title: The role of tumor necrosis factor-α and TNF-α receptors in cerebral arteries following cerebral ischemia in rat.
abstract: BACKGROUND
  Tumour necrosis factor-α (TNF-α) is a pleiotropic pro-inflammatory cytokine, which is rapidly upregulated in the brain after injury. TNF-α acts by binding to its receptors, TNF-R1 (p55) and TNF-R2 (p75), on the cell surface. The aim of this study was first to investigate if there is altered expression of TNF-α and TNF-α receptors in cerebral artery walls following global or focal ischemia, and after organ culture. Secondly, we asked if the expression was regulated via activation of the MEK-ERK1/2 pathway.
  
  
  METHODS
  The hypothesis was tested in vivo after subarachnoid hemorrhage (SAH) and middle cerebral artery occlusion (MCAO), and in vitro by organ culture of isolated cerebral arteries. The localization and amount of TNF-α, TNF-α receptor 1 and 2 proteins were analysed by immunohistochemistry and western blot after 24 and 48 h of organ culture and at 48 h following SAH or MCAO. In addition, cerebral arteries were incubated for 24 or 48 h in the absence or presence of a B-Raf inhibitor (SB386023-b), a MEK- inhibitor (U0126) or an NF-κB inhibitor (IMD-0354), and protein expression evaluated.
  
  
  RESULTS
  Immunohistochemistry revealed enhanced expression of TNF-α, TNF-R1 and TNF-R2 in the walls of cerebral arteries at 48 h after MCAO and SAH compared with control. Co-localization studies showed that TNF-α, TNF-R1 and TNF-R2 were primarily localized to the cell membrane and the cytoplasm of the smooth muscle cells (SMC). There was, in addition, some expression of TNF-R2 in the endothelial cells. Immunohistochemistry and western blot analysis showed that these proteins were upregulated after 24 and 48 h in culture, and this upregulation reached an apparent maximum at 48 h of organ culture. Treatment with U0126 significantly reduced the enhanced SMC expression of TNF-α, TNF-R1 and TNF-R2 immunoreactivities after 24 and 48 h of organ culture. The Raf and NF-κB inhibitors significantly reduced organ culture induced TNF-α expression while they had minor effects on the TNF-α receptors.
  
  
  CONCLUSIONS
  The present study shows that cerebral ischemia and organ culture induce expression of TNF-α and its receptors in the walls of cerebral arteries and that upregulation is transcriptionally regulated via the MEK/ERK pathway.
authors.affiliation: Department of Clinical Sciences, Division of Experimental Vascular Research, Lund University, Lund, Sweden. aida.maddahi@med.lu.se
authors.forename: Aida
authors.initials: A
authors.lastname: Maddahi
authors.forename: Lars S
authors.initials: LS
authors.lastname: Kruse
authors.forename: Qing-Wen
authors.initials: QW
authors.lastname: Chen
authors.forename: Lars
authors.initials: L
authors.lastname: Edvinsson
mesh_terms: D000818:Animals; D002545:Brain Ischemia / Q000378:metabolism* / Q000473:pathology; D002536:Cerebral Arteries / Q000033:anatomy & histology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020244:Infarction, Middle Cerebral Artery; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology; D008297:Male; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D048493:Proto-Oncogene Proteins B-raf / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism*; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000378:metabolism*; D046509:Tissue Culture Techniques; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D016328:NF-kappa B; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D014409:Tumor Necrosis Factor-alpha; D048493:Proto-Oncogene Proteins B-raf; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2