title: TNFR1-JNK signaling is the shared pathway of neuroinflammation and neurovascular damage after LPS-sensitized hypoxic-ischemic injury in the immature brain.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  To determine if TNFR1-JNK signaling is a shared pathway underlying neuroinflammation and neurovascular injury after lipopolysaccharide (LPS)-sensitized HI in the immature brain.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  TNFR1-JNK signaling is the shared pathway leading to neuroinflammation and neurovascular damage after LPS-sensitized HI in the immature brain.
authors.affiliation: Department of Pediatrics, Chi Mei Medical Center, Tainan, 710, Taiwan. lanwan@ms67.url.com.tw.|Department of Pediatrics, College of Medicine, Taipei Medical University, #250, Wu-Hsing Street, Taipei, 11031, Taiwan. lanwan@ms67.url.com.tw.|Department of Pediatrics, School of Medicine, Chung Shan Medical University, Taichung, 402, Taiwan. lanwan@ms67.url.com.tw.
authors.forename: Lan-Wan
authors.initials: LW
authors.lastname: Wang
authors.affiliation: Department of Pediatrics, Chang Gung Memorial Hospital - Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, 833, Taiwan. chao8725@ms16.hinet.net.
authors.forename: Ying-Chao
authors.initials: YC
authors.lastname: Chang
authors.affiliation: Department of Pediatrics, Tri-Service General Hospital, National Defense Medical Center, Taipei, 114, Taiwan. pedneuchen@hotmail.com.
authors.forename: Shyi-Jou
authors.initials: SJ
authors.lastname: Chen
authors.affiliation: Department of Pediatrics, College of Medicine, National Cheng Kung University Hospital, National Cheng Kung University, Tainan, 704, Taiwan. chiens16984129@gmail.com.
authors.forename: Chien-Hang
authors.initials: CH
authors.lastname: Tseng
authors.affiliation: Department of Pediatrics, College of Medicine, National Cheng Kung University Hospital, National Cheng Kung University, Tainan, 704, Taiwan. nckutu@gmail.com.
authors.forename: Yi-Fang
authors.initials: YF
authors.lastname: Tu
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, 115, Taiwan. mbfelix@imb.sinica.edu.tw.
authors.forename: Nan-Shih
authors.initials: NS
authors.lastname: Liao
authors.affiliation: Department of Pediatrics, College of Medicine, Taipei Medical University, #250, Wu-Hsing Street, Taipei, 11031, Taiwan. huangped@mail.ncku.edu.tw.|Department of Pediatrics, Wan-Fang Hospital, Taipei Medical University, Taipei, 110, Taiwan. huangped@mail.ncku.edu.tw.|Department of Pediatrics, College of Medicine, National Cheng Kung University Hospital, National Cheng Kung University, Tainan, 704, Taiwan. huangped@mail.ncku.edu.tw.
authors.forename: Chao-Ching
authors.initials: CC
authors.lastname: Huang
authors.affiliation: Department of Pediatrics, College of Medicine, National Cheng Kung University Hospital, National Cheng Kung University, Tainan, 704, Taiwan. z8408140@email.ncku.edu.tw.
authors.forename: Chien-Jung
authors.initials: CJ
authors.lastname: Ho
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D001921:Brain / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005260:Female; D020925:Hypoxia-Ischemia, Brain / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D007249:Inflammation / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D008070:Lipopolysaccharides / Q000633:toxicity*; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008297:Male; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000096:biosynthesis*
chemical_list: D008070:Lipopolysaccharides; D047888:Receptors, Tumor Necrosis Factor, Type I; C506236:Tnfrsf1a protein, mouse