Hypothermia at 35 A°C Reduces the Time-Dependent Microglial Production of Pro-inflammatory and Anti-inflammatory Factors that Mediate Neuronal Cell Death

Therapeutic hypothermia protects neurons after severe brain damage. This effect has been mainly achieved at the core temperatures of 32-34 A degrees C; however, the optimum temperature of therapeutic hypothermia is not fully defined. Here we studied whether hypothermic culture at 35 A degrees C had the same effects on the decrease of time-dependent expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, and nitric oxide (NO) by stimuli-activated microglia as that at 33 A degrees C, as determined in our previous reports, and whether these factors directly induced neuronal cell death. We 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 A degrees C), minimal hypothermic (35 A degrees C), and normothermic (37 A degrees C) conditions. We also determined the viability of rat neuronal pheochromocytoma PC12 cells treated with recombinant TNF-alpha or IL-10 or (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3, an NO donor). Production of TNF-alpha, 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-alpha, IL-10, and NOR3 caused the death of PC12 cells in a concentration-dependent manner after 24 h. Hypothermic culture at 35 A degrees C decreased the production of early-phase TNF-alpha and late-phase IL-10 and NO from ATP- and TLR-activated microglia as observed at 33 A degrees 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-alpha, IL-10, and NO by therapeutic hypothermia leads to the inhibition of neuronal cell death. Minimal hypothermia at 35 A degrees C may be sufficient to elicit neuroprotective effect.