vitamin e affects cell death in adult rat dentate gyrus

Journal of Neurocytology 32, 1155–1164 (2003)

Vitamin E affects cell death in adult rat dentate gyrusPAOLA FER R I 1,∗ , T I ZI AN A CECCHINI 1, SANDRA CIARONI 1,PATRIZIA AMBR O G I N I 2, R I CCARDO CUPPINI 2, SPARTACOSANTI 3, SER EN A BEN EDETTI 4, S ILVIA PAGLIARANI 4, PAOLO DELGRANDE 1 a n d S TEFAN O PAPA 1

1Institute of Morphological Sciences and 2Physiological Sciences, University of Urbino “Carlo Bo”, I-61029 Urbino, Italy;3Institute of N.P. Cytomorphology, CNR, I-40126 Bologna; 4Institute of Biological Chemistry “Giorgio Fornaini”, University of Urbino“Carlo Bo”, I-61029 Urbino, [email protected]

Received 6 August 2003; revised 14 November 2003; accepted 14 November 2003

Abstract

We have previously reported the presence of dying cells in the granule cell layer (GCL) of adult rat dentate gyrus (DG),where neurogenesis occurs. In particular, we found that cell death in the GCL increased in vitamin E deficiency and decreasedin vitamin E supplementation. These findings were regarded as related to changes in neurogenesis rate, which in turn wasinfluenced by vitamin E availability; a neuroprotective effect of vitamin E on cell death was also proposed. In order to verifythis latter hypothesis, we have studied cell death in all layers of DG in vitamin E-deficient and vitamin E-supplemented ratsand in control rats at different ages, using TUNEL and nick translation techniques. The phenotype of TUNEL-positive cellswas characterized and the existence of dying BrdU-positive cells was investigated. Dying cells with neuronal phenotype wereobserved throughout the DG in all experimental groups. The number of TUNEL-positive cells decreased from juvenile to adultage. A higher number of TUNEL-positive cells in vitamin E-deficient rats and a lower number in vitamin E-supplemented rats,with respect to age-matched controls, were found; moreover, in these groups, TUNEL-positive cells had a different percentagedistribution in the different layers of the DG. Our results confirm the occurrence of cell death in DG, demonstrate that cell deathaffects neuronal cells and support the hypothesis that the effect of vitamin E on cell death is not related to neurogenesis.

Introduction

Apoptosis has been observed in the central nervoussystem (CNS) of adult Mammals (White & Barone,2001). This programmed cell death has often been foundin regions in which neurogenesis persists in adulthood,such as olfactory bulb and dentate gyrus of the hip-pocampus (Biebl et al., 2000). Since a redundant numberof new neurons is produced during adult neurogene-sis and only some survive (Gould et al., 2001), in theseregions apoptosis has been proposed to be functionallyrelated to the neurogenetic process, as during develop-ment (Schlessinger et al., 1975).

Apoptosis is a complex process that can be inducedand regulated by numerous factors and/or conditions.Among the conditions involved in apoptosis, oxida-tive stress plays an important role: in fact, intracel-lularly produced reactive oxygen species (ROS) havebeen demonstrated to interact with apoptotic path-way via caspase activation (for a review, see Chandraet al., 2000). Consistent with these findings, oxidativedamage with unregulated production of ROS has been

∗To whom correspondence should be addressed.

shown to be involved in the slow progressive neuronaldeath characterizing neurodegenerative diseases suchas Alzheimer’s disease (AD) (Esposito et al., 2000).

Vitamin E is a family of molecules, among whichα-tocopherol is quantitatively and physiologically themost important (Kim et al., 1996). The antioxidant prop-erties of vitamin E are well known, tocopherols beingfree radical scavengers in biological membranes; vita-min E alone (Grundman, 2000) or together with othercompounds (Behl & Moosmann, 2002) has been demon-strated to have protective effects in AD patients. Besidesvitamin E antioxidant properties, several studies pointto alternative capabilities of this molecule in terms ofcell proliferation and survival regulation (for a review,see Brigelius-Flohe et al., 2002).

In previous work, we observed dying cells in thegranule cell layer (GCL) of adult rat dentate gyrus (DG),in accordance with other findings (Sloviter et al., 1993,1996; Young et al., 1999). We found that the number ofdying cells changed from juvenile to adult age (Ciaroni

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1156 FERRI, CECCHINI, CIARONI ET AL.

et al., 2002a, b) and when a condition of vitamin E de-ficiency or supplementation was induced in adult ani-mals (Ciaroni et al., 2002b; Cecchini et al., 2003). As ourinvestigations were limited to the GCL, i.e. the zone inwhich new neurons continue to be generated in adult-hood, the cell death process and changes in cell deathwere considered to be related to changes in neuroge-nesis rate, which in turn are affected by ageing andchanges in vitamin E levels.

Nevertheless, in considering a possible neuroprotec-tive effect of vitamin E, in this work a quantitative andqualitative analysis of cell death was extended to all DGlayers; it was performed in control young and adultrats and under vitamin E deficiency or supplementa-tion conditions. The TUNEL and nick translation tech-niques, which allow the visualization in situ of the DNAfragmentation, were carried out. The phenotype of dy-ing cells was characterized using neuronal and glialmarkers and the existence of dying BrdU-positive cellswas investigated.

Methods

ANIMALS

Sprague-Dawley male rats were used in accordance with Eu-ropean Union guidelines and Italian laws.

The animals were divided into three groups: α-tocopherol-supplemented rats, which received subcutaneously 2 mg/Kgbody weight of α-tocopherol in 4% ethyl alcohol and 1% Cre-mophor EL (BASF Actiengesellsdaft, Mannheim, Germany)aqueous solution every day for 45 days from 14 weeks of ageuntil death at 5 months of age (A5 group); vitamin E-deficientrats, fed a diet lacking in vitamin E (Bruno et al., 1990) from1 month of age until death at 5 months of age (D5 group);control rats, which were further divided into three groups:C1 group, rats killed at 1 month of age, when the postnataldevelopment of DG is terminated (Altman & Bayer, 1990), C2group, rats killed at 2 months of age, when they are thought tohave reached sexual maturity and C5 group, adult rats killedat 5 months of age. The TUNEL technique was used on C1 andC2 animals (n = 3, for both groups); C5, A5 and D5 rats wereused for different investigations (n = 3, for each group in eachinvestigation). Some animals were used for tocopherol assay,while others were used for the TUNEL qualitative and quanti-tative analysis. In addition, some rats were intraperitoneallyinjected with BrdU (50 mg/Kg b.w.) twice a day (8.30 a.m.and 6.30 p.m.) for three consecutive days, killed one day afterthe last administration and then used for double nick trans-lation/BrdU labeling.

TOCOPHEROL ASSAY

Alpha-tocopherol was assayed in brain and plasma of ratsfrom the C5, A5 and D5 groups, as described by Burtonet al. (1985). In short, 100 mg of brain were homogenized for1 minute with 100 µl of tocopherol acetate (68.7 µM) as an in-ternal standard and 2 ml of chilled distilled water. One ml ofSDS (50 mM), 3 ml of methanol and 3 ml of hexane were addedwhile the mixture was kept on ice, vortexing at each step. Thehomogenate was centrifuged at 3000 rpm at 4◦C for 10 min.

The hexane phase was drawn, concentrated to dryness undernitrogen current and resuspended with 0.4 ml of methanol.The assay was performed by HPLC with a reverse phase Al-tima C18 column (4.6 × 250 mm, 5 µm). The eluent phasewas constituted by methanol/water (98:2); the flow rate was1.6 ml/minutes. UV detection was carried out at 292 nm andthe retention time of α-tocopherol was 14.12 min. Recoverytrials, carried out with tocopherol acetate used as an internalstandard and extracted according to the described procedure,gave an average recovery of 95%. Values were expressed asµg of α-tocopherol per g of wet tissue.

A plasma sample (0.1 ml) from each rat was added to0.1 ml of tocopherol acetate (68.7 µM) and vortexed for 15 sec.0.5 ml of hexane were added and the mixture was vortexedtwice for 40 sec and then centrifuged at 3000 rpm at 4◦C for5 min. The hexane phase was drawn, concentrated to dry-ness under nitrogen current and resuspended with 0.4 ml ofmethanol. The HPLC assay was carried out according to theabove-mentioned procedure.

HISTOLOGICAL PROCEDURE

The rats used for cell death analysis were anaesthetized withsodium thiopental via i.p. (45 mg/Kg b.w.) and killed by anintracardial perfusion with normal saline followed by 4%paraformaldehyde in phosphate buffer saline (PBS; 0.01 M,pH = 7.4). The brain was then removed, dissected into twohemispheres and post-fixed in the same fixative for 5 hrs.The left hemisphere was embedded in Paraplast Plus (Sigma;melting point = 56◦–58◦C); 6-µm-thick serial coronal sectionswere cut and a series of 10 coronal sections of brain, spaced60 µm apart were selected for each animal.

Each investigation was performed in dorsal hippocampus,where the dentate gyrus is horizontally oriented beneath thecorpus callosum, starting at the junction of the GCL’s externaland internal blades at the crest until the superior colliculaappear.

TUNEL QUALITATIVE AND QUANTITATIVE ANALYSIS

For TUNEL staining, rehydrated sections were treated with0.02% proteinase K (20 mg/ml) in 5% Tris-HCl buffer (0.05 M;pH = 7.4) and 0.01% EDTA (0.2 M) in distilled water for 7min at room temperature and rinsed in water. Sections werethen treated with 1% hydrogen peroxide in 10% methyl al-cohol in PBS (0.01 M, pH = 7.4), to block endogenous per-oxidase, for 7 min at room temperature. After rinsing inPBS, dying cells were detected by DNA fragmentation us-ing the TUNEL method to specifically label the 3′ -hydroxyltermini of DNA strand breaks. For the TUNEL procedure,all reagents were part of a kit (Apoptag Plus, D.B.A., On-cor) that used a digoxigenin-conjugated dUTP followed bya peroxidase-conjugated anti-digoxigenin antibody and pro-cedures were performed according to the manufacturer’sinstructions. Finally, peroxidase was developed with 0.5mg/ml of 3,3′ -diaminobenzidine (DAB; Sigma) in Tris-HClbuffer with 0.01% hydrogen peroxide for 20 min at roomtemperature.

TUNEL-positive nuclei were counted in all layers of theDG (basal polymorphic, granule cell and plexiform layers)in at least 18 non-consecutive sections per animal; some sec-tions, adjacent to those in which TUNEL-positive cells were

Vitamin E and cell death 1157

counted, were stained with cresyl violet to test the tissue mor-phology. The quantitative results were calculated as the meannumber of TUNEL-positive cells/section as previously re-ported (Cheng et al., 2001; Ciaroni et al., 2002b).

In order to study the nucleus morphology of dying cells,some sections were deparaffinized, rehydrated, stained with5 µg/ml propidium iodide (PI) in PBS for 5 min at room tem-perature (Young et al., 1999) and observed using a BioRadRadiance 2000 laser scanning microscope (BioRad Laborato-ries, Richmond, CA).

DOUBLE-LABELINGS

To identify the phenotype of TUNEL-positive cells, DNA frag-mentation was shown in the same sections together with thefollowing markers: TUC 4, a neuronal transient antigen ex-pressed after the last mitotic division and before overt differ-entiation (Quinn et al., 1999), or NeuN, a protein expressedby fully differentiated neurons (Magavi et al., 2000), or GFAP,a glial cell marker.

For TUNEL/TUC 4 double-labeling, sections were incu-bated with normal goat serum (D.B.A., 1:10 in PBS) for10 min at room temperature and then with a rabbit anti-TUC 4 (Chemicon, 1:500 in PBS) overnight at 4◦C. Af-ter rinsing in PBS, sections were incubated with a CY3-conjugated goat anti-rabbit (Amersham, 1:100 in PBS)for 1 hr at room temperature. Sections were then post-fixed with 4% paraformaldehyde in PBS (0.1 M, pH =7.4) for 1 hr and then processed for TUNEL staining. TheTUNEL technique was essentially performed as previouslydescribed, but the kit for fluorescence (Apoptag Plus, D.B.A.,Oncor), which uses a digoxigenin-conjugated dUTP fol-lowed by a FITC-conjugated anti-digoxigenin antibody, wasadopted.

For TUNEL/NeuN double-labeling, sections were treatedwith normal goat serum (D.B.A., 1:10 in PBS) for 10 min andthen incubated with a mouse anti-NeuN (Chemicon, 1:100 inPBS) overnight at 4◦C, followed by incubation with a CY 3-conjugated goat anti-mouse (Amersham, 1:50 in PBS) for 1 hrSections were post-fixed with 4% paraformaldehyde in PBS(0.1 M, pH = 7.4) for 1 hr and then the TUNEL procedure wascarried out as previously described for double TUNEL/TUC4 labeling.

In order to verify the DNA fragmentation and death ofglial cells, double TUNEL/GFAP labeling was used: sectionswere incubated with normal goat serum (D.B.A., 1:10 in PBS)for 10 min at room temperature and then with a rabbit anti-GFAP (Sigma, 1:100 in PBS) overnight at 4◦C. Afterwards,sections were incubated with a CY 3-conjugated goat anti-rabbit (Amersham, 1:50 in PBS) for 1 hr. at room temperatureand were then post-fixed with 4% paraformaldehyde in PBS(0.1 M, pH = 7.4) for 1 hr Sections were processed for TUNELstaining for fluorescence as described above.

In order to determine whether proliferating precursorsdie early, a nick translation/BrdU double-labeling was em-ployed rather than the TUNEL method. The TUNEL method,which detects double-strand DNA breaking points, revealsthe late stages of cell death, when DNA fragmentation isadvanced, so it could mask BrdU incorporation. Conversely,nick translation labels early stages of cell death (Columbaro etal., 1998) and was thus more suitable. The nick translation pro-cedure was carried out in the same way as the TUNEL tech-

nique, but the TdT enzyme was substituted with 1% DNApolymerase I (5 U/µl) in the reaction mixture for 1 hr at37◦C. After FITC-conjugated anti-digoxigenin antibody incu-bation, sections were post-fixed with 4% paraformaldehydein PBS (0.1 M, pH = 7.4) for 1 hr. The detection of BrdU-labeled cells was then carried out. For DNA denaturation,sections were treated with 0.1 M HCl at 4◦C for 10 min, thenwith 4 M HCl at 37◦C for 30 min and rinsed with borate-buffered saline (0.1 M, pH = 8.5). After several rinses in 1%bovine albumin serum in PBS, sections were treated withnormal goat serum (D.B.A., 1:10 in PBS) for 10 min and in-cubated with a mouse anti-BrdU primary antibody (D.B.A.;1:300 in PBS) overnight at 4◦C. After rinsing in PBS, sec-tions were incubated with a CY 3-conjugated goat anti-mouse(Amersham; 1:50 in PBS) secondary antibody for 1 hr at roomtemperature.

A BioRad Radiance 2000 laser scanning microscope (Bio-Rad Laboratories, Richmond, CA) was used in the analy-sis of double-labeled cells. The percentage of a sample of30 TUNEL- or nick translation-positive cells which werepositive for each specific marker was determined in eachgroup.

STATISTICS

Values are expressed as mean ± S.D.; comparisons weremade with the non-parametric Kruskal-Wallis test, followedby post-hoc comparisons; the threshold of significance wasfixed at p = 0.05.

Results

PLASMA AND BRAIN TISSUE TOCOPHEROL LEVELS

The concentration of α-tocopherol in the brain tissueof control rats was 29.2 ± 1.5 µg/g. In α-tocopherol-supplemented rats it was 41.07 ± 1.21 µg/g after 45days of treatment, 28.9% higher than controls (Fig. 1A),consistent with what has been previously found(Cecchini et al., 2003). The level of α-tocopherol in braintissue was reduced in vitamin E-deficient rats after theywere fed a diet lacking in vitamin E for 4 months, in ac-cordance with previous findings (Ciaroni et al., 2002b).In fact, the concentration of α-tocopherol in the braintissue of these animals dropped to 4.6 ± 0.5 µg/g after4-month period, a decrease of 84.3% in comparison tocontrols (Fig. 1A). The differences among the groupswere statistically significant (Kruskal-Wallis test: p <

0.05; post-hoc comparisons: A5 vs. C5, p < 0.05; D5 vs.C5, p < 0.05; A5 vs. D5, p < 0.05).

The plasma level of α-tocopherol was 22.37 ±3.44 µM in C5 group rats, while it was 34.02 ± 1.66 µMin α-tocopherol-supplemented rats and 1.10 ± 0.50 µMin vitamin E-deficient rats, an increase of 34.2% and adecrease of 95.1% respectively in comparison with thecontrol value (Fig. 1B). The differences among the val-ues were statistically significant (Kruskal-Wallis test:p < 0.05; post-hoc comparisons: A5 vs. C5, p < 0.05; D5vs. C5, p < 0.05; A5 vs. D5, p < 0.05).


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Fig. 1. α-tocopherol levels in brain tissue (A) and plasma (B) in rats from the C5, A5 and D5 groups (for brain tissue and plasma,Kruskal-Wallis test: p < 0.05; post-hoc comparisons: A5 vs. C5, p < 0.05; D5 vs. C5, p < 0.05; A5 vs. D5, p < 0.05).

TUNEL STAINING

TUNEL-positive cells were found in all layers of theDG in every experimental group; TUNEL-positive cellswere found scattered in the plexiform and basal poly-morphic layers of the DG (Fig. 2A and B), whilethey were always located in the first rows in theGCL (Fig. 2C). TUNEL-positive nuclei were regularlyspheric or oval-shaped, intensely labeled, and almostalways single, rarely seen in groups of two or three el-ements (Fig. 2B).

Fig. 2. (A)TUNEL-positive cell in the plexiform layer. (B) TUNEL-positive cells in the basal polymorphic layer (arrow) and inthe GCL arranged in a group of two elements (arrowhead). (C) TUNEL-positive cell in the first rows of the GCL. (D) Dying cellin which micronuclei are visible (arrow) in cresyl violet stained section. In A, C and D, bar = 10 µm; in B, bar = 10 µm.

TUNEL-positive cells, identified by fluorescence, of-ten showed a typical apoptotic nuclear morphology.In fact, in some cases, more intensely labeled areasmarginated towards the nuclear periphery (Fig. 3A)could be observed. This rearrangement of the nucleusinto cup-shaped chromatin marginations was very sim-ilar to rearrangements shown by ultrastructural analy-sis of the apoptosis in hippocampus (Sloviter et al., 1993,1996) and in cell cultures (Falcieri et al., 1994). In othercases, TUNEL-positive cells showed the presence of


Fig. 3. (A and B) TUNEL-positive cells characterized by positive margination areas in the plexiform layer (A) and characterizedby the presence of micronuclei in the basal polymorphic layer (B). (C and D) PI-stained cells with nucleus split into micronucleiin the GCL (C) and plexiform layer (D). Bar = 5 µm.

micronuclei (Fig. 3B) scattered through the cytoplasm,resulting from nuclear modifications related to a latestage of apoptosis (Falcieri et al., 1994; Columbaro et al.,1998); micronuclei were also occasionally observed incresyl-violet stained sections (Fig. 2D).

The double-labeling analysis showed that about40% of TUNEL-positive cells were positive for TUC 4(Fig. 4A) and about 40% of TUNEL-positive cells wereNeuN-positive (Fig. 4B); no TUNEL-positive cell waslabeled for GFAP (Fig. 4C). No nick translation/BrdUdouble-labeled cells were found, but frameworks, inwhich one or two nick translation-positive cells were

part of a cluster of BrdU-positive cells, appearedvery frequently (Fig. 4D). No differences were notedamong the experimental groups in the double-labelinganalyses.

PI STAINING

PI staining confirmed the presence of apoptotic cellswithin the DG: the morphological characteristics ofthese cells suggested apoptotic cell death. In fact, theirnuclei often appeared to be split into various micronu-clei both in the GCL and in the other layers (Fig. 3Cand D).


Fig. 4. (A) Double staining confocal microscopy of TUNEL/TUC 4-double labeled cell in the GCL of the DG; merging ofthe two signals: TUNEL-positivity in green, TUC 4-immunopositivity in red. (B) Double staining confocal microscopy ofTUNEL/NeuN double-labeled cell in the plexiform layer of DG; merging of the two signals: TUNEL-positivity in green,NeuN-immunopositivity in red. (C) Double staining confocal microscopy of TUNEL/GFAP labeling; a TUNEL-positive cell inthe GCL and GFAP-positive cells in the basal polymorphic layer of DG: there are no TUNEL/GFAP double-labeled cells; mergingof the two signals: TUNEL-positivity in green, GFAP-immunopositivity in red. (D) Double staining confocal microscopy of nicktranslation/BrdU labeling; a cluster of BrdU-positive cells in the GCL in which a nick translation-positive but BrdU-negativecell is present: there are no nick translation/BrdU double-labeled cells; merging of the two signals, nick translation-positivityin green, BrdU-immunopositivity in red. In A and B, bar = 5 µm; in C and D, bar = 7 µm.

QUANTITATIVE ANALYSIS OF TUNEL POSITIVE CELLS

The mean number of TUNEL-positive cells/sectionwas 2.27 ± 0.43 throughout the DG at 1 month of age incontrols. They were found mostly in the GCL, and wereless numerous in the plexiform layer and even rarer inthe basal polymorphic layer (Fig. 5A).

As the age of animals increased, the number ofTUNEL-positive cells/section in the DG decreased,dropping to 1.85 ± 0.29 at 2 months of age and0.59 ± 0.10 at 5 months (Fig. 5A) (Kruskal-Wallis test:p < 0.05; post-hoc comparisons: C1 vs. C2, ns; C1vs. C5, p < 0.05; C2 vs. C5, p < 0.05). The observed


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Fig. 5. Quantitative analysis of cell death in the DG of rat. (A) effect of ageing on mean number of TUNEL-positive cells/sectionin DG (C1 = one-month-old rats; C2 = two month-old-rats; C5 = five month-old-rats; Kruskal-Wallis test: p < 0.05; post-hoccomparisons: C1 vs. C2, ns; C1 vs. C5, p < 0.05; C2 vs. C5, p < 0.05). (B) Effect of both vitamin E deficiency and supplementationon mean number of TUNEL-positive cells/section in the DG (D5 = adult vitamin E-deficient rats; A5 = adult α-tocopherol-supplemented rats; C5 = age-matched controls; Kruskal-Wallis test: p < 0.05; post-hoc comparisons: C5 vs. A5, p < 0.05; C5 vs.D5, p < 0.05; A5 vs. D5, p < 0.05). (C) Percentage distribution of TUNEL-positive cells in the three layers of DG in the differentexperimental groups (dark gray: GCL; light gray: plexiform layer; black: basal polymorphic layer).

decrease affected the different DG layers in the sameway, thus the percentage distribution of TUNEL-positive cells in plexiform, basal polymorphic andgranule cell layers did not change with ageing(Fig. 5C).

In vitamin E-deficient rats 0.94 ± 0.24 TUNEL-positive cells/section were found in the DG, whilein vitamin E-supplemented rats 0.32 ± 0.06 TUNEL-positive cells/section were detected (Fig. 5B). Bothvalues significantly differed from the values for age-matched controls (see above) (Kruskal-Wallis test: p <

0.05; post hoc comparisons: C5 vs. A5, p < 0.05; C5vs. D5, p < 0.05; A5 vs. D5, p < 0.05). The increasein cell death in vitamin E-deficient rats did not affectthe different DG layers in the same way. For exam-ple, we found a comparable number of TUNEL-positivecells in the GCL and plexiform layer of the vitamin E-deficient group, while in control group the number ofTUNEL-positive cells was higher in the GCL than inother layers. The variable effect of vitamin E on celldeath in the different DG layers was even clearer in vita-min E-supplemented rats. In fact, TUNEL-positive nu-clei were almost absent in the basal polymorphic layerwhile they were more numerous in both the GCL andplexiform layer (Fig. 5C).

Discussion

CELL DEATH ANALYSIS

The study of cell death in vivo is especially difficult be-cause cells die asynchronously and are engulfed by thesurrounding cells within a few hours after their death,and hence are only visible for a very brief period (Raffet al., 1993). Moreover, it has been demonstrated (Burschet al., 1990) that in a tissue region only 2–3% of cells aredying at one point in time, even though about 25% ofa cell population is eliminated in a single day (White &Barone, 2001). These findings may explain the very lowquantitative results in this study.

Our results in controls confirm the presence of dy-ing cells in the DG (Young et al., 1999). Cell deathaffects immature and fully differentiated neurons, asshown by the presence of double TUNEL/TUC 4-and TUNEL/NeuN-positive cells, but does not af-fects glial cells or proliferating precursors, as demon-strated by the absence of both TUNEL/GFAP- andnick-translation/BrdU-positive cells.

Dying cell nuclear morphology suggested that thenature of cell death in the DG may be apoptosis: in factdegenerating cells were identified using the TUNELtechnique, which labels DNA fragmentation, the main


characteristic of apoptosis (Arends et al., 1990). More-over, deep nuclear changes, i.e. typical cup-shaped ar-eas and micronuclei scattered through the cytoplasm,were also observed. In this regard, it is interestingto note that previous studies have demonstrated, bymeans of different technical approaches, that eithercup-shaped chromatin marginations or micronuclei,which appear to be made up of highly condensed chro-matin in ultrastructural investigations (Sloviter et al.,1993, 1996; Falcieri et al., 1994), were formed by vari-ably fractured highly supercoiled chromatine fibers andwere positive when tested by TUNEL and nick transla-tion techniques (Columbaro et al., 1998; Morioka et al.,1998).

The decrease in TUNEL-positive cells over time incontrols is consistent with previous findings (White &Barone, 2001), in which it was shown that cell death isa naturally occurring phenomenon which takes placenot only in the DG, but also in other regions of adultrat CNS, such as brainstem and neocortex, and the in-cidence of cell death decreases with ageing.

On the other hand, during DG postnatal develop-ment, cell proliferation and death are quantitatively rel-evant processes (Schlessinger et al., 1975) and, interest-ingly, neurogenesis also decreases with ageing (Kuhnet al., 1996). Thus, like in the development phase, a rela-tionship between cell proliferation and death probablyexists (Biebl et al., 2000) giving rise to cell turnover inthis anatomical structure and maintaining an adequatenumber of neuronal elements. The greater numbers ofdying cells found in the GCL than in other layers inboth young and adult controls supports this hypothe-sis, because the GCL is the region in which new neuronsare produced during adulthood, and the neurogenesisrate diminishes together with the cell death rate withageing (Ciaroni et al., 2002a, b).

VITAMIN E ACTION ON CELL DEATH

The number of dying cells in the DG was signifi-cantly higher in vitamin E-deficient rats and lower in α-tocopherol-supplemented rats in comparison with age-matched controls. The results show that vitamin E is afactor regulating cell death in all layers of the adult ratDG.

Nevertheless, the relative percentage of TUNEL-positive cells in the different layers was not maintainedin the vitamin E-deficient or supplemented rats. Theeffect of both the lack and the accumulation of vita-min E in the different layers suggests that α-tocopherolmay be able to exert a selective neuroprotective ef-fect on different cell populations. In particular, sincethe basal polymorphic layer appears to be more af-fected by α-tocopherol availability, we can hypothesizethat α-tocopherol-deficiency or supplementation has astronger influence on these neurons, which could bemore sensitive to oxidative stress. In fact, hilar neurons

have been previously described as particularly vulner-able towards various insults (Miki et al., 2003; Sloviteret al., 2003).

The hypothesis of a neuroprotective effect of vitaminE on DG cell death is consistent with its antioxidantproperties. In fact, in vitamin E deficiency, a greaternumber of free radicals may be present; free radicalsare considered intermediaries in the cascade of eventsthat lead to cell death (Ceballos-Picot, 1997). In particu-lar, it has been demonstrated that 4-hydroxynonenal,a lipid peroxidation product (Mattson, 1998), playsa crucial role in oxidative stress-induced apoptosis(Kruman et al., 1997). Moreover, the α-tocopherol sup-plementation could protect cells by death induced byfree radicals.

The molecular mechanisms through which vitamin Emay be able to protect cells from death may regard otherpathways, different from those involved in free radicalscavenging. In fact, the recently-demonstrated nonan-tioxidant properties of α-tocopherol, such as modu-lation of cellular signalling and transcriptional regu-lation (for a review, Azzi et al., 2002; Brigelius-Floheet al., 2002), must be taken into account. The mod-ulation of cellular signalling appears to be linked tothe inhibition of PKC; in particular, it has been shownthat α-tocopherol activates the protein phosphatasesPP2A, which prevents the phosphorylation of PKC-α(Brigelius-Flohe et al., 2002). This pathway underliesmany of the effects of vitamin E, such as the inhibi-tion of cell proliferation, which we have previously re-ported (Ciaroni et al., 2002b; Cecchini et al., 2003) tobe the mechanism of adult neurogenesis regulation byvitamin E. Moreover, nonantioxidant properties of vi-tamin E, regardless of whether these properties are re-lated to the inhibition of PKC, are still not fully under-stood, and therefore, we cannot rule out the possibilitythat vitamin E neuroprotective effect may be also dueto its nonantioxidant properties and not just its antiox-idant properties.

Alpha-tocopherol controls the expression of severalgenes (for a review, see Azzi et al., 2002; Brigelius-Floheet al., 2002) and it has been reported that in vitro α-tocopherol may increase NF-kB transcriptional activ-ity (Behl, 2000). An increase in the baseline activity ofNF-kB in neuronal cells may be responsible for theirresistance to oxidative stress and may also be involvedin neuroprotective pathways (Lezoualc’h et al., 1998).Recent studies have demonstrated that NF-kB inducesthe expression of anti-apoptotic and/or neurotrophicfactor genes (Yabe et al., 2001). These findings suggestthat vitamin E may be able to control gene expressionvia the activation of NF-kB (Behl, 2000).

Moreover, it has been demonstrated that vitamin Eis able to control the bcl 2 and gene-related expression(Ushakova et al., 1999) and that, in association with vi-tamin C, it is able to reduce apoptosis incidence in-creasing bcl 2-expression and decreasing bax protein


levels (Haendeler et al., 1996). Conversely, vitamin E de-ficiency, in association with selenium, causes a down-regulation of bcl 2-expression (Fischer et al., 2001). Ithas been reported that bcl 2 is able to inhibit cell deathfollowing a variety of insults both in vitro and in vivo(Allssopp et al., 1993; Zhong et al., 1993). It has alsobeen suggested that in vivo bcl 2 is responsible forneuronal survival both during development (Naruse& Keino, 1995) and under various experimental condi-tions (Martinou et al., 1994; Michaelidis et al., 1996). Inlight of these findings, we may hypothesize that vita-min E modulates bcl 2 expression in the DG and thatsuch regulation may help to protect cells against death.In this regard, it is interesting to note that the DG isone of the regions where bcl 2 expression is high notonly during development, but also during adulthood(Merry et al., 1994).

In conclusion, our results support the hypothesis of aneuroprotective effect of vitamin E in vivo. Because neu-ron death is believed to be involved in ageing (Coleman& Flood, 1987) and to underlie the pathogenesis of anumber of human neurodegenerative diseases, includ-ing Alzeheimer’s disease (Barinaga, 1998), an improvedunderstanding of exogenous factors, such as vitamin E,which are able to modulate this process may lead in duecourse to new therapeutic strategies (Behl, 2000).

Acknowledgments

We thank Mr. S. Cecchini for the digital image pro-cessing. This work was supported by a grant byMIUR (COFIN 2001).

References

ALLSOPP, T. E., WYATT, S., PATERSON, H. F. &DAVIES, A. M. (1993) The proto-oncogene bcl-2 can se-lectively rescue neurotrophic factor-dependent neuronsfrom apoptosis. Cell 73, 295–307.

ALTMAN, J. & BAYER, S. A. (1990) Migration and distri-bution of two populations of hippocampal granule cellprecursors during the perinatal and postnatal periods.Journal of Comparative Neurology 301, 365–381.

ARENDS, M. J., MORRIS, R. G. & WYLLIE, A. H. (1990)Apoptosis: The role of the endonuclease. American Journalof Pathology 136, 593–608.

AZZI, A., RICCIARELLI, R. & ZINGG, J. M. (2002) Non-antioxidant molecular functions ofα-tocopherol (vitaminE). FEBS Letters 519, 8–10.

BARINAGA, M. (1998). Is apoptosis key in Alzheimer’s dis-ease? Science 281, 1303–1304.

BEHL, C. (2000) Vitamin E protects neurons against oxidativecell death in vitro more effectively than 17-betaestradioland induces the activity of the transcription factor NF-kB.Journal of Neural Transmission 107, 393–407.

BEHL, C. & MOOSMANN, B. (2002) Oxidative nerve celldeath in Alzheimer’s disease and stroke: Antioxidantsas neuroprotective compounds. Biological Chemistry 383,521–536.

BIEBL, M., COOPER, C. M., WINKLER, J. & KUHN, H.G. (2000) Analysis of neurogenesis and programmed celldeath reveals a self-renewing capacity in the adult ratbrain. Neuroscience Letters 291, 17–20.

BRIGELIUS-FLOHE, R., KELLY, F. J., SALONEN, J. T.,NEUZIL, J., ZINGG, J. & AZZI, A. (2002) The Euro-pean perspective on vitamin E: Current knowledge andfuture research. American Journal of Clinical Nutrition 76,703–716.

BRUNO, C., CUPPINI, C. & CUPPINI, R. (1990) Matura-tion of the spontaneous transmitter release by regener-ated nerve endings in vitamin E-deficient rats. Journal ofNeural Transmission 81, 53–61.

BURSCH, W., KLEINE, L. & TENNISWOOD, M. (1990)The biochemistry of cell death by apoptosis. Biochemistryand Cell Biology 68, 1071–1074.

BURTON, G. W., WEBB, A. & INGOLD, K. U. (1985)A mild, rapid and efficient method of lipid extractionfor use in determining vitaminE/lipids ratio. Lipids 20,29–39.

CEBALLOS-PICOT, I. (1997) The Role of Oxidative Stress inNeuronal Death. Heidelberg: Springer, pp. 39–82.

CECCHINI, T., CIARONI, S., FERRI, P., AMBROGINI,P., CUPPINI, R., SANTI, S. & DEL GRANDE, P.(2003) α-Tocopherol, an exogenous factor of adult hip-pocampal neurogenesis regulation. Journal of Neuro-science Research 73, 447–455.

CHANDRA, J., SAMALI, A. & ORRENIUS, S. (2000) Trig-gering and modulation of apoptosis by oxidative stress.Free Radical Biology and Medicine 29, 323–333.

CHENG, C. M., COHEN, M., TSENG, V. & BONDY, C.A. (2001) Endogenous IGF1 enhances cell survival in thepostnatal dentate gyrus. Journal of Neuroscience Research64, 341–347.

CIARONI, S., CECCHINI, T., FERRI, P., AMBROGINI,P., CUPPINI, R., RICCIO, M., LOMBARDELLI, G.,PAPA, S. & DEL GRANDE, P. (2002a) Impairment ofneural precursor proliferation increases survival of cellprogeny in the adult rat dentate gyrus. Mechanisms ofAgeing and Development 123, 1341–1352.

CIARONI, S., CECCHINI, T., FERRI, P., CUPPINI, R.,AMBROGINI, P., SANTI, S., BENEDETTI, S., DELGRANDE, P. & PAPA, S. (2002b) Neural precursorproliferation and newborn cell survival in the adult ratdentate gyrus are affected by vitamin E deficiency. Neu-roscience Research 44, 369–377.

COLEMAN, P. D. & FLOOD, D. G. (1987) Neuron numbersand dendritic extent in normal aging and Alzheimer’sdisease. Neurobiology of Aging 8, 521–545.

COLUMBARO, M., GOBBI, P., RENO, F., LUCHETTI, F.,SANTI, S., VALMORI, A. & FALCIERI, E. (1998) Amultiple technical approach to the study of apoptotic cellmicronuclei. Scanning 20, 541–548.

ESPOSITO, E., ROTILIO, D., DI MATTEO, V., DIGIULIO, C., CACCHIO, M. & ALGERI, S. (2002) Areview of specific dietary antioxidants and the effects onbiochemical mechanisms related to neurodegenerativeprocesses. Neurobiology of Aging 23, 719–735.

FALCIERI, E., ZAMAI, L., SANTI, S., CINTI, C., GOBBI,P., BOSCO, D., CATALDI, A., BETTS, C. & VITALE,M. (1994) The behaviour of nuclear domains in the courseof apoptosis. Histochemistry 102, 221–231.


FISCHER, A., PALLAUF, J., GOHIL, K., WEBER, S. U.,PACKER, L. & RIMBACH, G. (2001) Effect of seleniumand vitamin E deficiency on differential gene expressionin rat liver. Biochemical and Biophysical Research Communi-cations 285, 470–475.

GOULD, E., VAIL, N., WAGERS, M. & GROSS, C. G.(2001) Adult-generated hippocampal and neocorticalneurons in macaques have a transient existence. Proceed-ings of the National Academy of Sciences of the United Statesof America 98, 10910–10917.

GRUNDMAN, M. (2000) Vitamin E and Alzheimer disease:The basis for additional clinical trials. American Journal ofClinical Nutrition 71, 630S–636S.

HAENDELER, J., ZEIHER, A. M. & DIMMELER, S. (1996)Vitamin C and E prevent lipopolysaccharide-inducedapoptosis in human endothelial cells by modulationof Bcl-2 and Bax. European Journal of Pharmacology 317,407–411.

KIM, H. S., ARAI, H., ARITA, M., SATO, Y., OGIHARA,T., TAMAI, H., INOUE, K. & MINO, M. (1996) Age-related changes of alpha-tocopherol transfer protein ex-pression in rat liver. Journal of Nutritional Science and Vi-taminology 42, 11–18.

KRUMAN, I., BRUCE-KELLER, A. J., BREDESEN, D. E.,WAEG, G. & MATTSON, M. P. (1997) Evidence that 4-hydroxynonenal mediates oxidative stress-induced neu-ronal apoptosis. Journal of Neuroscience 17, 5089–5100.

KUHN, H. G., DICKINSON-ANSON, H. & GAGE, F. H.(1996) Neurogenesis in the dentate gyrus of the adult rat:Age-related decrease of neuronal progenitor prolifera-tion. Journal of Neuroscience 16, 2027–2033.

LEZOUALC’H, F., SAGARA, Y., HOLSBOER, F. & BEHL,C. (1998) High constitutive NF-kB activity mediates re-sistance to oxidative stress in neuronal cells. Journal ofNeuroscience 18, 3224–3232.

MAGAVI, S. S., LEAVITT, B. R. & MACKLIS, J. D. (2000)Induction of neurogenesis in the neocortex of adult mice.Nature 405, 951–955.

MARTINOU, J. C., DUBOIS-DAUPHIN, M., STAPLE,J. K., RODRIGUEZ, I., FRANKOWSKI, H.,MISSOTTEN, M., ALBERTINI, A., TALABOT,D., CATSICAS, S., PIETRA, C. & HUARTE, J.(1994) Overexpression of bcl-2 in transgenic mice pro-tects neurons from naturally occurring cell death andexperimental ischemia. Neuron 13, 1017–1030.

MATTSON, M. P. (1998) Free radicals, calcium, and thesynaptic cell death continuum: Emerging roles of thetranscription factor NFkB. International Review of Neuro-biology 42, 103–168.

MERRY, D. E., VEIS, D. J., HICKEY, W. F. &KORSMEYER, S. J. (1994) Bcl-2 protein expression iswidespread in the developing nervous system and re-tained in the adult PNS. Development 120, 301–311.

MICHAELIDIS, T. M., SENDTNER, M., COOPER, J. D.,AIRAKSINEN, M. S., HOLTMANN, B., MEYER, M.& THOENEN, H. (1996) Inactivation of bcl-2 results inprogressive degeneration of motoneurons, sympatheticand sensory neurons during early postnatal develop-ment. Neuron 17, 75–89.

MIKI, T., HARRIS, S. J., WILCE, P. A., TAKEUCHI, Y.& BEDI, K. S. (2003) Effects of alcohol exposure dur-ing early life on neuron numbers in the rat hippocam-

pus. I. Hilus neurons and granule cells. Hippocampus 13,388–398.

MORIOKA, K., TONE, S., MUKAIDA, M. & TAKANO-OHMURO, H. (1998) The apoptotic and nonapoptoticnature of the terminal differentiation of erythroid cells.Experimental Cell Research 240, 206–217.

NARUSE, L. & KEINO, H. (1995) Apoptosis in the devel-oping CNS. Progress in Neurobiology 47, 135–155.

QUINN, C. C., GRAY, G. E. & HOCKFIELD, S. (1999)A family of proteins implicated in axon guidance andoutgrowth. Journal of Neurobiology 41, 158–164.

RAFF, M. C., BARRES, B. A., BURNE, J. F., COLES, H.S., ISHIZAKI, Y. & JACOBSON, M. D. (1993) Pro-grammed cell death and the control of cell survival:Lessons from the nervous system. Science 262, 695–700.

SCHLESSINGER, A. R., COWAN, W. M. & GOTTLIEB,D. I. (1975) An autoradiographic study of the time oforigin and the pattern of granule cell migration in thedentate gyrus of the rat. Journal of Comparative Neurology159, 149–176.

SLOVITER, R. S., DEAN, E. & NEUBORT, S. (1993) Elec-tron microscopic analysis of adrenalectomy-induced hip-pocampal granule cell degeneration in the rat: Apoptosisin the adult Central Nervous System. Journal of Compara-tive Neurology 330, 337–351.

SLOVITER, R. S., DEAN, E., SOLLA, A. L. &GOODMAN, J. H. (1996) Apoptosis and necrosis in-duced in different hippocampal neuron populations byrepetitive perforant path stimulation in the rat. Journal ofComparative Neurology 366, 516–533.

SLOVITER, R. S., ZAPPONE, C. A., HARVEY, B.D., BUMANGLAG, A. V., BENDER, R. A. &FROTSCHER, M. (2003) “Dormant basket cell’’ hypoth-esis revisited: Relative vulnerabilities of dentate gyrusmossy cells and inhibitory interneurons after hippocam-pal status epilepticus in the rat. Journal of ComparativeNeurology 459, 44–76.

USHAKOVA, T., MELKONYAN, H., NIKONOVA, L.,AFANASYEV, V., GRAZIEV, A. I., MUDRIK, N.,BRADBURY, R. & GOGVADZE, V. (1999) Modifi-cation of gene expression by dietary antioxidants inradiation-induced apoptosis of mice splenocytes. FreeRadical Biology and Medicine 26, 887–891.

WHITE, L. D. & BARONE, J. (2001) Qualitative and quan-titative estimates of apoptosis from birth to senescencein the rat brain. Cell Death and Differentiation 8, 345–356.

YABE, T., WILSON, D. & SCHWARTZ, J. P. (2001) Nfkap-paB activation is required for the neuroprotective effectsof pigment epithelium-derived factor (PEDF) on cerebel-lar granule neurons. Journal of Biological Chemistry 276,43313–43319.

YOUNG, D., LAWLOR, P. A., LEONE, P., DRAGUNOW,M. & DURING, M. J. (1999) Environmental enrichmentinhibits spontaneous apoptosis, prevents seizures and isneuroprotective. Nature Medicine 5, 448–453.

ZHONG, L. T., SARAFIAN, T., KANE, D. J., CHARLES,A. C., MAH, S. P., EDWARDS, R. H. & BREDESEN,D. E. (1993) Bcl-2 inhibits death of central neural cellsinduced by multiple agents. Proceedings of the NationalAcademy of Sciences of the United States of America 90,4533–4537.

vitamin e affects cell death in adult rat dentate gyrus

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