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Hyperbaric oxygen preconditioning protects rats against CNS oxygen toxicity

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Yehuda Arieli a,∗ , Doron Kotler a,b , Mirit Eynan a , Ayala Hochman b a b

Israel Naval Medical Institute, IDF Medical Corps, Haifa, Israel Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

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a b s t r a c t

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Article history: Accepted 18 March 2014

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Keywords: Central nervous system oxygen toxicity Hyperbaric oxidative stress Reactive oxygen species scavengers

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1. Introduction

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We examined the hypothesis that repeated exposure to non-convulsive hyperbaric oxygen (HBO) as preconditioning provides protection against central nervous system oxygen toxicity (CNS-OT). Four groups of rats were used in the study. Rats in the control and the negative control (Ctl−) groups were kept in normobaric air. Two groups of rats were preconditioned to non-convulsive HBO at 202 kPa for 1 h once every other day for a total of three sessions. Twenty-four hours after preconditioning, one of the preconditioned groups and the control rats were exposed to convulsive HBO at 608 kPa, and latency to CNS-OT was measured. Ctl− rats and the second preconditioned group (PrC−) were not subjected to convulsive HBO exposure. Tissues harvested from the hippocampus and frontal cortex were evaluated for enzymatic activity and nitrotyrosine levels. In the group exposed to convulsive oxygen at 608 kPa, latency to CNS-OT increased from 12.8 to 22.4 min following preconditioning. A significant decrease in the activity of glutathione reductase and glucose-6-phosphate dehydrogenase, and a significant increase in glutathione peroxidase activity, was observed in the hippocampus of preconditioned rats. Nitrotyrosine levels were significantly lower in the preconditioned animals, the highest level being observed in the control rats. In the cortex of the preconditioned rats, a significant increase was observed in glutathione S-transferase and glutathione peroxidase activity. Repeated exposure to non-convulsive HBO provides protection against CNS-OT. The protective mechanism involves alterations in the enzymatic activity of the antioxidant system and lower levels of peroxynitrite, mainly in the hippocampus. © 2014 Published by Elsevier B.V.

Hyperbaric hyperoxia can lead to central nervous system oxygen toxicity (CNS-OT), resulting in grand mal convulsions, loss of consciousness, and in extreme cases, death. It is as yet unknown whether repetitive exposure to hyperbaric oxygen (HBO), which is common practice in hyperbaric medicine (Gesell, 2008) and military diving, has a cumulative effect. It is widely accepted that increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as peroxynitrite or NO2 , makes a major contribution to the development of CNS-OT (Oury et al., 1992; Beckman and Koppenol, 1996; Chavko and Harabin, 1996; Demchenko et al., 2001). The tissues

∗ Corresponding author at: Israel Naval Medical Institute, Box 22, Rambam Health Care Campus, P.O. Box 9602, 3109601 Haifa, Israel. Tel.: +972 4 8693257; fax: +972 4 8693258. E-mail addresses: [email protected], [email protected], [email protected] (Y. Arieli).

affected in the brain are the frontal cortex, the striatum, and the hippocampus (Wang et al., 1998; Garcia et al., 2010). Cells have evolved defense strategies against ROS, including antioxidant enzymes such as superoxide dismutase to scavenge superoxide, catalases and peroxidases to break down hydrogen peroxide, and glutathione S-transferase to neutralize lipid peroxides, as well as their auxiliary enzymes, glutathione reductase and glucose-6-phosphate dehydrogenase. Low levels of ROS stimulate adaptive responses by increasing the cellular activity of these enzymatic antioxidants, and of various low molecular weight antioxidants. Higher levels of ROS, such as are produced under conditions of hyperoxia, can overwhelm the antioxidative capacity of the cells and cause oxidative injury. This manifests as protein oxidation, DNA damage with increased mutational rates, and lipid peroxidation, resulting in membrane damage, metabolic perturbation and death. The general term used to describe these phenomena is “oxidative stress”. Nitric oxide (NO) is known to be a powerful vasodilator, and therefore a regulator of cerebral blood flow. It is inactivated by interaction with superoxide, which converts it to peroxynitrite.

http://dx.doi.org/10.1016/j.resp.2014.03.006 1569-9048/© 2014 Published by Elsevier B.V.

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Exposure to HBO is associated with increased levels of NO and its metabolites in the brain, which correlate positively with CNSOT (Oury et al., 1992; Beckman and Koppenol, 1996; Chavko and Harabin, 1996; Demchenko et al., 2001). Previous investigations demonstrated shortening of the latency to CNS-OT when rats were exposed to convulsive oxygen at 608 kPa once a day over an extended period, indicating a cumulative effect of the HBO exposure (Harel et al., 1969; Arieli and Hershko, 1994). On the other hand, there is a large body of evidence showing that environmental effectors such as heat acclimation (Arieli et al., 2003; Ay et al., 2007a) or preconditioning by exposure to non-convulsive HBO (Yu et al., 2005; Niu et al., 2007; Zhou et al., 2007; Li et al., 2008; Cimino et al., 2012), provide protection against hyperoxic injury, while the latter was also shown to protect against various aspects of hypoxic injury (Wada et al., 2001). However, studies of the possible beneficial effect of hyperoxic preconditioning on CNSOT are few and far between. In the present study, we examined the hypothesis that under well defined conditions of pressure, exposure duration and frequency, repetitive exposure to non-convulsive HBO as a preconditioning procedure may have a protective effect against CNS-OT.

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2. Materials and methods

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All experiments were approved by the Israel Ministry of Defense Animal Care Committee, and animals were handled in accordance with internationally accepted humane standards.

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2.1. Experimental animals

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EEG electrode implanted in the rat’s skull to monitor the first electrical discharge may affect the brain tissue attached to the skull. Immediately thereafter the gas flow to the chamber was switched back to air, and pressure was reduced at a rate of 101 kPa per min. As a result, convulsions lasted from 15 to 60 s. Rats in the second preconditioned group (PrC−) were not exposed to convulsive HBO. Rats in the negative control group (Ctl−) were not subjected to any HBO exposure (n = 5). At the end of each treatment protocol, the animals were anesthetized using a mixture of 1 ml xylazine (20 mg/ml) and 4 ml ketamine (50 mg/ml) in a dose of 1 ml/1500 g body weight, until no pain signals were monitored. The hippocampus and frontal cortex were quickly removed on ice, washed in ice-cold 10 mM phosphate buffer saline (pH 7.4), promptly frozen in liquid nitrogen, and stored at –80 ◦ C for biochemical analysis. 2.3. Preparation of crude extracts For preparation of homogenates, the brain samples were thawed, resuspended (1 g/2.5 ml) in 50 mM phosphate buffer (pH 6.8) containing 1 mM PMSF, 1 ␮g/ml leupeptin, 1 mM EDTA and 0.1% triton X-100, and homogenized by a hand-driven plastic homogenizer followed by sonication for 4 × 15 s using an ultrasonic processor (Misonix XL-2020, Misonix, Farmingdale, NY, USA). The extracts were centrifuged at 150,000 × g for 1 h at 4 ◦ C, and the resulting supernatant was stored at –80 ◦ C. 2.4. Assessment of antioxidant enzyme activity

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Three-month-old male Sprague-Dawley rats weighing 256–310 g were housed in plastic cages under standard conditions, with free access to drinking water and standard chow. They were kept in an 8 h light, 16 h dark cycle and the ambient temperature was maintained at 21–22 ◦ C.

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2.2. Preconditioning protocol and experimental procedure

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Rats were divided randomly into 4 groups, 15 animals in three of the groups, and 5 in the fourth group (Table 1). The animals in two of the groups were exposed one at a time to non-convulsive HBO at 202 kPa for 1 h, once every other day for a total of three sessions as preconditioning (Wada et al., 2001). Rats in the control (Ctl) group were kept in air under normobaric conditions. Twentyfour hours after the preconditioning procedure, the rats in one of the preconditioned groups (PrC) and the Ctl animals were exposed to convulsive HBO at 608 kPa, and latency to the appearance of CNS-OT convulsions was measured. All of the rats in these two groups exhibited seizures, whose occurrence was determined by observation of tail stiffness, sometimes associated with fast movement of the forelimbs (clearly not a “face-washing” procedure) or the back limbs, rolling around the chamber, sudden, uncontrolled movements or jumping, etc. When at least one of these symptoms persisted for more than 5 s, we measured the latency from the start of the exposure (O2 level > 95%). This method was used in an attempt to avoid contamination of the brain tissues to be examined, because we found, in previous investigations, that using an Table 1 List of experimental groups and treatments. Group #

Group name

N

Preconditioning

HBO

1 2 3 4

PrC PrC− Ctl Ctl−

15 15 15 5

+ + − −

+ − + −

Enzyme activity (except for catalase) was assessed spectrophotometrically in a micro-plate reader (Spectramax 190, Molecular Devices Co., Sunnyvale, CA, USA) using 96-well micro-titer plates (Costar, Corning, Inc., Corning, NY, USA), in a volume of 200 ␮l at 30 ◦ C. Glutathione peroxidase (GSH-Px) activity was determined as described elsewhere (Flohé and Günzler, 1984), with t-butyl hydroperoxide as a substrate. Briefly, the assay is based on the determination of oxidation of NADPH at 340 nm, in a reaction mixture containing 50 mM potassium phosphate (pH 7.0), 1 mM GSH, 0.12 mM t-butyl hydroperoxide, 0.15 mM NADPH, and 0.24 units of glutathione reductase. One unit of enzyme activity results in the oxidation of 1 ␮mol GSH/min. Glutathione S-transferase (GST) activity was determined spectrophotometrically according to Habig et al. (1974) using 1-chloro-2,4-dinitrophenol as a substrate. The reaction mixture contained 70 mM potassium phosphate (pH 6.5), 2.1 mM 1-chloro2,4-dinitrophenol, and 10 mM GSH. One unit of enzyme activity corresponds to the binding of 1 ␮mol GSH/min. Glutathione reductase (GRX) activity was assayed by measuring the rate of NADPH oxidation (Carlberg and Mannervik, 1985). The reaction mixture contained 0.2 mM NADPH, 100 mM potassium phosphate buffer (pH 7.4), 1 mM EDTA, and 2 mM GSSG. One unit of enzyme activity was defined as the amount of enzyme that catalyzes the oxidation of 1 ␮mol of NADPH per minute. Glucose-6-phosphate dehydrogenase (G6PD) activity was assayed based on the reduction of NADP+ to NADPH at 30 ◦ C (BenBassat and Goldberg, 1980). The reaction mixture contained 4 mM glucose 6-phosphate, 40 mM Tris–HCl (pH 8.2), 5 mM MgCl2 , and 0.6 mM NADP+ One unit of enzyme activity was defined as the amount of enzyme that catalyzes the reduction of 1 ␮mol of NADP+ per minute. Catalase activity was assayed polarographically using a Clarktype oxygen electrode (Yellow Springs Instruments, Inc., Yellow Springs, OH, USA) by following the initial, linear rate of oxygen production from H2 O2 (Goldberg and Hochman, 1989). The assay was performed at 30 ◦ C in a reaction mixture containing 100 mM

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potassium phosphate buffer (pH 6.3) and 20 mM H2 O2 . One unit enzyme activity was defined as the amount of enzyme that catalyzes the decomposition of 1 ␮mol H2 O2 /min at an initial concentration of 20 mM H2 O2 .

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2.5. Western immunoblotting

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Brain samples were homogenized with SDS buffer (20% glycerol and 6% SDS in 0.12 M Tris buffer with a pH of 6.8), centrifuged at 14,000 rpm for 20 min at 4 ◦ C, and the supernatant was boiled for 10 min. The protein concentration of the specimens was quantified by the Bradford method (Bio-Rad Laboratories, Richmond, CA, USA). Prepared samples were further diluted in sample buffer to allow loading of a total 50 ␮g protein in each well. Protein was separated on 10% polyacrylamide gel under denaturing conditions according to the method of Laemmli (1970). The samples were diluted in dissociation buffer (10% SDS, 200 mM EDTA, 10% glycerol, 5% 2-mercaptoethanol, and 0.001% bromphenol blue), blended, and heated again to a temperature of 95 ◦ C for 2 min. Electrophoresis was conducted at 100 mA (150 V) for 2 h and the separated proteins were transferred onto nitrocellulose membrane (400 mA, 4 ◦ C, 2 h). The nitrocellulose membranes were blocked in TBS containing 5% milk powder for 1 h, after which they were incubated at 4 ◦ C overnight with polyclonal IgG cross-reactive to the nitrotyrosine antibody diluted 1:1000 (Cell Signaling Technology, Sidney, BC, Canada). After repeated washing in TBS with 0.2% Tween 20, the membranes were incubated at room temperature for 1 h with horseradish peroxidase-conjugated rabbit anti-rabbit IgG diluted 1:1000 (Sigma, St. Louis, MO, USA). The membrane was then developed to enhance detection by chemiluminescence (Amersham, Bucks, UK) and exposed to X-ray film (Kodak, Rochester, NY, USA). The level of nitrotyrosine was measured by scanning the immunoblots with a laser densitometer (Vilber Leurmat, Torcy, France). For each gel, the band densities were calculated by integrating the area in pixels, and were normalized to the mean level of a band from the Ctl− rats. The normalized values for each group were then averaged. For each point we used five samples taken from five different rats. Protein level was quantified by the Bradford method (Bio-Rad Laboratories, Richmond, CA, USA) with bovine serum albumin as a standard.

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2.6. Statistical analysis

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Data are expressed as mean ± SD Statistical analyses were performed using one-way ANOVA and the Shapiro–Wilk test was used to ascertain normality. Statistical significance was defined as P < 0.05.

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3. Results

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Following non-convulsive HBO preconditioning, there was a significant increase in latency to CNS-OT, from 12.8 ± 8.5 min in the Ctl group to 22.4 ± 10.2 min in the PrC group (P < 0.02, Fig. 1). Enzymatic activity in the brain is presented in Figs. 2–6. In the hippocampus (Figs. 2–4), there was a significant increase in the activity of GSH-Px following preconditioning (0.23 ± 0.04 vs. 0.37 ± 0.02 ␮mol GSH/min in the Ctl and PrC groups, respectively; P < 0.01). In contrast, there was a significant decrease in the activity of GRX (0.252 ± 0.073 vs. 0.145 ± 0.04 ␮mol GSH/min in the Ctl and PrC groups, respectively; P < 0.05). There were no significant differences in the activity of GST and catalase. In the frontal cortex of the PrC rats (Figs. 5 and 6), we observed a significant increase in the activity of GSH-Px (0.28 ± 0.04 vs. 0.34 ± 0.04 ␮mol GSH/min in the Ctl and PrC groups, respectively; P < 0.015, Fig. 5) and GST (1.37 ± 0.28 vs. 1.64 ± 0.18 ␮mol GSH/min in the Ctl and PrC groups,

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Fig. 1. Latency to CNS-OT in the preconditioned (PrC) and control rats. *Significant difference between the groups, P < 0.02. In all figures results are presented as mean ± SD.

Fig. 2. Activity of three ROS scavengers, glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase (GRx), and glutathione peroxidase (GSH-Px), in the hippocampus of preconditioned (PrC) and control rats. *Significant difference between the groups: G6PD, P < 0.05; GRx, P < 0.04; GSH-Px, P < 0.01.

Fig. 3. Activity of catalase in the hippocampus of preconditioned (PrC) and control rats. No significant difference was found between the groups.

Fig. 4. Activity of glutathione S-transferase (GST), in the hippocampus of preconditioned (PrC) and control rats. No significant difference was found between the groups.

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Fig. 5. Activity of three ROS scavengers, glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase (GRx), and glutathione peroxidase (GSH-Px), in the frontal cortex of preconditioned (PrC) and control (Ctl) rats. *Significant difference between the groups, P < 0.015.

Fig. 7. Nitrotyrosine levels in a hippocampal protein of 150 kDa for all four groups. Ctl−, negative control group (no preconditioning, no convulsive HBO exposure); Ctl, control group (no preconditioning, convulsive HBO exposure); PrC−, preconditioned group without exposure (preconditioning, no convulsive HBO exposure); PrC, preconditioned group with exposure (preconditioning + convulsive HBO exposure). This pattern represents all six marked proteins. Significant differences were found between the PrC group and the Ctl− and Ctl groups.

Table 3 The eight proteins marked by the nitrotyrosine-specific antibody in the frontal cortex. Protein size (kDa) 31 37 45 51 61 Fig. 6. Glutathione S-transferase (GST) activity in the frontal cortex of preconditioned (PrC) and control (Ctl) rats. *Significant difference between the groups, P < 0.04.

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respectively; P < 0.04, Fig. 6). There were no significant differences in the activity of catalase, GRX and G6PD. Using Western blot technique, we found differences between the groups for proteins labeled by a specific antibody to nitrotyrosine. In the hippocampus, we observed changes in nitrotyrosylation of six proteins (Table 2, Fig. 7). Analysis of the data from the hippocampus revealed a similar trend in all six marked proteins. Nitrotyrosine levels in the Ctl group (non-preconditioned, exposed Table 2 The six proteins marked by the nitrotyrosine-specific antibody in the hippocampus. Protein sizeCtl−

31 37 50 75 150

PrC−

PrC 2

(kDa) 21.5

Ctl

(pixel/mm ratio) 1.00 ± 0.50 *P < 0.03 1.00 ± 0.61 *P < 0.04 1.00 ± 0.67 *P < 0.05 1.00 ± 0.45 *P < 0.02 1.00 ± 0.66 *P < 0.05 1.00 ± 0.38 *P < 0.004

2.78 ± 0.73 *P < 0.0005 2.19 ± 1.09 *P < 0.01 2.12 ± 1.20 *P < 0.009 0.97 ± 0.49 *P < 0.02 1.54 ± 0.99 *P < 0.03 1.18 ± 0.32 *P < 0.0002

0.95± 046 *P < 0.04 0.54 ± 0.36 #P < 0.04 0.86 ± 0.42 0.57 ± 0.34 *P < 0.02 0.61 ± 0.57 0.73 ± 0.42 *P < 0.01

0.22 ± 0.06 0.18 ± 0.05 0.18 ± 0.06

150

Ctl−

Ctl

1.00 ± 0.61 1.00 ± 0.67 1.00 ± 1.07 1.00 ± 0.45 *P < 0.0003 1.00 ± 0.59 *P < 0.04 1.00 ± 0.66 *P < 0.04 1.00 ± 0.33

2.19 ± 1.09 2.12 ± 1.06 0.52 ± 0.43 0.27 ± 0.18

1.00 ± 0.42 *P < 0.02

PrC− (pixel/mm2 ratio)

0.2 ± 0.01 *P < 0.005 1.54 ± 0.99 *P < 0.02 0.13 ± 0.08 *P < 0.003 0.39 ± 0.16

PrC

0.36 ± 0.16 0.86 ± 0.42 0.29 ± 0.16 0.23 ± 0.14 #P < 0.005 1.97 ± 2.05

0.18 ± 0.06 0.18 ± 0.06 1.50 ± 1.14 0.17 ± 0.1

0.61 ± 0.57

0.17 ± 0.09

0.48 ± 0.27 $P < 0.003 0.35 ± 0.09

1.35 ± 0.96

0.48 ± 0.19

0.57 ± 0.32

Values are mean ± s.d. Ctl−, negative control group (no preconditioning, no toxicityinducing HBO exposure); Ctl, control group (no preconditioning, toxicity-inducing HBO exposure); PrC−, preconditioned group without exposure (preconditioning, no toxicity-inducing HBO exposure); PrC, preconditioned group with exposure (preconditioning + toxicity-inducing HBO exposure). No significant trend was demonstrated between groups. *Significant difference compared with PrC; #, significant difference compared with Ctl−; $ significant difference compared with Ctl.

to convulsive HBO) increased in comparison with the Ctl− rats (not exposed at all to HBO). A similar trend toward a decrease in the levels of these proteins was found in both of the preconditioned groups (PrC and PrC−). This was significant only in the PrC group (preconditioned, exposed to convulsive HBO). In the cortex, there were changes in protein nitrotyrosylation compared with the animals in the Ctl− group (not exposed at all to HBO), but unlike in the hippocampus, in some there was a decrease and in others an increase (Table 3, Fig. 8). We did not note any obvious trend for each group of bands (proteins having a similar molecular weight).

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0.17 ± 0.10 0.17 ± 0.09

4. Discussion

0.1 ± 0.01

In the present study, we have shown that repeated preconditioning by exposure to non-convulsive HBO provides protection against CNS-OT, as seen in the increased latency. This treatment also induced alterations in the activity of enzymes related to the antioxidant system, and resulted in changes in protein nitrotyrosine. Studies on the biological effects of HBO have been conducted in different models (humans, animals, and cultured cells), at various oxygen pressures and for one or more exposures and different

Values are mean ± s.d. Ctl−, negative control group (no preconditioning, no toxicityinducing HBO exposure); Ctl, control group (no preconditioning, toxicity-inducing HBO exposure); PrC−, preconditioned group without exposure (preconditioning, no toxicity-inducing HBO exposure); PrC, preconditioned group with exposure (preconditioning + toxicity-inducing HBO exposure). All six proteins demonstrated the same trend of differences between groups. *Significant differences compared with PC; # significant difference compared with Ctl−.

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Fig. 8. Nitrotyrosine levels in a cortical protein of 150 kDa for all four groups. Ctl−, negative control group (no preconditioning, no convulsive HBO exposure); Ctl, control group (no preconditioning, convulsive HBO exposure); PrC−, preconditioned group without exposure (preconditioning, no convulsive HBO exposure); PrC, preconditioned group with exposure (preconditioning + convulsive HBO exposure). Significant differences were found between the Ctl− and the other three groups.

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durations. Consequently, the results vary considerably from study to study and are sometimes hard to compare. It is well documented that HBO treatment may cause increased production of ROS and RNS, in addition to changes in the expression of antioxidants and nitric oxide synthase, resulting in damage associated with oxidative and nitrosative stress (Jamieson et al., 1986; Ay et al., 2007b; Gröger et al., 2009). However, non-convulsive HBO has also been reported to promote protective preconditioning against further HBO insults and ischemia-reperfusion injury in the brain, as well as in the spinal cord, heart, and liver (Rothfuss et al., 1998; Wang et al., 1998; Freiberger et al., 2006; Nie et al., 2006; Gröger et al., 2009). We have shown here that subjecting rats to non-convulsive oxygen at 202 kPa for 1 h once every other day for a total of three sessions provided protection when the animals were further exposed to convulsive HBO at 608 kPa. This was manifested by a modest and significant increase in the latency to CNS-OT. Previous studies conducted on human subjects and rodents reported both beneficial and deleterious effects of pre-exposure to nonconvulsive HBO on the brain and other organs. The positive or negative conclusion in these studies depended mainly on the oxygen pressure and the duration and number of treatments employed, and whether subsequent exposure ended in seizures. Gröger et al. (2009) examined repetitive exposure of human subjects to non-convulsive HBO during combat activity or underwater demolition work. Rothfuss et al. (1998) exposed subjects to three 20-min sessions of non-convulsive HBO at 2.5 atmospheres absolute (ATA) under experimental conditions. Both studies found resultant DNA damage, which not only rapidly disappeared after the end of the exposure (Rothfuss et al., 1998), but was also absent on subsequent exposure (Speit et al., 1998). Harabin et al. (1990) exposed rats and guinea pigs to O2 at 2.8 ATA delivered either continuously, or intermittently in repeated cycles of 10 min on 100% O2 followed by 2.5 min on air. In both species, the time required to produce convulsions and death was significantly prolonged by intermittency. In a more complex study, Wada et al. (2001) showed that pretreatment of gerbils with non-convulsive HBO at 2 ATA once every other day for three or five sessions induced tolerance to forebrain ischemia. However, one pretreatment session of this kind, five sessions on hyperbaric air at 2 ATA once every other day, or ten daily sessions of HBO at 3 ATA, all failed to accomplish this. Fenton and Robinson (1993) showed that rats repeatedly exposed to convulsive HBO at 4 ATA for 1 or 1.5 h on 3 consecutive days after 5 or 10 exposures to non-convulsive HBO at 2 ATA for 2 h, developed convulsions earlier than naive controls. A similar decrease in

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latency was reported by Chavko et al. (2001), when they exposed rats twice to convulsive HBO at 5 ATA with intervals of 1, 2, and 6 days between exposures. The data cited above may lead one to conclude that if HBO pretreatments do not end in seizures, they will generally (but not always) provide protection against or reduce sensitivity to CNSOT, and that if they do end in seizures they will fail to provide protection. The simple and comprehensible reasons for such contradictory results would be the different experimental protocols or different animal models. We suggest, however, that this may also reflect the complexity of the mechanisms involved in CNSOT, where a small change in the exposure protocol is sufficient to provoke a completely different, or even the opposite physiological response. The following aspects of HBO and oxidative stress may serve further to illustrate the complexity of the situation. To the best of our knowledge, there are no reports in the literature on the response of G6PD to HBO treatment. G6PD catalyzes the oxidation of glucose to generate NADPH from NADP. Since NADPH is required for the recycling of GSH by GRX, it was expected that its activity would increase in response to HBO. However, in the present study we found that G6PD activity decreased in the hippocampus and was without change in the cortex. This corroborates the findings of a previous investigation, which demonstrated significantly longer latency to CNS-OT in G6PD knock-out mice compared with the wild type (Eynan et al., 2012). It was also shown that increased G6PD activity stimulated oxidative stress in adipocytes (Park et al., 2006), whereas G6PD deficiency decreased vascular superoxide in mice (Matsui et al., 2006). Gupte et al. (2007) compared heart tissues taken from healthy subjects with those taken from subjects who suffered from congestive heart failure. They found a high correlation between G6PD activity, NADPH-oxidase activity, and the oxidative stress level. This response may be related to another cellular function of NADP, which also serves as a substrate for NADPH-oxidase. This last enzyme catalyzes the production of superoxide by oxidation of NADPH. Hence, down regulation of G6PD activity may contribute to the reduction of oxidative stress. Taking all of this into consideration, it would not be unreasonable to expect a decrease in the activity of GRX (as we indeed observed in the present study) associated with down regulation of G6PD activity, due to the conversion by GRX of the oxidized form of glutathione (GSSG) to glutathione by oxidizing NAPH to NADP+ . We have also shown that preconditioning resulted in an increase in GSH-Px activity in the hippocampus, whereas there were no significant changes in the activity of GST and catalase. In the cortex, however, the response was different; there was an increase in GST and GSH-Px, with no significant changes in the activity of catalase, GRX and G6PD. These findings suggest that the antioxidant enzymes differ in their response to ROS (and perhaps also RNS) in different regions of the CNS. An increase in GSH-Px activity was noted in both the hippocampus and the cortex, suggesting a rise in the steady-state concentration of H2 O2 as a result of HBO treatment. A similar response of the antioxidant system to HBO treatment, with increases, decreases, or no change in activity, has been reported in previous studies. Healthy human subjects and patients receiving HBO treatment were repeatedly exposed to HBO on a routine therapy protocol (Dennog et al., 1999; Benedetti et al., 2004). This resulted in a significant decrease in erythrocyte superoxide dismutase and catalase, without any change in the activity of GSHPx, when compared with the first HBO exposure. A rise in catalase activity was noted in lymphocytes during the recovery period after scuba diving, whereas GSH-Px activity increased both immediately after the scuba diving session and after HBO exposure in resting conditions (Ferrer et al., 2007). When rats were exposed to HBO, GSH-Px activity in the cerebral cortex increased after a short exposure but decreased after a longer one. This is borne out by other

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studies, which showed that exposure for more than 2 h caused a decrease in the activity of GSH-Px (Jenkinson et al., 1988; Harabin et al., 1990). Exposure of rats and guinea pigs to O2 at 2.8 ATA delivered either continuously or intermittently, resulted in an increase in catalase and GSH-Px activity in the brain, but no change in superoxide dismutase (Harabin et al., 1990). NO and its metabolites play a major role in CNS-OT. NO is a powerful vasodilator and therefore a regulator of cerebral blood flow, but it also antagonizes the vasoconstrictive effects of O2 . It is highly diffusible, and combines very rapidly with superoxide to form peroxynitrite. Peroxynitrite is a potent oxidant which induces neurotoxicity by oxidation of cellular targets such as nucleic acids, lipids, and proteins. One of the end products of such reactions is nitration of tyrosine residues on proteins to produce 3-nitrotyrosine. The detection of nitrotyrosine has been used as a peroxynitrite marker in several pathological conditions such as traumatic brain injury, cerebral ischemia, and neurodegenerative disorders (Coeroli et al., 1998; Mésenge et al., 1998; Torreilles et al., 1999). Using this indirect method, we found a clear trend in all six hippocampal proteins. This indicates that treatment of rats with non-convulsive HBO at 202 kPa resulted in a decrease in protein nitrosylation, whereas further exposure at a higher pressure had the opposite effect. This decrease was most profound in the PrC group, and together with our findings regarding the activity of ROS scavenger enzymes, it further supports the increased latency in the animals from that group. Previous studies have shown that exposure of rats to HBO was sometimes associated with increased levels of NO and its metabolites in the brain, including peroxynitrite (Chavko et al., 2001; Thom et al., 2002, 2003; Chavko et al., 2003; Akgül et al., 2007; Ay et al., 2007a), and that this was dependent on neuronal nitric oxide synthase (Thom et al., 2002). It was shown that such treatments resulted in protein nitrosylation (Chavko et al., 2003). These higher levels correlate positively with the increase in regional cerebral blood flow that precedes the EEG discharges associated with CNSOT (Akgül et al., 2007). In other studies, however, NO production was shown to decrease as a result of HBO treatment (Ito et al., 1996; Demchenko et al., 2000). Sometimes the outcome was even more complex. It was shown that shorter exposure to HBO at a relatively lower pressure resulted in a decrease in NO production and cerebral blood flow, whereas an increase in these parameters was observed with longer exposure at a higher pressure (Demchenko et al., 2000, 2001, 2003; Allen et al., 2009). It was found in these studies that the early vasoconstrictive phase is dependent on endothelial nitric oxide synthase, whereas the late vasodilatative and toxic stage depends on both endothelial and neuronal nitric oxide synthase. Another interesting angle on this intriguing issue is the relative hypoxic state induced by HBO preconditioning. Balestra et al. (2006) and Cimino et al. (2012) have reported that levels of hypoxicinducible factor 1 alpha (HIF 1␣) and erythropoietin, both known to have a neuroprotective effect, increase after breathing pure normobaric or hyperbaric oxygen.

5. Conclusion In conclusion, the present study demonstrates that repeated exposure to non-convulsive oxygen at 202 kPa in the rat has a preconditioning effect, providing protection against CNS-OT. It is probably the interplay between reactive species (ROS and RNS) and a number of other factors that is responsible for the varied response. At higher levels, they contribute to enzyme inactivation and additional forms of cellular damage, as well as CNS-OT. At lower, non-physiological concentrations, however, they induce the antioxidant system, mainly in the hippocampus.

Acknowledgements The opinions and assertions contained herein are the private ones of the authors, and are not to be construed as official or as reflecting the views of the Israel Naval Medical Institute. The authors wish to thank Mr. Richard Lincoln for skillful editing of the manuscript.

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