Oxidative stress in head trauma in aging

Free Radical Biology & Medicine 41 (2006) 77 – 85 www.elsevier.com/locate/freeradbiomed

Original Contribution

Oxidative stress in head trauma in aging ChangXing Shao a , Kelly N. Roberts b , William R. Markesbery b,c , Stephen W. Scheff b , Mark A. Lovell a,b,⁎ b

a Department of Chemistry, University of Kentucky, Lexington, KY 40536, USA Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY 40536, USA c Neurology and Pathology, University of Kentucky, Lexington, KY 40536, USA

Received 23 November 2005; revised 6 March 2006; accepted 10 March 2006 Available online 3 April 2006

Abstract Oxidative damage is proposed as a key mediator of exacerbated morphological responses and deficits in behavioral recovery in aged subjects with traumatic brain injury (TBI). In the present study, we show exacerbated loss of tissue in middle aged (12 months) and aged (22 months) Fisher-344 rats compared to young animals (3 months) subjected to moderate TBI. Analysis of 4-hydroxynonenal (4-HNE) and acrolein, neurotoxic by-products of lipid peroxidation, shows significant (P < 0.05) age-dependent increases in ipsilateral (IP) hippocampus 1 and 7 days post injury. In IP cortex, 4-HNE was significantly elevated 1 day post injury in all age groups, and both 4-HNE and acrolein were elevated in middle aged and aged animals 7 days post injury. Comparison of antioxidant enzyme activities shows significant (P < 0.05) age-dependent decreases of manganese superoxide dismutase in IP hippocampus and cortex 1 and 7 days post injury. Glutathione reductase activity also showed an age-dependent decrease. Overall, our data show increased levels of oxidative damage, diminished antioxidant capacities, and increased tissue loss in TBI in aging. © 2006 Elsevier Inc. All rights reserved. Keywords: Acrolein; Glutathione peroxidase; Glutathione reductase; Glutathione transferase; 4-Hydroxynonenal; Traumatic brain injury; Superoxide dismutase

Introduction Traumatic brain injury (TBI) results in delayed dysfunction and death of neuron populations near and distant to the site of injury through secondary processes [1]. Current data show that neurons undergo a progressive degeneration involving increased excitatory amino acids, loss of ion equilibrium, decreased ATP production, aberrant proteolytic enzyme activity, reduction of reactive oxygen species (ROS) scavenAbbreviations: CON, contralateral; Cu/ZnSOD, copper/zinc superoxide dismutase; GPx, glutathione peroxidase; GRed, glutathione reductase; GST, glutathione transferase; 4-HNE, 4-hydroxynonenal; IP, ipsilateral; MnSOD, manganese superoxide dismutase; NBD-Cl, 7-chloro-4-nitrobenzo-2-oxa-1,3diazole; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; PUFA, polyunsaturated fatty acids; ROS, reactive oxygen species; TBI, traumatic brain injury. ⁎ Corresponding author. 135 Sanders-Brown Building, 800 South, Limestone Street, Lexington, KY 40536-0230, USA. Fax: +1 859 323 2866. E-mail address: [email protected] (M.A. Lovell). 0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.03.007

gers, and increased free radical formation [2–7]. Increased generation of ROS following TBI could lead to lipid peroxidation and formation of neurotoxic aldehydic products. Previous studies show increased lipid peroxidation, including significant elevations of thiobarbituric acid-reactive substances (TBARS) [8] and 8-isoprostaglandin F2α [9] following TBI in young animals. Studies of age-related changes following TBI indicate an exacerbated morphological response and a deficit in behavioral recovery, which is proposed to be mediated by secondary damage [10–12]. Although oxidative stress has been suggested to contribute to neuron loss following TBI, the study of oxidative stress in aging is still limited. Recently, 4-hydroxynonenal (4-HNE) and acrolein [13,14], two neurotoxic byproducts of lipid peroxidation of arachidonic acid, have become of interest in a variety of neurological diseases involving oxidative stress [15–20]. To assess the possible involvement of oxidative damage in TBI in aging, we utilized a unilateral controlled cortical impact model of TBI in young (3 months),

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middle aged (12 months), and aged (22 months) Fisher-344 rats and measured tissue sparing, and 4-HNE and acrolein levels 1 and 7 days post injury. Additional studies were performed to evaluate antioxidant enzyme activities in aging following TBI. These studies may provide insight regarding secondary injury in TBI in aging and may provide potential targets for clinical therapeutic intervention. Materials and methods

assessment of spared tissue employed the Cavalieri method as described previously [22,23]. The amount of tissue sparing was expressed as percentage of the total cortical volume of the injured hemisphere compared to the uninjured hemisphere. In this way each animal is used as its own control. These methods obviate a need to adjust values due to possible differential shrinkage resulting from fixation and tissue processing. A total of 12 evenly spaced sections were used for each animal. All sections were assessed blind with respect to age group.

Subjects and surgical procedures Tissue processing for lipid peroxidation and enzyme assay The present studies were conducted in adult male Fisher344 rats. All injury procedures were approved by the Institutional Animal Care and Use Committee of the University of Kentucky. Every effort was made to minimize both the possible suffering and the number of animals used. All animals were housed 2/cage on a 12 h light/dark cycle with free access to water and food. Twenty-one young (3 months), 24 middle aged (12 months), and 21 aged (22 months) rats (205–500 g/animal) were subjected to a unilateral cortical impact on the surface of the brain utilizing an electronic controlled pneumatic impact device (ECPI) as previously described [21]. Briefly, each animal was anesthetized with 2% isofluorane and placed in a Kopf stereotaxic frame with the incisor bar set at −5. Following a midline incision and retraction of the skin, a 6-mm-diameter craniotomy was made approximately midway between bregma and lamda with a Michele hand trephine (Miltex, NY). The skull disk was removed without disturbing the dura. The exposed brain was injured using the ECPI. The impact rod had a 5-mm-diameter beveled tip that was used to compress the cortex to a depth of 1.5 mm at 3.5 m/s. Following injury Surgicel (Johnson & Johnson, TX) was placed over the injury site, and the skull disk was replaced and sealed with a thin layer of dental acrylic. The incision was closed with surgical staples and the animals maintained at 35° to 37°C with a heating pad. Ten young, 10 middle aged, and 8 aged animals were allowed to survive for 1 day. Eleven young, 14 middle aged, and 13 aged animals were euthanized 7 days post injury. Method for estimating tissue sparing For studies of tissue sparing, 6 young, 6 middle aged, and 5 aged animals 7 days post injury were overdosed with sodium pentobarbital and transcardially perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in PBS, and decapitated. Brains were removed, postfixed in 4% paraformaldehyde containing 10% sucrose at 4°C overnight, and subsequently cryoprotected in a 20% sucrose/4% paraformaldehyde solution for an additional 24 h. Coronal 50-μm sections were cut with a cryostat throughout the rostral-caudal extent of the injured cortex extending from the septal area to the posterior aspect of the hippocampus. Sections were stained with cresyl violet and subjected to image analysis (NIH Image v. 1.62). Quantitative

For enzymatic and oxidative stress assays, animals were anesthetized using 5% isofluorane and subjected to cervical dislocation. Ipsilateral (IP) and contralateral (CON) hippocampi and cortices were dissected, immediately frozen in liquid nitrogen, and stored at −80°C until used for analysis. For analysis tissue samples were homogenized using a chilled Dounce homogenizer on ice in 1 ml Hepes buffer (pH 7.4) containing 137 mM NaCl, 4.6 mM KCl, 1.1 mM KH2PO4, 0.6 mM MgSO4, pepstatin A, leupeptin, aprotinin, and PMSF. Samples were centrifuged at 100,000g for 1 h at 4°C and the supernatant was used for analyses. Total protein concentrations were determined using the Pierce BCA method (Sigma, St. Louis, MO). 4-Hydroxynonenal and acrolein assay Dot-blot analyses of 4-HNE and acrolein-modified proteins were carried out using a Schleicher & Schuell dot-blot apparatus as described by Saiki et al. [24] with modification. Briefly, 20 μg of 100,000g supernatant protein was loaded in triplicate onto nitrocellulose in a 40–50 μl volume. Vacuum was applied until the solution was evacuated from the wells. After air drying, blots were incubated overnight in 5% dry milk in 0.05% Tween 20/Tris buffered saline (TTBS) to block nonspecific binding of the primary antibody. Blots were probed with a 1:2000 dilution of anti-4-HNE (Alpha Diagnostic, San Antonio, TX) or anti-acrolein (United States Biological, Swampscott, MA) polyclonal antibodies for 1 h at room temperature, washed in TTBS 3 × 10 min, and then incubated with a 1:3000 dilution of horseradish peroxidaseconjugated goat anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA) for 1 h. Detection of bound antibodies was achieved by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) and exposure of the membrane to Hyperfilm (Amersham). Staining intensity was quantified using Scion Image (NIH). The average intensity of triplicate dots was calculated for 4-HNE or acrolein content in an individual sample. To validate linearity of response of the dot blots, aliquots of 100,000g protein from a representative rat were incubated for 4 h at 37°C with increasing concentrations of 4-HNE (10, 25, 50, 100 μM) or acrolein (1.0, 5.0, 10, 25 μM). The acrolein and 4-HNEmodified proteins were subjected to dot-blot analyses as described above.

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Enzyme assays The method for assay of total SOD (Cu/Zn and MnSOD) activity was that of Misra and Fridovich [25] as modified by Mizuno [26]. Briefly, 70-μl aliquots of the 100,000g supernatant were placed in UV-grade cuvettes and 1.46 ml 68 mM NaH2PO4 containing 1.35 mM EDTA (pH 7.8), 100 μl 4 mM xanthine, and 170 μl 3.53 mM epinephrine (pH 11.5) was added. Following incubation at 30°C for 5 min, 10 μl xanthine oxidase (Calbiochem) was added to the solution and the absorbance followed at 320 nm (Genesys 10 UV, Thermospectronic) for 3 min. The blank for the assay contained 70 μl supernatant heated at 100°C for 5 min. For MnSOD, the same protocol was followed with the addition of 200 μl 20 mM KCN. Enzyme activities were expressed as units per microgram protein, where 1 unit reduces the absorbance change by 50%. Activities are expressed as mean ± SD percentage of contralateral activity. Cu/Zn activity was obtained by subtracting MnSOD activity from total activity. Glutathione transferase (GST) activity was assayed using the method of Ricci et al. [27], which measures the rate of conjugation of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBDCl) with glutathione catalyzed by glutathione transferase. The NBD-Cl complex is a stable, yellow compound that strongly absorbs at 419 nm with a molar absorption coefficient of 14.5 mM–1 cm–1. For the assay, 1.6 ml of 0.1 M sodium acetate buffer (pH 5.0) was mixed with 200 μl 0.2 mM NBD-Cl and 100 μl 0.5 mM reduced glutathione. The reaction was initiated by the addition of 100 μl aliquots of the 100,000g tissue supernatant, and the absorbance was followed for 3 min. Enzyme activity was expressed in units per microgram total protein, with 1 unit = 1 nmol NBD-Cl complex formed per minute. Results are expressed as mean ± SD percentage of contralateral activity. For glutathione peroxidase (GPx) activity, the method of Paglia and Valentine [28] as modified by Mizuno [26] was followed, which measures the rate of oxidation of reduced glutathione to oxidized glutathione by H2O2 as catalyzed by GPx present in the tissue supernatant. For the assay, 100-μl aliquots of the 100,000g supernatant were added to 1.68 ml 68 mM KH2PO4 buffer (pH 7.0) containing 1 mM EDTA, 100 μl 2 mM NADPH, 10 μl 66 U/ml glutathione reductase (GRed), and 10 μl 200 mM sodium azide. The assay was initiated with 100 μl 15 mM H2O2. The decrease in absorbance was followed for 2 min at 340 nm. Quantification was based on a molar absorption coefficient of 6270 M–1 cm–1 for NADPH [29]. The blank in the enzyme assay consisted of 100 μl supernatant heated at 100°C for 5 min, and its value was subtracted from the sample value to correct for nonenzymatic oxidation of glutathione. Activity of the enzyme was expressed in units per microgram total protein, with 1 unit = 1 nmol NADPH oxidized per minute. Activities are expressed as mean ± SD percentage of contralateral activity. The method of assay for GRed is that of Mizuno [26] and is based on the reduction of oxidized glutathione by GRed and exogenous NADPH. The assay measures a loss in absorbance at 340 nm as NADPH is converted to NADP+. One hundred-

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microliter aliquots of the 100,000g supernatant were added to cuvettes containing 1.58 ml 68 mM KH2PO4 buffer, 1 mM EDTA, and 100 μl 2 mM NADPH. The absorbance was followed at 340 nm for 3 min following initiation of the reaction by the addition of 220 μl 13.6 mM oxidized glutathione. Enzyme activities were calculated as described for GPx and expressed as mean ± SD percentage of contralateral activity. Statistical analysis One-way ANOVA and the Student-Newman-Keuls test were used for age-dependent differences in enzyme activity, oxidative stress, and tissue sparing using the commercially available ABSTAT software (Anderson Bell, Arvada, CO). The two-tailed Student t test was used for time course differences in oxidative stress and enzyme activities. Significant differences were set at P < 0.05. Results Assessment of tissue sparing At 7 days post injury, rats from each age group were assessed for cortical tissue sparing immediately below the site of impact. ANOVA showed a significant difference among the group means [F(2,14) = 17.36, P < 0.001]. Post hoc analysis revealed significant differences (P < 0.05) between young and middle aged, and young and aged groups. Young animals showed 89.4 ± 4.4% tissue sparing whereas middle aged and aged animals showed 77.1 ± 3.9 and 77.0 ± 3.8% tissue sparing, respectively (P < 0.05) (Fig. 1). There were no significant differences in tissue sparing between middle aged and aged animals. 4-HNE and acrolein dot-blot calibration To validate antibody response for dot-blot assays, representative protein samples were incubated with increasing concentrations of 4-HNE or acrolein and subjected to dot-blot analysis.

Fig. 1. The percentage of tissue sparing in Fisher-344 rats subjected to 1.5 mm injury. ANOVA showed a significant difference between the three age groups. Bars represent the mean ± SD. Student-Newman-Keuls, *P < 0.05.

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Figs. 2A and B show a statistically significant linear response between immunostaining intensity and increasing 4-HNE levels (r = 0.95, P < 0.0001). Figs. 2C and D show similar results for acrolein (r = 0.84, P = 0.0006). 4-HNE and acrolein relative content in hippocampus To evaluate oxidative damage in IP hippocampus 1 and 7 days post injury, we measured levels of 4-HNE and acrolein, markers of lipid peroxidation. Because the controlled cortical impact injury model produces a gradient of secondary injury with no significant changes of antioxidant response in distal area, we used CON tissue as internal controls to limit animal to animal variability, and focused on oxidative response in IP tissues [30]. The data in IP tissues are expressed as percentage of CON tissues. Our data show that 4-HNE levels in IP hippocampus significantly (P < 0.05) increase 1 day post injury in young (230.1 ± 53.4%), middle aged (253.2 ± 45.1%), and aged animals (288.6 ± 46.3%) (Fig. 3). Statistical analyses showed an age-dependent difference [F(2,27) = 3.2334, P < 0.05], with aged animals showing significantly higher 4-HNE levels than young animals (P < 0.05, Student-NewmanKeuls test) 1 day post injury. Another lipid peroxidation marker, acrolein, showed a similar trend with a significant agedependent increase 1 day post injury [F(2,27) = 3.5055, P < 0.05], although the differences were more modest (Fig. 4).

Fig. 3. 4-Hydroxynonenal levels in IP hippocampus following TBI. Bars represent the mean ± SD. *P < 0.05 compared with young animals at corresponding time point.

At 7 days post injury, 4-HNE levels showed an age-dependent increase [F(2,19) = 9.598, P < 0.01], although overall levels decreased significantly for young (104.3 ± 10.3%) and middle aged (138.8 ± 55.6%) animals compared to levels observed 1 day post injury. No significant changes were observed for acrolein in hippocampus 7 days post injury for any age group.

Fig. 2. Correlation of 4-HNE and acrolein immunohistochemical staining of protein samples incubated with increasing concentrations of 4-HNE or acrolein. Each sample was loaded in triplicate. (A) Calibration curve of 4-HNE concentration vs immunohistochemical staining, r = 0.9532. (B) Representative dot blot with increasing 4-HNE concentrations. (C) Calibration curve of acrolein concentration vs staining intensity, r = 0.8414. (D) Representative dot blot with increasing acrolein concentrations.

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Antioxidant enzyme activities in IP hippocampus and cortex

Fig. 4. Acrolein levels in IP hippocampus following TBI. Bars represent mean ± SD. *P < 0.05 compared with young animals in corresponding time point.

4-HNE and acrolein relative content in cortex In cortex, mechanical injury triggers cell necrosis and apoptosis with concomitant production of ROS and toxic byproducts of lipid peroxidation including 4-HNE and acrolein. Our data from IP cortex are expressed as percentage of CON cortex. Levels of 4-HNE-modified proteins in IP cortex in young, middle aged, and aged animals 1 day post injury showed no age-dependent changes, although 4-HNE protein adducts were increased in young (166.5 ± 17.8%), middle aged (166.5 ± 29.9%), and aged animals (160.8 ± 20%) compared to corresponding CON cortex (Table 2). Acrolein levels in IP cortex did not change significantly in any age group 1 day post injury (Table 2). In contrast, 4-HNE levels increased for middle aged animals (371.7 ± 28.7%), but decreased for young (110.7 ± 16.0%) and aged (148.2 ± 52.9%) animals at 7 days post injury relative to levels at 1 day post injury. Post hoc testing following ANOVA [F(2,17) = 87.037, P < 0.001] showed that 4-HNE levels at 7 days post injury were significantly higher in middle aged animals compared with young animals. Our data also showed significantly increased acrolein in middle aged (179.3 ± 38.9%) and aged animals (169.2 ± 72.8%) compared with young animals (104.7 ± 10.6%) 7 days post injury.

Measurement of markers of lipid peroxidation showed increased oxidative stress in aging. To determine if alterations in antioxidant enzyme capacity occurred as a function of age, Cu/Zn and MnSOD, GPx, GRed, and GST activities were determined in IP and CON hippocampus and cortex 1 day and 7 days post injury. The data from IP tissues were expressed as percentage of those from corresponding CON tissues. At 1 day post injury, IP hippocampus showed decreased MnSOD activity for aged animals (83.9 ± 17.9%) (Table 1), a slight decrease for young (97.7 ± 16.3%), and a slight increase for middle aged (111.8 ± 24.1%) animals. ANOVA showed a significant age-dependent decrease of MnSOD in IP hippocampus [F(2,27) = 4.54, P < 0.05], with more pronounced loss of MnSOD activity (P < 0.05) in aged animals compared with middle aged animals. At 7 days post injury, our data showed that MnSOD activities in middle aged (66.7 ± 38.9%) and aged (71.2 ± 8.9%) animals decreased significantly compared with activities 1 day post injury, but not for young animals (95.8 ± 8.9%). ANOVA [F(2,20) = 3.5004, P < 0.05] showed an age-dependent decrease of MnSOD activity, with aged animals showing significantly lower activities compared with young animals. In IP cortex, our data showed MnSOD activity decreased significantly (P < 0.05) in middle aged (63.5 ± 27.2%) and aged animals (60.7 ± 20.8%) compared with young animals (98.7 ± 28.2%) 1 day post injury [F(2,25) = 5.9072, P < 0.01] (Table 2). At 7 days post injury, young and middle aged animals showed significant depletion of MnSOD compared to 1 day. Analysis of Cu/ZnSOD activities showed no significant changes in IP hippocampus or cortex among the three ages of animals at any time point. However, Cu/ZnSOD activity decreased significantly at 7 days post injury (54.1 ± 11.6%) compared to 1 day post injury (92.4 ± 22.6%) in IP cortex of aged animals (Table 2). Levels of GPx, which reduces oxidative stress by converting H2O2 and a variety of organic peroxides to stable alcohols and water, showed an increase in young IP hippocampus 1 day (169.3 ± 68.9%) and 7 days post injury (143.9 ± 88.0%), although the increases were not statistically significant. Only slight changes were observed in response to injury in middle aged and aged IP hippocampus (Table 1). Statistical analysis

Table 1 Antioxidant enzyme activities in IP hippocampus in aging Young

MnSOD Cu/ZnSOD GPx GRed GST

Middle aged

Aged

1 day

7 days

1 day

7 days

1 day

7 days

97.7 ± 19.8 104.3 ± 22.5 169.3 ± 68.9 111.2 ± 13.6 95.9 ± 8.1

95.8 ± 24.6 95.8 ± 10.1 143.9 ± 88.0 98.1 ± 24.2 94.3 ± 16.6

111.8 ± 23.5 115.8 ± 50.5 111.0 ± 39.7 ⁎ 107.4 ± 8.8 92.8 ± 12.6

66.7 ± 38.9 # 129.5 ± 71.6 113.4 ± 13.1 89.3 ± 6.1 # 84.8 ± 14.6

83.9 ± 17.9 $ 113.4 ± 24.7 99.6 ± 20.0 ⁎ 108.4 ± 18.0 94.1 ± 13.5

71.2 ± 8.9⁎,# 105.1 ± 12.1 107.2 ± 14.0 87.5 ± 8.7 # 87.7 ± 5.7

All values are mean ± SD. The value is percentage of that of CON tissue. # P < 0.05 compared with 1 day in the same age group. $ P < 0.05 compared with middle aged IP tissues at the same time point. * P < 0.05 compared with young IP tissues at the same time point.

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Table 2 4-HNE, acrolein and antioxidant enzyme activities in IP cortex in aging Young 1 day 4-HNE Acrolein MnSOD Cu/ZnSOD GPx GRed GST

166.5 ± 17.8 103.1 ± 19.5 98.7 ± 28.2 71.9 ± 21.1 116.4 ± 35.3 99.8 ± 20.0 81.5 ± 17.7

Middle aged 7 days 110.7 ± 16.0 104.7 ± 10.6 73.2 ± 12.3 # 77.7 ± 12.7 149.2 ± 38.8 93.2 ± 22.5 65.3 ± 23.3

Aged

1 day

7 days

1 day

7 days

166.5 ± 29.9 104.4 ± 19.3 63.5 ± 27.2 ⁎ 97.7 ± 37.0 106.2 ± 30.3 81.5 ± 14.1 ⁎ 69.6 ± 20.8

371.7 ± 28.7 ⁎ 179.3 ± 38.9 ⁎ 61.1 ± 16.5 63.8 ± 23.7 180.7 ± 106.3 66.0 ± 33.8 43.3 ± 18.6 #

160.8 ± 22.7 115.2 ± 30.0 60.7 ± 20.8 ⁎ 92.4 ± 22.6 94.8 ± 40.2 82.0 ± 16.6 ⁎ 70.5 ± 20.7

148.2 ± 52.9 $ 169.2 ± 72.8 ⁎ 48.4 ± 20.3 ξ 54.1 ± 11.6 ξ 101.8 ± 31.0 102.2 ± 29.4 87.7 ± 28.9 $

All values are mean ± SD. The value is percentage of that of CON tissue. * P < 0.05 compared with young IP tissues at the same time point. $ P < 0.05 compared with middle aged IP tissues at the same time point. # P < 0.05 compared with 1 day in the same age group. ξ P < 0.01 compared with 1 day in the same age group.

showed that GPx activity was significantly lower (P < 0.05) in middle aged (111.0 ± 39.7%) and aged animals (99.6 ± 20.0%) compared to young animals 1 day post injury in IP hippocampus (Table 1). In IP cortex, GPx activity increased in young and middle aged animals 7 days post injury, but the changes did not reach statistical significance (Table 2). Glutathione transferase activities showed a significant decrease (P < 0.05) in IP cortex in middle aged animals (43.3 ± 18.6%) compared with aged animals (87.7 ± 28.9%) 7 days post injury. Statistical analyses showed GST activity also decreased significantly 7 days post injury compared to 1 day post injury in middle aged animals (Table 2). In IP hippocampus, GST activities did not show age and post injury time-dependent changes (Table 1). Another antioxidant enzyme, GRed, which functions to reduce oxidized glutathione to reduced glutathione for participation in the GPx reaction, was significantly depleted in IP cortex (P < 0.05) of middle aged (81.5 ± 14.1%) and aged animals (82.0 ± 16.6%) compared to young animals (99.8 ± 20.0%) 1 day post injury (Table 2). In IP hippocampus, our data showed no age-dependent differences at either time point (Table 1). However, IP hippocampal GRed activity decreased significantly (P < 0.05) 7 days post injury (89.3 ± 6.1% for middle aged, 87.5 ± 8.7% for aged) compared to 1 day (107.4 ± 8.8% for middle aged, 108.4 ± 18.0% for aged) post injury (Table 1). Young animals showed no significant change in IP hippocampal GRed activity. Discussion Aging is associated with an increased mortality rate and greater acute neurological deficits following TBI, but the mechanism is not well understood [10,11,31–34]. There has been little study of markers of oxidative stress or antioxidant capacities following TBI in aged animals [35]. The present studies demonstrate an age-dependent decrease in tissue sparing, increased lipid peroxidation, and diminished antioxidant enzyme capacities 1 and 7 days following TBI. Previous studies show that alterations in glutamate, an excitatory amino acid, coupled with increased oxidative stress were major factors contributing to the progressive neuropa-

thology in TBI [36]. Mitochondria are the power source of cells, and are thought to be major sources of free radical production following TBI [6,7,37]. There are several antioxidant enzymes in neurons that decrease oxidative stress, including Cu/ZnSOD, MnSOD, catalase, GPx, and GST [38–43]. Cu/ZnSOD, a cytosolic enzyme and MnSOD a mitochondrial enzyme, catalyze the dismutation of superoxide radical to hydrogen peroxide, which is then converted to water by catalase and GPx. Glutathione transferase can detoxify aldehydic by-products of lipid peroxidation through conjugation to glutathione. Another antioxidant enzyme, GRed, functions to regenerate reduced glutathione for GPx. Although these enzymes work in concert to protect the cell, cell death can result if ROS production exceeds the defense capacity. Free radical species can cause extensive damage to biological macromolecules, including peroxidation of membrane polyunsaturated fatty acids (PUFA) [44–46]. PUFA peroxidation ultimately leads to loss of both structural and functional integrity of the cell with generation of toxic aldehydic byproducts, such as 4-HNE and acrolein [47], which may further contribute to neuronal cell death by protein and DNA modification [48]. Neuron death can lead to tissue shrinkage or loss following TBI. In an aging study, Hoane et al. [33] showed that middle aged animals exhibited increased tissue loss compared to young animals following TBI, although aged animals were not investigated in their study. Our current data show that middle aged and aged animals both experience significant tissue loss compared to young animals with no significant difference between middle aged and aged animals 7 days post injury. Although the hippocampus does not exhibit significant tissue loss in cortical TBI, neuron degeneration has been detected up to 7 days post injury, which may contribute to memory deficit [49]. Oxidative damage is suggested to be an important factor contributing to those neurological deficits [49–51] following TBI except for tissue loss, and is hypothesized to play a role in diminished recovery from TBI in aged subjects. There has been limited study of oxidative damage in the hippocampus following TBI. Using immunohistochemistry Wang et al. [39] showed that 4-HNE levels increased following TBI in hippocampus in young

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animals. Using dot-blot immunochemistry, we show that 4HNE levels increased significantly in IP hippocampus 1 day post injury in all age groups, suggesting severe oxidative damage at this time point. The intensive oxidative damage could lead to delayed neuron death in IP hippocampus. Acrolein, another neurotoxic marker of lipid peroxide showed a similar trend as 4-HNE with aged animals showing higher levels in IP hippocampus compared to CON hippocampus. At 7 days post injury, 4-HNE levels were reduced to basal levels in young animals compared to 1 day post injury. Although middle aged animals also showed reduced levels of 4-HNE compared to 1 day post injury, the levels were elevated compared to CON hippocampus. Levels of 4-HNE remained elevated in aged animals at 7 days post injury. The agedependent oxidative damage in hippocampus may help explain increased functional deficits following TBI in aged subjects. Differences in oxidative damage in aging may result from diminished antioxidant capacities. Superoxide dismutases function to convert superoxide ion to less active hydrogen peroxide and avoid the formation of further ROS, and have been widely investigated in neurodegenerative diseases. Transgenic mice overexpressing SOD showed increased tissue sparing and improved neurologic function compared with their WT littermates following TBI [52]. In addition, DeKosky et al. [30] reported that SOD activity decreased significantly 6 h after TBI and 20% of activity was lost 1 day and 30% lost 7 days post injury in young rats subjected to severe injury in the anterior hippocampus. However, this study did not include middle aged or aged animals. Our data show that there are no significant changes in hippocampal Cu/ZnSOD activity for any age or time point studied. Although hippocampal MnSOD does not change significantly in young and middle aged animals 1 day post injury, it decreased significantly (20%) in aged animals compared to middle aged animals. In contrast to the study of DeKosky et al. [30], we did not observe significant losses of SOD even 7 days post injury in young animals. However, we observed a 30% loss in IP hippocampus MnSOD activity in middle aged and aged animals. Statistical analysis showed an age-dependent decrease of MnSOD activity in aging. The decrease of the mitochondrial MnSOD could compromise the cell's ability to remove superoxide generated after the TBI and lead to increased lipid peroxidation [53], increased neuron loss, and increased cognitive deficits in older animals. In the antioxidant defense system, GPx follows SOD and reduces hydrogen peroxide to water with consumption of glutathione. Table 1 shows that GPx increased in young IP hippocampus 1 and 7 days post injury with little change in middle aged or aged animals. A significant age-dependent decrease of GPx activity was observed 1 day post injury in middle aged and aged animals compared to young animals. At 7 days post injury, GPx activities were lower in middle aged and aged IP hippocampus compared with young IP hippocampus, although the differences did not reach statistical significance. These data are consistent with those of DeKosky et al. [30] who showed increased GPx activity in hippocampus in young TBI animals 7 days post injury. Our data further show that GPx

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protein expression increased significantly (data not shown) in IP cortex and hippocampus, which is consistent with the theory that this enzyme is upregulated by oxidative stress [54]. However higher abundance of this enzyme did not improve neuron antioxidant capacity in middle aged and aged animals. Young animals showed elevated GPx activities compared with older animals that may help to limit oxidative damage. Previous studies show GST overexpression is neuroprotective in cell culture [38]. Glutathione transferase can alleviate damage from lipid peroxidation by-products (4-HNE and acrolein) by catalyzing their conjugation with glutathione. GST depletion could result in increased protein modification and lead to increased protein dysfunction, which could contribute to further oxidative stress. Our data show no significant differences in GST activity at any time point or for any age in IP hippocampus. In IP cortex, GST activity was significantly decreased in middle aged animals 7 day post injury. In contrast, GST levels of aged cortex reached levels approaching those of CON cortex. Glutathione reductase, which functions to regenerate reduced glutathione, has been studied in neurodegenerative disease [29], but not in TBI. Our study shows a significant decrease in GRed in middle aged and aged IP hippocampus at 1 and 7 days post injury, although there were no age-dependent differences. In cortex, primary mechanical brain injury causes subdural hematoma, laceration, diffuse vascular injury, and cell death in the impact zone that involves extensively complicated processes [55,56]. ROS generation in cortex of young animals following TBI was shown to increase with time in the early stages [57– 59]. In addition, lipid peroxidation products were significantly increased immediately following injury, and remained elevated for several days in young animals [45,60,61]. In our study, 4HNE levels increased in IP cortex in each age group at 1 day post injury, but did not show age-dependent differences (Table 2). At 7 days post injury, 4-HNE levels decreased in IP cortex in young animals, and kept increasing in middle aged animals. Aged animals did not show more oxidative damage 7 days post injury compared to 1 day post injury. Unlike oxidative stress in hippocampus, there is no clear age-dependent change in cortex, especially at 1 day post injury. Hall et al. [58] reported that a major source of ROS is microvasculature, and saline perfusion of the injured mice eliminated ROS levels measured in their study. It is possible that blood in the cortex tissue masked the difference in our study. Antioxidant enzyme activities have not been studied in cortex in TBI even though oxidative stress has been well documented over the last several decades. Previous studies show that secondary damage can regulate gene expression and inflammatory response following TBI, especially genes related to metabolism and antioxidant enzymes [62–64]. However, proteins are the units that function directly in cell defense, and gene transcription change may not indicate antioxidant enzyme defense capacity since oxidative damage may inactivate enzymes following TBI. In our study, antioxidant enzymes were investigated to evaluate possible changes in antioxidant capacity as a function of age. Our data show that MnSOD activity in IP cortex was depleted in an age-dependent manner 1

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day post injury (Table 2) and that the decrease correlated with increased tissue loss. Diminished antioxidant activity may compromise defense capacities against oxidative stress [6] and may contribute to neuron death in the impact zone. At 7 days post injury, MnSOD activity in IP cortex significantly decreased in young animals. MnSOD also decreased in aged animals, but did not show an age-dependent difference. Activities of Cu/ ZnSOD were decreased in IP cortex of all three age groups 7 days post injury and of young animals 1 day post injury, but they did not show significant age-dependent differences. GPx activity in IP cortex did not change significantly 1 day post injury in any age group studied, although young and middle aged animals showed increased activity at 7 days post injury. Comparison of data for an age-dependent effect showed no agedependent differences at either time point. GST activity decreased 1 day post injury in IP cortex in each age group with more pronounced losses occurring in middle aged animals 7 days post injury. GST depletion in cortex may result in intensive biochemical modification of biomolecules by toxic aldehydic by-products from lipid peroxidation, suggesting middle aged animals may suffer further oxidative damage. GRed decreased significantly in middle aged and aged IP cortex compared to young animals 1 day post injury, although no significant differences were observed 7 days post injury. GRed depletion 1 day post injury could contribute to depletion of glutathione, a critical low molecular weight antioxidant, and contribute to cell death and tissue loss in later days. In summary, our data show increased tissue loss and alterations in antioxidant capacity in cortex in TBI in aging. Examination of IP hippocampus showed that 4-HNE and acrolein levels increase and antioxidant enzyme decrease in an age-dependent manner. The tissue loss coupled with increased levels of lipid peroxidation may contribute to deficit in behavioral recovery in TBI in aging. Acknowledgments This work was supported by the Kentucky Spinal Cord and Head Trauma Research Trust (No.1-10A), Abercrombie Foundation, and NIH AG21981. The authors thank Paula Thomason for editorial assistance. References [1] Cooper, P. Central nervous system trauma status reportIn: Povlishhock J.; Becker, D., eds. Delayed brain injury. Washington, DC: National Institutes of Health; 1985:217–282. [2] Faden, A. I. Experimental neurobiology of central nervous system trauma. Crit. Rev. Neurobiol. 7 (3-4):175–186; 1993. [3] Mattson, M. P.; Scheff, S. W. Endogenous neuroprotection factors and traumatic brain injury: mechanisms of action and implications for therapy. J. Neurotrauma 11 (1):3–33; 1994. [4] Siesjo, B. K.; Katsura, K.; Zhao, Q.; Folbergrova, J.; Pahlmark, K.; Siesjo, P.; Smith, M. L. Mechanisms of secondary brain damage in global and focal ischemia: a speculative synthesis. J. Neurotrauma 12 (5):943–956; 1995. [5] Tator, C. The neurobiology of central nervous system traumaIn: Salzman, S., Faden, A., eds. Ischemia as a secondary neural injury. Oxford: Oxford; 1994.

[6] Azbill, R. D.; Mu, X.; Bruce-Keller, A. J.; Mattson, M. P.; Springer, J. E. Impaired mitochondrial function, oxidative stress and altered antioxidant enzyme activities following traumatic spinal cord injury. Brain Res. 765 (2):283–290; 1997. [7] Sullivan, P. G.; Keller, J. N.; Mattson, M. P.; Scheff, S. W. Traumatic brain injury alters synaptic homeostasis: implications for impaired mitochondrial and transport function. J. Neurotrauma 15 (10):789–798; 1998. [8] Hsiang, J. N.; Wang, J. Y.; Ip, S. M.; Ng, H. K.; Stadlin, A.; Yu, A. L.; Poon, W. S. The time course and regional variations of lipid peroxidation after diffuse brain injury in rats. Acta Neurochir. (Wien) 139 (5):464–468; 1997. [9] Hoffman, S. W.; Roof, R. L.; Stein, D. G. A reliable and sensitive enzyme immunoassay method for measuring 8-isoprostaglandin F2 alpha: a marker for lipid peroxidation after experimental brain injury. J. Neurosci. Methods 68 (2):133–136; 1996. [10] Hamm, R. J.; Jenkins, L. W.; Lyeth, B. G.; White-Gbadebo, D. M.; Hayes, R. L. The effect of age on outcome following traumatic brain injury in rats. J. Neurosurg. 75 (6):916–921; 1991. [11] Hamm, R. J.; White-Gbadebo, D. M.; Lyeth, B. G.; Jenkins, L. W.; Hayes, R. L. The effect of age on motor and cognitive deficits after traumatic brain injury in rats. Neurosurgery 31 (6):1072–1077 (discussion 1078); 1992. [12] Susman, M.; DiRusso, S. M.; Sullivan, T.; Risucci, D.; Nealon, P.; Cuff, S.; Haider, A.; Benzil, D. Traumatic brain injury in the elderly: increased mortality and worse functional outcome at discharge despite lower injury severity. J. Trauma 53 (2):219–223 (discussion 223–214); 2002. [13] Medina-Navarro, R.; Duran-Reyes, G.; Diaz-Flores, M.; Hicks, J. J.; Kumate, J. Glucose-stimulated acrolein production from unsaturated fatty acids. Hum. Exp. Toxicol. 23 (2):101–105; 2004. [14] Esterbauer, H.; Schaur, R. J.; Zollner, H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11 (1):81–128; 1991. [15] Springer, J. E.; Azbill, R. D.; Mark, R. J.; Begley, J. G.; Waeg, G.; Mattson, M. P. 4-hydroxynonenal, a lipid peroxidation product, rapidly accumulates following traumatic spinal cord injury and inhibits glutamate uptake. J. Neurochem. 68 (6):2469–2476; 1997. [16] Baldwin, S. A.; Broderick, R.; Osbourne, D.; Waeg, G.; Blades, D. A.; Scheff, S. W. The presence of 4-hydroxynonenal/protein complex as an indicator of oxidative stress after experimental spinal cord contusion in a rat model. J. Neurosurg. 88 (5):874–883; 1998. [17] Lovell, M. A.; Ehmann, W. D.; Mattson, M. P.; Markesbery, W. R. Elevated 4-hydroxynonenal in ventricular fluid in Alzheimer's disease. Neurobiol. Aging 18 (5):457–461; 1997. [18] Lovell, M. A.; Xie, C.; Markesbery, W. R. Acrolein is increased in Alzheimer's disease brain and is toxic to primary hippocampal cultures. Neurobiol. Aging 22 (2):187–194; 2001. [19] Liu, Y.; Xu, G.; Sayre, L. M. Carnosine inhibits (E)-4-hydroxy-2-nonenalinduced protein cross-linking: structural characterization of carnosineHNE adducts. Chem. Res. Toxicol. 16 (12):1589–1597; 2003. [20] Engle, M. R.; Singh, S. P.; Czernik, P. J.; Gaddy, D.; Montague, D. C.; Ceci, J. D.; Yang, Y.; Awasthi, S.; Awasthi, Y. C.; Zimniak, P. Physiological role of mGSTA4-4, a glutathione S-transferase metabolizing 4-hydroxynonenal: generation and analysis of mGsta4 null mouse. Toxicol. Appl. Pharmacol. 194 (3):296–308; 2004. [21] Baldwin, S. A.; Scheff, S. W. Intermediate filament change in astrocytes following mild cortical contusion. Glia 16 (3):266–275; 1996. [22] Michel, R. P.; Cruz-Orive, L. M. Application of the Cavalieri principle and vertical sections method to lung: estimation of volume and pleural surface area. J. Microsc. 150 (Pt. 2):117–136; 1988. [23] Sullivan, P. G.; Bruce-Keller, A. J.; Rabchevsky, A. G.; Christakos, S.; Clair, D. K.; Mattson, M. P.; Scheff, S. W. Exacerbation of damage and altered NF-kappaB activation in mice lacking tumor necrosis factor receptors after traumatic brain injury. J. Neurosci. 19 (15):6248–6256; 1999. [24] Saiki, R. K.; Bugawan, T. L.; Horn, G. T.; Mullis, K. B.; Erlich, H. A. Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature 324 (6093):163–166; 1986.

C. Shao et al. / Free Radical Biology & Medicine 41 (2006) 77–85 [25] Misra, H. P.; Fridovich, I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247 (10):3170–3175; 1972. [26] Mizuno, Y. Changes in superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities and thiobarbituric acidreactive products levels in early stages of development in dystrophic chickens. Exp. Neurol. 84 (1):58–73; 1984. [27] Ricci, G.; Caccuri, A. M.; Lo Bello, M.; Pastore, A.; Piemonte, F.; Federici, G. Colorimetric and fluorometric assays of glutathione transferase based on 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Anal. Biochem. 218 (2):463–465; 1994. [28] Paglia, D. E.; Valentine, W. N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70 (1):158–169; 1967. [29] Lovell, M. A.; Ehmann, W. D.; Butler, S. M.; Markesbery, W. R. Elevated thiobarbituric acid-reactive substances and antioxidant enzyme activity in the brain in Alzheimer's disease. Neurology 45 (8):1594–1601; 1995. [30] DeKosky, S. T.; Taffe, K. M.; Abrahamson, E. E.; Dixon, C. E.; Kochanek, P. M.; Ikonomovic, M. D. Time course analysis of hippocampal nerve growth factor and antioxidant enzyme activity following lateral controlled cortical impact brain injury in the rat. J. Neurotrauma 21 (5):491–500; 2004. [31] Maughan, P. H.; Scholten, K. J.; Schmidt, R. H. Recovery of water maze performance in aged versus young rats after brain injury with the impact acceleration model. J. Neurotrauma 17 (12):1141–1153; 2000. [32] Klein, M.; Houx, P. J.; Jolles, J. Long-term persisting cognitive sequelae of traumatic brain injury and the effect of age. J. Nerv. Ment. Dis. 184 (8):459–467; 1996. [33] Hoane, M. R.; Lasley, L. A.; Akstulewicz, S. L. Middle age increases tissue vulnerability and impairs sensorimotor and cognitive recovery following traumatic brain injury in the rat. Behav. Brain Res. 153 (1):189–197; 2004. [34] Teasdale, G.; Skene, A.; Parker, L.; Jennett, B. Age and outcome of severe head injury. Acta Neurochir. Suppl. (Wien) 28 (1):140–143; 1979. [35] Wagner, A. K.; Bayir, H.; Ren, D.; Puccio, A.; Zafonte, R. D.; Kochanek, P. M. Relationships between cerebrospinal fluid markers of excitotoxicity, ischemia, and oxidative damage after severe TBI: the impact of gender, age, and hypothermia. J. Neurotrauma 21 (2):125–136; 2004. [36] Faden, A. I.; Demediuk, P.; Panter, S. S.; Vink, R. The role of excitatory amino acids and NMDA receptors in traumatic brain injury. Science 244 (4906):798–800; 1989. [37] Matsushita, M.; Xiong, G. Projections from the cervical enlargement to the cerebellar nuclei in the rat, studied by anterograde axonal tracing. J. Comp. Neurol. 377 (2):251–261; 1997. [38] Xie, C.; Lovell, M. A.; Xiong, S.; Kindy, M. S.; Guo, J.; Xie, J.; Amaranth, V.; Montine, T. J.; Markesbery, W. R. Expression of glutathione-Stransferase isozyme in the SY5Y neuroblastoma cell line increases resistance to oxidative stress. Free Radic. Biol. Med. 31 (1):73–81; 2001. [39] Wang, H.; Cheng, E.; Brooke, S.; Chang, P.; Sapolsky, R. Overexpression of antioxidant enzymes protects cultured hippocampal and cortical neurons from necrotic insults. J. Neurochem. 87 (6):1527–1534; 2003. [40] Okado-Matsumoto, A.; Fridovich, I. Subcellular distribution of superoxide dismutases (SOD) in rat liver: Cu,Zn-SOD in mitochondria. J. Biol. Chem. 276 (42):38388–38393; 2001. [41] Valentine, J. S.; Wertz, D. L.; Lyons, T. J.; Liou, L. L.; Goto, J. J.; Gralla, E. B. The dark side of dioxygen biochemistry. Curr. Opin. Chem. Biol. 2 (2):253–262; 1998. [42] Ho, Y. S.; Magnenat, J. L.; Bronson, R. T.; Cao, J.; Gargano, M.; Sugawara, M.; Funk, C. D. Mice deficient in cellular glutathione peroxidase develop normally and show no increased sensitivity to hyperoxia. J. Biol. Chem. 272 (26):16644–16651; 1997. [43] Fridovich, I. Superoxide anion radical (O2– ), superoxide dismutases, and related matters. J. Biol. Chem. 272 (30):18515–18517; 1997.

.

85

[44] Beckman, J. S.; Carson, M.; Smith, C. D.; Koppenol, W. H. ALS, SOD and peroxynitrite. Nature 364 (6438):584; 1993. [45] Hall, E. D.; Detloff, M. R.; Johnson, K.; Kupina, N. C. Peroxynitritemediated protein nitration and lipid peroxidation in a mouse model of traumatic brain injury. J. Neurotrauma 21 (1):9–20; 2004. [46] McCall, J. M.; Braughler, J. M.; Hall, E. D. Lipid peroxidation and the role of oxygen radicals in CNS injury. Acta Anaesthesiol. Belg. 38 (4): 373–379; 1987. [47] Uchida, K.; Kanematsu, M.; Morimitsu, Y.; Osawa, T.; Noguchi, N.; Niki, E. Acrolein is a product of lipid peroxidation reaction. Formation of free acrolein and its conjugate with lysine residues in oxidized low density lipoproteins. J. Biol. Chem 273 (26):16058–16066; 1998. [48] Graham, D. I.; Adams, J. H.; Doyle, D. Ischaemic brain damage in fatal non-missile head injuries. J. Neurol. Sci. 39 (2–3):213–234; 1978. [49] Hall, E. D.; Sullivan, P. G.; Gibson, T. R.; Pavel, K. M.; Thompson, B. M.; Scheff, S. W. Spatial and temporal characteristics of neurodegeneration after controlled cortical impact in mice: more than a focal brain injury. J. Neurotrauma 22 (2):252–265; 2005. [50] Camandola, S.; Poli, G.; Mattson, M. P. The lipid peroxidation product 4hydroxy-2,3-nonenal increases AP-1-binding activity through caspase activation in neurons. J. Neurochem. 74 (1):159–168; 2000. [51] Anderson, K. J.; Miller, K. M.; Fugaccia, I.; Scheff, S. W. Regional distribution of fluoro-jade B staining in the hippocampus following traumatic brain injury. Exp. Neurol. 193 (1):125–130; 2005. [52] Maier, C. M.; Chan, P. H. Role of superoxide dismutases in oxidative damage and neurodegenerative disorders. Neuroscientist 8 (4):323–334; 2002. [53] Awasthi, D.; Church, D. F.; Torbati, D.; Carey, M. E.; Pryor, W. A. Oxidative stress following traumatic brain injury in rats. Surg. Neurol. 47 (6):575–581 (discussion 581–572); 1997. [54] Baud, O.; Greene, A. E.; Li, J.; Wang, H.; Volpe, J. J.; Rosenberg, P. A. Glutathione peroxidase-catalase cooperativity is required for resistance to hydrogen peroxide by mature rat oligodendrocytes. J. Neurosci. 24 (7):1531–1540; 2004. [55] Raghupathi, R. Cell death mechanisms following traumatic brain injury. Brain Pathol. 14 (2):215–222; 2004. [56] Biros, M. Emergency medicine: concepts and clinical practice, In: Rosen, P., Barkin, R.M., Danzi, D.F., et al. (Eds.), Head trauma, 4th Ed. Mosby Year Book Inc., St. Louis; 1998. [57] Nishio, S.; Yunoki, M.; Noguchi, Y.; Kawauchi, M.; Asari, S.; Ohmoto, T. Detection of lipid peroxidation and hydroxyl radicals in brain contusion of rats. Acta Neurochir. Suppl. 70:84–86; 1997. [58] Hall, E. D.; Andrus, P. K.; Yonkers, P. A. Brain hydroxyl radical generation in acute experimental head injury. J. Neurochem. 60 (2):588–594; 1993. [59] Smith, S. L.; Andrus, P. K.; Zhang, J. R.; Hall, E. D. Direct measurement of hydroxyl radicals, lipid peroxidation, and blood-brain barrier disruption following unilateral cortical impact head injury in the rat. J. Neurotrauma 11 (4):393–404; 1994. [60] Inci, S.; Ozcan, O. E.; Kilinc, K. Time-level relationship for lipid peroxidation and the protective effect of alpha-tocopherol in experimental mild and severe brain injury. Neurosurgery 43 (2):330–335 (discussion 335–336); 1998. [61] Tyurin, V. A.; Tyurina, Y. Y.; Borisenko, G. G.; Sokolova, T. V.; Ritov, V. B.; Quinn, P. J.; Rose, M.; Kochanek, P.; Graham, S. H.; Kagan, V. E. Oxidative stress following traumatic brain injury in rats: quantitation of biomarkers and detection of free radical intermediates. J. Neurochem. 75 (5):2178–2189; 2000. [62] Shetty, A. K.; Rao, M. S.; Hattiangady, B.; Zaman, V.; Shetty, G. A. Hippocampal neurotrophin levels after injury: relationship to the age of the hippocampus at the time of injury. J. Neurosci. Res. 78 (4):520–532; 2004. [63] Matzilevich, D. A.; Rall, J. M.; Moore, A. N.; Grill, R. J.; Dash, P. K. Highdensity microarray analysis of hippocampal gene expression following experimental brain injury. J. Neurosci. Res. 67 (5):646–663; 2002. [64] Long, Y.; Zou, L.; Liu, H.; Lu, H.; Yuan, X.; Robertson, C. S.; Yang, K. Altered expression of randomly selected genes in mouse hippocampus after traumatic brain injury. J. Neurosci. Res. 71 (5):710–720; 2003.