Background: Ischemic brain injury results in the release of damage-associated molecular patterns
(DAMPs) and endogenous mediators of sterile inflammation. Targeted temperature management
(TTM) is neuroprotective via induced intracellular cold-shock RBM3 and CIRBP expression.
However, extracellular CIRBP has been implicated as a mediator of sterile inflammation.
Microglia mediated neuroinflammation has been well investigated as both detrimental
and regenerative properties have been described for these cells. Still the effect
of TTM on microglia activation and DAMPs release after ischemia/reperfusion (I/R)
injury remains to be elucidated. Comprehension of the role of microglia in I/R-induced
sterile inflammation is needed to improve the efficacy of TTM. Therefore, we investigated
the effect of cooling on cell viability, DAMPs release, and inflammation in I/R injured
microglia.
Method: BV-2 microglia were exposed to 2 to 6 hours of oxygen-glucose deprivation (OGD, 0.2%
oxygen in glucose/serum-free medium) followed by 3 to 19 hours of reperfusion (21%
oxygen in complete medium). Cells were maintained at either normothermia (37°C) or
cooled to 33.5°C (TTM) 1 hour after experimental start. Necrosis was assessed by LDH
release and apoptosis by Caspase-3/-8/-9 cleavage. Intra/extracellular DAMPs (CIRBP,
Hsp70, and HMGB-1) and antiapoptotic RBM3 were evaluated by the Western Blot. Oxidative
stress (iNOS), inflammatory cytokines (TNF-a, IL-1a/-1b, IL-6, and IL-10), and microglial
activation markers (AIF1 and MCP-1) were assessed by RT-qPCR.
Results: TTM attenuated I/R-induced necrosis but not OGD-induced oxidative stress (iNOS) in
the microglia. We also observed significant inductions of neuroprotective RBM3 and,
even more rapidly, CIRBP by cooling. Moreover, OGD-induced DAMPs release was also
attenuated by cooling and correlated with degree of necrosis, suggesting a passive
secretion mechanism in the microglia. Exposure to OGD suppressed most cytokine gene
expressions independently of temperature management. Interestingly, cooled Normoxia
groups had significant increase of microglial activation marker, Iba1, IL-1b, as well
as TNF-a gene expressions.
Conclusion: TTM conveyed neuroprotection as observable in increased cell viability, attenuated
DAMPs release, and induced expression of cold shock proteins in I/R injured microglial
cells. As cooling alone resulted in both upregulated expression of proinflammatory
cytokines, as well as of neuroprotective cold shock proteins, TTM might have ambiguous
effects in sterile inflammation.
