Briefly, the fractional anisotropy (FA) maps were created by fitting a tensor model to the raw diffusion data using FDT (Functional MRI of the Brain Diffusion Toolbox) and then brain extracted with BET (FMRIB Brain Extract Tool;Smith, 2002). better preservation of growth and white matter functions. The FTY720 treatment also provided dose-dependent reduction of brain atrophy, rescuing >90% of LPS/HI-induced brain tissue loss. Interestingly, FTY720 neither opposed pure-HI brain injury nor directly inhibited microglia in bothin vivoandin vitromodels, highlighting its unique mechanism against inflammation-sensitized HI injury. Together, these results suggest that the dual hit of systemic inflammation and neonatal HI injury triggers early onset of the TH17/IL-17-mediated immunity, which causes severe brain destruction but responds remarkably to the therapeutic blockade of lymphocyte trafficking. Keywords:adaptive immunity, chorioamnionitis, choroid plexus, Fingolimod, FTY720, Th17 == Introduction == Intrauterine contamination (chorioamnionitis) is Nrp2 usually a high risk factor for cerebral palsy in term and near-term neonates (Wu et al., 2003). The combination of perinatal contamination/inflammation and hypoxicischemic (HI) insult also causes greater brain injury and poorer response to therapeutic hypothermia (Wintermark et al., 2010;Hagberg et al., 2012;Osredkar et al., 2014). The true incidence of chorioamnionitis, ranging from 3 to 30% in a meta-analysis (Wu, 2002), is usually unclear because of heterogeneity in the criteria for clinical diagnosis. These concerns highlight the need for better understanding and more effective therapy of infection-sensitized HI injury in newborns. In addition, it remains a puzzle how remote maternal contamination aggravates neonatal HI brain injury (Leviton et al., 2005), because newborns were often considered immunodeficient, lacking the adaptive immunity to initiate systemic anti-pathogen responses. However, it has been reported that chorioamnionitis induces circulating CD45RO-positive (CD45RO+) effector/memory CFTR-Inhibitor-II T-cells associated with brain damage in preterm neonates (Duggan et al., 2001). Because the traditional T-helper 1 (TH1) or TH2 cells are yet to mature in neonates (Sharma et al., 2012), we hypothesized that this early-onset active T-cells after contamination/HI injury may be TH17-like lymphocytes that have a high propensity to induce autoimmune and inflammatory disorders (Korn et al., 2009). This hypothesis is in accord with recent findings in rhesus macaques showing that experimental chorioamnionitis alters the regulatory T-cell/L-17 balance (Kallapur et al., 2013). Furthermore, several critical factors for TH17-lymphocyte differentiation, including interleukin-6 (IL-6) and IL-1, are induced by neonatal HI injury (Bettelli et al., 2006;Ghoreschi et al., 2010). If this hypothesis is usually accurate, inflammation-sensitized neonatal HI brain injury may be treated by systemic administration of FTY720 (fingolimod), a sphingosine-1-phosphate receptor (S1PR) agonist that prevents lymphocytes from exiting the lymph nodes and reaching the target organs (Brinkmann et al., 2010). To test this hypothesis, we compared the expression of TH17-lymphocyte markers in cord blood mononuclear cells (CBMCs) among late-preterm neonates with and without chorioamnionitis. We then tested the effects of FTY720 in a lipopolysaccharide (LPS)-sensitized HI brain injury model, in which rodent neonates were exposed to low-dose LPS to mimic subclinical contamination and convert a moderate or subthreshold HI insult to CFTR-Inhibitor-II severe brain damage in a Toll-like receptor-4 and myeloid differentiation primary response gene 88 (MyD88)-dependent manner (Lehnardt et al., 2003;Eklind et al., 2005;Wang et al., 2009). The dual LPS/HI insult also accelerates microglial activation and the brain nuclear factor-B (NF-B) signaling (Yang et al., 2013a,b), making it a unique paradigm to investigate the mechanisms that connectin systemic inflammatory responses CFTR-Inhibitor-II to HI brain injury. Our experiments showed that this dual hit of systemic LPS exposure and HI triggers early influx of leukocytes and severe brain damage in rodent neonates. However, systemic administration of FTY720 markedly attenuated the LPS/HI-induced brain damage and neuroinflammation, despite CFTR-Inhibitor-II its inability to block pure-HI insult or directly inhibit microglial activation. These results suggest unique pathological mechanisms and a novel therapy of inflammation-sensitized HI brain injury in newborns. == Materials and Methods == == == == == == LPS/HI surgery and drug treatment. == The experimental paradigm of LPS-sensitized HI was performed as described previously (Eklind et al., 2005;Wang et al., 2009;Yang et al., 2013a,b). Briefly, LPS (0.3 mg/kg body weight) was injected intraperitoneally to 7-d-old Wistar rat pups 4 h before the induction of the Vannucci HI procedure. Low-dose LPS was used to mimic subclinical contamination, which by itself caused no apparent brain damage but converted a moderate or subthreshold HI insult to severe brain damage (Eklind et al., 2005;Yang et al., 2013b). Rat pups were anesthetized by 3% CFTR-Inhibitor-II isoflurane mixed with room air when the right common carotid artery was ligated. After 1 h recovery, pups were placed in glass chambers infused with 10% oxygen/90% nitrogen gas and submerged in a 37C water bath. For.