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Journal of Neurochemistry, 2002, 80, 894–898

Local and systemic increase in lipid peroxidation after moderate experimental traumatic brain injury Domenico Pratico`,* Peter Reiss, Lina X. Tang,* Syan Sung,* Joshua Rokachà and Tracy K. McIntosh ,§ *Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, USA Department of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, USA àClaude Pepper Institute, Department of Chemistry, Florida Institute of Technology, Melbourne, Florida, USA §Veteran Administration Medical Center, Philadelphia, USA

Abstract Traumatic brain injury is a common event associated with neurological dysfunction. Oxidative damage, may contribute to some of these pathologic changes. We used a specific and sensitive marker of lipid peroxidation, the isoprostane 8,12iso-iPF2a-VI, to investigate whether local and also systemic lipid peroxidation were induced following lateral fluid percussion (FP) brain injury in the rat. Animals were anesthetized and subjected to lateral FP brain injury of moderate severity, or to sham injury as controls. Urine was collected before anesthesia (baseline), 6 and 24 h after injury. Blood was collected at baseline, 1, 6 and 24 h after injury. Animals were killed 24 h after surgery and their brains removed for biochemical analysis. No significant difference was observed

at baseline (preinjury) for urine and plasma 8,12-iso-iPF2a-VI levels between injured and sham-operated animals. By contrast, plasma and urinary levels increased significantly already at 1 and further increased 24 h following brain injury, when compared to sham-operated animals. Finally, compared with sham, injured animals had a significant increase in brain 8,12iso-iPF2a-VI levels. These results demonstrate that moderate brain injury induces widespread brain lipid peroxidation, which is accompanied by a similar increase in urine and plasma. Peripheral measurement of 8,12-iso-iPF2a-VI levels after brain injury may be a reliable marker of brain oxidative damage. Keywords: isoprostanes, lipid peroxidation, oxidative stress, traumatic brain injury. J. Neurochem. (2002) 80, 894–898.

Traumatic brain injury (TBI) is an extremely common occurrence in the United States of America with over 250 000 individuals admitted to hospitals each year (Kraus et al. 1994). TBI has long been recognized as a form of central nervous system (CNS) injury that is associated with long-term cognitive and neurologic motor dysfunction (Kurth et al. 1994; Klein et al. 1996). Although the cellular and molecular substrates for post-traumatic changes have not yet been fully elucidated, it is known that TBI triggers a cascade of events that results in alteration of cerebral blood flow and metabolism (Yamakani and McIntosh 1991; Yoshino et al. 1991), tissue edema (Soares et al. 1992), neuronal degeneration and finally cell death (Hicks et al. 1996). Extensive experimental evidence exists for the early occurrence and importance of reactive oxygen species formation and subsequent oxidative damage as major candidates responsible for various pathological responses in

the pathogenesis of TBI (Ikeda and Long 1990; Hall et al. 1993). Oxidative damage in the CNS manifests itself primarily as lipid peroxidation since this organ is rich in peroxidizable fatty acids and has a relative scarcity of antioxidant defense systems (Floyd 1999). Isoprostanes are

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Received October 12, 2001; revised manuscript received December 4, 2001; accepted December 10, 2001. Address correspondence and reprint requests to Domenico Pratico`, Center for Experimental Therapeutics, University of Pennsylvania, BRB II/III, room 812, 421 Curie Blvd., Philadelphia, PA 19014, USA. E-mail: [email protected] Abbreviations used: AD, Alzheimer’s disease; FP, fluid percussion; LA, left cortex adjacent region; LC, left injured cortex; LH, left injured hippocampus; MDA, malondialdehyde; PBS, phosphate-buffered saline; RA, adjacent area in the right cortex; RC, right controlateral cortex; RH, right controlateral hippocampus; TBARS, thiobarbituric acid-reacting substances; TBI, traumatic brain injury; RA, right adjacent cortex region.

Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry, 80, 894–898

Oxidative damage in traumatic brain injury 895

chemically stable end-products of oxidative modification of lipids, are formed in vivo, accumulate in tissue, circulate in plasma and are excreted in urine. A consistent amount of data have shown that they represent specific and sensitive markers of in vivo lipid peroxidation and oxidative damage (Pratico` et al. 2001a). In the present study, we use an established clinically relevant model of TBI (McIntosh et al. 1989) to assess levels of a major isoprostane, 8,12-iso-iPF2a-VI (Pratico` et al. 2001a) in brain, plasma and urine of adult rats. We show that this model induces a significant increase in 8,12-iso-iPF2a-VI levels in brain tissue as well as in plasma and urine, which is detected as early as 1 h after TBI. This is coincidental with a significant reduction in plasma levels of vitamin E and ascorbic acid. We conclude that TBI induces a widespread increase in brain lipid peroxidation, which is reflected in the periphery by a similar increase in plasma and urine. Measurement of 8,12-iso-iPF2a-VI in urine or plasma after TBI could afford a noninvasive surrogate marker to study brain oxidative damage and to investigate the therapeutic effects of antioxidants in such a clinical condition.

Materials and methods Animal preparation and induction of the injury Adult, male Sprague–Dawley CD rats (Harlan, Indianapolis, IN, USA) (n ¼ 16, weight 305–400 g) were given access to food and water ad libitum. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania and were in accordance with federal guidelines (National Research Council, Guide for the Care and Use of Laboratory Animals, National Academy Press, Washington, DC, USA). Animals were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and then surgically prepared for lateral fluid percussion (FP) brain injury or sham operation as previously described in detail (McIntosh et al. 1989). In brief, after placement in a stereotactic frame, the scalp and temporal muscles were reflected, and a 5.00-mm craniotomy was performed over the left parietal cortex, between lambda and bregma sutures, leaving the dura mater intact. A hollow female Luer–Lok fitting was positioned over the craniotomy and held in place with dental cement. Animals were attached to the FP device (a saline-filled cylinder) via the female Luer–Lok. In a subset of animals (n¼8) moderate (2.4–2.7 atmospheres) brain injury was then induced by a rapid injection of a pressure pulse of saline into the closed cranial cavity. Sham-injured animals (n¼8) were anesthetized and surgically prepared as above described, but were not subjected to brain injury. After brain injury or sham injury, the Luer–Lok and the dental cement were removed, and the skin sutured. Animals recovered after injury or sham injury on heating pads to maintain normothermia. Urine samples were collected before surgery (baseline), and at 6 and 24 h after brain injury or sham injury. Blood was collected at baseline, 1, 6 and 24 h after brain injury or sham injury. Injured and sham control animals were killed at 24 h after surgery. Animals were perfused intracardially for 10 min with ice-cold phosphate-

buffered saline (PBS) containing 2 mm/L EDTA and 20 mM/L of the antioxidant butylated hydroxytoluene, pH 7.4. They were then decapitated and brains were quickly removed. Brains were dissected in the following regions, according to the technique of Glowinski and Iversen (1996) with reference to the atlas of Paxinos and Watson (1986): left injured parietal cortex (LC), corresponding area in the controlateral right cortex (RC), cortex adjacent to injured parietal cortex (LA), corresponding adjacent area in the right cortex (RA), left hippocampus (LH) and right hippocampus (RH). Samples were immediately frozen on dry-ice and kept at )80°C until analysis. Isoprostane analyses Collected urine was spiked with a fixed amount of internal standard, extracted on a C18 column, then purified by thin layer chromatography and finally assayed by negative ion chemical ionization gas chromatography mass spectrometry as previously described (Pratico` et al. 2001b). A urine aliquot (0.1 mL) was used for measurement of creatinine levels by a commercially available, standardized, automated colorimetric assay (Sigma Chemical Co., St Louis, MO, USA). Urine levels were expressed as nanograms per milligram of creatinine. Blood was anticoagulated with trisodium citrate (3.8%) and centrifuged at 1.8 g for 15 min at 4°C to obtain plasma. Plasma was spiked and treated as described above. Levels were expressed as nanograms per milliliter. Brain tissues were homogenized, and total lipids were extracted using Folch solution (chloroform/methanol 2 : 1 v/v), as previously described (Pratico` et al. 2001b). Next, base hydrolysis was performed using 15% KOH at 45°C for 1 h, and the total 8,12iso-iPF2a-VI levels were measured as described above. All of the assays were always performed without knowledge of the time-point or treatment. Vitamins measurements Plasma levels of vitamin E were assayed by HPLC after hexane extraction, on a reverse phase C18 column as previously described (Pratico` et al. 1998a). For ascorbic acid levels, plasma samples were deproteinized with 10% metaphosphoric acid and assayed by HPLC with electrochemical detection as previously described (Pratico` et al. 1998a). All of the assays were always performed without knowledge of the time-point or treatment. Statistical analysis Data are expressed as the mean ± standard error of the mean (SEM). Isoprostane and vitamin levels were assessed by ANOVA and subsequent Student’s unpaired two-tailed t-test. Significance was set at p < 0.05.

Results

We found that, compared with baseline, urinary levels of 8,12-iso-iPF2a-VI were significantly increased at 6 h (452 ± 39 versus 322.5 ± 23 pg/mg creatinine, p ¼ 0.02) and continued to rise at 24 h (580 ± 31 pg/mg creatinine, p < 0.01) post-injury (Fig. 1). By contrast, no significant changes were observed in sham-operated animals (295 ± 17, 322 ± 23 and 345 ± 28 pg/mg creatinine; at base, 6 and 24 h post-injury) (Fig. 1).


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Fig. 1 Urinary levels of 8,12-iso-iPF2a-VI in FP-injured (d) or shamtreated animals (j) before (base), at 6 and 24 h after injury (n ¼ 8 for each group)(*p ¼ 0.02; **p < 0.01).

Fig. 2 Plasma levels of 8,12-iso-iPF2a-VI in FP-injured (d) or shamtreated animals (j) before (base), at 1, 6 and 24 h after injury (n ¼ 8 for each group) (*p < 0.01).

No difference in 8,12-iso-iPF2a-VI plasma levels were found at baseline between FP injured and sham-treated animals (0.88 ± 0.05 versus 0.92 ± 0.06 pg/mL) (Fig. 2). After the injury, while no significant change was observed in sham-operated animals at any time point, FP-treated animals showed a significant increase in plasma levels of 8,12-isoiPF2a-VI as early as 1 h post-injury (1.42 ± 0.1 versus 0.94 ± 0.05 pg/mL, p < 0.01). These levels further increased at 6 (1.79 ± 0.08 versus 1.02 ± 0.07 pg/mL, p < 0.01) and 24 h after surgery (2 ± 0.07 versus 1.03 ± 0.07 pg/mL, p < 0.001) (Fig. 2). A direct correlation between urine and plasma levels was observed (data not shown), suggesting a common origin and mechanism of formation for 8,12-isoiPF2a-VI. Plasma vitamin E and ascorbic acid levels were significantly reduced 24 h after TBI (18 ± 2 versus 10 ± 1.5 lM, 51 ± 4 versus 32 ± 5 lM, respectively; p < 0.001 for both). Their levels inversely correlated with plasma levels of 8,12-iso-iPF2a-VI (r2¼) 0.70; r2¼ ) 0.68, p < 0.01 for both). By contrast, no significant change of vitamin levels was observed in sham-treated animals (not

Fig. 3 Brain levels of 8,12-iso-iPF2a-VI in FP-injured (j) or shamtreated animals (h) 24 h after brain injury (n ¼ 8 for each group). Brains were divided in the following regions: LC, left injured cortex; LA, left cortex adjacent region; LH, left injured hippocampus; RC, right controlateral cortex; RA, right adjacent cortex region; RH, right controlateral hippocampus (*p < 0.0001).

shown). Since the animals were not ventilated, they all had a transient episode of post-injury apnea. However, the average duration was 15–35 s, and during this time no significant change in arterial blood gases or pH values was noticed in any animals (not shown). Animals were killed 24 h after injury or sham operation and brains were quickly removed. Compared with shamoperated, injured animals had a significant increase in 8,12iso-iPF2a-VI levels in all three brain regions considered (Fig. 3). The increase was much higher in the ispsilateral hemisphere, with this order of magnitude for the three regions: injured cortex higher than adjacent cortex (45 ± 1.6 versus 36 ± 1.3 ng/g tissue), and adjacent cortex higher than injured hippocampus (36 ± 1.3 versus 31 ± 2.3 ng/g tissue). However, compared with sham-injured we observed significantly elevated 8,12-iso-iPF2a-VI levels also in the controlateral brain hemisphere (Fig. 3) of the injured animals, suggesting that even focal TBI induced a generalized increase in brain oxidative damage. Discussion

This study provides direct evidence of diffuse increased brain lipid peroxidation after moderate experimental TBI in rats. We also show, for the first time, that this increase in parenchymal lipid peroxidation is accompanied by a similar increase in peripheral blood as well as in urine, and a decrease in circulating levels of endogenous antioxidants. TBI is a very common event with immediate and delayed neurological consequences. However, the pathophysiological mechanisms underlying acute and delayed secondary effects after TBI are not completely understood. Oxidative damage has been implicated in many of the pathological changes that occur after traumatic brain injury (Hsiang et al. 1997; Ercan et al. 2001). However, most of the published results have


Oxidative damage in traumatic brain injury 897

often relied on measurement performed in ex vivo systems with unclear relevance to in vivo brain injury (DeZwart et al. 1999). In general, commonly employed indices of oxidant stress are constrained by such issues as the nonsensitivity or instability of the target anylate, contamination of the anylate by events ex vivo, and nonspecificity of analytical methodology (Pratico` 2001). Several studies have indirectly demonstrated the early generation of superoxide radicals in injured brains, which resulted in secondary damage to the brain microvasculature (Povlishock and Kontos 1992). Some investigators have used spin trap probes or salicylate trapping methods to demonstrate an early post-traumatic occurrence of hydroxyl radical formation in injured brain (Torbati et al. 1991; Hall et al. 1993), which also correlated with the development of brain–blood barrier disruption (Smith et al. 1994). Others have relied on even less-specific and sensitive assays for measuring lipid peroxidation, such as thiobarbituric acid-reacting substances (TBARS), malondialdehyde (MDA), and have concluded that oxidative stress occurs after TBI (Inci et al. 1998). Isoprostanes are a new class of lipids, isomers of prostaglandins, formed by a free radical-catalyzed peroxidation of polyunsaturated fatty acids independently of the cyclooxygenase activity. They are generated in vivo, stored in tissue, circulate in plasma and excreted in urine. Abundant data in the literature have shown that they represent sensitive and specific markers of in vivo lipid peroxidation (Pratico` et al. 2001a). Previously, it has been reported that total brain levels of the isoprostane iPF2a-III (also known as 8-isoPGF2a, or 15-F2t-isoprostane) increased after cortical contusion compared to control animals (Hoffman et al. 1996; Tyurin et al. 2000). We confirmed and extended this observation by measuring a more abundant isoprostane, 8,12-iso-iPF2a-VI, we showed that not only it is increased in specific brain regions, but also in the whole body after moderate experimental TBI. To the best of our knowledge the precise cellular source of post-traumatic brain isoprostane generation is not completely known. Using a cell culture model of traumatic injury, it has been shown that astrocytes can generate isoprostanes in response to trauma (Hoffman et al. 2000). Interestingly, these compounds are not only specific markers of lipid peroxidation but also possess biological activities. Among them, they are potent vasoconstrictors of cerebral arterioles (Hoffman et al. 1997) and periventricular brain microvessels (Hou et al. 2000). Taken together, these observations suggest that isoprostanes could play also an important role in the reduction of cerebral blood flow following brain injury. Previously, we have shown that isoprostanes are increased in brain of aging mice deficient of the apolipoprotein E, which are known to have elevated circulating indices of oxidative damage (Pratico` et al. 1999). Further, we have shown that they are increased in selective brain regions of Alzheimer’s disease (AD) and in patients with clinical

diagnosis of AD, a disease putatively associated with an increase in brain oxidative damage (Pratico` et al. 1998b, 2000). These data, along with our current observation, further support the hypothesis that 8,12-iso-iPF2a-VI may be a sensitive and specific marker of brain oxidative damage. Since TBI is a known environmental risk factor for AD (Plassman et al. 2000), we speculate that the increase in brain lipid peroxidation observed after TBI could represent a pathogenetic link between these two conditions. In our study the increase in lipid peroxidation was observed not only in ipsilateral brain regions where the trauma occurred, but also in the adjacent areas and, surprisingly, in controlateral brain regions. This suggests that a moderate brain trauma can induce a widespread and bilateral increase in brain lipid peroxidation. Remarkably, we observed that, coincident with the increase in CNS tissue concentrations, urinary and plasma isoprostane levels were also significantly higher than baseline and shaminjured animals. Finally, we found that systemic levels of two major endogenous antioxidants, vitamin E and ascorbic acid, were significantly reduced in injured but not in shamoperated animals, and that these levels inversely correlated with the increase in isoprostane. These findings suggest that the consumption of endogenous antioxidants reflect an attempt of the whole body to neutralize excessive reactive oxygen species formation following TBI (Shohami et al. 1999). In conclusion, these results demonstrate that experimental TBI induces widespread brain oxidative damage, which is also reflected in the periphery by a similar increase in plasma and urine. These data support the hypothesis that noninvasive measurement of 8,12-iso-iPF2a-VI urinary levels could be used after traumatic brain injury as reliable marker of brain oxidative damage and to monitor therapeutic effects of antioxidants in clinical trials. Acknowledgements This work was supported by grants from the American Heart Association, the National Institute of Health (NS08803, GM34690) and a Merit Review Award grant to TKM from the Veteran Administration.

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