Effect of microglial derived extracellular vesicles on neuronal damage after heat stress

Objective To investigate the effect of microglial derived extracellular vesicles on neuronal damage in the context of heat stress. Methods After BV2 microglial cells were exposed to heat stress, the supernatant was collected and subjected to ultracentrifugation at different speeds to obtain large and small vesicles, respectively. Nano Particle Tracking and Zeta Potential Distribution Analyzer was used to measure and analyze the size distribution of the large vesicles and small vesicles. Western blotting was used to detect the expression of specific vesicle surface markers, TSG101, CD63 and flotillin-1. Microglial extracellular vesicles were labeled with PKH67 dye and then co-cultured with N2a cells to examine the uptake by capacity the neurons. After large and small vesicles derived from microglia after heat stress stimulation were co-cultured with N2a cells, respectively, CCK-8 assay, lactate dehydrogenase (LDH) assay, Trypan blue staining and TUNEL assay were employed to evaluate heat stress induced neuronal damage. Results The small vesicles were in a particle size of 30~120 nm, and highly expressed TSG101 and CD63, whereas the large vesicles, in a size of 90~1 000 nm, highly expressed flotillin-1. The BV2-derived extracellular vesicles could be taken up by N2a cells and were proved to be involved in the modulation of N2a cell injury caused by heat stress. CCK-8 assay showed that both large and small vesicles of microglial cells inhibited the viability of N2a cells after heat exposure (P<0.05). The results of LDH assay, Trypan blue staining and TUNEL assay showed that both large (P<0.05) and small vesicles (P<0.01) significantly enhanced the LDH release, blue stain intensity and apoptosis of N2a cells after heat exposure, and the release, intensity and apoptosis were stronger in the cells treated with small vesicles than those group of large vesicles. Conclusion Microglia aggravate heat stress-induced neuronal damage through releasing extracellular vesicles.