Hyperoxia-induced immature brain injury through the TLR4 signaling pathway in newborn mice

Abstract

Toll-like receptor 4 (TLR4), a pathogen-associated molecular pattern receptor, is known to initiate an inflammatory cascade in response to certain stimuli within the central nervous system (CNS). Although TLR4 activation is known to be a first-line response of the innate immune system, whether and how hyperoxia influences TLR4 signaling in an immature brain remains unclear. In this study, TLR4 wild-type (W) and TLR4 knock-out(M) mice were exposed to 100% oxygen (the WO2 and MO2 groups, respectively), and control groups were exposed to ambient air (the WA and MA groups, respectively) for 48h after postnatal-day (PND) 3. Next, neuronal apoptosis was quantified, and Morris water maze assays were conducted. The WO2 mice showed increased TLR4 expression compared with the WA mice, additionally, the expression level of Tumor Necrosis Factor-α (TNF-α) in the WO2 mice was significantly increased compared with the levels in the WA, MA and MO2 mice. Electron microscopy and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assays showed a significant increase, compared to the WO2 mice, in neuronal apoptosis within the prefrontal cortex and hippocampal CA1 region in the WO2 mice. In contrast, there were no obvious differences in neuronal apoptosis between the MO2 and MA groups. The results of the Morris water maze tests demonstrated marked deficits in learning and memory in the WO2 mice but much milder deficits in the MO2 mice compared to the WA and MA groups, respectively. Moreover, cultured N9 (TLR4 wild-type, derived from ICR/CD1 mice) microglia exposed to hyperoxia showed an immediate increase in the expression of TLR4 mRNA, followed by an increase in the expression of both TNF-α and reactive oxygen species (ROS), but this increase was abrogated by the loss of TLR4 signaling in TLR4-knockout microglia (primary cells from a C3H/HeJ strain defective in TLR4). Taken together, these data suggest that 1) TLR4 signaling is involved in hyperoxia-induced immune responses in the immature brain and 2) the loss of TLR4 activation may abrogate the neuronal apoptosis and cognitive deficits following hyperoxia exposure in newborn mice.