Significance was defined asp< 0.05. Images were created using Coreldraw (v. and its amplification by inhibition of coincident anti-inflammatory cytokine signaling, may be opportune targets for the development of novel therapeutics that can mimic the protection seen in cold pre-conditioning. Keywords:hippocampus, hormesis, innate immunity, interleukin-1, microglia, slice culture Cold pre-conditioning effectively reduces brain injury in experimental animals (Nishioet al.2000;Yunokiet al.2002). However, no clinical pre-conditioning treatment strategies based on hypothermia have been developed to reduce neurological complications associated with general anesthesia and related surgical procedures (Molleret al.1998;Bendszus and Stoll 2006;McKhannet al.2009). This void likely results from the inherent difficulties in administering this form of therapy, which to date has only been applied after the onset of brain disease (for review seeSchaller and Graf 2003;Tang and Yenari 2010). In addition, the underlying mechanisms of cold pre-conditioning are unknown. This precludes the development of effective cold pre-conditioning mimetics, although evidence suggests involvement of cytokines. Fairchildet al.(2000)show thatin vitroexposure of monocytes pre-activated by lipopolysaccharide ARQ 197 (Tivantinib) to hypothermia triggers enhanced production of tumor necrosis factor alpha (TNF-) and interleukin (IL)-1. Microglia, although perhaps not solely derived from monocytes (Simard and Rivest 2004;Chanet al.2007), are a similar predominant source of cytokines (Hanisch 2002) including TNF- in uninjured brain (Hulseet al.2008). Furthermore, microglia are activated by synaptic activity (Zivet al.2006), which may act as an adequate pre-activating stimulus for TNF- production (Kraiget al.2010) necessary for cold preconditioning to be effective. Second, a wide array of preconditioning stimuli evoke subsequent neuroprotection via mechanisms involving TNF- and microglia (for review seeHallenbeck 2002;Kraiget al.2010). Accordingly, we examined brain cytokine signaling in cold pre-conditioning using hippocampal slice cultures from rats. Slice cultures are ideally suited to this purpose because, while deafferented, they are a mature and functionally intact area of brain that remains viable and stable for weeksin vitro. Importantly, slice culture longevity allows microglia time to become quiescent after 10 days in culture, making the preparation ideal for study of neural immune signalingin vitro, where environmental conditions ARQ 197 (Tivantinib) can be accurately controlled (Ransohoff and Perry 2008). Our results confirmed that cold pre-conditioning neuroprotection involved increased expression of TNF- from microglia. Considerable evidence points to the involvement of TNF- in an array of pre-conditioning paradigms. However, IL-11 inhibits TNF- production. Accordingly, we also focused to the potential involvement of IL-11 in cold pre-conditioning. Our results provide the first evidence that removal of an anti-inflammatory cytokine, namely IL-11, enhances cold pre-conditioning protection. This work has appeared in preliminary form (Kraiget al.2008;Mitchellet al.2009). == Materials and methods == == Culture preparation and maintenance == We prepared slice cultures and initially maintained them in media containing 23% horse serum (#26050088; Invitrogen, Carlsbad, CA, USA;Kunkler and Kraig 1997) with transfer to serum-free media after 7 daysin vitroand experimental use between 18 and 24 dayin vitro. Cultures maintained in serum-free media showed ~90% vitality (Appendix S1). == Experimental manipulations == We administered cold pre-conditioning at several temperatures and over various time periods to establish doseresponse patterns. Sixwell trays containing serum-free media were allowed to equilibrate to hypothermic temperatures (25.5, 28, 30 and 32C) for at least 20 min prior to cold pre-conditioning at these temperatures in an incubator (5% CO2balance air). Slice cultures were transferred from normal incubation conditions to cold pre-conditioning trays for 20, 40, 60, ARQ 197 (Tivantinib) 90, 120, 150, or 180 min. Cultures were then transferred back to media equilibrated at normal incubation conditions for 24 h before excitotoxic damage (defined below). Using the neuroprotection response design set up (Fig. 1), we performed all the frosty preconditioning at 30C for 90 min because this heat range demonstrated FGF22 the broadest effective range and 90 min was about mid-range. == Fig. 1. == Cool pre-conditioning was neuroprotective and implemented a U-shaped heat range and period doseresponse design. (a) Immunostaining for NeuN displays the typical primary neuron cytoarchitecture.
