeed, magnetic stimulation has been demonstrated to modulate neuronal excitability through the induction of currents²⁰. Consequently, it cannot be disregarded that the interaction between neurons and microglia may influence microglia function. Consequently, in vitro experimentation is necessary to ascertain whether the impact of magnetic stimulation on microglia is independent of the alteration in neuronal excitability. Recent studies have identified a modulatory effect of repetitive magnetic stimulation on microglia in experimental models. Some studies demonstrated that repetitive magnetic stimulation could promote microglia polarization towards M2^19,21. However, further exploration into its microglia function remains to be elucidated. Notably, Amelie Eichler et al. intervened in brain tissue slices in vitro using 10 Hz magnetic stimulation and found that 10 Hz magnetic stimulation was able to promote the release of cytokines from microglia to influence neural excitability and plasticity²². While this study expanded the understanding of magnetic stimulation's effects on microglia, further investigation at the cellular level is necessary to fully elucidate its mechanisms.

Thus, we established a protocol to verify whether magnetic stimulation can promote the phagocytosis of myelin debris in vitro. Unlike myelin debris after disease or tissue damage, which is a product of injury, the myelin debris used in this experiment is the shape of the myelin sheath after it breaks down. In addition, the concept of myelin debris is a product of myelin disintegration. As the primary phagocytic cells of the brain, microglia play a pivotal role in the clearance of damaged cells and myelin debris²³, which is associated with diseases such as multiple sclerosis and Alzheimer's^24,25. The accumulation of myelin debris has been demonstrated to exacerbate neuroinflammation and hinder regeneration²⁶. The objective of the study is to investigate the potential of magnetic stimulation to enhance microglial phagocytosis of myelin debris, thereby reducing neuroinflammation and supporting CNS recovery in neurodegenerative and traumatic conditions. In summary, the study employs a co-culture approach, whereby microglia are co-cultured with myelin debris and repeated magnetic stimulation is performed in order to verify the effect of repetitive magnetic stimulation on the phagocytic capacity of microglia.

Protocol

Here, three SD female rats (aged 2-3 weeks) were used to extract myelin debris by sucrose-gradient centrifugation. All rats were purchased from the Animal Core Facility of Nanjing Medical University, Nanjing, China (Animal license: SYXK (Su) 2021-0023). All rats were raised under control conditions (temperature 22 ± 2 °C, a 12 h/12 h light/dark period, 55% ± 5% relative humidity). All animal experiments were approved by the Nanjing Medical University Animal Care and Use Committee (No. IACUC-1903031 and No. IACUC-2005001) and were aimed to minimize suffering, and the number of animals used.

1. Myelin isolation

NOTE: Myelin debris was extracted as described previously, and some modifications were done²⁷. Ensure that all steps were performed at 4 °C and under sterile conditions.

1. Isolation of crude myelin
   1. Brain tissue extraction
      1. Following anesthesia with pentobarbital (50 mg/kg, intraperitoneal injection), assess depth of anesthesia by toe pinch response. Decapitate the female rats. Dissect the whole head on ice and bisect the skull with scissors to fully expose the brain and facilitate the removal of the complete brain.
      2. Excise the membranes, cerebellum, and hippocampus with scissors and forceps in a meticulous and aseptic manner to prevent contamination of the brain tissue sucrose mixture. Clean the brain 3x with PBS to remove any residual blood or tissue.
      3. Transfer the cleaned brains to 10 mL of 0.32 M sterile sucrose solution and cut the brain tissue with the microsurgical scissors into 5 mm³ pieces to obtain a brain tissue sucrose mixture.
   2. Brain tissue homogenization
      1. Transfer the brain tissue sucrose mixture to a 50 mL sterile homogenizer and add 30 mL of 0.32 M sterile sucrose solution. Use a 50 mL glass homogenizer to homogenize for 2 min to obtain a smooth brain tissue homogenate.
      2. Dilute the brain tissue homogenate to 90 mL with 0.32 M sterile sucrose solution, mix, and slowly dispense into the upper part of six 38.5 mL sterile thin-walled polypropylene ultracentrifuge tubes containing 20 mL of 0.83 M sterile sucrose solution. Add 15 mL of homogenate to each centrifuge tube and then level with 0.32 M sterile sucrose solution.
   3. Crude myelin debris collection
      1. Precool an ultracentrifuge rotor at 4 °C, and centrifuge the sample at 75,000 x g for 45 min. Following the centrifugation process, collect the myelin debris from the interface between the two sucrose densities using a sterile Pasteur tube.
         NOTE: It is imperative during the collection process to exercise caution and avoid inadvertently collecting other components of the contaminated sample.
2. First isotonic separation and purification
   1. Transfer the collected myelin debris solution to a 50 mL centrifuge tube and adjust the volume to 35 mL with pre-cooled sterile phosphate-buffered saline (PBS) buffer. Transfer the solution to a new 50 mL sterile homogenizer and homogenize for 2 min to obtain a homogenate.
   2. Distribute the myelin debris homogenate evenly in six 38.5 mL sterile thin-walled polypropylene ultracentrifuge tubes, adding sterile PBS buffer. Subject the tubes to centrifugation at 75,000 x g for 45 min at 4 °C.
3. Second isotonic separation and purification
   1. Following centrifugation, discard the supernatant and resuspend the solid white pellet in 10 mL of pre-cooled sterile PBS buffer in order to obtain a myelin debris suspension. Distribute the suspension evenly in two 38.5 mL sterile thin-walled polypropylene ultracentrifuge tubes, with the volume balanced by the addition of sterile PBS buffer.
   2. Centrifuge the samples at 4 °C at 75,000 x g for 45 min.
4. Purified myelin collection
   1. Following centrifugation, discard the supernatant and resuspend the solid white pellet in 6 mL of sterile PBS buffer. Divide the myelin debris suspension into six 1.5 mL centrifuge tubes and then subject to centrifugation at 4 °C and 12,000 x g for 10 min.
   2. Following centrifugation, discard the supernatant and resuspend the solid white pellet in 100 µL of pre-cooled sterile phosphate-buffered saline (PBS).
5. Quantification of myelin debris
   1. Take 3 µL of the myelin debris suspension for quantification of the protein content using a BCA protein quantification kit. Store the remaining myelin debris suspension at -80 °C.

2. Fluorescent labeling of myelin debris

1. Incubation of Carboxyfluorescein diacetate N-succinimidyl ester
   1. Thaw the requisite myelin debris at 4 °C or ice water and centrifuge at 12,000 x g for 10 min at 4 °C. Discard the supernatant and resuspend the myelin debris in 200 µL of Carboxyfluorescein diacetate N-succinimidyl ester solution (CFSE; 50 µM).
   2. Incubate at room temperature in the dark for 30 min. Following the incubation period, centrifuge at 12,000 x g for 10 min at 4 °C.
   3. Discard the supernatant and wash the samples 3x with 500 µL of sterile PBS buffer. Following the washing process, resuspend the myelin debris in 100 µL of pre-cooled sterile PBS buffer in order to obtain a suspension of fluorescently labeled myelin debris, which can be stored at -80 °C.

3. Microglia culture

1. Culture BV-2 cells in humidified 5% CO₂ at 37 °C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) FBS, penicillin (100 U/mL), and streptomycin (100 U/mL). Seed nearly 50% Bv-2 cells in the 12-well plate and 24-well plate for further testing.

4. In vitro repeated magnetic stimulation

1. Perform the in vitro repeated magnetic stimulation intervention with the following setting: treatment frequency of 20 Hz, treatment intensity of 1% of the maximum output intensity (i.e., 0.035 Tesla), stimulation time of 5 s, rest period of 20 s. Administer 600 pulses with a single treatment time of 2.5 min.
2. During magnetic stimulation, position the center of the coil in alignment with the center of the culture plate and ensure the coil fits snugly to the bottom of the culture plate.
   1. Predetermine the parameters of magnetic stimulation and fix the direction of the magnetic stimulation coil to be perpendicular to the ground and oriented in an upward direction. Sterilize the magnetic stimulation coil with alcohol to prevent contamination from cells.
   2. Following this, retrieve the cell culture plate from the incubator and position it above the magnetic stimulation coil for magnetic stimulation. Sterilize the plate by spraying alcohol on it, and then return it to the incubator. Keep the plate covered throughout the process.

5. Testing of microglial phagocytosis of myelin debris

1. Repeated magnetic stimulation treatment for LPS+rMS group
   1. Use CFSE (50 µM) to label myelin debris to assess phagocytosis. Plate 1 x 10⁴ BV-2 cells in 24-well plates overnight. Change the medium to serum-free medium and treat cells withlipopolysaccharide (LPS; 1 µg/mL) for 12 h. Gently aspirate the old medium using a ppipette and then add the new serum-free medium immediately using the pipette.
   2. Following a 12 h period of LPS intervention, add the myelin debris (100 µg/mL) to the medium for co-culture under dark conditions. Subject the cells to repeated magnetic stimulation interventions as described in step 4.2.
2. Immunofluorescence and Imaging
   1. Wash the non-adsorbed myelin debris with PBS gently and fix the cells with 4% PFA for 15 min. Block the cells with PBS containing 10% donkey serum albumin and 0.3% Triton X-100 for 1 h at room temperature, then wash with PBS and incubate with primary antibody (IBA-1, 1:200) at 4 °C overnight.
   2. Incubate the cells with secondary antibodies (1: 300, mouse) for 2 h at room temperature. Capture images using a confocal microscope at 40x and 20x.

Results

In this study, we initially fed BV-2 microglia cells in vitro by extracting brain-derived myelin debris and utilizing repetitive magnetic stimulation to enhance their phagocytic capacity. Subsequently, BV-2 microglia cells were cultured and subjected to LPS intervention prior to magnetic stimulation, thereby emulating the in vivo state of microglia under rigorous neurological validation. Subsequently, rMS intervention was applied following LPS stimulation. To validate the phagocytosis of cells, immunofl...

Discussion

Repetitive magnetic stimulation (rMS) is a non-invasive neurostimulation technique that has been widely applied in the treatment of various neurological and psychiatric disorders, including cognitive impairments, Alzheimer's disease (AD), stroke, brain injury, and spinal cord injury (SCI). rMS utilizes a pulsed magnetic field to modulate the membrane potential of neurons, selectively influencing neural excitability within the nervous system¹¹. In addition to its effects on neurons, rMS has bee...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antifade Mounting Medium with DAPI	Beyotime, China	P0131
BCA Kit	ThermoFisher Scientific, America	23227
Bv-2 cell	ATCC	CBP60922
Cell culture 12-well plates	Biofil, China
Cell culture 24-well plates	Biofil, China
Confocal Laser Scanning Microscope	Zeiss, Germany	800
Constant temperature incubator	ThermoFisher Scientific, America
CFSE	MedChemExpress, America	HY-158820
DMEM-Culture medium	KeyGen, China	KGL1202-500
FBS	Gibco, America	16250078
Iba1	Abcam, America	ab48004
Magnetic stimulation apparatus	Yiruide, China	CCY-IA
Magnetic stimulation coil	Yiruide, China
Paraformaldehyde	BioSharp, China
PBS	KeyGen, China	KGL2210-500
Penicillin-Streptomycin	Gibco, America	15140-122
Sucrose	MedChemExpress, America	HY-B1779
Trypsin	KeyGen, China	KGL2107-100
Ultracentrifuge	Beckman, America	L-100XP