title: Quantitative real-time RT-PCR analysis of inflammatory gene expression associated with ischemia-reperfusion brain injury.
abstract: Ischemia-reperfusion brain injury initiates an inflammatory response involving the expression of adhesion molecules and cytokines, some of which are regulated by the nuclear transcription factor NF-kappaB. In this study the authors examined mRNA expression levels for several important genes associated with inflammation at five time points (3, 6, 12, 24, and 72 hours) after transient middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. A sensitive and quantitative technique (TaqMan real-time QRT-PCR) was used to simultaneously measure mRNA levels for key cell adhesion molecules and inflammatory cytokines. Gene expression increased significantly in the injured hemisphere for interleukin (IL)-1beta (12-fold increase at 24 hours), IL-6 (25-fold increase at 6 hours) and ICAM-1 (4-fold increase at 24 hours), and the interhemispheric differences for these genes were significant for every time point examined (P < 0.05 for all values). Tumor necrosis factor-alpha mRNA was upregulated in the injured versus uninjured hemisphere from 3 to 24 hours (5-fold increase at 6 hours), while E-selectin showed a significant increase in mRNA levels from 6 to 24 hours after MCAO (10-fold increase at 6 hours) (P < 0.05 for all values). VCAM-1 mRNA levels did not respond differentially to injury at any time point between the two brain hemispheres. At all time points examined, activated NF-kappaB immunoreactivity was observed in cells throughout the infarct-damaged tissue. These results are consistent with the proinflammatory properties of the induced molecules, which are involved in the initiation of the inflammatory cascade, and may thus contribute to secondary cellular responses that lead to further brain damage.
authors.affiliation: Neuropharmacology and Molecular Biology Department, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.
authors.forename: Rossana
authors.initials: R
authors.lastname: Berti
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Williams
authors.forename: John R
authors.initials: JR
authors.lastname: Moffett
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Hale
authors.forename: Luisa C
authors.initials: LC
authors.lastname: Velarde
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Elliott
authors.forename: Changping
authors.initials: C
authors.lastname: Yao
authors.forename: Jitendra R
authors.initials: JR
authors.lastname: Dave
authors.forename: Frank C
authors.initials: FC
authors.lastname: Tortella
mesh_terms: D000818:Animals; D001483:Base Sequence; D003342:Corpus Striatum / Q000276:immunology / Q000473:pathology; D017931:DNA Primers; D019040:E-Selectin / Q000235:genetics; D005786:Gene Expression Regulation; D007249:Inflammation / Q000235:genetics*; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics*; D015850:Interleukin-6 / Q000235:genetics*; D002546:Ischemic Attack, Transient / Q000235:genetics* / Q000503:physiopathology; D008297:Male; D020768:Middle Cerebral Artery; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000235:genetics* / Q000503:physiopathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics*
chemical_list: D017931:DNA Primers; D019040:E-Selectin; D015850:Interleukin-6; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1