Neurological deficits in children, including cerebral palsy, are associated with prior infection during the perinatal period. Experimentally, we have shown that pre-exposure to the Gram-negative component LPS potentiates hypoxic-ischemic (HI) brain injury in newborn animals. LPS effects are mediated by binding to TLR4, which requires recruitment of the MyD88 adaptor protein or Toll/IL-1R domain-containing adapter inducing IFN-β for signal transduction. In this study, we investigated the role of MyD88 in neonatal brain injury. MyD88 knockout (MyD88 KO) and wild-type mice were subjected to left carotid artery ligation and 10% O2 for 50 min on postnatal day 9. LPS or saline were administered i.p. at 14 h before HI. At 5 days after HI in wild-type mice, LPS in combination with HI caused a significant increase in gray and white matter tissue loss compared with the saline-HI group. By contrast, in the MyD88 KO mice there was no potentiation of brain injury with LPS-HI. MyD88 KO mice exhibited reduced NFκB activation and proinflammatory cytokine-chemokine expression in response to LPS. The number of microglia and caspase-3 activation was increased in the brain of MyD88 KO mice after LPS exposure. Collectively, these findings indicate that MyD88 plays an essential role in LPS-sensitized HI neonatal brain injury, which involves both inflammatory and caspase-dependent pathways.

Susceptibility and progression of brain injury in the newborn is closely associated with an exacerbated innate immune response, but the underlying mechanisms are often unclear. Toll-like receptors (TLRs) are important innate immune sensors that may influence the vulnerability of the developing brain. In the current study, we provide novel data to show that activation of the viral innate immune receptor TLR-3 sensitizes the neonatal brain to subsequent hypoxic-ischemic (HI) damage. Poly inosinic:poly cytidylic acid (Poly I:C), a synthetic ligand for TLR-3, was administered to neonatal mice 14 h before cerebral HI. Activation of TLR-3 before HI increased infarct volume from 3.0 Ϯ 0.5 to 15.4 Ϯ 2.1 mm 3 and augmented loss of myelin basic protein from 13.4 Ϯ 6.0 to 70.6 Ϯ 5.3%. The sensitizing effect of Poly I:C was specific for the TLR-3 pathway because mice deficient in the TLR-3 adaptor protein Toll/IL-1R domain-containing adaptor molecule-1 (TRIF) did not develop larger brain damage. The increased vulnerability was associated with a TRIF-dependent heightened inflammatory response, including proinflammatory cytokines, chemokines, and the apoptosis-associated mediator Fas, whereas there was a decrease in reparative M2-like CD11b ϩ microglia and phosphorylation of Akt. Because TLR-3 is activated via double-stranded RNA during most viral infections, the present study provides evidence that viral infections during pregnancy or in the neonate could have great impact on subsequent HI brain injury.

BackgroundHypoxic-ischemic (HI) brain injury remains a major problem in newborns, resulting in increased risk of neurological disorders. Neonatal HI triggers a broad inflammatory reaction in the brain, including activation of the innate immune system. Toll-like receptors (TLRs), which are key components of the innate immune system, are believed to play a role in adult cerebral ischemic injury. The expression of TLRs in the neonatal brain and their regulation after HI is unknown.MethodsWild type C57BL/6, TLR 1 knockout (KO) and TLR 2 KO mice were subjected to HI at postnatal day 9 and sacrificed 30 min, 6 h, 24 h or 5 days after HI. TLR mRNA expression was determined by RT-qPCR and protein and cell type localisation by immunohistochemistry (IHC). To evaluate brain injury, infarct volume was measured in the injured hemisphere.ResultsmRNA expression was detected for all investigated TLRs (TLR1-9), both in normal and HI exposed brains. After HI, TLR-1 was down-regulated at 30 min and up-regulated at 6 h and 24 h. TLR-2 was up-regulated at 6 h and 24 h, and TLR-7 at 24 h. Both TLR-5 and TLR-8 were down-regulated at 24 h and 30 min respectively. IHC showed an increase of TLR-1 in neurons in the ipsilateral hemisphere after HI. TLR-2 was constitutively expressed in astrocytes and in a population of neurons in the paraventricular nucleus in the hypothalamus. No changes in expression were detected following HI. Following HI, TLR-2 KO mice, but not TLR-1 KO, showed a decreased infarct volume compared to wild type (p = 0.0051).ConclusionsThis study demonstrates that TLRs are regulated after HI in the neonatal brain. TLR-1 protein was up-regulated in injured areas of the brain but TLR-1 KO animals were not protected from HI. In contrast, TLR-2 was constitutively expressed in the brain and TLR-2 deficiency reduced HI injury. These data suggest that TLR-2, but not TLR-1, plays a role in neonatal HI brain injury.

The choroid plexus is the site of the blood-cerebrospinal fluid (CSF) barrier (BCSFB) and has also been considered as a possible route for peripheral immune signals and cells to transfer to the central nervous system. Infection/ inflammation stimulates innate and subsequent adaptive immune responses via Toll-like receptors (TLRs). In this study, we have investigated the mRNA expression of TLRs, cytokines, and tight junction proteins in the choroid plexus in the immature brain after systemic inflammation, as well as accumulation of immune cells into the CSF. Specific ligands for TLR-1/2, TLR-3, and TLR-4 were administered to postnatal day 8 mice and mRNA expression for the targeted genes was examined in the choroid plexus. We found that mRNA for all four TLRs was detected in the choroid plexus under control conditions. Following immune stimulation, expression of all the TLRs was upregulated by their respective ligands, except for TLR-4 mRNA, which was downregulated by Pam 3 CSK 4 (PAM; a TLR-1/2 ligand). In addition, we investigated BCSFB regulation after TLR stimulation and found that TLR-1/2 and TLR-4 activation was associated with changes in mRNA expression of the tight junction protein occludin in the choroid plexus. PAM induced choroid plexus transcription of TNF-α and resulted in the most dramatic increase in numbers of white blood cells in the CSF. The data suggest a possible mechanism whereby systemic inflammation stimulates TLRs in the choroid plexus, which may lead to disturbances in choroid plexus barrier function, as well as infiltration of immune cells through the plexus.

Toll-like receptor (TLR)3 activation during the neonatal period produces responses linked to the origins of neuropsychiatric disorders. Although there is sexual dimorphism in neuropsychiatric disorders, it is unknown if brain responses to TLR3 activation are sex-specific. We hypothesized that poly I:C in a post-natal day (P)8 model induces a sexually dimorphic inflammatory responses. C57BL6 mice received intraperitoneal injection of poly I:C (10 mg/kg) or vehicle [normal saline (NS)] at P8. Pups were killed at 6 or 14 h for caspase 3 and 8 activity assays, NFkB ELISA, IRF3, AP1, and GFAP western blotting and cytokines/chemokines gene expression real time qRT-PCR (4–6/group). A second group of pups were killed at 24 h (P9) or 7 days (P15) after poly I:C to assess astrocytic (GFAP) and microglia (Iba1) activation in the hippocampus, thalamus and cortex using immunohistochemistry, and gene and protein expression of cytokines/chemokines using real time RT-PCR and MSD, respectively (4–6/group). Non-parametric analysis was applied. Six hours after poly I:C, caspase-3 and -8 activities in cytosolic fractions were 1.6 and 2.8-fold higher in poly I:C-treated than in NS-treated female mice, respectively, while gene expressions of pro-inflammatory cytokines were upregulated in both sexes. After poly I:C, IRF3 nuclear translocation occurred earlier (6 h) in female mice and later (14 h) in male mice. At 14 h after poly I:C, only male mice also had increased nuclear NFκB levels (88%, p < 0.001) and GFAP expression coinciding with persistent IL-6 and FAS gene upregulation (110 and 77%, respectively; p < 0.001) and IL-10 gene downregulation (-42%, p < 0.05). At 24 h after poly I:C, IL-1β, CXCL-10, TNF-α, and MCP-1 were similarly increased in both sexes but at 7 days after exposure, CXCL-10 and INFγ were increased and IL-10 was decreased only in female mice. Accordingly, microglial activation persisted at 7 days after poly I:C in the hippocampus, thalamus and cortex of female mice. This preliminary study suggests that TLR3 activation may produce in the developing neonatal mouse brain a sexually dimorphic response with early activation of caspase-dependent pathways in female mice, activation of inflammatory cascades in both sexes, which then persists in female mice. Further well-powered studies are essential to confirm these sex-specific findings.