Oxidative Stress Following Traumatic Brain Injury in Rats: Quantitation of Biomarkers and Detection of Free Radical Intermediates *†Vladimir A. Tyurin, *†Yulia Y. Tyurina, *Grigory G. Borisenko, *†Tatiana V. Sokolova, *Vladimir B. Ritov, †Peter J. Quinn, §Marie Rose, Patrick Kochanek, §¶Steven H. Graham, and *#**Valerian E. Kagan Departments of *Environmental and Occupational Health, §Neurology, Anesthesiology and Critical Care Medicine, and #Pharmacology and **Cancer Institute, University of Pittsburgh; ¶Geriatric Research Educational and Clinical Center, V.A. Pittsburgh Health System, Pittsburgh, Pennsylvania, U.S.A.; ‡Division of Life Sciences, King’s College, London, England; and †Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia Abstract: Oxidative stress may contribute to many pathophysiologic changes that occur after traumatic brain injury. In the current study, contemporary methods of detecting oxidative stress were used in a rodent model of traumatic brain injury. The level of the stable product derived from peroxidation of arachidonyl residues in phospholipids, 8-epi-prostaglandin F 2 , was increased at 6 and 24 h after traumatic brain injury. Furthermore, relative amounts of fluorescent end products of lipid per- oxidation in brain extracts were increased at 6 and 24 h after trauma compared with sham-operated controls. The total antioxidant reserves of brain homogenates and wa- ter-soluble antioxidant reserves as well as tissue concen- trations of ascorbate, GSH, and protein sulfhydryls were reduced after traumatic brain injury. A selective inhibitor of cyclooxygenase-2, SC 58125, prevented depletion of ascorbate and thiols, the two major water-soluble anti- oxidants in traumatized brain. Electron paramagnetic res- onance (EPR) spectroscopy of rat cortex homogenates failed to detect any radical adducts with a spin trap, 5,5-dimethyl-1-pyrroline N-oxide, but did detect ascor- bate radical signals. The ascorbate radical EPR signals increased in brain homogenates derived from trauma- tized brain samples compared with sham-operated con- trols. These results along with detailed model experi- ments in vitro indicate that ascorbate is a major antioxi- dant in brain and that the EPR assay of ascorbate radicals may be used to monitor production of free rad- icals in brain tissue after traumatic brain injury. Key Words: Traumatic brain injury—Oxidative stress—Ascor- bate —Spin traps—Thiols— 8-epi-Prostaglandin F 2 . J. Neurochem. 75, 2178 –2189 (2000). Interplay of three major deleterious pathways— gluta- mate excitotoxicity, Ca 2+ overload, and oxidative stress—is believed to be responsible for the damage and neuronal death following traumatic brain injury (TBI) (Juurlink and Paterson, 1998). It has been suggested that oxidative stress plays a key role both in primary damage following acute TBI (Kontos and Povlishock, 1986; Ikeda and Long, 1990) and in secondary deleterious processes associated with inflammatory mediators and neutrophil-mediated inflammation (Feuerstein et al., 1997; Juurlink and Paterson, 1998). Furthermore, reac- tive oxygen species may accelerate axonal damage after TBI (Povlishock and Kontos, 1992). Although this hy- pothesis has been strongly supported by successful use of different classes of antioxidants and spin traps as protec- tors against TBI (Chan et al., 1987; Ikeda and Long, 1990; Sen et al., 1994; Inci et al., 1998; Lewen and Hillered, 1998; Zhang et al., 1998), the sources and mechanisms of oxidative stress induced by TBI, how- ever, have not been yet identified (Awasthi et al., 1997). Furthermore, despite the prevailing opinion on the im- portant role of free radicals in TBI, the direct experimen- tal data supporting this notion remain controversial. Cyclooxygenase is the enzyme that catalyzes the forma- tion of prostaglandins (PGs) from arachidonic acid. The enzyme also has peroxidase activity and can induce forma- tion of radicals and dopamine quinones independent of its Resubmitted manuscript received July 4, 2000; accepted July 6, 2000. Address correspondence and reprint requests to Dr. V. E. Kagan at Department of Environmental and Occupational Health, 260 Kappa Drive, University of Pittsburgh, Pittsburgh, PA 15238, U.S.A. E-mail: kagan@pitt.edu Abbreviations used: AAPH, 2,2'-azobis(2-aminodinopropane) dihy- drochloride; COX-2, cyclooxygenase; DMPO, 5,5-dimethyl-1-pyrro- line N-oxide; DTPA, diethylenetriaminepentaacetic acid; EPR, electron paramagnetic resonance; GS • , thiyl radical; LPO, lipid peroxidation; • OH, hydroxyl radical; PDAM, 1-pyrenyldiazomethane; PG, prosta- glandin; 8-epi-PGF 2 , 8-epi-prostaglandin F 2 (F 2 -isoprostane); SC 58125, 1-[(4-methylsulfonyl)phenyl]-3-trifluoromethyl-5-[(4-fluoro)- phenyl]pyrazole; SDS, sodium dodecyl sulfate; TBI, traumatic brain injury; VP-16, etoposide. 2178 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 2000 International Society for Neurochemistry