Hypothermia Reduces Toll-Like Receptor 3-Activated Microglial Interferon-βand Nitric Oxide Production

Abstract

Therapeutic hypothermia protects neurons after injury to the central nervous system (CNS). Microglia express toll-like receptors (TLRs) that play significant roles in the pathogenesis of sterile CNS injury. To elucidate the possible mechanisms involved in the neuroprotective effect of therapeutic hypothermia, we examined the effects of hypothermic culture on TLR3-activated microglial release of interferon (IFN)-β and nitric oxide (NO), which are known to be associated with neuronal cell death. When rat or mouse microglia were cultured under conditions of hypothermia (33°C) and normothermia (37°C) with a TLR3 agonist, polyinosinic-polycytidylic acid, the production of IFN-β and NO in TLR3-activated microglia at 48 h was decreased by hypothermia compared with that by normothermia. In addition, exposure to recombinant IFN-β and sodium nitroprusside, an NO donor, caused death of rat neuronal pheochromocytoma PC12 cells in a concentration-dependent manner after 24 h. Taken together, these results suggest that the attenuation of microglial production of IFN-β and NO by therapeutic hypothermia leads to the inhibition of neuronal cell death.

Original article Effects of hypothermia on ex vivo microglial production of pro- and anti-inflammatory cytokines and nitric oxide in hypoxic-ischemic brain-injured mice

“…With the aim of elucidating the possible mechanisms underlying the neuroprotective effects of therapeutic hypothermia, we have previously shown that hypothermia reduces the production of earlyphase and late-phase inflammatory factors as well as that of late-phase anti-inflammatory factor in primary microglia in vitro [33][34][35][36]. We considered that an approach using microglia removed directly from injured brains may yield more clinically relevant information.…”

“…*p < 0.05 compared with 37°C. therapeutic hypothermia [33][34][35][36]. IL-10 has been considered to be neuroprotective because it reduces the production of pro-inflammatory cytokines [8,26].…”

“…These doses were in accordance with our previous in vitro studies including those of IL-10 and NO [33,36]. Because the present study was designed to exploit our previous in vitro findings in terms of the temporal change of TNF-α, IL-10, and NO production [33,35,36], we selected each culture period above for the cytokines/NO as representative time points.…”

“…A colorimetric assay with Griess reagent (Sigma-Aldrich) was performed, as described in our previous reports [33][34][35][36].…”

“…Previously, we and other groups have demonstrated a reduced production of tumour necrosis factor-alpha (TNF-α: another pro-inflammatory cytokine that is associated with neuronal injury [3,40]), IL-6, interferon-beta (IFN-β, which is known to be associated with neuronal cell death [15]), interleukin-10 (IL-10: an anti-inflammatory cytokine), and NO in hypothermic culture conditions [20,31,[33][34][35][36]42]. In particular, we proposed that these effects were temporally related to the reduction of early-phase and late-phase inflammatory factors as well as that of late-phase anti-inflammatory factor(s) [33][34][35][36].…”