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 pre conditioning) results in s ignificant brain protection to subsequent severe ischemic insult. Because p revious studies suggest that tumor necrosis factor-alpha (TNF-alpha) plays a role in both promoting ischemic damage and neuroprotection, the present w ork aimed to evaluate the expression of TNF-alpha mRNA in an established mo del of ischemic preconditioning using a transient 10-minute occlusion of th e 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 a bsolute copy number of TNF-alpha transcript in rat brain after the precondi tioning 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 t o 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 potenti al role of TNF-alpha in the phenomenon of ischemic preconditioning.