Background: White matter injury is the major form of brain damage in very preterm infants. Selective white matter injury in the immature brain can be induced by lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI) in the postpartum (P) day 2 rat pups whose brain maturation status is equivalent to that in preterm infants less than 30 weeks of gestation. Neuroinflammation, blood–brain barrier (BBB) damage and oligodendrocyte progenitor apoptosis may affect the susceptibility of LPS-sensitized HI in white matter injury. c-Jun N-terminal kinases (JNK) are important stress-responsive kinases in various forms of insults. We hypothesized that LPS-sensitized HI causes white matter injury through JNK activation-mediated neuroinflammation, BBB leakage and oligodendroglial apoptosis in the white matter of P2 rat pups. Methods: P2 pups received LPS (0.05 mg/kg) or normal saline injection followed by 90-min HI. Immunohistochemistry and immunoblotting were used to determine microglia activation, TNF-α, BBB damage, cleaved caspase-3, JNK and phospho-JNK (p-JNK), myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP) expression. Immunofluorescence was performed to determine the cellular distribution of p-JNK. Pharmacological and genetic approaches were used to inhibit JNK activity. Results: P2 pups had selective white matter injury associated with upregulation of activated microglia, TNF-α, IgG extravasation and oligodendroglial progenitor apoptosis after LPS-sensitized HI. Immunohistochemical analyses showed early and sustained JNK activation in the white matter at 6 and 24 h post-insult. Immunofluorescence demonstrated upregulation of p-JNK in activated microglia, vascular endothelial cells and oligodendrocyte progenitors, and also showed perivascular aggregation of p-JNK-positive cells around the vessels 24 h post-insult. JNK inhibition by AS601245 or by antisense oligodeoxynucleotides (ODN) significantly reduced microglial activation, TNF-α immunoreactivity, IgG extravasation, and cleaved caspase-3 in the endothelial cells and oligodendrocyte progenitors, and also attenuated perivascular aggregation of p-JNK-positive cells 24 h post-insult. The AS601245 or JNK antisense ODN group had significantly increased MBP and decreased GFAP expression in the white matter on P11 than the vehicle or scrambled ODN group. Conclusions: LPS-sensitized HI causes white matter injury through JNK activation-mediated upregulation of neuroinflammation, BBB leakage and oligodendrocyte progenitor apoptosis in the immature brain.

Hypoxic-ischemia (HI) and inflammation are the two major pathogenic mechanisms of brain injury in very preterm infants. The neurovascular unit is the major target of HI injury in the immature brain. Systemic inflammation may worsen HI by up-regulating neuroinflammation and disrupting the blood–brain barrier (BBB). Since neurons and oligodendrocytes, microvascular endothelial cells, and microglia may closely interact with each other, there may be a common signaling pathway leading to neuroinflammation and neurovascular damage after injury in the immature brain. TNF-α is a key pro-inflammatory cytokine that acts through the TNF receptor (TNFR), and c-Jun N-terminal kinases (JNK) are important stress-responsive kinases. To determine if TNFR1-JNK signaling is a shared pathway underlying neuroinflammation and neurovascular injury after lipopolysaccharide (LPS)-sensitized HI in the immature brain. Postpartum (P) day-5 mice received LPS or normal saline (NS) injection before HI. Immunohistochemistry, immunoblotting and TNFR1- and TNFR2-knockout mouse pups were used to determine neuroinflammation, BBB damage, TNF-α expression, JNK activation, and cell apoptosis. The cellular distribution of p-JNK, TNFR1/TNFR2 and cleaved caspase-3 were examined using immunofluorescent staining. The LPS + HI group had significantly greater up-regulation of activated microglia, TNF-α and TNFR1 expression, and increases of BBB disruption and cleaved caspase-3 levels at 24 hours post-insult, and showed more cortical and white matter injury on P17 than the control and NS + HI groups. Cleaved caspase-3 was highly expressed in microvascular endothelial cells, neurons, and oligodendroglial precursor cells. LPS-sensitized HI also induced JNK activation and up-regulation of TNFR1 but not TNFR2 expression in the microglia, endothelial cells, neurons, and oligodendrocyte progenitors, and most of the TNFR1-positive cells co-expressed p-JNK. Etanercept (a TNF-α inhibitor) and AS601245 (a JNK inhibitor) protected against LPS-sensitized HI brain injury. The TNFR1-knockout but not TNFR2-knockout pups had significant reduction in JNK activation, attenuation of microglial activation, BBB breakdown and cleaved caspase-3 expression, and showed markedly less cortical and white matter injury than the wild-type pups after LPS-sensitized HI. TNFR1-JNK signaling is the shared pathway leading to neuroinflammation and neurovascular damage after LPS-sensitized HI in the immature brain.

Background: Apoptosis, neuroinflammation and blood-brain barrier (BBB) damage affect the susceptibility of the developing brain to hypoxic-ischemic (HI) insults. c-Jun N-terminal kinase (JNK) is an important mediator of insulin resistance in obesity. We hypothesized that neonatal overweight aggravates HI brain damage through JNK hyperactivation-mediated upregulation of neuronal apoptosis, neuroinflammation and BBB leakage in rat pups. Methods: Overweight (OF) pups were established by reducing the litter size to 6, and control (NF) pups by keeping the litter size at 12 from postnatal (P) day 1 before HI on P7. Immunohistochemistry and immunoblotting were used to determine the TUNEL-(+) cells and BBB damage, cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP), and phospho-JNK and phospho-Bim_EL levels. Immunofluorescence was performed to determine the cellular distribution of phospho-JNK. Results: Compared with NF pups, OF pups had a significantly heavier body-weight and greater fat deposition on P7. Compared with the NF-HI group, the OF-HI group showed significant increases of TUNEL-(+) cells, cleaved levels of caspase-3 and PARP, and ED1-(+) activated microglia and BBB damage in the cortex 24 hours post-HI. Immunofluorescence of the OF-HI pups showed that activated-caspase 3 expression was found mainly in NeuN-(+) neurons and RECA1-(+) vascular endothelial cells 24 hours post-HI. The OF-HI group also had prolonged escape latency in the Morris water maze test and greater brain-volume loss compared with the NF-HI group when assessed at adulthood. Phospho-JNK and phospho-Bim_EL levels were higher in OF-HI pups than in NF-HI pups immediately post-HI. JNK activation in OF-HI pups was mainly expressed in neurons, microglia and vascular endothelial cells. Inhibiting JNK activity by AS601245 caused more attenuation of cleaved caspase-3 and PARP, a greater reduction of microglial activation and BBB damage post-HI, and significantly reduced brain damage in OF-HI than in NF-HI pups. Conclusions: Neonatal overweight increased HI-induced neuronal apoptosis, microglial activation and BBB damage, and aggravated HI brain damage in rat pups through JNK hyperactivation.

Background: Hypoxia/ischemia and inflammation are two major mechanisms for cerebral palsy (CP) in preterm infants. Objective: To investigate whether hypoxia/ischemia- and infection-related events in the perinatal and neonatal periods had cumulative effects on CP risk in very-low-birth-weight (VLBW) premature infants. Methods: From 1995 to 2005, 5,807 VLBW preterm infants admitted to Taiwan hospitals were enrolled. The cumulative effects of hypoxic/ischemic and infectious events during the perinatal and neonatal periods on CP risk at corrected age 24 months were analyzed. Results: Of the 4,355 infants with 24-month follow-up, 457 (10.5%) had CP. The CP group had significantly higher incidences of hypoxia/ischemia-related events in the perinatal and neonatal periods, and sepsis in the neonatal period than the normal group. Three hypoxic/ischemic events, including birth cardiopulmonary resuscitation (OR 2.25; 95% CI 1.81-2.82), patent ductus arteriosus (PDA) ligation (2.94; 1.35-5.75) and chronic lung disease (3.14; 2.61-3.85) had the most significant contribution to CP. Relative to CP risk for infants with neither the three hypoxic/ischemic events nor sepsis, the CP odds increased 1.98-, 2.26- and 2.15-fold for infants with birth cardiopulmonary resuscitation, PDA ligation and chronic lung disease, respectively; while the combination with sepsis further increased the odds to 3.18-, 3.83- and 3.25-fold, respectively. Using the three hypoxic/ischemic events plus sepsis, CP rates were 10.0, 16.7, 26.7, 40.0 and 54.7% for infants with none, one, two, three and four events, respectively. Conclusions: Hypoxic/ischemic and infectious events across the perinatal and neonatal periods exerted cumulative effects on CP risk in VLBW premature infants.

Background: Cystic periventricular leukomalacia (cPVL) is the most severe white matter injury and is often associated with intraventricular hemorrhage (IVH) in preterm infants. Objective: The aim of this study was to investigate the prevalence, risk factors and neurodevelopmental outcomes of isolated cPVL and cPVL with low-grade and high-grade IVH in premature infants. Methods: From 2001 to 2012, 9,964 infants with <31 weeks' gestational age (GA) admitted to Taiwan hospitals were enrolled. cPVL was classified into three groups: isolated cPVL, cPVL with low-grade (I/II) IVH, and cPVL with high-grade (III) IVH. Results: Of 7,805 infants with complete ultrasound data, 286 (3.7%) had cPVL. Among the cPVL infants, 93 (32.5%) were isolated, 118 (41.3%) had low-grade IVH and 75 (26.2%) had high-grade IVH. The risk of cPVL with IVH was significantly higher among infants with <27 weeks' GA than those with ≥27 weeks' GA, in contrast to that of isolated cPVL. Using infants without cPVL and IVH as the reference group, the most significant predictor of isolated cPVL was neonatal sepsis (odds ratio 2.39; 95% confidence interval 1.52-3.77), while 5-min Apgar score <5 (2.50; 1.48-4.21) and prolonged mechanical ventilation (2.19; 1.42-3.42) were associated with cPVL with low-grade IVH, and GA <27 weeks (2.63; 1.56-4.42), pneumothorax (3.04; 1.40-6.65) and prolonged mechanical ventilation (3.36; 1.88-6.01) contributed to cPVL with high-grade IVH. cPVL infants with low-grade and high-grade IVH had a higher risk of abnormal neurodevelopmental outcomes than infants with isolated cPVL at the age of 24 months. Conclusions: Isolated cPVL, cPVL with low-grade IVH and cPVL with high-grade IVH had different risk factors and neurodevelopmental outcomes, suggestive of different causal pathways.