Int. J. Devl Neuroscience 23 (2005) 663–671 www.elsevier.com/locate/ijdevneu Total antioxidant capacity is impaired in different structures from aged rat brain Ionara Rodrigues Siqueira b,c,*, Cı́ntia Fochesatto a, Aline de Andrade a, Melissa Santos a, Martine Hagen a, Adriane Bello-Klein b, Carlos Alexandre Netto a,b b a Departamento de Bioquı́mica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil Programa de Pós-Graduação em Ciências Biológicas-Fisiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil c Centro Universitário UNIVATES, Lajeado, RS, Brazil Received 1 December 2004; received in revised form 28 February 2005; accepted 4 March 2005 Abstract Our data support a disproportion between free radicals levels and scavenging systems activity in different cerebral regions of the aging rat. We investigated the total reactive antioxidant potential and reactivity levels, which represent the total antioxidant capacity, in different cerebral regions of the aging rat (cortex, striatum, hippocampus and the cerebellum). In addition, we have determined several oxidative stress parameters, specifically the free radicals levels, the macromolecules damage (lipid peroxidation and carbonyl content), as well as the antioxidant enzymes activities in different cerebral areas from young (2 months-old), mature adult (6 months-old) and old (24 months-old) male Wistar rats. Free radicals levels, determined by 20 ,70 -dichlorofluorescein diacetate probe, were higher in striatum, cerebellum and hippocampus from aged rats. There was an age-related increase in lipoperoxidation in hippocampus and cerebral cortex. In the cerebellum, a high activity of superoxide dismutase and a decrease of catalase activity were observed. The striatum exhibited a significant catalase activity decrease; and glutathione peroxidase activity was diminished in the hippocampus of mature and aged rats. There was a marked decrease of total antioxidant capacity in hippocampus in both reactivity and potential levels, whereas striatum and cerebral cortex displayed a reduction on reactivity assay. We suggest that age-related variations of total antioxidant defenses in brain may predispose structures to oxidative stressrelated neurodegenerative disorders. # 2005 ISDN. Published by Elsevier Ltd. All rights reserved. Keywords: Aging; Brain; Free radical content; Lipid peroxidation; Total reactive antioxidant potential; Total antioxidant reactivity Aging is a complex biological process characterized by a gradual decline in biochemical and physiological functions of most organs. The free radical theory of aging (Harman, 1992), one of the numerous constructs developed to explain Abbreviations: ABAP, 2,20 -azo-bis-(2-amidinopropane); CAT, catalase; DCFH-DA, 20 ,70 -dichlorofluorescein diacetate; DNPH, 2,4-dinitrophenylhydrazine; GPx, glutathione peroxidase; H2O2, hydrogen peroxide; LPO, lipid peroxidation; O2 , superoxide radical; OS, oxidative stress; SOD, superoxide dismutase; TAR, total antioxidant reactivity; TBARS, thiobarbituric acid-reactive substances; TRAP, total radical-trapping antioxidant potential * Corresponding author at: Centro III, Centro Universitário UNIVATES, Rua Barbedo 426, 03, 90110-260, Porto Alegre, RS, Brazil. Tel.: +55 51 3714700; fax: +55 51 3316 4085. E-mail address: ionara@ufrgs.br (I.R. Siqueira). the aging process, states that free radicals, such as the superoxide radical anion (O2 ), hydrogen peroxide (H2O2) and the highly reactive hydroxyl radicals, may produce oxidative damage directly to critical biological molecules. In order to handle that, organisms utilize antioxidant defenses, including enzymes like superoxide dismutase (SOD), which converts O2  into H2O2, catalase (CAT), that is responsible for the detoxification of H2O2, and glutathione peroxidase (GPx) that breaks down peroxides, notably those derived from the oxidation of membrane phospholipids, as well as non-enzymatic antioxidant molecules (carotenoids, vitamin E, GSH) (Halliwell, 1992). Cellular damage arising from the oxidative stress (OS), an imbalance between the activity of free radicals generation and scavenging systems, has been implicated in neuronal 0736-5748/$30.00 # 2005 ISDN. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdevneu.2005.03.001 664 I.R. Siqueira et al. / Int. J. Devl Neuroscience 23 (2005) 663–671 degeneration associated with normal aging (Davies et al., 2001; Dogru-Abbasoglu et al., 1997; Evans, 1993). Supporting this idea, a long term supplementation of various antioxidants has been found to retard the loss of spatial memory and decreases damage to brain proteins in aged gerbils and rats (Carney et al., 1991; Bickford et al., 2000). Previous works have shown that antioxidant functions decline in almost all aged mammals studied (Harman, 1992). Although age-related changes in the antioxidant enzymatic system have been described, results on the SOD, GPx, CAT and glutathione reductase activities are contradictory (Benzi and Moretti, 1995), pointing to a rather complex relationship to link brain antioxidative enzymes with the aging process. Although the OS has been suggested to be a possible cause of brain aging process, only a few studies have directly assessed the total antioxidant capacity. The main goal of the present study was to investigate the total reactive antioxidant potential (TRAP) and total antioxidant reactivity (TAR) levels, measurements that express the total antioxidant capacity, in different cerebral regions of the aging rat (cortex, striatum, hippocampus and the cerebellum). Additionally, in an effort to compare our findings with results reported in the literature, we also investigated others parameters of oxidative stress, namely the free radicals content, indexes of damage to macromolecules—lipid peroxidation (LPO) and protein carbonyl content, along with antioxidant enzyme activities: superoxide dismutase, glutathione peroxidase and catalase. 1.2. Free radicals levels 1. Experimental 1.4. Protein carbonyl content 1.1. Animals and tissue preparation Protein carbonyl content was determined by the use of 2,4dinitrophenylhydrazine (DNPH) (Levine et al., 1990). Aliquots of samples were incubated with streptomycin sulfate solution (1%) for 15 min. The mixture was centrifuged at 3600  g; the supernatant was divided and incubated for 1 h, at room temperature, with 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2 M HCl, or only with 2 M HCl (blank). The protein was precipitated by adding an equal volume of 20% TCA. After centrifugation (8600  g), the pellet was washed three times with ethanol: ethyl acetate (1:1) to remove excesses DNPH. The precipitated protein was redissolved in 6 M guanidine HCl and the absorbance of solutions was measured at 370 nm. Carbonyl content was calculated taking the extinction coefficient of 22 mM 1 cm 1 mg 1 of protein. Male Wistar rats of 2–3 months, 6 months and 20 months of age, obtained from the Central Animal House of the Department of Biochemistry, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, were maintained under standard conditions (12 h light/12 h dark), with room temperature of 22  2 8C and food (20%, w/w protein commercial chow, Germani, Porto Alegre, RS, Brazil) and water ad libitum. All experiments were approved by the Local Animal Care Committee. Rats were decapitated; the brain regions (hippocampi, frontal cortices, striata and cerebella) were quickly dissected out and instantaneously placed in liquid nitrogen and stored at 70 8C until biochemical assays. On the day of the experiments, brain tissue were homogenized in 10 volumes of ice-cold phosphate buffer (0.1 M, pH 7.4) containing KCl (140 mM) and EDTA (1 mM) in a Teflon-glass homogenizer. To assay carbonyl levels and antioxidant enzymes activities, samples were homogenized in the presence of PMSF. The homogenate was centrifuged at 960  g for 10 min to remove nuclei and cell debris; the supernatant, a suspension of mixed and preserved organelles, was used for the assays. The procedures were performed at 4 8C. To assess the free radicals content we used 20 ,70 dichlorofluorescein diacetate (DCFH-DA) as a probe (Lebel et al., 1990). An aliquot of the sample was incubated with DCFH-DA (100 mM) at 37 8C for 30 min. The reaction was terminated by chilling the reaction mixture in ice. The formation of the oxidized fluorescent derivative (DCF) was monitored at excitation and emission wavelengths of 488, 525 nm, respectively, using a fluorescence spectrophotometer (Hitachi F-2000). The free radicals content was quantified using a DCF standard curve and results were expressed as pmol of DCF formed/mg protein. All procedures were performed in the dark, and blanks containing DCFH-DA (no homogenate) were processed for measurement of autofluorescence (Driver et al., 2000; Sriram et al., 1997). 1.3. Thiobarbituric acid reactive substances (TBARS) LPO was evaluated by thiobarbituric acid reactive substances (TBARS) test (Bromont et al., 1989). Aliquots of samples were incubated with 10% trichloroacetic acid (TCA) and 0.67% thiobarbituric acid (TBA). The mixture was heated (30 min) on a boiling water bath. Afterwards, nbutanol was added and the mixture was centrifuged. The organic phase was collected to measure fluorescence at excitation and emission wavelengths of 515 and 553 nm, respectively. 1,1,3,3-tetramethoxypropane, which is converted to malondialdehyde (MDA), was used as standard. 1.5. Total reactive antioxidant potential (TRAP) assay TRAP and TAR are based on luminol-enhanced chemiluminescence measurement, induced by an azo initiator (Evelson et al., 2001; Lissi et al., 1992; Lissi et al., 1995). The reaction mixture contained 2,20 -azobis (2-amidinopropane) dihydrochloride (ABAP) 10 mM, source of peroxyl radicals, and luminol (4 mM) in glycine buffer (0.1 M, pH 8.6). The chemiluminescence (CL) generated was measured in a I.R. Siqueira et al. / Int. J. Devl Neuroscience 23 (2005) 663–671 scintillation counter (Beckman) working in the out of coincidence mode. The addition of Trolox (antioxidant standard, 200 nM) or samples (5.0 ml of sample) decreases CL to basal levels for a period (induction time) proportional to the concentration of antioxidants. The TRAP values were calculated as equivalents of Trolox concentration per mg of protein. 1.6. Total antioxidant reactivity (TAR) assay The reaction mixture contained 2 mM ABAP and 6 mM luminol in glycine buffer. TAR values were determined by assessing the initial decrease of luminescence calculated as the ratio ‘Io/I’, where ‘Io’ is the CL in the absence of additives, and ‘I’ is the CL after addition of the 20 nM Trolox, or the samples (1 ml). TAR values were expressed as equivalents of Trolox concentration per mg of protein (Lissi et al., 1995). 1.7. Superoxide dismutase (SOD) activity SOD activity was determined using a RANSOD kit (Randox Labs, USA). This method employs xanthine and xanthine oxidase to generate O2  that react with 2-(4iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a red formazan dye, which is assayed spectrophotometrically at 505 nm at 37 8C. The inhibition on production of the chromogen is proportional to the activity of SOD present in the sample. 1.8. Catalase (CAT) activity The homogenate was incubated with ethanol (10%) and Triton (10%). The activity was assayed at 25 8C by determining the rate of degradation of H2O2 at 240 nm in 10 mM potassium phosphate buffer (pH 7.0). The extinction coefficient of 43.6 mM 1 cm 1 was used for calculation. One unit is defined as 1 pmol of H2O2 consumed per minute and the specific activity is reported as units per mg protein (Aebi, 1984). 1.9. Glutathione peroxidase (GPx) activity The reaction was carried out at 25 8C in 600 ml of solution containing 100 mM potassium phosphate buffer pH 7.7, 1 mM EDTA, 0.4 mM sodium azide, 2 mM GSH, 0.1 mM NADPH, 0.62 U GSH reductase. The activity of seleniumdependent GPx was measured taking tert-butyl-hydroperoxide as the substrate at 340 nm. The contribution of spontaneous NADPH oxidation was always subtracted from the overall reaction rate. GPx activity was expressed as nmol NADPH oxidized per minute per mg protein (Wendel, 1981). 665 1.11. Statistical analysis Data were evaluated by one-way analysis of variance (ANOVA) followed by the Duncan’s multiple range test when appropriate. Analysis was performed using the Statistical Package for the Social Sciences (SPSS) software in a PC-compatible computer. A difference was considered significant when P < 0.05. Values are expressed as mean  standard error mean (S.E.M.). 2. Results Our results corroborate that an imbalance occurs between free radicals levels and scavenging systems activity. There was a significant increase of DCF levels in hippocampus (F (2.14) = 5.91; p < 0.01), striatum (F (2.15) = 5.51; p < 0.01) and cerebellum (F (2.14) = 6.63; p < 0.009) of old rats, as compared to those of young ones; that effect is already present in the hippocampus of mature rats (Fig. 1A). As regards to lipid peroxidation, there was an augment in TBARS levels in hippocampus (F (2.15) = 3.73; p < 0.04) and cerebral cortex (F (2.16) = 6.42; p < 0.009) from mature and old rats (Fig. 1B). On the other hand, no differences on the protein carbonyl content were found in any brain region (data not shown). The effects of aging on the antioxidant brain enzymes studied are presented in Fig. 2. SOD activity was almost 35% greater in cerebellum (F (2.13) = 3.86; p < 0.04, Fig. 2A), whereas CAT activity was significantly reduced in striatum (about 25%, F (2.15) = 3.88; p < 0.04) and cerebellum (F (2.14) = 5.69; p < 0.01) from senescent rats (Fig. 2B). The activity of GPx was decreased in the hippocampus of mature adult and aged rats, as compared to young ones (F (2.15) = 3.71; p < 0.04, Fig. 2C). The [SOD]/ [CAT] activity ratio was significantly higher in the cerebellum of senescent animals (Fig. 3, F (2.13) = 5.18; p < 0.02). Fig. 4 illustrates the effect of aging on total antioxidant reactivity and potential in brain tissue. Significant agedependent decreases on both TRAP (F (2.17) = 35.09; p < 0.001) and TAR (F (2.21) = 9.48; p < 0.001) levels were observed in the hippocampus of aged rats (about 40 and 50%, respectively). A decrease in TRAP and TAR (20 and 45%, respectively) in striatum (F (2.21) = 4.28; p < 0.02) and a decrease in TAR in the frontal cortex of senescent rats (F (2.14) = 3.51; p < 0.05) were also shown. These results indicate that the antioxidant defenses are significantly diminished in aged brain. 3. Discussion 1.10. Protein determination The total protein concentration was determined using the method described by Lowry et al. (1951). This study adds evidence for the role of OS in the brain aging process. Surprisingly, little attention has been paid to the total antioxidant activity during brain aging. This 666 I.R. Siqueira et al. / Int. J. Devl Neuroscience 23 (2005) 663–671 Fig. 1. Effects of aging on free radical content (A), using DCFH-DA as a probe, and lipid peroxidation through TBARS (B). Results are expressed as percentage of control (mean  S.E.M.) for six to eight experiments. The mean DCF values from young groups were 2.92  0.39 (hippocampus), 2.48  0.16 (frontal cortex), 1.84  0.13 (striatum) and 3.23  0.14 (cerebellum) nmol DCF formed per mg protein. The TBARS indexes were 0.11  0.01 (hippocampus), 0.12  0.02 (frontal cortex), 0.27  0.05 (striatum) and 0.14  0.02 (cerebellum) pmol MDA per mg protein. ANOVA followed by Duncan’s test ( p < 0.05). * Significantly different, as compared to young and mature adult groups, #significantly different, as compared to the young group. perspective is essential because a substantial proportion of cellular antioxidants may be composed by unidentified compound(s). Mendoza-Nunez and collaborators (2001) described DNA damage in lymphocytes and low levels of serum total antioxidant levels of the elderly. In addition, in vivo total antioxidant status was related with the age-related differential gene expression as well as expression of heat shock proteins (Visala Rao et al., 2003). During the skin aging process a significant decrease in the levels and activity of the water-soluble low molecular weight antioxidant was detected (Kohen and Gati, 2000). Moreover reducing equivalents, interpreted as low molecular weights antioxidant molecules, was altered in liver, lungs and kidneys, but not in heart and brain during the aging process (Kohen et al., 1997). To our knowledge, this is the first report demonstrating age-related changes on cerebral total antioxidant status. Aging changes both total antioxidant reactivity and potential indexes in distinct areas of the rat brain. TRAP measurements determine the quantity of antioxidants present in the sample, whereas the TAR assay indicates its reactivity (Lissi et al., 1995; Desmarchelier et al., 1997). TRAP and TAR assays of chemiluminescence production, in reaction of luminol with ABAP-generated peroxyl radical, may indicate the ability of a tissue, or a compound, to scavenge peroxyl radical, O2  and/or luminol-derived radicals (Lissi et al., 1992). Evelson and colleagues (2001) have evaluated the total amount of antioxidants in rat brain, liver, kidney and heart homogenates and concluded that such procedures seem to evaluate enzymatic and nonenzymatic defenses. TAR may be taken as a useful index of the capacity of a given sample to modulate the damage associated with enhanced production of free radicals (Lissi et al., 1992). Our data show that the antioxidant reactivity (TAR) was the most sensitive biochemical parameter; in addition, the decrease on TAR indexes in brain aging might be responsible for its higher susceptibility to the oxidative events in the aging brain. The alterations on TRAP and TAR levels did not are attributed to some known antioxidant, since there is an indicative of the existence of unidentified and specifically unmeasured antioxidant molecules (Evelson et al., 2001). The major finding here reported is that the hippocampus, one of the most vulnerable brain regions to oxidative stress (Candelario-Jalil et al., 2001), was the structure with I.R. Siqueira et al. / Int. J. Devl Neuroscience 23 (2005) 663–671 667 Fig. 2. Effects of aging on activities of antioxidant enzymes, SOD (panel A), GPx (panel B) and CAT (panel C). Results are expressed as mean  S.E.M. for six to eight experiments. ANOVA followed by Duncan’s test ( p < 0.05). *Significantly different, as compared to young and mature adult groups, #significantly different, as compared to the young group. impaired antioxidant function already in mature age, suggesting that this may be implicated with the beginning of the aging process. The time-course of TRAP and TAR records showed a progressive decrease with age in hippocampus; both indexes were almost 30% lower in mature animals when compared to younger rats, and fall to about 40 and 50%, respectively, in the aged group. The association of these antioxidant indexes with those of free radical content and lipid peroxidation assays reveals the existence of a marked oxidative stress in the aging hippocampus. It follows that the cellular redox status, defined as the balance between intracellular oxidants and antioxidants (Castagne et al., 1999) is not within an optimal range for cell survival; the age-related variations in the antioxidant defenses can be suggested to be the cause of increased susceptibility to disease related to redox imbalance in advanced age. Indeed, that can be the case for disorders 668 I.R. Siqueira et al. / Int. J. Devl Neuroscience 23 (2005) 663–671 Fig. 3. Effects of aging on [SOD]/[CAT] activity ratio in rat brain. Columns represent mean  S.E.M. of six to eight experiments. ANOVA followed by Duncan’s test ( p < 0.05). *Significantly different, as compared to young and mature adult groups. related to excitotoxicity since LPO products impair glutamate uptake and mitochondrial function in synaptosomes, so favoring glutamate overactivity (Keller et al., 1997). The OS may lead to several alterations in membranes, including its molecular structure, producing significant changes in the biophysical properties such as asymmetry and fluidity. In addition, OS increases calcium dysfunction in compromised cells, resulting in long-lasting increases of cytosolic calcium what contributes to cell death by calciumdependent mechanisms (Joseph et al., 2000). We found increases in free radical content in the striatum, hippocampus and cerebellum from rats of 20 months of age. Driver and colleagues (2000) also showed that there was an age-related increase (24 months, Long-Evans) in basal free radicals production using DCF assay in these cerebral areas, as well as suggested that DCFH could be used as a sensitive Fig. 4. Effects of aging on total antioxidant capacity in rat brain regions. Columns represent mean  S.E.M. for percentage of young group values, of six to eight experiments. Total antioxidant reactivity (TAR, panel A) mean values from the young group were 88  14 (hippocampus), 28  4.3 (frontal cortex), 48  5.3 (striatum) and 80  17 (cerebellum) pmol eq. Trolox per mg protein. Total antioxidant potential (TRAP, panel B) values from the young group were 41  5.9 (hippocampus), 50  8.1 (frontal cortex), 31  4.2 (striatum) and 54  9.4 (cerebellum) pmol eq. Trolox per mg protein. ANOVA followed by Duncan’s test ( p < 0.05). *Significantly different, as compared to young and mature adult groups, #significantly different, as compared to the young group. I.R. Siqueira et al. / Int. J. Devl Neuroscience 23 (2005) 663–671 probe for detection of species reactive of oxygen in crude brain homogenates. There are reports that TBARS levels do not change with cerebral aging (Cand and Verdetti, 1989), however others have shown an increase in LPO levels (Farooqui et al., 1997; Sawada and Carlson, 1987). Our result from cerebral cortex is in accordance with those obtained by McGahon et al. (1999), who observed an increase in old Wistar rats (22 months), as compared to young (4 months) ones. We found no changes on protein-bound carbonyl levels, although we measured it in homogenates after light centrifugation to remove nuclei and cell debris. This procedure was taken because the use of crude homogenates may mask the changes in distinct cellular fractions, since it has been proposed that protein susceptibility to oxidative damage vary among different subcellular fractions (Pleshakova et al., 1998). Moreover, carbonyl compounds are just a fraction of oxidized amino acids and various noncarbonyl products may be formed (Requena et al., 2001). Interestingly, Davies and colleagues (2001) found slight increases in the amount of protein carbonyls in cytosolic and mitochondrial fractions of aging brain, while no effect was detected in whole tissue homogenates. On the other hand, conflicting results on the antioxidant enzymatic systems in the aging process are available in literature. Antioxidant enzymes are considered to be a primary defense that prevents biological macromolecules from oxidative damage. SOD is mainly located in neurons whereas GPx, the major protective enzyme against the action of H2O2, is mostly present in astrocytes. The brain has a much higher SOD to GPx activity ratio than other organs of the rat (Benzi and Moretti, 1995). This, together with lower CAT activity, makes the brain the most vulnerable organ to H2O2. Several reports have been published on age-related changes of brain total SOD activity in rodents, sometimes with conflicting results. Brain SOD activities were reported to be unchanged as a result of aging in whole brain from older Wistar rats (Sahoo and Chainy, 1997) and in older Fisher 344 rats (Tian et al., 1998), although the Cu Zn SOD activity was unaffected in several structures, namely cortex, hippocampus, striatum, hypothalamus and cerebellum, from Wistar rats (Danh et al., 1983). Interestingly, it decreased in brain areas, cortex, hippocampus, hypothalamus and cerebellum from Charles Foster rats (Gupta et al., 1991) and in whole brain from Fischer 344 rats (Semsei et al., 1991). Such conflicting results of SOD activity may arise from differences in strains, conditions of animal maintenance, experimental procedures or reflect superoxide formation, since SOD activity can be induced by peroxidative stress (Benzi and Moretti, 1995). After the removal of superoxide radical by SOD there is the H2O2 production, which is further degrades by other antioxidant enzymes, CAT and GPx, to water. Catalase activities in the whole brain significantly decreased in older Fisher 344 rats (Tian et al., 1998) and Wistar rats (Sahoo and Chainy, 1997). Because of 669 catalase is very low activity in the brain, it seems to play a secondary role in H2O2 compared with other organs. Brain GPx activities were reported to be unchanged in cortex (Cand and Verdetti, 1989; Dogru-Abbasoglu et al., 1997) and whole brain (Sahoo and Chainy, 1997) from Wistar rats and whole brain (Tian et al., 1998) and brain regions (Carrillo et al., 1992) in Fischer 344, although a decrease also been reported (Benzi et al., 1989). It is important to note that brain regions with high DCF levels, e.g. hippocampus, striatum and cerebellum, also display changes in at least one antioxidant enzyme activity. In addition, the O2 , one of the molecules accumulating with oxidative stress, has been reported to directly interact and inactivate both CAT and GPx (Kono and Fridovich, 1982). Accordingly, we found that CAT activity was decreased in striatum and cerebellum from senescents and that of GPx was decreased in the hippocampus of mature adult and aged rats, what would produce a hazardous state due to presence from H2O2. This species combined to metal iron would originate hydroxyl radicals, since hydrogen peroxide is not effectively transformed into water. The activity ratios between antioxidant enzymes namely SOD/CAT and CAT/GPx ratios have been considered as an index of oxidative status (Palomero et al., 2001). The high activity of SOD, combined with the decrease of CAT function in the older cerebellum, might allow accumulation of H2O2. The augment of SOD activity may be resulting from the fact that peroxidative stress induces SOD activity (Benzi and Moretti, 1995). Additionally, the fact of TRAP and TAR levels were unaltered in cerebellum might be explained by higher SOD activity, which may mask a decrease of other antioxidant level. This result may be involved with impaired cerebellar functions of aged rats, given that the motor learning on a runway task are impair (Bickford, 1993; Bickford et al., 1992). Moreover, GPx and CAT activities were markedly decreased in the hippocampus as compared to other areas in young age. Yet, the gerbil model of transient cerebral ischemia leads to a persistent and marked decrease in hippocampal GPx and glutathione reductase activities (Candelario-Jalil et al., 2001), what indicates that lowered antioxidant capacity in this brain region is possibly related to their greater vulnerability towards ischemia. Interestingly, the effect of aging on GPx activity could be involved in the decline of learning and memory abilities with advancing age. The age-related spatial learning and memory deficits have been connected, at least in part, to change in the synaptic connections and function of hippocampal formation. McGahon and colleagues (1999) have been suggested that the impairment of maintenance of long-term potentiation (LTP) in aged rats is involved with a decrease in arachidonic acid concentration, a proposed retrograde messenger in LTP in hippocampus. It is important to note that the arachidonate metabolism seems be related to GPx activity. GPx is able to reduce peroxides, including phospholipid hydroperoxides and other lipid hydroperox- 670 I.R. Siqueira et al. / Int. J. Devl Neuroscience 23 (2005) 663–671 ides, considering a reduction aging-related of activity GPx, we might suppose that occur an enhanced the basal level of cellular hydroperoxides and consequently a reduction of arachidonic acid content and the impairment in LTP (McGahon et al., 1999). As regards to ischemia injury, the regional heterogeneity to neutotoxicants might be related to dissimilar antioxidant capacity in different cerebral areas. For example, organometal toxicant trimethyltin induces a prominent neurodegeneration in the hippocampi (especially CA1) in rats (Scallet et al., 2000). Various factors, including the distribution of these chemicals, may be responsible to differences susceptibility; the inequality antioxidant capacity and consequently differences on cellular redox condition, which may modulate thiol status, could contribute to tissue sensitivity to neurotoxicants. Taken together, our results support the idea that the loss of proper antioxidant defense in the rat appears to be highly involved in the brain aging process. In conclusion, our results show the existence of oxidative stress, an imbalance between free radicals content and scavenging systems, during cerebral aging process, what may relate to its vulnerability to oxidative events. This susceptibility to oxidative stress is present in the hippocampus already in mature age, what may explain its increased vulnerability to excitotoxic events like brain ischemia and to neurodegenerative disorders. Additional studies are necessary to test whether this phenomenon is causally related to normal aging. 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