Detection of tumor necrosis factor-alpha mRNA induction in ischemic brain tolerance by means of real-time polymerase chain reaction.
A short duration of ischemia (i.e., ischemic preconditioning) results in significant brain protection to subsequent severe ischemic insult. Because previous studies suggest that tumor necrosis factor-alpha (TNF-alpha) plays a role in both promoting ischemic damage and neuroprotection, the present work aimed to evaluate the expression of TNF-alpha mRNA in an established model of ischemic preconditioning using a transient 10-minute occlusion of the middle cerebral artery. Because the level of TNF-alpha mRNA expression in the brain was too low to be consistently detected by Northern technique, a real-time polymerase chain reaction method was applied to quantitate the absolute copy number of TNF-alpha transcript in rat brain after the preconditioning procedure. TNF-alpha mRNA was induced in the ipsilateral cortex as early as 1 hour (27 +/- 1 copies of mRNA per microgram of tissue compared to 11 +/- 3 copies in sham-operated samples) after preconditioning, reached a peak level at 6 hours (49 +/- 10 copies of transcript, n = 4, P < 0.01), and persisted up to 2 days. These data not only demonstrate the utility of real-time polymerase chain reaction for sensitive and accurate measurement of mRNA expression in normal and injured tissues but also suggest a potential role of TNF-alpha in the phenomenon of ischemic preconditioning.
Wang
X
X
Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania, USA.
Li
X
X
Erhardt
J A
JA
Barone
F C
FC
Feuerstein
G Z
GZ
D000818:Animals; D001921:Brain / Q000098:blood supply* / Q000378:metabolism*; D003001:Cloning, Molecular; D003199:Computer Systems; D004247:DNA / Q000235:genetics; D019194:Ischemic Preconditioning; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D012269:Ribosomal Proteins / Q000235:genetics; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D012333:RNA, Messenger; D012269:Ribosomal Proteins; D014409:Tumor Necrosis Factor-alpha; C041806:ribosomal protein L32; D004247:DNA