Ascorbate uptake is decreased in the hippocampus of ageing rats

Neurochemistry International 58 (2011) 527–532

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Ascorbate uptake is decreased in the hippocampus of ageing rats Ionara Rodrigues Siqueira a,b,*, Viviane Rostirolla Elsner b, Marina Concli Leite c, Cla´udia Vanzella a, Felipe dos Santos Moyse´s b, Christiano Spindler a, Grac¸a Godinho c, Cı´ntia Battu´ c, Suzana Wofchuk c, Diogo Onofre Souza c, Carlos Alberto Gonc¸alves c, Carlos Alexandre Netto c a b c

Departamento de Farmacologia, Instituto de Cieˆncias Ba´sicas da Sau´de, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil Programa de Po´s-Graduac¸a˜o em Cieˆncias Biolo´gicas: Fisiologia, Rua Sarmento Leite, 500, 90050-170 Porto Alegre, RS, Brazil Departamento de Bioquı´mica, Rua Ramiro Barcelos, 2600, CEP 90035-003 Porto Alegre, RS, Brazil

A R T I C L E I N F O

A B S T R A C T

Article history: Received 8 November 2010 Received in revised form 7 January 2011 Accepted 10 January 2011 Available online 14 January 2011

Ascorbate, an intracellular antioxidant, has been considered critical for neuronal protection against oxidant stress, which is supported especially by in vitro studies. Besides, it has been demonstrated an age-related decrease in brain ascorbate levels. The aims of the present study were to investigate ascorbate uptake in hippocampal slices from old Wistar rats, as well as its neuroprotective effects in in vitro and in vivo assays. Hippocampal slices from male Wistar rats aged 4, 11 and 24 months were incubated with radiolabeled ascorbate and incorporated radioactivity was measured. Hippocampal slices from rats were incubated with different concentrations of ascorbate and submitted to H2O2induced injury, cellular damage and S100B protein levels were evaluated. The effect of chronic administration of ascorbate on cellular oxidative state and astrocyte biochemical parameters in the hippocampus from 18-months-old Wistar rats was also studied. The ascorbate uptake was decreased in hippocampal slices from old-aged rats, while supplementation with ascorbate (2 weeks) did not modify any tested oxidative status in the hippocampus and the incubation was unable to protect hippocampal slices submitted to oxidative damage (H2O2) from old rats. Our data suggest that the decline of ascorbate uptake might be involved in the brain greater susceptibility to oxidative damage with advancing age and both in vitro and vivo assays suggest that ascorbate supplementation did not protect hippocampal cells. ß 2011 Elsevier Ltd. All rights reserved.

Keywords: Ascorbate Ageing process Rats Oxidative stress Neuroprotection Hippocampus

1. Introduction Ascorbate is an important component of neuronal antioxidant network and an enzyme cofactor in the synthesis of catecholamines and certain peptide hormones (Chatterrjee et al., 1975). Ascorbate scavenges oxygen or nitrogen-centered radical species generated during normal metabolism, such as peroxyl radicals, superoxide, singlet oxygen, peroxynitrite and hydroxyl radicals. Several lines of evidence indicate that ascorbate is localized preferentially in neurons (Rice, 2000). It has been suggested that ascorbate acts in the recovery of damage induced by pathological conditions especially related to oxidative stress, such as, those induced by severe antioxidant depletion and ischemia-reperfusion injury. The idea that intracellular ascorbate is crucial for neuronal protection against oxidant stress is supported by several studies. Ascorbate prevents lipid

* Corresponding author at: Departamento de Farmacologia, Instituto de Cieˆncias Ba´sicas da Sau´de, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, 500 Sala 202, 90050-170 Porto Alegre, RS, Brazil. Tel.: +55 51 3308 3121; fax: +55 51 3308 3121. E-mail address: [email protected] (I.R. Siqueira). 0197-0186/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2011.01.011

peroxidation induced by oxidizing agents in cultured cells, brain slices and brain microssomes (Seregi et al., 1978; Kovachich and Mishra, 1983); an increase of intracellular ascorbate content in rat brain slices reduces the swelling caused by oxidant stress (Brahma et al., 2000). In addition to its actions against free radicals, ascorbate also can contribute to protection via the glutamate–ascorbate heteroexchange, which could minimize the excitotoxic consequences of glutamate release (Gru¨newald and Fillenz, 1984; Cammack et al., 1991) and act at the redox modulatory site of receptors, as NMDA receptor (Majewska et al., 1990). It is important to note that several studies show an age-related decrease of brain ascorbate levels. Ageing is associated with a significant reduction in a-tocopherol, ascorbate and glutathione contents (Sahoo and Chainy, 1997); and it has been proposed that the mechanisms for age-related reduction of ascorbate levels include loss of ascorbate synthesis and/or altered ascorbate transport characteristics. On the other hand, Lykkesfeldt et al. (1998) reported that rat hepatic synthesis rates did not change with age. Besides, it is possible that intracellular ascorbate may be influenced by several mechanisms, namely, recycling and uptake dehydroascorbate and ascorbate efflux.

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Ascorbate is transported into mammalian cells by two types of proteins: the sodium-ascorbate co-transporters (SVCTs), SVCT1 and SVCT2, which cause Na+-dependent uptake of L-ascorbate. In the brain, SVCT2 is expressed in neuroepithelial cells of the choroid plexus, allowing ascorbate transport in cerebrospinal fluid, as well as in neurons (Tsukaguchi et al., 1999). SVCT2 is found in larger densities in cortex, hippocampus and cerebellum (Mun et al., 2006). Lykkesfeldt and Moos (2005) suggested that the decrease of ascorbate levels may either contribute to, or result from, the ageing process. Michels et al. (2003) demonstrated a drastic decrease on ascorbate concentration and expression of isoform sodiumdependent vitamin C transporters 1 (SVCT1) in freshly isolated hepatocytes from old Fischer 344 rats, concluding that the agerelated decline in hepatic ascorbate content in rats was associated with altered ascorbate uptake. To the best of our knowledge, there is a lack of studies describing age-related modifications on ascorbate uptake in any brain region, as well as the effects of ascorbate on astrocyte biochemical parameters, as assessed by glial fibrillary acidic protein (GFAP) and a calcium-binding protein (S100B) content, the major markers of astrocytes in CNS (Walz and Lang, 1998; Savchenko et al., 2000). Moreover, it is known that the astrocytes have a role restoring ascorbate (Wilson et al., 2000; Daskalopoulos et al., 2002). Considering that the hippocampus is a vulnerable brain region to excitotoxic events, like brain ischemia and neurodegenerative disorders (Candelario-Jalil et al., 2001), the aims of the present study were to evaluate (1) the effect of ageing process on ascorbate uptake abilities in hippocampal slices, (2) the effect of chronic administration of ascorbate on cellular oxidative state and astrocyte biochemical parameters in the hippocampus from 18months-old Wistar rats and (3) the protective role of ascorbate on H2O2-induced injury to hippocampal slices. 2. Materials and methods 2.1. Animals Male Wistar rats were obtained from our breeding colony and maintained under controlled light and environmental conditions (12 h light/12 h dark cycle at 22  2 8C) with food and water ad libitum in Biote´rio Setorial of Departamento de Farmacologia, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS. The Ethics Local Committee approved all handling and experimental conditions. 2.2. Ascorbate uptake in hippocampal slices Male Wistar rats aged 4 (n = 5), 12 (n = 5) and 24 (n = 5) months were used. Rats were decapitated, hippocampi were quickly dissected out and transverse sections (400 mm) were prepared using a McIlwain tissue chopper. L-[14C]ascorbarte was dissolved in 20 mM HEPES, pH 7.4, with 0.4 mM homocysteine to prevent oxidation (Wilson, 1989) and stored in aliquots at 80 8C for up to 2 months. To evaluate the optimal time for assaying L-[14C]ascorbarte uptake, an incubation time curve using slices from hippocampus was employed (data not shown); the time chosen was 60 min at 37 8C. Briefly, slices were pre-incubated in Hank’s balanced salt solution (HBSS) at 37 8C for 15 min, followed by the addition of 0.08 mCi L-[14C]ascorbate and 188 mM ascorbate (final concentration). Incubation was stopped after 60 min with three ice-cold washes of 1 mL HBSS, immediately followed by the addition of 0.5 N NaOH. Sodium-independent uptake was determined on ice (4 8C), using N-methylD-glucamine instead of sodium chloride. Both the specific and non-specific uptakes were performed in triplicate. Incorporated radioactivity was measured using a liquid scintillation counter. Protein content was measured by Lowry method (Lowry et al., 1951). Radioactivity per well was determined, standardized by protein, the values were obtained as pmol ascorbate uptake/min/mg protein. Results are expressed as percentage of control (mean  S.E.M.) for experiments in triplicates.

experiments, brain tissue was homogenized in 10 volumes of ice-cold phosphate buffer (0.1 M, pH 7.4) containing EDTA and phenylmethylsulfonyl fluoride (PMSF, 1 mM) in a Teflon-glass homogenizer. 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. 2.3.2. Free radical levels 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 nm and 525 nm, respectively. 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). 2.3.3. Thiobarbituric acid reactive substances (TBARS) Lipid peroxidation (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, n-butanol 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. 2.3.4. Oxidation of protein tryptophan residues Samples were solubilized in sodium dodecyl sulfate (SDS) at final concentration of 0.1%. The intrinsic tryptophan fluorescence was determined at excitation and emission wavelengths of 280 and 345 nm, respectively (Gusow et al., 2002). 2.3.5. Total thiol content Cellular thiols, as glutathione and protein thiols, were measured. Aliquots of samples were incubated with 100 mM DTNB (final concentration) for 15 min in darkness. Absorbance of the reaction mixture was measured at 412 nm (Khajuria et al., 1999); results are expressed as nmoles SH per mg protein. 2.3.6. 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(4-iodophenyl)-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. 2.3.7. Glutathione peroxidase (GPx) activity The reaction was carried out at 25 8C in 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 selenium-dependent 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). 2.3.8. Quantification of S100B and GFAP S100B content in the hippocampus was measured by ELISA (Leite et al., 2008) as described further (Section 2.4.3). ELISA for GFAP was carried out by coating the microtiter plate with 100 mL samples containing 30 mg of protein for 48 h at 4 8C. Incubation with polyclonal anti-GFAP from rabbit for 2 h was followed by incubation with a secondary antibody conjugated with peroxidase for 1 h, at room temperature; the standard GFAP curve ranged from 0.1 to 10 ng/mL (Tramontina et al., 2007). 2.3.9. Protein determination Protein was measured by the Coomassie blue method using bovine serum albumin as standard (Bradford, 1976). 2.4. Effect of in vitro ascorbate on H2O2-treated in hippocampal slices

2.3. Effect of chronic administration of ascorbate on several cellular oxidative state 2.3.1. Animals, treatment and tissue preparation Male Wistar rats of 18 months of age were treated daily with saline or ascorbate (50, 500 or 1000 mg/kg) by gavage during 2 weeks. Rats were decapitated; hippocampus were quickly dissected out and instantaneously placed in liquid nitrogen and stored at 70 8C until biochemical assays. On the day of the

2.4.1. Preparation and incubation of brain slices The rats (aged 4–24 months) were sacrificed by decapitation, and brains were removed and placed in cold saline medium with the following composition (in mM): 120, NaCl; 2, KCl; 1, CaCl2; 1, MgSO4; 25, HEPES; 1, KH2PO4 and 10, glucose adjusted to pH 7.4 and previously aerated with O2. The hippocampi were dissected out and transverse slices (400 mm) were obtained using a McIlwain tissue chopper.

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Table 1 The content of free radicals (DCF), thiobarbituric acid reactive substance (TBARS) levels, oxidation of protein tryptophan residues (TRI) and total thiol content, in hippocampus of 18-months old rats treated with ascorbate (50, 500 or 1000 mg/kg) or saline (control). No significant differences between control and ascorbate group were detected. Data are expressed as mean  S.E.M.; analyzed by ANOVA p > 0.05. DCF (pmol DCF formed/mg protein); TBA (pmol MDA formed/mg protein); TRI (% control); total thiois (nmoles SH formed/mg protein). DCF 0.13 0.11 0.13 0.13

TBA (0.015) (0.012) (0.020) (0.020)

2.73 1.78 1.61 2.58

Slices were then transferred immediately into 24-well culture plates, each well containing 0.3 mL of saline medium and only one slice. The medium was changed every 15 min with fresh saline medium at room temperature. Following 120 min of equilibration period, the medium was removed and replaced with fresh saline containing, or not, 200, 400 or 1000 mM ascorbate for 30 min at 30 8C. After this period, the medium was changed and H2O2 (1 mM) was added for 60 min at 30 8C (de Almeida et al., 2008). 2.4.2. Cellular damage Cellular damage was quantified by measuring both intracellular and that released into the medium lactate dehydrogenase (LDH) (Koh and Choi, 1987). LDH activity was determined using a commercial kit (Doles Reagents, Goiaˆnia, Brazil). Results obtained from control slices were taken as 100% and data were analyzed by ANOVA followed by Duncan multiple range test. 2.4.3. S100B levels S100B levels were determined in the slice medium at 15 min, 30 min and at end of incubation time (60 min) with H2O2. S100B levels in the slice medium were measured by ELISA (Leite et al., 2008). Briefly, 50 mL of sample plus 50 mL of Tris buffer were incubated for 2 h on a microtiter plate previously coated with monoclonal anti-S100B (SH-B1). Polyclonal anti-S100B was incubated for 30 min and then peroxidase-conjugated anti-rabbit antibody was added for a further 30 min. A colorimetric reaction with o-phenylenediamine was measured at 492 nm. The standard S100B curve ranged from 0.025 to 2.5 ng/mL. Results obtained from control slices were taken as 100%. 2.5. Statistical analysis Data were evaluated by two-way analysis of variance (ANOVA) followed by the Duncan’s multiple range test when appropriate. The in vitro H2O2-treated in hippocampal slices results considered dose and time as factors. 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.).

3. Results 3.1. Ascorbate uptake in hippocampal slices Fig. 1 illustrates the effect of ageing on uptake of 14C-ascorbate in hippocampus slices. A significant decrease (about 40%) in the uptake of 14C-ascorbate in hippocampal slices from rats aged 11 and 24 months, as compared to young ones, was observed (ANOVA followed by Duncan, F(2.15) = 3.71; p < 0.05), what may be related to decline in ascorbate levels during the brain ageing process. 3.2. Effect of chronic administration of ascorbate on cellular oxidative state in the hippocampus Surprisingly, the chronic treatment with ascorbate during 2 weeks did not modify any oxidative status parameters evaluated in the hippocampus from 18-months-old rats. Ascorbate did not modify the content of free radicals, in addition to indexes of damage to macromolecules, lipid peroxidation, thiobarbituric acid reactive substance (TBARS) levels, and protein oxidative damage – total thiol content, and tryptophan residues content (Table 1). Also, there were no changes on the activities of the antioxidant enzymes SOD and GPx were found, as depicted in Table 2. SOD converts superoxide radical into H2O2 and GPx breaks down peroxides, notably those derived from the oxidation of membrane phospho-

TRI (0.93) (0.35) (0.24) (0.38)

100.00 85.10 83.68 101.12

Total thiois (14.10) (12.03) (6.58) (8.78)

5.72 7.12 6.74 6.56

(0.39) (0.99) (0.67) (1.15)

Table 2 The activities of the enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) in hippocampus of 18-months old rats treated with ascorbate (50, 500 or 1000 mg/kg) or saline (control). There were no differences between the groups. Data are expressed as mean  S.E.M.; analyzed by ANOVA p > 0.05. SOD (U/ mg protein); GPx (nmol NADPH oxidized/min/mg protein). Group/enzyme

SOD

Saline 50 mg/kg 500 mg/kg 1000 mg/kg

0.38 0.38 0.35 0.38

GPx (0.031) (0.024) (0.010) (0.021)

36.9 32.8 32.7 34.4

(3.38) (2.49) (2.12) (3.06)

lipids. This result might suggest no effect of chronic administration of ascorbate on antioxidant defenses. Moreover, in order to evaluate whether the chronic administration of ascorbate alters some astrocyte biochemical parameters, the contents of S100B and GFAP proteins, the major markers of astrocytes in CNS (Walz and Lang, 1998; Savchenko et al., 2000), were determined. However, no alterations on these parameters in in vivo treatment were observed, as demonstrated in Table 3. Even, no interaction between the cellular oxidative state parameters evaluated in our study and astrocyte biochemical parameters were found. 3.3. Effect of in vitro ascorbate on H2O2-treated in hippocampal slices In order to investigate the injury caused by H2O2, we employed released lactic dehydrogenase (LDH) into the media as an index of cell damage or lysis, which is used commonly as markers for brain cell damage (Gabryel et al., 2002; Noraberg et al., 1999). The increase on the leakage of LDH indicates loss of membrane integrity. The incubation with ascorbate did not change the intracellular (IC) nor the released (EC) LDH activities in hippocampal slices submitted to H2O2 (Fig. 2). Interestingly, the ascorbate decreased 120

uptake ascorbate (% control)

Group/parameter Saline 50 mg/kg 500 mg/kg 1000 mg/kg

100 80

*

*

11 months

24 months

60 40 20 0

4 months

Fig. 1. Effect of ageing on uptake of radiolabeled ascorbate in hippocampus slices. Columns represent mean  S.E.M. for percentage of young group values. ANOVA followed by Duncan (p < 0.05); *significantly different as compared to the young group.

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Table 3 Content of proteins S100B and GFAP in hippocampus of 18-months old rats treated with ascorbate (50, 500 or 1000 mg/kg) or saline (control). No significant differences between control and ascorbate group were detected. Indeed, no interaction between the cellular oxidative state parameters evaluated and astrocyte biochemical parameters were found. Data are expressed as mean  S.E.M.; analyzed by ANOVA p > 0.05. S100 (mg/mg protein); GFAP (mg/mg protein). Group/protein

S100

Saline 50 mg/kg 500 mg/kg 1000 mg/kg

21.32 21.30 21.84 26.18

GFAP (1.18) (1.61) (1.90) (1.65)

0.66 0.72 0.69 0.87

(0.060) (0.071) (0.057) (0.120)

LDH activity (% of control)

140 120 100 80 60 40 20 0

200 uM

400 uM

1000 uM

200 uM

IC

400 uM

1000 uM

EC

Fig. 2. Effect of incubation with ascorbate (200, 400 or 1000 mM) on cellular damage in hippocampal slices submitted to H2O2, evaluated by intracellular (IC) and released (EC) lactate dehydrogenase (LDH). Results are expressed as percentage of the control group. Two-way ANOVA.

S100B activity (% of control)

100 90

*

*

*

*

80

*

70

*

60

*

* *

50 40 30 20 10 0

200

400 1000

15 minutes

200

400 1000

30 minutes

200

400 1000

60 minutes

Fig. 3. Effect of different ascorbate concentrations (200, 400 and 1000 mM) on S100B released in hippocampal slices submitted to H2O2. The S100B was evaluated 15, 30 and 60 min after the incubation. Horizontal lines represent results obtained from control slices (0 mM) at each time point which were taken as 100%. Two-way ANOVA followed by Duncan (p < 0.05); *significantly different as compared to its control (0 mM).

significantly the released S100B in hippocampal slices in all concentrations and incubation times evaluated (ANOVA followed by Duncan, F(3.24) = 5.285; p < 0.05; Fig. 3). However released S100B did not differ among any ascorbate concentrations in all time points studied. 4. Discussion Our data demonstrated that radiolabeled ascorbate uptake was decreased in hippocampal slices from old-aged rats. On the other

hand, the chronic administration of ascorbate (2 weeks) did not modify any oxidative status in the hippocampus, as well ascorbate incubation was unable to protect hippocampal slices submitted to oxidative damage (H2O2) from old rats. To our knowledge, this is the first evidence that showed the effect of ageing on ascorbate uptake in the brain. In fact, uptake ascorbate and consequently its levels decreased might be related to increased cell death in hippocampus exposed to ischemia and excitotoxic injury. This result is relevant since ascorbate has several intracellular functions as an enzyme cofactor and also contributes to antioxidant defense. It is interesting to note that our previous data showed that hippocampal slices from old animals was more susceptible to oxygen and glucose deprivation condition, an ischemia–reoxygenation injury (Siqueira et al., 2004) and hippocampal neurons from old animals are more susceptible to glutamate and b-amyloid toxicity than young neurons (Brewer, 2000). Indeed, considering that neurons have tenfold higher ascorbate levels than glia, it seems reasonable to suppose that especially neurons are sensitive to ascorbate deficiency. Besides, decrease in the uptake of 14C-ascorbate in hippocampal slices from aged rats might be related to previous data demonstrating a relevant reduction on total antioxidant capacity in older hippocampus (Siqueira et al., 2005), given that ascorbate is an important water-soluble antioxidant. It is important to note that the reduction on hippocampal ascorbate uptake is especially relevant, since humans have lost the ability to synthesize ascorbate, which is available from dietary sources then, evenly important, is the ability to absorb by the gut (Kallner et al., 1977). Humans, nonhuman primates and guinea pigs have a nonfunctional gene for the enzyme, L-gulono-g-lactone oxidase, the last step of ascorbate biosynthesis. The lack of this ability makes the humans susceptible to ascorbate deficiency (Nishikimi et al., 1994). Indeed, some studies strongly support that dietary ascorbate uptake is impaired with age in both humans (Brubacher et al., 2000), and rats (Michels et al., 2003). Michels et al. (2003) suggest a reduced ascorbate transport into the plasma and into target cells with age. Although a number of studies (Lykkesfeldt et al., 1998) have reported that there are no age-associated alterations in hepatic ascorbate synthesis in rats, the decline in ascorbate levels during the ageing process has been reported (Sahoo and Chainy, 1997; Lykkesfeldt et al., 1998). In the present study older hippocampal slices demonstrated a decrease on uptake of radiolabeled ascorbate. Our result is in agreement with those obtained by Michels et al. (2003) that describe a sodium-dependent ascorbate transport declines during the ageing process in hepatocytes. Considering ascorbate properties, we have hypothesized that ascorbate supplementation would protect hippocampal cells in both in vitro and vivo assays. Although, the incubation with ascorbate did not alter the cellular damage in hippocampal slices exposed to oxidative damage in all tested concentrations, similar brain concentration (200 mM), or only slightly higher (400 mM), and a higher concentration (1000 mM) than those in the cerebrospinal fluid, concentrations considered sufficient to saturate high affinity Na+-ascorbate co-transport systems (Wilson, 1989). This result is in agreement with our in vivo assay findings, since no effect of supplemental ascorbate on oxidative stress in the hippocampus was observed. In the present study we demonstrated a decrease (about 40%) in the uptake of 14C-ascorbate already in hippocampal slices from 11 months-old rats as compared to young. This result is in agreement with our previous results (Siqueira et al., 2005) that have reported that the hippocampus was the structure with impaired antioxidant function already in 6 months-old rats, suggesting that this may be implicated with the beginning of the ageing process. Hippocampi from 6-months-old rats demonstrated a decrease in TRAP (Total

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Reactive Antioxidant Potential) and TAR (Total Antioxidant Reactivity) levels, both indexes were almost 30% lower when compared to younger rats. While these parameters fall to about 40 and 50%, respectively, in the aged group (24 months-old rats). In addition the changes on cerebral total antioxidant status may be related to vulnerability of hippocampus to oxidative stress. Although we have not investigated, we can suppose that the decrease on uptake of 14C-ascorbate in hippocampal slices from 6months-old rats may be similar to TRAP and TAR indexes. Moreover, we may also speculate that a compensatory mechanism was responsible for similar levels of uptake of 14C-ascorbate in hippocampal slices from 11- and 24-months-old rats. Nevertheless, present data does not agree with those with liver tissue (Michels et al., 2003), where the incubation with increasing amounts of radiolabeled ascorbate and higher ascorbate concentration increased its uptake in hepatocytes from both young and old rats, although in cells from old rats remained lower than from young ones. This difference could be explained by transporter isoforms distribution, since liver expresses preferentially SVCT1, only SVCT2 is found in the brain tissues (Tsukaguchi et al., 1999). Considering that there are strong evidence that uptake on the SVCT2 is one route for ascorbate entry into neural tissues (Sotiriou et al., 2002) we might suggest that altered SVCT2 activity is related to ageing process. It is interestingly to cite that plasma ascorbate levels are dependent of dietary intake and, in rats, endogenous synthesis. Previous findings suggest that the ascorbate synthesis does not change in ageing process; however we can suppose that decreased SVCT1 expression in hepatocytes (Michels et al., 2003) may lead to alterations on efflux of ascorbate from these cells, reducing the plasma ascorbate levels. As stated in previous work, the dietary ascorbate uptake is impaired with age (Brubacher et al., 2000). The plasma ascorbate concentration after specific loading dose of ascorbate was significantly lower in the elderly when compared to young and middle-aged adults (Brubacher et al., 2000). Considering that the expression of SVCT1 was reduced in hepatocytes from old Fischer 344 rats, we might suppose that SVCT1 downregulation also occurs in the intestinal lumen, expressed on the apical membrane, which transports ascorbate across the intestinal barrier (MacDonald et al., 2002). Certainly it is possible that both ageing process and ascorbate administration influence ascorbate levels in liver, kidney and serum, as well as brain. It is clear that much additional work will be required before a complete conclusion be done, such as molecular biological, immunological, and pharmacological approaches. The future potential strategies might be used to determine the ascorbate levels. Ascorbate decreased S100B secretion in hippocampal slices exposed to H2O2. It is known that secreted S100B acts as a cytokine for neighboring cells (astrocytes, neurons and microglia), depending on its concentration; being neurotrophic at nanomolar levels and apoptotic at micromolar levels (Donato, 2001; Van Eldik and Wainwright, 2003). Our results might support the modulation of S100B secretion as a critical component of the molecular mechanisms induced by ascorbate. Previous data support the hypothesis of an imbalance between free radical levels and scavenging systems in ageing rat brain, particularly in the hippocampus. It was observed a marked decrease of total antioxidant capacity and glutathione peroxidase activity, as well as an increase on free radicals levels, determined by 20 -70 -dichlorofluorescein diacetate probe and on lipoperoxidation. Accordingly it has been suggested that age-related variations of total antioxidant defenses in brain may predispose structures to oxidative stress-related neurodegenerative disorders (Siqueira et al., 2005). A long term dietary supplementation of various antioxidants has been found to retard the onset of age-related

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deficits in neural functioning (Bickford et al., 2000; Villeponteau et al., 2000; Carney et al., 1991; Socci et al., 1995). Surprisingly, there are few studies on the effects of exogenous ascorbate on oxidative status in hippocampus, one of the most vulnerable brain regions to oxidative stress (Candelario-Jalil et al., 2001). Although the chronic treatment with ascorbate did not alter the oxidative status parameters evaluated in hippocampus from 18months-old rats, a role for ascorbate in antioxidant network as a critical component of the molecular mechanisms cannot be excluded. Other experimental models were unable to demonstrate a functional neuroprotective property of ascorbate per se. Our results are in agreement with those obtained by Harrison and colleagues (2009) where ascorbate did not affect both oxidative stress markers, malondialdehyde and glutathione levels of frontal cortex and cerebellum from mice. Accordingly they demonstrated also that brain ascorbate levels did not increase significantly following i.p. administration, while it increased twofold in the liver. Some studies have demonstrated that ascorbate associated with tocopherol improves learning and memory in aged mice (15 months), while this effect was not observed in 3-monthold mice or when ascorbate was administered alone (Arzi et al., 2004). This finding corroborates the hypothesis that association of antioxidants improving the antioxidant network efficacy is crucial to reduce the damage caused by oxidative stress. It is possible also to speculate that there were undetected alterations in other oxidative stress parameters as well as compensatory mechanism associated with chronic administration. Neuron–glia interaction plays an essential role in neuronal survival. It has been described an astrocytic reactivity in the cortex and hippocampus of aged rats (Baydas and Tuzcu, 2005) and in senescence-accelerated mice hippocampus (Wu et al., 2005). Baydas et al. (2003) suggest that gliosis occurring in diabetes mellitus may be related, at least in part, with free radicals formation and antioxidants may prevent reactive gliosis possibly by antioxidant effect. In present study, the chronic administration of ascorbate did not alter the expression of S100B and GFAP proteins in hippocampal astrocytes from aged rats. Summarizing, our data propose that the decline of ascorbate uptake might be involved in the brain greater susceptibility to oxidative damage with advancing age and both in vitro and vivo assays suggest that ascorbate supplementation did not protect hippocampal cells. The detailed mechanism by which ageing modify ascorbate uptake remains a subject for further investigations. Acknowledgements We gratefully acknowledge financial support by PRONEX, CAPES, CNPq. Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico – CNPq (Dr. I.R. Siqueira; Felipe Moyse´s-2009, Viviane Elsner-2009); Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior – CAPES (Felipe Moyse´s-2008). References Arzi, A., Hemmati, A.A., Razian, A., 2004. Effect of vitamins C and E on cognitive function in mouse. Pharmacol. Res. 49, 249–252. Baydas, G., Reiter, R.J., Yasar, A., Tuzcu, M., AKdemir, I., Nedzvetskii, V.S., 2003. Melatonin reduces glial reactivity in the hippocampus, cortex, and cerebellum of streptozotocin - induced diabetic rats. Free. Radic. Biol. Med. 35, 797–804. Baydas, G., Tuzcu, M., 2005. Protective effects of melatonin against ethanol-induced reactive gliosis in hippocampus and cortex of young and aged rats. Exp. Neurol. 194, 175–181. Bickford, P.A., Gould, T., Briederick, L., Chadman, K., Pollock, A., Young, D., ShukittHale, B., Joseph, J., 2000. Antioxidant-rich diets improve cerebellar physiology and motor learning in aged rats. Brain Res. 866, 211–217. Bradford, M.M., 1976. A rapid and sensitive method for the quantication of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254.

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