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Advanced Neurology Proteomic analysis of microglia exosomes

8 weeks old, were purchased from the Animal Model and IL-4-treated microglia (M2-EXO) were subjected to
Center of Nanjing University and housed under controlled tandem mass tag (TMT)-based quantitative proteomics
conditions with standard diets provided. The transient at Jingjie PTM BioLab, China. The exosome samples in
middle cerebral artery occlusion (tMCAO) model was lysis buffer (8 M urea, 1% protease inhibitor cocktail)
established as previously described. After anesthetizing were sonicated on ice 3 times and then centrifuged at
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the mice, the middle cerebral artery was occluded using 12,000 × g for 10 min to collect the supernatant. Following
a 6-0 surgical monofilament nylon suture (Doccol the determination of concentration using a BCA kit, the
Corporation, USA). Approximately 60 min later, the suture protein samples were digested with trypsin and labeled
was withdrawn to allow reperfusion. Following suture with TMT. High pH reverse-phase HPLC was used to
removal, exosomes from different phenotypes of microglia fractionate the tryptic peptides using an Agilent 300
were promptly administered through the tail vein with a Extend-C18 column (5 μm particles, 4.6 mm ID, 250 mm
daily dosage of 100 μg for 3 consecutive days. length). The peptides were then analyzed using the EASY-
The post-tMCAO brain tissue infarct volume was nLC 1000 UPLC system (Thermo Fisher Scientific, Inc.,
evaluated using 2% TTC (2,3,5 triphenyltetrazolium USA) for liquid chromatography-mass spectrometry
chloride; Sigma-Aldrich, USA). Briefly, following (LC-MS)/MS analysis. The acquired MS/MS data were
euthanasia, the brains were rapidly removed and sliced processed with the Maxquant search engine (v.1.5.2.8,
coronally into consecutive 1 mm sections. These slices were Max Planck Institute of Biochemistry, Germany). Proteins
then immersed in 2% TTC at 37°C for 20 min. Images of were deemed significantly differentially expressed if
the stained slices were captured and analyzed using ImageJ the fold change in expression was >1.3 and the P < 0.05.
software. To avoid data errors induced by cerebral edema, Gene ontology (GO) and Kyoto Encyclopedia of Genes
the infarct volume was computed as previously described 15 and Genomes (KEGG) analyses were performed to assess
(Equation I): molecular functions, cellular components, biological
processes, and signaling pathway enrichment.
( )
Infarct volume % =
Contralateral hemisphere volume- 2.10. Statistical analysis
ipsilateral hemisphere non-infarct volume (I) The data in this study were analyzed using Statistical
× 100 Package for the Social Sciences 18.0 software and were
Contralateral hemisphere volume × 2
found to be normally distributed. All data were presented
as the mean ± standard deviation. Appropriate statistical
2.8. Behavioral tests methods were applied to determine significance. Student’s
The neurological function of tMCAO mice was assessed t-test was used to evaluate differences between two groups,
using the modified neurological severity scores (mNSS), while comparisons between more than two groups were
grip test, and rotarod test as previously described. determined using one-way analysis of variance with
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The mNSS assessed motor, sensory, reflex, and balance Bonferroni post hoc test. A P < 0.05 was considered
functions, with scores ranging from 0 to 18, where a higher significantly different.
score indicates a more pronounced impairment. 3. Results
Rotarod tests were used to evaluate the motor and
coordination abilities of the mice. Before tMCAO, mice 3.1. Successful in vitro induction of detrimental and
protective phenotypes of microglia
were trained on a rotarod (RWD Life Science, China) twice
daily for 3 days at varying speeds (10, 20, 30, and 40 rpm). Primary cultured and isolated microglia were initially
After tMCAO induction, mice were positioned on a rotarod treated with LPS and IL-4 for 24 h to establish stable
set at 40 rpm, and the latency to fall was recorded with a detrimental and protective phenotypes in vitro. The
maximum time of 300 s. Furthermore, a grip strength meter phenotypes of the induced microglia were characterized
(GS3, Bioseb, France) was used to measure the forelimb grip through fluorescence microscopy, qPCR, Western blotting,
strength of the mice. Mice were positioned with their paws and immunofluorescence staining. Cell morphology
on a metal bar, and their tail was gently pulled backward. results revealed that the cell bodies of the resting
The peak grip strength value was recorded. microglia were small with a smooth surface, whereas the
cell bodies of the detrimental M1 microglia stimulated
2.9. Exosomal proteomics with LPS exhibited enlargement, edema, a rough surface,
Exosomes isolated from the conditioned media of untreated numerous branches, and irregular shapes. Conversely,
microglia (M0-EXO), LPS-treated microglia (M1-EXO), IL-4 promoted the generation of beneficial M2 microglia

Volume 3 Issue 3 (2024) 4 doi: 10.36922/an.3166