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Research Report

Chronic green tea catechins administration prevents oxidativestress-related brain aging in C57BL/6J mice

Qiong Lia, Haifeng Zhaob, Ming Zhaoa, Zhaofeng Zhanga, Yong Lia,⁎aDepartment of Nutrition & Food Hygiene, School of Public Health, Peking University, Beijing 100191, PR ChinabDepartment of Nutrition and Food Hygiene, School of Public Health, Shanxi Medical University, Taiyuan 030001, Shanxi, PR China

A R T I C L E I N F O

⁎ Corresponding author. Fax: +86 10 82801177E-mail address: liyongbmu@163.com (Y. LAbbreviations: ROS, reactive oxygen spec

peroxidase; TBARS, thiobarbituric acid reactPSD95, post-synaptic density 95; NMDAR1, N

0006-8993/$ – see front matter © 2010 Elsevidoi:10.1016/j.brainres.2010.07.074

A B S T R A C T

Article history:Accepted 21 July 2010Available online 1 August 2010

As the organism ages, production of reactive oxygen species (ROS) increases whileantioxidants defense capability declines, leading to oxidative stress in critical cellularcomponents, which further enhances ROS production. In the brain, this vicious cycle ismoresevere as brain is particularly vulnerable to oxidative damage. In our study, 14-month-oldfemale C57BL/6J mice were orally administered 0.05% green tea catechins (GTC, w/v) indrinking water for 6 months. We found that GTC supplementation prevented the decreasein total superoxide dismutase and glutathione peroxidase activities in serum as well asreduced the thiobarbituric acid reactive substances and protein carbonyl contents in thehippocampus of aged mice. The activation of transcriptional factor nuclear factor-kappa Band lipofuscin formation in pyramidal cells of hippocampal CA1 region, which are all relatedto oxidative stress, was also reduced after GTC treatment.We also found that long-term GTCtreatment prevented age-related reductions of two representative post-synaptic proteinspost-synaptic density 95 and N-methyl-D-aspartate receptor 1 in the hippocampus. Theseresults demonstrated that chronic 0.05% green tea catechins administration may preventoxidative stress related brain aging in female C57BL/6J mice.

© 2010 Elsevier B.V. All rights reserved.

Keywords:Green tea catechinsAgingOxidative stress

1. Introduction

Aging is characterized by a progressive impairment ofbiological systems leading to an increase in age-relatedmortality (Murali et al., 2008). The reactive oxygen species(ROS)-elicited oxidative stress is one of the major factorsinvolved in the aging process (Harman, 1956; Senthil Kumaranet al., 2008; Serrano and Klann, 2004), and may account for theunderlining mechanisms responsible for age-related degen-erative diseases (Lu et al., 2007; Multhaup et al., 1997). As a

.i).ies; GTC, green tea cateive substances; NF-κB, n-methyl-D-aspartate rece

er B.V. All rights reserved

post-mitotic organ, brain has multiple factors that contributeto oxidative damage, including high utilization of inspiredoxygen, large amount of easily oxidizable polyunsaturatedfatty acids, abundance of redox active transition metal ionsand relative deficiency of antioxidant defense systems (Balu etal., 2005; Floyd and Hensley, 2002; Kaur et al., 2008; Unno et al.,2004). Increased ROS production damages almost everyintracellular macromolecules in neurons including protein,lipid and DNA, promotes dysfunction of variousmetabolic andsignaling pathways in vulnerable brain tissues (Levites et al.,

chins; T-SOD, total superoxide dismutase; GSH-Px, glutathioneuclear factor-kappa B; GAP43, 43 kDa growth-associated protein;ptor 1; PSD, post-synaptic density

.

Table 1 – The T-SOD and GSH-Px activities in serum ofmice.

Group T-SOD (U/ml) GSH-Px (U/ml)

Aged control 331.87±11.94 804.14±105.79Young control 391.92±8.69⁎⁎ 1200.94±50.11⁎⁎

GTC-treated 385.77±13.96⁎⁎ 1181.15±53.14⁎⁎

All values in the table were expressed as Mean±SEM. Each grouphad 10 subjects.**P<0.01 versus aged control group.

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2002; Lu et al., in press). In fact, it has been suggested that thebehavioral and neuronal deficits in normal aging and neuro-degenerative diseases seen in the elderly population are theresult of an increasing vulnerability to the long-term effects ofoxidative stress (Floyd, 1999; Lau et al., 2005; Shukitt-Hale etal., 2007).

In recentyears,naturaldietarycomponentswithantioxidantactivity have received particular attention because of their rolein modulating oxidative stress associated with brain aging andchronic conditions (Joseph et al., 2009; Kaur et al., 2008; Lu et al.,2007). Green tea, a pleasant, popular and safe beverage, isparticularly rich in catechins which constitute about 30% of thegreen tea solid extract. HPLC analysis of catechins in green teashows that (−)-epigallocatechin-3-gallate (EGCG) is the majorconstituent (over 60%), followed by (−)-epigallocatechin (EGC),(−)-epicatechin (EC) and (−)-epicatechin-3-gallate (ECG) (Li et al.,2009; Ramassamy, 2006). Green tea catechins (GTC) have beendemonstrated to act directly as radical scavengers of ROS andmay also exert indirect antioxidant effects through activation oftranscription factors and antioxidant enzymes (Assuncao et al.,2010; Guo et al., 1996; Higdon and Frei, 2003; Srividhya et al.,2008; Weinreb et al., 2004). In addition, GTC possess well-established iron-chelating and anti-inflammatory activities(Guo et al., 1996; Mandel et al., 2006; Mandel et al., 2007). Brainpenetrating property of GTC also makes such compounds animportant class of functional food for postponing brain aging(Abd ElMohsen et al., 2002; Kaur et al., 2008; Mandel et al., 2007).Hence, the present study was initiated to evaluate whetherchronic green tea catechins administration could prevent theoxidative damage induced brain aging in female C57BL/6J mice.

Table 2 – The TBARS and Protein carbonyl levels in thehippocampus and cerebral cortex of mice.

TBARS(nmol/mg protein)

Protein carbonyl(nmol/mg protein)

Hippocampus Cerebralcortex

Hippocampus Cerebralcortex

Agedcontrol

0.58±0.02 0.16±0.02 4.56±0.56 2.95±0.52

Youngcontrol

0.16±0.01⁎⁎ 0.14±0.01 2.59±0.30⁎⁎ 2.38±0.18

GTC-treated

0.21±0.05⁎⁎ 0.13±0.01 2.96±0.20⁎ 2.45±0.13

All values in the table were expressed as Mean±SEM. Each grouphad 4 mice.*P<0.05, **P<0.01 versus the aged control group.

2. Results

2.1. GTC intake and body weights

No deaths or obvious clinical signs were found in all groupsthroughout the experimental period. Daily water intake permousedidnot differ among the aged control (4.27±0.06 ml/day),young control (4.33±0.05 ml/day) and GTC-treated (4.37±0.04 ml/day) group. The mean dose of GTC was thereforecalculated to be about 80mg/kg/day. Body weights of micesubmitted to GTC treatment were similar to that of the agedcontrol mice both at the beginning (Aged control: 26.75±0.83 g;GTC: 26.74±0.75 g) and at the end of the study (Aged control:27.01±1.07 g; GTC: 27.49±0.71 g), indicating that chronic ad-ministration of GTC did not alter their appetite or provokemalnutrition.

2.2. Effect of GTC on total superoxide dismutase (T-SOD)and glutathione peroxidase (GSH-Px) activities in serum

T-SOD and GSH-Px are two important antioxidant enzymesinvolved in scavenging ROS. Table 1 shows that the T-SOD andGSH-Px activities of aged mice were significantly lower thanyoung animals (T-SOD: P<0.01; GSH-Px: P<0.01). However,mice administrated with GTC had higher activities of bothenzymes (T-SOD: P<0.01; GSH-Px: P<0.01) comparedwith agedcontrol mice.

2.3. Effect of GTC on thiobarbituric acid reactivesubstances (TBARS) and protein carbonyl levels in thehippocampus and cerebral cortex

The TBARS and protein carbonyl content are good indicatorsof lipid peroxidation and protein oxidation in oxidative stressprocess. As in Table 2, in the hippocampus, aged mice havesignificantly higher both TBARS and protein carbonyl contentsthan young control mice (TBARS: P<0.01; Protein carbonyl:P<0.01). These increase could be prevented by chronic GTCtreatment (TBARS: P<0.01; Protein carbonyl: P<0.05). In thecerebral cortex, there was no significant difference among allthe three groups on the level of TBARS and protein carbonyl[TBARS: F (2,9)=0.962, P>0.05; Protein carbonyl: F (2,9)=0.884,P>0.05].

2.4. Effect of GTC on nuclear factor-kappa B (NF-κB)activation in the hippocampus and cerebral cortex of aged mice

Analysis of western blot results revealed no difference of NF-κB p65 subunit in the whole homogenates of hippocampusand cortex. However, the nuclear p65 level was significantlyhigher in the aged control mice compared with the youngsubjects in the hippocampus (P<0.05), and GTC treated miceshowed significantly lower nuclear p65 expression than agedcontrol (P<0.05). Conversely, hippocampal cytoplasmic p65level decreased significantly in aged mice (P<0.01) and wasincreased in response to GTC treatment (P<0.05). As to thecerebral cortex, there were no differences in nuclear andcytoplasmic expressions of NF-κB p65 subunit among all the

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three groups (nuclear: F (2,9)=1.729, P>0.05; cytoplasmic: F(2,9)=0.691, P>0.05) (Fig. 1).

2.5. Effect of GTC on the volume density of lipofuscin inhippocampal CA1 pyramidal cells

Using electron microscopy, lipofuscin granules appear asosmiophilic, preferentially perinuclear and irregularly shapedbodies of variable electron density. Aged control mice showedmarked accumulations of lipofuscin in the cytoplasm of CA1pyramidal neurons, its volume density (Vv) was significantlyhigher than young control (P<0.05). However, long-termtreatment of GTC could significantly prevent the formationof the lipofuscin in CA1 pyramidal cells (P<0.05) (Fig. 2).

2.6. Effect of GTC on the expression of synapse-associatedproteins

Weanalyzed the relativeabundanceof twopre-synapticproteinsincluding 43 kDa growth-associated protein (GAP43) and synap-

Fig. 1 – Levels of NF-κB p65 subunit in the hippocampus andcerebral cortex of mice. (A) Representative immunoblotsshowing nuclear, cytoplasmic and whole NF-κB p65 subunit(65 kDa) levels of all groups of mice are presented. (B) Thequantifications for the density of the bands from the scannedautoradiographic films, the whole p65 was probed as aninternal control. The relative density of the aged control wasset as 1.0. ResultswereMean±SEMof four determinations onhippocampus or cortex homogenates from four individualmice in each group. *P<0.05, **P<0.01 versus the aged controlgroup.

tophysin, as well as two post-synaptic proteins including post-synaptic density 95 (PSD95) andN-methyl-D-aspartate receptor 1(NMDAR1) in the hippocampus and cortex of mice. The resultsindicated that there were no significant differences in theexpression of GAP43 and synaptophysin among all these threegroups in both hippocampus and cortex (GAP43: F (2,9)=0.526,P>0.05 for hippocampus, F (2,9)=0.164, P>0.05 for cortex;Synaptophysin: F (2,9)=0.108, P>0.05 for hippocampus, F (2,9)=0.158, P>0.05 for cortex). As to the post-synaptic proteins, thePSD95 and NMDAR1 protein levels in the hippocampus de-creasedmarkedly in the aged controlmice relative to youngmice(PSD95: P<0.01; NMDAR1: P<0.05), andGTC administration couldsignificantly prevent the reductions of there twoproteins (PSD95:P<0.05; NMDAR1: P<0.05). However, No significant differenceswere found in the cerebral cortex of these two postsynapticproteins among all three groups (PSD95: F (2,9)=0.291, P>0.05;NMDAR1: F (2,9)=0.186, P>0.05) (Fig. 3).

3. Discussion

Green tea catechins are powerful hydrogen-donating antiox-idants and free radical scavengers in a number of in vitrosystems and in vivo models (Levites et al., 2001; Srividhya etal., 2008; Srividhya et al., 2009). It has been found that GTChave much higher antioxidant activity on a molar basis thanvitamins C or E in vitro (Hagerman et al., 2003; Unno et al.,2004). The chemical structures contributing to these effectsinclude the vicinal dihydroxy, trihydroxy structure and gallatemoiety, which can chelate metal ions and prevent thegeneration of free radicals, as well as allow electron delocal-ization and confer high reactivity to quench free radicals (Baluet al., 2005; Khan and Mukhtar, 2007; Mandel et al., 2006;Senthil Kumaran et al., 2008). Furthermore, GTC may alsofunction indirectly by inducing the expression of someantioxidant protective enzymes (Higdon and Frei, 2003;Srividhya et al., 2008; Unno et al., 2004; Weinreb et al., 2004).

In the present study, chronic GTC treatment could preventthe decline of T-SOD and GSH-Px activities in the serum ofaged mice, and it could also significantly prevent the age-related increasing of TBARS and protein carbonyl content inthe hippocampus. It has been reported that GTC can distributeinto brain so they are able to directly scavenge ROS in neurons(Abd El Mohsen et al., 2002; Mandel et al., 2006). Moreover, theserum with increased antioxidative activity was alwayssupplied to the brain, which can also prevent oxidativedamage in the brain. Regular consumption of GTC seems tobe important for long-term therapeutic applications becauseneurons are constantly exposed to oxidative stress duringaging process (Lau et al., 2005; Peng et al., 2008; Unno et al.,2004, 2007). During the experimental period, GTC were mixedwith drinking water, this feeding regimen is well tolerated byanimals and has been used in mice in many previous studies(Adhami et al., 2004; Li et al., 2009). As the mice were housedfive per cage, it was difficult to determine if individual subjectreceived comparable amounts of GTC, thus, we could onlycalculate the mean dose to be about 80 mg /kg/day.

The transcription factorNF-κB isamaster regulatormediatingcellular defense against infectious agents and environmental

Fig. 2 – Representative Electron micrographs of the pyramidal neurons in CA1 region of the hippocampus in aged control (A),Young control (B) andGTC-treatedmice (C). Themagnifications are 8000×. Although lipofuscin granules (arrows) can be found inthe cytoplasmof all groups, there are large and complex conglomerates of irregular configurationswith peripheral vacuoles onlyin (B). (D) Comparisons of the volume density of lipofuscin granules (%) in each group. All valueswere expressed asMean±SEM.Each group had 3 subjects. *P<0.05, versus the aged control group.

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and cellular stress. However, recent studies have indicated thatconstitutive activation of NF-κB is a ubiquitous phenomenonamong various cell types including neurons in the aging process,contributing deleteriously to many age-related chronic diseases(Gosselin and Abbadie, 2003; Kriete and Mayo, 2009; Lane, 2003).Oxidative stress, mitochondrial dysfunction, endoplasmic stressresponse, organelle cross-talk and DNA damage are all involvedin the mechanisms of NF-κB activating during aging (Kriete andMayo, 2009). Activation of NF-κB requires its translocation intothe nucleus where it activates target genes (Hayden and Ghosh,2004; Pahl, 1999). The most abundant form of NF-κB in thenervous system is primarily the p50/p65 heterodimer, in whichthe p65 subunit contains the transcriptional activation domain(Kaltschmidt et al., 1994; Kaltschmidt et al., 1995; Longpre et al.,2006;Memet, 2006). In our study,we found that p65protein levelsin thenuclear of thehippocampus in agedmicewerehigher thanyoungsubjects, andGTCtreatment significantlydecreased them.It is possible that the antioxidant and anti-inflammatory abilityof GTC could be related to its inhibitory effect. In fact, similarinhibitory effects of green tea extracts on NF-κB translocationhave been observed by other investigators in several in vitro andin vivo models of neurodegenerative diseases (Kim et al., 2009;Levites et al., 2002; Toldy et al., 2005).

It seems that only the hippocampus was severely damagedin the aged female C57BL/6J mice in our study. The hippo-campus always appears to be an early target of age-relatedstructural and physiological changes, probably because it is

more prone to oxidative damage due to a higher oxygenconsumption rate and more ROS generation than other brainregions (Balu et al., 2005; Peters, 2006; Rosenzweig and Barnes,2003). We assumed that the 20 months old mice we used werein the early stage of aging in which the cerebral cortex was notyet involved. We then explored the ultrastructural changes ofpyramidal cells in the hippocampal CA1 region, which is a verysensitive area of the hippocampus and is a highly consistentanatomicmarker of hippocampal aging (Kerr et al., 1991;Millerand O'Callaghan, 2005; Wu et al., 2006).

Lipofuscin, an undegradable intralysosomal substance accu-mulated over time within postmitotic cells such as neurons, isoften considered a hallmark of aging and a prominent structuralmarker of cellular oxidative damage (Murali et al., 2008; Termanand Brunk, 2004; Wu et al., 2006). It is primarily composed ofoxidatively modified protein and lipid degradation residues,besides, metals especially iron are also found in considerablequantities (Brun and Brunk, 1970; Brunk and Terman, 2002).Although lipofuscinhas longbeen thoughtaharmlesswear-and-tear product, there is accumulating evidence of its harmfulproperties. Due to high content of iron, lipofuscin seems tosensitize lysosomes and cells to oxidative stress (Terman et al.,1999a; Terman and Brunk, 2006). Most importantly, amassedlipofuscin interferes with autophagy, decreases lysosomal deg-radation, eventually decreases cellular adaptability and favorsthe appearance of various pathologies in old age (Brunk andTerman, 2002; Terman et al., 1999b; Terman and Brunk, 2006). Of

Fig. 3 – Levels of synapse-associated proteins in thehippocampus and cerebral cortex of mice. (A) Representativeimmunoblots showing GAP43 (43 kDa), synaptophysin(38 kDa), PSD95 (95 kDa) and NMDAR1 (105 kDa) levels of allgroups of mice are presented. (B) The quantifications for thedensity of the bands from the scanned autoradiographicfilms, β-actin was probed as an internal control. The relativedensity of the aged control was set as 1.0. Results were Mean±SEM of four determinations on hippocampus or cortexhomogenates from four individual mice in each group.*P<0.05, **P<0.01 versus the aged control group.

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significance, the prevention of lipofuscin accumulation inneurons has been suggested to slow age- or disease-relatedimpairments of neuronal functions (Assuncao et al., in press;Double et al., 2008).Considering the role of intralysosomaloxidative stress in lipofuscinogenesis, it would be attractive todevelop antioxidants and iron chelators with lysosomotropicproperties to retard lipofuscinaccumulation (Porta, 2002;Termanand Brunk, 1998; Terman and Brunk, 2006). In our study, wefound that GTC-treated mice had significantly lower lipofuscinvolumedensity in the cytoplasmofCA1pyramids than their age-matched controls. This effect of GTC revealed once more theirneuroprotective ability, which may well be a consequence oftheir radical-scavenging and iron-chelating properties to protectproteins and lipids against oxidative alterations in the hippo-campal formation (Assuncao et al., in press).

Increased oxidative stress and accumulation of oxidativelydamaged macromolecules in the brain may contribute to theneurochemical and cognitive deficits seen in normal aging as

well as in neurodegenerative diseases (Haque et al., 2008; Lu etal., in press; McDonald et al., 2005; Shukitt-Hale et al., 2007). Ithas been suggested that the behavioral and neuronal deficitssuch as learning and memory decline seen in the elderlypopulation are the result of an increasing susceptibility to thelong-term oxidative stress (Kaur et al., 2008; Lau et al., 2005;Serrano and Klann, 2004; Shukitt-Hale et al., 2006). We thenexamined the expression pattern of several synapse-associ-ated proteins in the hippocampus and cortex. We found thatthe levels of post-synaptic proteins PSD95 and NMDAR1 weresignificantly decreased in the hippocampus of aged mice andincreased after GTC treatment. NMDAR1 is a subtype receptorof glutamate on the post-synaptic membrane, which caninitiate the long-term potentiation (Burgdorf et al., in press;Magnusson, 2001; Zhao et al., 2009). PSD95 is core scaffoldcomponent in the architecture of post-synaptic density (PSD),which is very important in neurotransmission and synapticplasticity (Nyffeler et al., 2007). Recent data demonstrated thataged memory-impaired rats exhibited a marked reduction inthe PSD area in hippocampal excitatory synapses, a change inPSD size is therefore expected to reflect a correspondingchange in the composition and content of PSD proteins(Nicholson et al., 2004). Our results indicated that GTC mayregulate the neuronal post-synapse morphology, leading tothe increasing efficiency of neuronal communication in thehippocampus and prevent the memory decline during aging.Previous studies have also found that supplement the diets ofanimals with antioxidants reversed age-dependent decline inlong-term potentiation and learning and memory, and thisimprovement was correlated with a reduction in lipidperoxidation, protein oxidation, and levels of oxidized nucleicacids in the aged mice, indicating a link between antioxidantlevels and the preservation of adequate cognition relatedexpression/function (Christie et al., 2009; Dyall et al., 2007;Haque et al., 2008; Serrano and Klann, 2004).

In conclusion, the primary finding of this study is thatchronic green tea catechins administration could effectivelyprevent the age-related oxidative damage in female C57BL/6Jmice. Thus, drinking tea every day might be an effective habitto prevent brain aging in old people and improve the quality oftheir life.

4. Experimental procedures

4.1. Animals and supplementation

Female C57BL/6J mice were provided by the Department ofLaboratory Animal Science of Peking University HealthScience Center and were housed five per cage with a 12 hdark/light cycle under controlled temperature (23±2 °C) andhumidity (50±10%). The animals had free access to food andwater. After a seven day acclimatization to the laboratoryconditions, 14-month-old mice (n=30) were randomly dividedinto two groups: aged control group (n=15) and GTC-supple-mented group (n=15). Moreover, we assigned 1-month-oldmice (n=15) as the young control group. GTC-treated micewere orally administered green tea catechins (purity: 93%;EGCG: 71%; Orient Tea Development Co., LTD, China) mixed

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with drinking water (0.5 g/l) for 6 months. Water containingGTC was freshly prepared at about 17:00 pm everyday, and atthe same time water left in the bottle was measured todetermine amounts consumed. During the experimentalperiod, the animal's weight and food consumption weremeasured once a week. All experiments were performed inaccordance with the National Institute of Health Guide for theCare and Use of Laboratory Animals (NIH Publications No. 80-23), and with the guidelines established by the InstitutionalAnimal Care and Use Committee of Peking University,ensuring that animal numbers and suffering were kept to aminimum.

4.2. Tissue preparation

After the chronic GTC treatment, 12 mice in each group wereextirpated eyes to sample blood after deeply anesthesia, andthen sacrificed by cervical displacement. Serum was collectedto analyze T-SOD and GSH-Px activities. The hippocampusand cerebral cortex of each mouse were immediately dissect-ed out and stored at −80 °C for biochemical studies.

4.3. Detection of T-SOD and GSH-Px activity in serum

T-SOD and GSH-Px assays in serumwere carried out accordingto the manufacturer's protocols (Jiancheng Institute of Bio-technology, Nanjing, China). The principles of these kits are asfollows. The method which was used to measure T-SODemploys xanthine and xanthine oxidase to generate superox-ide radicals. Activity of GSH-Px was reflected by the speed ofenzymatic reaction, in which GSH-Px promote glutathioneenzyme to generate oxidized form glutathione.

4.4. Detection of TBARS and protein carbonyl levels in thehippocampus and cerebral cortex of mice

The level of TBARS in brain tissue homogenates wasdetermined using the method previously described (Lu et al.,2007). 0.5 ml of each homogenate was mixed with 3 ml ofH3PO4 solution (1%, v/v) followed by addition of 1 ml ofthiobarbituric acid solution (0.67%, w/v). The mixture wasincubated at 95 °C in a water bath for 45 min. The coloredcomplex was extracted into n-butanol, and the absorption at532 nm was measured using tetramethoxypropane as stan-dard. TBARS levels were expressed as nmol per milligram ofprotein.

Protein carbonyl content was measured by the methoddescribed previously (Balu et al., 2005). Briefly, 100 μl of thecortex or hippocampus homogenates was incubated with0.5 ml 2,4-dinitrophenylhydrazone (DNPH) for 60 min. Subse-quently, the protein was precipitated from the solution usingof 20% TCA. The pellet was washed after centrifugation(3400 g) with ethyl acetate:ethanol (1:1, v/v) mixture threetimes to remove excess of DNPH. The final protein pellet wasdissolved in 1.5 ml of 6 M guanidine hydrochloride. Thecarbonyl content was evaluated in a spectrophotometer atwavelength of 370 nm. A standard curve of bovine serumalbuminwas included in each assay to determine the linearityand to measure the extent of derivatization. The results werepresented in nmol/mg protein.

4.5. Preparation of tissue sections and transmissionelectron microscopy (TEM)

Under anesthesia, 3 mice in each group were perfused by 4%paraformaldehyde and 2.5% glutaraldehyde in phosphatebuffer saline (PBS). The brains were removed and immediatelyimmersed in 2.5% glutaraldehyde in 0.1 M PBS at pH 7.4 on ice,with shaking for 6 h. After sufficient washing in 0.1 M PBS, twoblocks (approximately 1 mm wide, 5 mm long, and 1 mmthick) containing the hippocampal formation were sliced fromthe right and left hemisphere of the brain in each mouse. Allblocks were post-fixed in 1% osmium tetroxide for 2 h at 4 °C.They were rinsed in distilled water for several times,dehydrated in graded series (20–100%) of ethanol and then inpropylene oxide, infiltrated with Epon 812, and finallypolymerized in pure Epon 812 for 48 h at 65 °C. Afterpolymerization, selected areas of CA1 region including thepyramidal layer of the hippocampuswere identified, trimmed,and mounted on blank plastic blocks. From each block, 20serial ultrathin sections containing the CA1 pyramidal celllayers were cut on an ultramicrotome using diamond knives.After the random selection of the first ultrathin section, one ineach third section was sampled, collected on copper grids,thus, there were 6 ultrathin sections on the copper grid of eachblock. All the sections were then stained with 4% uranylacetate and Reynolds lead citrate, and observed with a PhilipsEM208s transmission electron microscope.

Twelve CA1 pyramidal cells in which the nucleus wasvisualized were randomly photographed per animal fromultrathin sections at primary magnification of ×2500 andobserved at a final magnification of ×8000. The fraction of cellcytoplasm occupied by lipofuscin granules (volume density,Vv) was estimated by using point counting techniques. Thisfraction was calculated in each micrograph with an appropri-ate plastic replica by counting the number of points that fell onlipofuscin granules and the number of points hitting theneuronal cytoplasm (Assuncao et al., 2007).

4.6. Western immunoblotting

Nuclear and cytoplasmic proteins from hippocampus andcortex were extracted by Nuclear-Cytosol Extraction Kit(Applygen Technologies Inc, Beijing, China). As to the wholeproteins, the hippocampus and cortex of each mouse werehomogenized with five volumes (w/v) of tissue lysis solution:Tris (50 mM), Triton X-100 (0.1%), NaCl (150 mM), and EGTA/EDTA (2 mM), pH 7.4, containing mammalian protease inhib-itor cocktail (1:100 dilution), sodium pyrophosphate (1 mM),PMSF (10 μg/ml), sodium vanadate (1 mM), and sodiumfluoride (50 mM). Homogenates were then centrifuged at12,000g for 15 min at 4 °C, and the supernatants werecollected. All the protein concentrations were determinedusing the BCA assay kit (Pierce Biotechnology, USA). Samplescontaining equal protein amounts (50 μg /lane) and prestainedmolecular weight standards were separated by SDS-PAGE (NF-κB, GAP43, synaptophysin, β-actin: 10% gel; PSD95: 8% gel;NMDAR1: 6% gel), transferred to PVDF membranes (Millipore,USA). The membranes were blocked by 5% non-fat milk inTris-buffered saline (TBS) with 0.1% Tween-20 for 2 h atambient temperature, incubated overnight at 4 °C with the

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following antibodies: polyclonal anti-NF-κB (1:500; Santa Cruz,USA), polyclonal anti-GAP43 (1:500; Millipore, USA), polyclonalanti- synaptophysin (1:2000; Millipore, USA), monoclonal anti-PSD95 (1:500; Chemicon, Canada), polyclonal anti-NMDAR1(1:500; Millipore, USA). Then, membranes were washed andincubated for 1 h at 37 °Cwith the appropriate HRP-conjugatedsecondary antibody, developed with enhanced chemilumi-nescence (ECL kit; Millipore, USA) and visualized using Kodakfilms. The autoradiographic films were scanned and densito-metric analyses were performed using public domain NIHImage Program (developed at the U.S. National Institutes ofHealth and available on the internet at http://rsb.info.nih.gov/nih-image/).

4.7. Statistical analysis

One-way analysis of variance was used to analyze groupdifferences followed by LSD (equal variances assumed) orTamhane's T2 (equal variances not assumed) post-hoc tests. Acriterion of P<0.05 was considered significant. The resultswere expressed as mean±SEM.

Acknowledgments

Thisworkwas supportedby the foundation (No. 2006BAD27B08)from the Ministry of Science and Technology of the People'sRepublic of China.

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