Journal of Cerebral Blood Flow and Metabolism 20:15-20 2000 The International Society for Cerebral Blood Flow and Metabolism Published by Lippincott Williams & Wilkins, Inc., Philadelphia Short Communication Detection of Tumor Necrosis Factor-a mRNA Induction in Ischemic Brain Tolerance by Means of Real-Time Polymerase Chain Reaction Xinkang Wang, Xiang Li, Joseph A. Erhardt, Frank C. Barone, and Giora Z. Feuerstein Department of Cardiovascu lar Pharmacol ogy, SmithK l ine Beecham Pharmaceuticals, King of Prussia, Pennsylvania, U.S.A. mmry: A short duration of ischemia (i.e., ischemic pre conditioning) results in significant brain protection to subse quent severe ischemic insult. Because previous studies suggest that tumor necrosis factor-x (TNF-x) plays a role in both pro moting ischemic damage and neuroprotection, the present work aimed to evaluate the expression of TNF-x mRNA in an es tablished model of ischemic preconditioning using a transient 10-minute occlusion of the middle cerebral artery. Because the level of TNF-x mRNA expression in the brain was too low to be consistently detected by Northen technique, a real-time polymerase chain reaction method was applied to quantitate the absolute copy number of TNF-x transcript in rat brain after the Tumor necrosis factor a (TNF-a) is a pleitrophic cy tokine with diverse biologic activities. TNF-a is ex pressed in monocytes/macrophages as well as many other cells including glia and neurons (Akira et al., 1990; Liu et al., 1994; Munozfernandez and Fresno, 1998). Whereas normal brain tissue has little or no TNF-a ex pression, this cytokine is significantly induced ater tran sient or permanent focal cerebral ischemia (Liu et al., 1994; Wang et al., 1994; Buttini et al., 1996). In addition, TNF-a receptors are also present in the brain and may mediate neuronal and glial activation, proliferation, dif ferentiation, and survival (Munozfernandez and Fresno, Received April 21, 1999; final revision received July 23, 1999; ac cepted July 28, 1999. Address correspondence and reprint requests to Xinkang Wang, PhD, Department of Cardiovascular Sciences, Dupont Pharmaceuticals Company, Experimental Station, E400/3420B, Wilmington, DE 19880- 0400, U.S.A. Current address for X. Wang and G. . Feuerstein is Department of Cardiovascular Sciences, DuPont Pharmaceuticals Company, Wil mington, DE 19880-0400, U.S.A. Abbreviations used: Ct, threshold cycle; MCAO, occlusion of the middle cerebral artery; PCR, polymerase chain reaction; TNF-x, tumor necrosis factor-x. 15 preconditioning procedure. TNF-x mRNA was induced in the ipsilateral cortex as early as 1 hour (27 ± I copies of mRNA per microgram of tissue compared to II ± 3 copies in sham operated samples) ater 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 in jured tissues but also suggest a potential role of TNF-x in the phenomenon of ischemic preconditioning. y r: TNF x-Ischemic tolerance-Ischemic preconditioning-Focal stroke-TaqMan-Polymerase chain reaction. 1998). Furthermore, the expression of TNFR2 was dem onstrated to be upregulated during cerebral infection (Lucas et al., 1997). Although the precise role of TNF-a in brain function and especially in stroke is not fully understood, it has been suggested that TNF-a expression in brain ischemia is detrimental to neurons because the administration of anti-TNF-a antibodies or TNF receptor linked to poly ethylene glycol (TNFbp, which binds and inhibits TNF a) significantly reduced focal cerebral ischemic injury (Dawson et al., 1996; Brone et al., 1997; Nawashiro et al., 1997a). In addition,  vv administration of TNF-a into brain parenchyma produced significant inlamma tory reaction in brain capillaries including pericapillary edema and significant leukocyte accumulation (Feuer stein et al., 1994; Liu et al., 1994). TNF-a administration into the cerebroventricular space before ischemia aug ments the extent of tissue damage and neurologic deficits (Barone et al., 1997). These results are in agreement with early studies that suggested TNF-a might play a crucial role in tissue injury during disease processes when it is released from activated lymphocytes, macrophages of