• Tomohiro Matsui, Yusuke Yoshida, Masashi Yanagihara, and Hiromi Suenaga.
• Department of Laboratory Sciences, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-kogushi, Ube, Yamaguchi, 755-8505, Japan, giants@yamaguchi-u.ac.jp.
• Neurocrit Care. 2014 Apr 1;20(2):301-10.

BackgroundTherapeutic hypothermia protects neurons after severe brain damage. This effect has been mainly achieved at the core temperatures of 32-34 °C; however, the optimum temperature of therapeutic hypothermia is not fully defined. Here we studied whether hypothermic culture at 35 °C had the same effects on the decrease of time-dependent expression of tumor necrosis factor (TNF)-α, interleukin (IL)-10, and nitric oxide (NO) by stimuli-activated microglia as that at 33 °C, as determined in our previous reports, and whether these factors directly induced neuronal cell death.MethodsWe determined the levels of cytokines and NO produced by microglia cultured with adenosine triphosphate (ATP), a toll-like receptor (TLR)2 agonist (N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2R,S)-propyl)-(R)-cysteinyl-seryl-(lysyl)3-lysine, Pam(3)CSK(4)), or a TLR4 agonist (lipopolysaccharide) under mild hypothermic (33 °C), minimal hypothermic (35 °C), and normothermic (37 °C) conditions. We also determined the viability of rat neuronal pheochromocytoma PC12 cells treated with recombinant TNF-α or IL-10 or (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3, an NO donor).ResultsProduction of TNF-α, as well as that of IL-10 and NO were decreased by minimal hypothermia at 1.5-6, and 24-48 h, respectively, compared with normothermia, although some effects were diminished as compared with those by mild hypothermia. Exposure to TNF-α, IL-10, and NOR3 caused the death of PC12 cells in a concentration-dependent manner after 24 h.ConclusionHypothermic culture at 35 °C decreased the production of early-phase TNF-α and late-phase IL-10 and NO from ATP- and TLR-activated microglia as observed at 33 °C, albeit with diminished effects. Moreover, these factors caused the death of neuronal cells in a concentration-dependent manner. These results suggest that the attenuation of microglial production of TNF-α, IL-10, and NO by therapeutic hypothermia leads to the inhibition of neuronal cell death. Minimal hypothermia at 35 °C may be sufficient to elicit neuroprotective effect.

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