JOURNAL OF NEUROTRAUMAVolume 19, Number 4, 2002© Mary Ann Liebert, Inc.Increased Protein Oxidation and Decreased Creatine Kinase BB Expression and Activity after Spinal Cord Contusion InjuryMARINA AKSENOVA,1,2D. ALLAN BUTTERFIELD,2,3SHU-XIN ZHANG,2MARK UNDERWOOD,2and JAMES W. GEDDES2,4ABSTRACTTraumatic injury to the spinal cord triggers several secondary effects, including oxidative stress andcompromised energy metabolism, which play a major role in biochemical and pathological changesin spinal cord tissue. Free radical generation and lipid peroxidation have been shown to be earlyevents subsequent to spinal cord injury. In the present study, we demonstrated that protein oxida-tion increases in rat spinal cord tissue after experimental injury. As early as 1 h after injury, thelevel of protein carbonyls at the injury epicenter was significantly higher than in control (169%,p,0.05) and increased gradually over the next 4 weeks to 1260% of control level. Both caudal androstral parts of the injured spinal cord demonstrated a mild increase of protein carbonyls by 4 weekspostinjury (135–138%, p,0.05). Immunocytochemical analysis of protein carbonyls in the spinalcord cross-sections showed increased protein carbonyl immunoreactivity in the epicenter sectioncompared to rostral and caudal sections of the same animal or control laminectomy animals. In-creased protein carbonyl formation in damaged spinal cord tissue was associated with changes inactivity and expression of an oxidative sensitive enzyme, creatine kinase BB, which plays an im-portant role in the maintenance of ATP level in the CNS tissue. Damage to CK function in the CNSmay severely aggravate the impairment of energy metabolism. The results of our study indicate thatevents associated with oxidative damage are triggered immediately after spinal cord trauma butcontinue to occur over the subsequent 4 weeks. These results suggest that antioxidant therapeuticstrategies may be beneficial to lessen the consequences of the injury and potentially improve therestoration of neurological function.Key words: creatine kinase; oxidative stress; protein oxidation; spinal cord injury491The 1Department of Pharmacology, 2Sanders-Brown Center on Aging, 3Department of Chemistry and Center on MembraneSciences, and 4Spinal Cord and Brain Injury Research Center, University of Kentucky, Lexington, Kentucky.INTRODUCTIONTHERE ARE TWO MECHANISMS of damage to the spinalcord after acute spinal cord injury: the primary me-chanical injury and a secondary injury due to many ad-ditional damaging processes, including vascular, bio-chemical, and ionic changes (Anderson and Hall, 1993;Tator and Fehlings, 1991; Young, 1993). Much of thedamage that follows acute trauma is due to secondary ef-fects such as ischemia, edema, glutamate excitotoxicity,Ca21overload, compromised energy metabolism, andoxidative stress (Hall and Braughler, 1989; Juurlink andPaterson, 1998; Siesjo et al., 1989). The injured spinalcord provides a fertile environment for the generation ofoxygen radicals and lipid peroxidation reactions thoughtto contribute to neuronal dysfunction and cell loss fol-lowing traumatic damage to the CNS (Awasthi et al.,1997; Springer et al., 1997; Hall and Braughler, 1986;Hall, 1989). A number of studies indicate that activationof glutamate receptors and the influx of Ca21lead to theimpaired function of the mitochondrial electron transportsystem and ROS formation (Happel et al., 1981; Moriyaet al., 1994). Increased ROS production that overwhelmendogenous scavenging mechanisms contributes to thedamage of critical cellular components including nucleicacids, proteins, and phospholipids. Oxygen free radicalsalso may trigger inflammation (for review, see Dirnaglet al., 1999), which is considered one of the leading sec-ondary events in spinal cord injury.During the past decade, free radical generation was es-tablished to be an early biochemical event subsequent tospinal cord injury. Most reports concentrated on free-rad-ical-induced lipid peroxidation and membrane damage,although it is acknowledged that free radicals damageproteins and nucleic acids as well (Anderson and Hall,1994; Barut et al., 1993; Braughler et al., 1985; Butter-field and Stadtman, 1997; Butterfield et al., 2001; Floydand Carney, 1992). The important role of free radicals intissue damage associated with spinal cord injury is un-derscored by the fact that treatment with free radical scav-engers can be effective. Iwasa et al. (1989) demonstratedthe protective effect of vitamin E on experimental com-pression injury of the rat spinal cord by inhibiting dam-age induced by lipid peroxidation. Protective effects ofvitamin E, methylprednisolone, and tirilazad mesylateagainst spinal cord injury was demonstrated by Koc etal. (1999). Cyclosporin-A and methylprednisolone wereeffective upon inhibition of lipid peroxidation after spinalcord injury as was demonstrated by Diaz-Ruiz et al.(2000). Melatonin has been shown to be very effectivein protecting the injured spinal cord from lipid peroxi-dation (Kaptanoglu et al., 2000). Promoting glutathionesynthesis after spinal cord trauma was shown to decreaseoxidative stress and to allow tissue preservation in spinalcord after compression injury (Kamencic et al., 2001),possibly by blocking damaging effects of lipid peroxida-tion products (Subramaniam et al., 1997; Pocernich et al.,2001). It has also been shown that the generation of ROS,impaired mitochondrial function, and lipid peroxidationoccur very early following spinal cord injury (Kamencicet al., 2001; Azbill et al., 1997; Hall, 1989). In the pre-sent study, we examined the oxidation of cytoplasmicspinal cord proteins and the localization of protein oxi-dation following spinal cord injury in rats up to 4 weekspostinjury.Related to the ROS-mediated oxidative damage arechanges in energy metabolism after acute spinal cordtrauma (Anderson et al., 1980a; Anderson and Hall, 1993;Tator and Fehlings, 1991; Zhang et al., 1993). The phos-phocreatine/creatine kinase (PCr/CK) system plays a keyrole in buffering of ATP levels in the cell under stressconditions (Bessman and Carpenter, 1985; Hemmer andWallimann, 1993), and creatine kinase isoenzymes arevery sensitive to oxidative modifications (Hensley et al.,1994;