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Advanced Neurology Proteomic analysis of microglia exosomes

tissue. This deprivation results in irreversible damage and methods. Briefly, cortical tissues underwent enzymatic
neuronal death within the core infarct area. The current digestion with TrypLE at 37°C, followed by centrifugation
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stroke management emphasizes preserving the ischemic and filtration to isolate the glial cells. After 10 – 14 days in
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penumbra surrounding this core area. Although nerve cells a 5% CO humidified atmosphere at 37°C, microglia were
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in this region may be dysfunctional, they remain viable, harvested by shaking the culture flasks and then seeded
and timely intervention holds the potential to enhance the into 12- or 24-well plates for subsequent experiments.
recovery of nerve function. Shortly after stroke, microglia The primary microglia were maintained in DMEM
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activate and play a crucial role in this process. These innate medium (Thermo Fisher, United States of America [USA])
immune cells differentiate into either M1 (neurotoxic) or containing 10% fetal bovine serum (Biological Industries,
M2 (neuroprotective) phenotypes. However, the precise Israel), penicillin (100 U/mL; Thermo Fisher, USA), and
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mechanisms underlying their effects require further research. streptomycin (100 μg/mL; Thermo Fisher, USA). To
activate M1 and M2 microglia, primary microglia were
Exosomes are small extracellular vesicles (30 – 200 nm) treated with 100 ng/mL lipopolysaccharide (LPS) (Sigma-
secreted by various cell types, carrying RNA, DNA, proteins, Aldrich, USA) or 10 ng/mL interleukin (IL)-4 (Sigma-
and lipids, thereby enabling intercellular communication. Aldrich, USA) for 24 h, respectively. Primary cortical
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Recently, exosomes have gained attention for their neurons were isolated from E15 – 17 embryos and plated
potential impact on disease prognosis and for their role as on poly-d-lysine-coated plates (Sigma-Aldrich, USA).
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targets for disease monitoring and drug delivery. Notably, These neurons were cultured in a neurobasal medium
exosomes can cross the brain-blood barrier, making them containing B27, GlutaMAX (Thermo Fisher, USA), and
applicable in ischemic stroke treatment. For instance, penicillin/streptomycin.
the exosomal circRNA BBS2 from umbilical cord-
mesenchymal stem cells (UC-MSCs) inhibits ferroptosis 2.2. Isolation, characterization, and labeling of
and alleviates ischemic stroke. In addition, MSC-derived exosomes
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exosomes promote neurological recovery and brain tissue Exosomes were isolated from microglial-conditioned media
remodeling in an elderly rat ischemic stroke model. 10 using differential centrifugation. Briefly, the culture media
Microglia communicate with neurons and other cells and were centrifuged consecutively at 500 × g for 5 min, 3,000 × g
release exosomes containing diverse contents. Depending for 25 min, and 12,000 × g for 60 min to remove debris. The
on the stimulus, these exosomes can either be harmful or exosomes were then harvested through two successive ultra-
beneficial to brain tissue. For example, exosomal miR-423-5p centrifugations at 120,000 × g and 100,000 × g for 70 min
from oxygen-glucose deprivation/reoxygenation each. The resulting precipitated exosomes were suspended in
(OGD/R)-activated microglia exacerbates damage to brain phosphate-buffered saline (PBS) for further characterization.
microvascular endothelial cells and contributes to vascular Exosomes were visualized using transmission electron
loss. Conversely, exosomal miR-124 from M2 microglia microscopy (TEM). In addition, nanoparticle tracking
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protects against ischemic brain injury and neuronal death by analysis (NTA) was employed to determine the concentration
targeting ubiquitin-specific protease 14 (USP14). Although and size of exosomes using a nanoparticle-tracking analyzer
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studies have explored the effects of exosomes derived from (Malvern Panalytical, UK [UK]).
microglia in different activation states on neuronal survival Following the manufacturer’s guidelines, exosomes
after ischemic stroke, the differences between microglia of were stained with Dil dye (5 μM) for 10 min (Beyotime,
different phenotypes have not been fully explored. China). The supernatant was discarded after centrifugation
In this study, we investigated the effects of exosomes (110,000 g, 1 h, 4°C) to eliminate excess Dil dye.
derived from distinct phenotypes of microglia on ischemic Subsequently, the exosome pellets were resuspended in
brain injury. Our research focused on a proteomic sterile PBS for neuronal uptake studies.
analysis, comparing the expression and functions of the 2.3. Western blotting
two microglial exosomal proteins. These findings provide
valuable insights into the role of microglia in various Protein extracted from primary microglia and exosomes
activation states following an ischemic stroke. was quantified using a bicinchoninic acid (BCA) assay. The
quantified proteins were then separated on 12.5% Sodium
2. Materials and methods dodecyl-sulfate polyacrylamide gel electrophoresis gels
and transferred onto 2-μm polyvinylidene difluoride
2.1. Primary cell culture
membranes. The PVDF membranes were blocked with
Primary microglia were isolated from the cerebral cortex 5% skimmed milk powder in 0.1% TBST for 60 min and
of neonatal C57/BL6J mice using previously established then incubated with specific primary antibodies against

Volume 3 Issue 3 (2024) 2 doi: 10.36922/an.3166