ECAM2014-813672

Research ArticleMultifunctional Effects of Mangosteen Pericarp on Cognition inC57BL6J and Triple Transgenic Alzheimerrsquos Mice

Hei-Jen Huang1 Wei-Lin Chen2 Rong-Hong Hsieh3 and Hsiu Mei Hsieh-Li2

1 Department of Nursing Mackay Junior College of Medicine Nursing and Management Taipei 112 Taiwan2Department of Life Science National Taiwan Normal University Taipei 116 Taiwan3 School of Nutrition and Health Sciences Taipei Medical University Taipei 110 Taiwan

Correspondence should be addressed to Hsiu Mei Hsieh-Li hmhsiehntnuedutw

Received 21 September 2014 Revised 6 November 2014 Accepted 6 November 2014 Published 1 December 2014

Academic Editor Ki-Wan Oh

Copyright copy 2014 Hei-Jen Huang et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Mangosteen- (Garcinia mangostana-) based nutraceutical compounds have long been reported to possess multiple health-promoting properties The current study investigated whether mangosteen pericarp (MP) could attenuate cognitive dysfunctionFirst we found that treatment with MP significantly reduced the cell death and increased the brain-derived neurotrophic factor(BDNF) level in an organotypic hippocampal slice culture (OHSC) We then investigated the effects of age and MP diet on thecognitive function of male C57BL6J (B6) mice After 8-month dietary supplementation the MP diet (5000 ppm) significantlyattenuated the cognitive impairment associated with anti-inflammation increasing BDNF level and decreasing p-tau (phospho-tau S202) in older B6 mice We further applied MP dietary supplementation to triple transgenic Alzheimerrsquos disease (3timesTg-AD)mice from5 to 13months oldTheMPdiet exerted neuroprotective antioxidative and anti-inflammatory effects and reduced theA120573deposition and p-tau (S202S262) levels in the hippocampus of 3timesTg-ADmice which might further attenuate the deficit in spatialmemory retrieval Thus these results revealed that the multifunctional properties of MP might offer a promising supplementarydiet to attenuate cognitive dysfunction in AD

1 Introduction

Alzheimerrsquos disease (AD) is a major public health crisis inthe elderly The hallmark pathologic features of AD are theaccumulation of extracellular senile plaques and intraneu-ronal neurofibrillary tangles (NFT) in the brain parenchyma[1] The senile plaques consist of 120573-sheet aggregated amyloidpeptides (A120573) from misprocessing of the amyloid precursorprotein (APP) while the NFT are formed by hyperphos-phorylation of tau proteins in paired helical filaments [2]Detection of A120573 oligomers in the cerebrospinal fluid of thepostmortem AD brain suggests that soluble A120573 oligomersbut not fibrillar A120573 are the neurotoxic species in the patho-logical progression of AD thus providing a potential targetfor AD therapy [3] However therapeutic strategies targetingA120573 or tau protein within the brain have failed to demonstrateefficacy

The development of multifunctional therapy is a cur-rent trend against multifactorial neurodegenerative disorders

such as AD [4] Polyphenols in natural plants are receivingattention for theirmultifunction in terms of neuroprotectionantioxidant and anti-inflammation activities Evidence sug-gests that a combination of phytomedicines showed neuro-protective efficacy in preventing neurodegenerative disease[5] Some natural substances that upregulate the expressionof brain-derived neurotrophic factor (BDNF) [6] and anti-inflammation activity [7] could potentially slow the progres-sion of AD

Mangosteen (Garcinia mangostana L) is a tropical treenative to Southeast Asia that produces a fruit whose peri-carp is a rich source of xanthones which have shownremarkable pharmacological activities [8] Many studies havesuggested that extracts of mangosteen pericarp (MP) suchas 120572-mangostin and 120574-mangostin have biological functionsin terms of anti-inflammation antioxidation anticancerantimicrobial and neuroprotective activities [9ndash13] Recentevidence further suggests that polyphenols in combinationinduce better protection against cognitive impairment than

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 813672 18 pageshttpdxdoiorg1011552014813672

2 Evidence-Based Complementary and Alternative Medicine

individual polyphenols [14] These putative health claimsare based on in vitro observations and anecdotal reports ofthe top-selling botanical supplement containing mangosteenfruit in the United States [15] However the proposed healthbenefits of MP have only received very limited attention interms of cognitive dysfunction in neurodegenerative diseasessuch as AD Therefore in the present study we evaluatedthe effects of MP by in vitro organotypic hippocampal sliceculture (OHSC) and by using in vivo B6 and AD mousesystems

2 Materials and Methods

21 Mice B6 and 3timesTg-AD (harboring PS1M146V APPSweand TauP30IL transgenes) mice were purchased from theNational Breeding Center for Laboratory Animals Taiwanand the Jackson Laboratory (004807) respectively B6 malemice (5-week-old) were used to measure the neuroprotec-tion of MP in the organotypic hippocampal slice cultureIn addition the supplement of regular diet and MP dietwas administrated in the B6 male (aged 3 weeks and 5months) and 3timesTg-AD (aged 5 months) mice The micewere housed in individual-ventilation cages and maintainedon a 12 h lightdark cycle at a controlled room temperatureand humidity in accordance with standard use protocols andanimal welfare regulations The mice consumed food andwater ad libitum All experiments were performed duringthe light phase between 7 am and 7 pm All study protocolswere also approved by the Institutional Animal Care and UseCommittee of National Taiwan Normal University TaipeiTaiwan

22 Organotypic Hippocampal Slice Culture (OHSC) OHSCswere conducted in a slightly modified manner from theprevious report [16] Young adult male B6 mice (5-week-old) were anesthetized by Avertin (04 gkg body weightSigma St LouisMOUSA) and decapitatedThe hippocampiwere rapidly dissected in ice-cold artificial cerebrospinalfluid (aCSF) consisting of the following (in mM) NaCl 118KCl 25 MgSO

43 NaH

2PO411 NaHCO

326 CaCl

21

and glucose 11 (all reagents from Sigma) bubbled with 95O25CO

2 Subsequently coronal slices (350 120583m thick) were

cut with a vibratome (VT1200S Leica Wetzlar Germany)The sliced hippocampi were transferred onto sterile 04 120583mporousmembrane confetti (Millicell-PCFMillipore Ireland)and cultured with medium consisting of 50 MEM 25horse serum 18 HBSS 4mM L-glutamine 12mM glucose45mM NaHCO

3 20mM sucrose 100UmL penicillin and

100mgmL streptomycin (all reagents from Gibco or Sigma)in a humidified 5 CO

2atmosphere at 37∘C The medium

was changed three times a week On day in vitro (DIV) 5slices were treated with MP (10 120583M) or vehicle (DMSO) for12 hr The cell death level of the slices was then examined byPI staining Furthermore the BDNF level was examined toevaluate the neuroprotective effect of MP treatment on thehippocampal slices

23 PIUptakeAssay inOHSC PI uptake is an effective assess-ment of cell damage At the stated time point slices were

incubated with 5120583gmL PI (Sigma) in culture medium for30minThe stained slices were observed using a fluorescencemicroscope (Leica Wetzlar Germany) and the fluorescentintensity was quantified using AxioVision software (CarlZeiss Jena Germany)

24 Diets The male B6 (aged 3 weeks and 5 months) and3timesTg-AD (aged 5 months) mice were randomly divided intotwo groups with 15sim17 animals in each group (i) regular diet(LabDiet 5010 calories provided by protein 275 fat 135and carbohydrate 59) and (ii) regular diet with 5000 ppmof MP (Lord Duke Biotechnology Company containing3560mgg 120572-mangostin 063mgg 120573-mangostin 146mgg3-isomangostin 142mgg 8-deoxygartanin 155mgg gar-tanin and 132mgg 9-hydroxycalabaxanthone) supplementas in previous studies [15 17] Baseline behavioral assessmentwas conducted in themale 3timesTg-ADmice at 5 months of ageMice were maintained on MP dietary supplementation for 8months

25 Morris Water Maze Task (MWM) The Morris watermaze task was performed as previously described [18] Inbrief it consists of pretraining training testing and probingAll trials lasted for a maximum of 60 sec On the day priorto spatial training all mice underwent pretraining in orderto assess their swimming ability and to acclimatize them tothe pool In the three 60 sec pretraining trials the mousewas released into the water facing the wall of the poolfrom semirandomly chosen cardinal compass points Afterthree trials of acclimatization each mouse was placed onthe invisible platform located at the center of the targetquadrant and allowed to stay there for 20 sec The mice weregiven a 4-day training session consisting of four 60-secondtraining trials (intertrial interval 20ndash30min) per day Thehidden platformwas always placed at the same location of thepool (northeast quadrant as the target quadrant) throughoutthe training period During each trial from semirandomlychosen cardinal compass points the mouse was releasedinto the water facing the pool wall After climbing onto theplatform the mouse was allowed to rest on it for 20 sec Ifthe mouse failed to swim to the platform within 60 sec itwould be placed on the platform to stay on it for 20 sec by anexperimenter Twenty-four hours after the last training trialall mice were given three testing trials to assess the time takento climb onto the hidden platform Two and forty-eight hoursafter the last testing trial all mice were given two probe trialsto evaluate the retrieval of the short- and long-term spatialmemory of the platform

26 ELISA Analyses Blood samples were collected from theretroorbital plexus of anesthetized mice and centrifuged at2000timesg for 15min at 4∘C after the MWM (119899 = 3sim5 for eachgroup) Sera were collected and stored at minus80∘C until useThe levels of glutathione (GSH) and IL-6 in the serum weremeasured using a Glutathione assay kit (Cayman ChemicalMI USA) and an IL-6 ELISA kit (RampD Systems MN USA)respectively For the BDNF assay hippocampal slices werehomogenized in 8 times volume of lysis buffer containing 50mM

Evidence-Based Complementary and Alternative Medicine 3

Tris buffer pH 74 150mM NaCl 1 nonidet P-40 1mMEDTA 1mM phenylmethanesulfonyl fluoride 1mM sodiumvanadate 1mM sodium fluoride 10120583M aprotinin 10 120583Mpepstatin and 100 120583M leupeptin The protein extracts werediluted with an equal volume of DPBS and further acidifiedwith 1NHCl to pH2-3 for 15min Subsequently sampleswereneutralized with 1NNaOH In addition the concentration ofBDNF in the hippocampal slices wasmeasured using a BDNFELISA kit (PromegaWI USA)These assays were performedfollowing the manufacturerrsquos instructions

27 Western Blot Analysis Proteins were extracted fromthe mouse hippocampus (119899 = 3ndash5 per group) The proteinconcentration was determined using a bicinchoninic acid(BCA) protein assay kit (Pierce Rockford IL USA) Proteins(25 120583g) were separated by SDS-PAGE and transferred toPVDF membranes The blots were probed with variousprimary antibodies (Table 1) and secondary antibodies anti-rabbit and anti-mouse IgG HRP-linked antibody (1 10000Amersham Pharmacia Biotech MA USA) The specificantibody-antigen complex was detected by an enhancedchemiluminescence detection system (AmershamPharmaciaBiotech) The same blot was stripped and reprobed for thehousekeeping protein 120573-actin to serve as a loading controlQuantitationwas performed using the LAS-4000 chemilumi-nescence detection system (Fujifilm Tokyo Japan) and thetarget protein density was normalized to the 120573-actin internalcontrol

28 Immunostaining Mice (119899 = 3ndash5 per group) were anes-thetized and transcardially perfused with 09 NaCl fol-lowed by 4paraformaldehydeMouse brainswere sectionedinto 30 120583m slices For immunohistochemical staining anendogenous peroxidase block was performed for 10min in3 H2O2PBS Nonspecific epitopes of free-floating sections

were blocked by incubation in 3 normal horsegoatrabbitserum and 01 triton X-100 in PBS After blocking sectionswere incubated in primary antibodies (Table 1) overnight atroom temperature washed with PBS and incubated with thesecondary antibodies (1 200 dilution in blocking solutionVector Laboratories CA USA) for 1 h and then they wereincubated in an avidin-biotin complex for 1 h at room temper-ature The reaction was developed using a diaminobenzidine(DAB) kit (Vector Laboratories)

For immunofluorescent staining nonspecific epitopesof free-floating sections were blocked by incubation in 1BSA in TBST In addition sections were hybridized withprimary antibodies (Table 1) in blocking solution overnightat 4∘C washed with TBST and incubated in the secondaryantibodies (1 500 dilution in blocking solution InvitrogenAlexaFlour Eugene Oregon USA) for 2 h at 37∘C followedby washing and staining with DAPI (1 1000) for another10min at room temperature

All sections were mounted on gelatin-coated slides andcover-slipped for fluorescencelight microscopic observationusing a confocal spectral microscope imaging system (LeicaTCS SP2 Wetzlar Germany) The positive staining signal ina specific area was first selected as the standard signal Then

the number of cells stained positive was counted using digitalimage analysis software (Image Pro Plus Media CyberneticsMD) Pixel counts were taken as the average from threeadjacent sections per animal

29 Statistical Analysis Two-way ANOVA was used toanalyze ldquoagerdquo ldquodietrdquo and the interactions between themregarding influences on the behavioral andneuropathologicalmeasures in the B6 mice One-way ANOVA was conductedto determine the main effect of diet on the behavioralmeasures for the 3timesTg-AD mice ANOVA analyses followedby post hoc LSD multiple comparisons when significantwere carried out with the most relevant variables from eachmeasurement In addition an independent sample 119905-test wasused to compare the differences between MP and vehicle inthe analysis of neuropathological characterization for OHSCand the 3timesTg-AD mice Whether the swimming velocityaffected the cognitive performance was analyzed by covariateanalysis of variance (ANCOVA) with swimming velocity asa covariate All statistical analyses were performed usingSPSS160 software The results are represented as the mean plusmnSEM Differences were considered statistically significantwhen 119875 lt 005

3 Results

31 MP Reduced the Cell Death Level Associated with Increas-ing BDNF Level in Adult Mouse Hippocampal Slices Toexamine the neuroprotective effect of MP slices at DIV5were incubated with 10 120583M MP After 12 h the hippocampalslices were examined using PI staining and BDNFELISAMPtreatment significantly attenuated the cell death level (119875 lt001 Figures 1(a) and 1(b)) and increased the BDNF level (119875 lt005 Figure 1(c)) in the hippocampal slices These resultssuggested that MP reduced the cell death level associatedwith upregulation of the BDNF level in the hippocampal slicecultures

32MP SupplementaryDiet Attenuated Cognitive Impairmentin Older B6 Mice The effects of age and an MP diet onspatial learning and memory were evaluated by the MWMtask During the training period the latency to reach theplatform in the 4 trials of each training day was averagedand assessed as the spatial learning ability (Figure 2(a)) Wefound that the older B6 mice that received a regular diet (119875 =061 Figure 2(a)) or an MP diet (119875 = 084 Figure 2(a)) didnot exhibit decreased latencies onto the platform followingtraining days 1 to 4 However the younger B6 mice had agood spatial learning ability on a regular diet (119865

359= 488

119875 lt 001 Figure 2(a)) and theMPdiet (119865359= 325119875 lt 005

Figure 2(a)) In addition there were no significant differencesin theMP diet or the interaction between age andMP diet onthe spatial learning ability (119875 gt 005 Figure 2(a))

During the test period the time spent searching for theplatform was assessed as the acquisition of spatial learn-ing The escape latencies of the older B6 mice were notsignificantly different from those of the younger B6 mice(119875 = 052 Figure 2(b)) In addition the escape latencies of


Table 1 List of the primary antibodies used in this study

Antibodies Species Supplier WB dilution IHC dilution Epitope specificityAmyloid beta antibodies

A1205731ndash40 Rabbit Invitrogen mdash 1 2000 Amyloid beta


APP Rabbit Sigma-Aldrich mdash 1 500 Amyloid precursor proteinBACE Rabbit Cell Signaling 1 1000 mdash Beta secretase

Neurotransmission antibodies5-HT Rat Millipore mdash 1 100 Serotonergic neuronsCalbindin Mouse Sigma-Aldrich mdash 1 1000 Calcium binding proteinChAT Rabbit Millipore mdash 1 500 Cholinergic neurons

NeuN Mouse Millipore mdash 1 10001 500 (IF) Neuronal cells

NR2A Rabbit Millipore 1 1000 mdash NMDA receptor 2ANR2B Rabbit Millipore 1 1000 mdash NMDA receptor 2BTH Rabbit Millipore mdash 1 1000 Noradrenergic neurons

Inflammation antibodiesCOX2 Rabbit Millipore 1 1000 mdash Prostaglandin synthase-2

GFAP Mouse Millipore mdash 1 10001 500 (IF) Astrocytes

Iba-1 Rabbit Wako mdash 1 1000 MicrogliaSignaling antibodies

Akt 12 Rabbit Cell Signaling 1 1000 mdash Total AktpAkt 12 Rabbit Cell Signaling 1 1000 mdash Akt phosphorylated at Thr450

CDK5 Mouse Millipore 1 1000 mdash Cyclin-dependent kinase-5Erk 12 Rabbit Cell Signaling 1 1000 mdash Total Erk 12pErk 12 Rabbit Cell Signaling 1 1000 mdash Erk 12 phosphorylated at Thr202Tyr204

GSK3120572 Rabbit Cell Signaling 1 1000 mdash Total GSK3120572pGSK3120572 Rabbit Cell Signaling 1 1000 mdash GSK3120572 phosphorylated at Ser21

GSK3120573 Rabbit Epitomics 1 1000 mdash Total GSK3120573pGSK3120573 Rabbit Epitomics 1 1000 mdash GSK3120573 phosphorylated at Ser9

JNK Rabbit Cell Signaling 1 1000 mdash Total JNKpJNK Rabbit Cell Signaling 1 1000 mdash JNK phosphorylated at Thr183Tyr185

p38 Rabbit Cell Signaling 1 1000 mdash Total p38pp38 Rabbit Cell Signaling 1 1000 mdash p38 phosphorylated at Thr180Tyr182

Tau antibodiespTau Rabbit AnaSpec mdash 1 1000 Tau hyperphosphorylated at Ser202

pTau Rabbit Millipore 1 1000 mdash Tau hyperphosphorylated at Ser262

Tau1 Mouse Millipore 1 1000 mdash Tau unphosphorylation formOther antibodies120573-Actin Mouse Millipore 1 2000 mdash 120573-ActinBDNF Rabbit Millipore mdash 1 500 (IF) Brain-derived neurotrophic factor

WB Western blot IHC immunohistochemistry IF immunofluorescence

the mice that were administered MP diet were also notdifferent to those of themice that were administered a regulardiet (119875 = 006 Figure 2(b)) There was also no age timesMP dietinteraction during the testing period (119875 = 096 Figure 2(b))Post hoc LSD multiple analyses further showed that the MPdiet significantly decreased the escape latencies in the olderB6 mice (119875 lt 001 Figure 2(b))

Two hours after the last testing trial the time spent in thetarget quadrant was assessed as the retrieval of short-termmemory The older B6 mice exhibited a significantly reducedamount of time spent in the target quadrant as comparedwiththe younger B6 mice (119865

142= 861 119875 lt 001 Figure 2(c))

TheMPdiet significantly increased the retrieval of short-termmemory as compared with the regular diet (119865

142= 613


MPDMSO

500120583m

(a)

PI u

ptak

e (

)

0

25

50

75

100

MPDMSOTreatment

lowastlowast

(b)

BDN

F le

vel (

ngm

g)

0

200

400

600

800

TreatmentMPDMSO

lowastlowast

(c)

Figure 1 MP induced neuroprotection through increasing BDNF in adult mouse hippocampal slices (a)The cell death level of hippocampalslices was characterized with PI staining after treatment with MP (10120583M) or DMSO The scale bar of PI staining is 500 120583m (b) Thequantification of PI staining TreatmentwithMP significantly decreased the cell death level (c)The results of BDNFELISA in the hippocampalslices Treatment with MP increased the level of BDNF in the hippocampal slices Data are expressed as means plusmn SEM 119899 = 9ndash12 slicesgrouplowastlowast119875 lt 001 compared with the DMSO group

119875 lt 005 Figure 2(c)) Furthermore the retrieval of short-term memory was also observed in the interaction betweenage and MP diet (119865

142= 732 119875 lt 001 Figure 2(c)) Post

hoc analyses further showed that the MP diet significantlyincreased the time spent in the target quadrant for the olderB6 mice (119875 lt 0001 Figure 2(c)) Forty-eight hours after thelast testing trial the time spent in the target quadrant wasassessed as the retrieval of long-termmemoryWe found thatthe older B6 mice exhibited a significantly reduced retrievalof long-termmemory as compared with the younger B6mice(119865142= 421 119875 lt 005 Figure 2(d)) However there were no

significant differences in theMP diet (119875 = 031) and the age timesMP diet interaction (119875 = 057) for the retrieval of long-termmemory

Furthermore the swimming velocity was significantlydecreased in the older B6mice as compared with the youngerB6 mice (119865

140= 3705 119875 lt 0001 Figure 2(e)) There was

no significant difference in the MP diet as compared with theregular diet in swimming velocity (119875 = 016 Figure 2(e))However there was a significant difference in the interactionbetween age and MP diet on swimming velocity (119865

140=

1363 119875 lt 0001 Figure 2(e)) According to post hoc analysisthe MP diet significantly increased the swimming velocityin the older B6 mice (119875 lt 005 Figure 2(e)) Thereforethe swimming velocity was significantly correlated with ageThese were then entered as covariates in an ANCOVA toexamine whether their inclusion was associated with anattenuated cognitive dysfunction on age followingMPdietary


1 2 3 4 1 2 3 4

Late

ncie

s (s)

0

10

20

30

40

50

60

Regular diet MP diet

YoungerDay

Older

(a)

Younger Older

Late

ncie

s (s)

0

10

20

30

40

50

60lowastlowast

(b)

Younger Older

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60

2hr after acquisition

lowast

(c)

Younger Older

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60



(d)

Younger Older

Swim

min

g ve

loci

ty (c

ms

)

0

6

12

18

24

30

Regular dietMP diet

lowast

(e)

Figure 2 MP diet attenuated the impairments in spatial learning and memory in older B6 mice (a) The spatial learning ability of B6 miceat different ages and with dietary supplementation The older B6 mice showed a poor spatial learning ability as compared with the youngermice and MP dietary supplementation had no influence on the improvement of spatial learning ability (b) The spatial learning acquisitionof B6 mice at different ages and with dietary supplementation The MP diet increased the spatial learning acquisition in the older B6 mice(c) The short-term memory retrieval in mice measured 2 h after the last testing trial The older B6 mice showed impairment in short-termmemory retrieval and MP dietary supplementation attenuated the impairment in the older B6 mice (d) The long-term memory retrievalin mice measured 48 h after the last testing trial The older B6 mice showed impairment in long-term memory retrieval and MP dietarysupplementation showedno influence on the impairment (e)The swimming velocity of the B6mice at different agesThedecreased swimmingvelocity of the older B6 mice was rescued by the MP diet Data are expressed as mean plusmn SEM 119899 = 15group 119875 lt 005 and

119875 lt 0001comparison between the older and younger groups lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 comparison between the regular and MP dietgroups


BDN

FBD

NF

CA1

DG

Younger OlderMP minus + minus +

(A) (B) (C) (D)

(E) (F) (G) (H)

50120583m

5120583m

(a)

CA1

DG

pTau

Ser

202

pTau

Ser

202

(A) (B) (C) (D)

(E) (F) (G) (H)

50120583m

(b)

GFA

P

DG

(A) (B) (C) (D)

50120583m

(c)

Iba-1

(A) (B) (C) (D)

50120583m

5120583m

(d)

Figure 3 Continued


IL-6

leve

l in

seru

m (p

gm

L)0

350

700

1050

1400

Younger Older

Regular dietMP diet

lowast

(e)

Figure 3 Molecular effects of the MP diet in B6 mice Representative immunostaining images of BDNF (a) p-tau (S202) (b) activatedastrocytes (c) andmicroglia (d) in the B6mouse hippocampus (e)The systemic IL-6 level was determined by ELISA with mouse serumTheIL-6 level was greatly increased in the older B6 mice which was significantly decreased by the MP diet All of the deregulations in the oldermice were ameliorated after MP treatment 119899 = 3ndash5group Arrowheads indicate positive staining signals 119875 lt 001 comparison betweenthe older and younger groups lowast119875 lt 005 comparison between the regular and MP diet groups

Table 2 The results of immunostaining in C57BL6 mice after dietary supplementation

Target Younger OlderRegular diet MP diet Regular diet MP diet

BDNF 31 plusmn 094 45 plusmn 160lowastlowastlowastb 21 plusmn 181lowastlowastlowasta 34 plusmn 123lowastlowastlowastb

Tau pSer202 206 plusmn 821 116 plusmn 1748lowastlowastb 281 plusmn 2255lowasta 171 plusmn 773lowastlowastlowastb

GFAP 39 plusmn 287 35 plusmn 052 46 plusmn 253 36 plusmn 236lowastb

Iba-1 44 plusmn 116 35 plusmn 072lowastlowastlowastb 43 plusmn 050 39 plusmn 051lowastlowastlowastb

Each value represents the mean plusmn SEM (119899 = 3ndash5 for each group)aOlder mice compared with younger micebRegular diet compared with MP diet grouplowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001

supplementation Levenersquos test of equality of error varianceswas 017 From the results of the two-way ANCOVA analysisthere was no influence to attenuate the cognitive dysfunctionon age followingMP diet with swimming velocity as a covari-ant In addition after 8-month dietary supplementation withan MP diet or a regular diet the gain of body weight wasincreased in both the younger and older B6 mice (data notshown) The blood glucose did not differ among groupsseparated according to age MP diet and age times MP diet(data not shown) Therefore these results showed that theolder B6 mice exhibited impairments in spatial learning andmemory However the MP supplementary diet attenuatedthe deficits in spatial learning acquisition and short-termmemory retrieval in the older B6 mice

321 MP Supplementary Diet Upregulated the BDNF Leveland Decreased p-Tau (Phospho-Tau S202) and InflammatoryResponse in Older B6 Mice Age is a risk factor for theprogressive development of AD In the older B6 mice theBDNF level was significantly reduced (119865

127= 5408 119875 lt

0001 Figure 3(a) and Table 2) and the p-tau (S202) was

significantly increased (119865125= 1320 119875 lt 001 Figure 3(b)

and Table 2) as compared with younger B6 mice HowevertheMP diet significantly increased the level of BDNF (119865

127=

9293 119875 lt 0001 Figure 3(a) and Table 2) and decreased thelevel of p-tau (S202) (119865

125= 3136 119875 lt 0001 Figure 3(b)

andTable 2) in the older B6mice For gliosis therewas no sig-nificant difference between older and younger B6 mice MPdiet significantly decreased the activated astrocytes (119865

111=

1058 119875 lt 005 Figure 3(c) and Table 2) and microglia(119865119= 7162 119875 lt 0001 Figure 3(d) and Table 2) There

was also a significant interaction of age times diet in activatedmicroglia (119865

119= 1009 119875 lt 001 Figure 3(d) and Table 2)

When assessing the systematic inflammatory response theIL-6 level was found to be significantly increased in the olderB6 mice as compared with the younger mice (119865

119= 1747

119875 lt 0001 Figure 3(e)) while theMP diet effectively reducedthe level (119865


also a significant interaction in age timesMP diet for the B6 mice(119865119= 1535119875 lt 001 Figure 3(e)) A post hoc LSDmultiple

comparison showed that the MP diet significantly decreasedthe IL-6 level in the older B6 mice (119875 lt 005 Figure 3(e))We also characterized several AD-related targets however


the molecules involved in the deposition of A120573 (APP BACE1A12057340 and A120573

42) and tau-related kinases (CDK5 GSK3120573

JNK p38 and ERK)were not significantly altered in the olderB6mice (data not shown)These results showed that the olderB6 mice exhibited an increased systematic inflammatoryresponse increased p-tau level (S202) and decreased BDNFlevel The MP diet attenuated these impairments in the olderB6 mice

33 MP Supplementary Diet Attenuated the Deficit in SpatialMemory Retrieval in 3timesTg-AD Mice During the trainingperiod we found that a good spatial learning ability wasmaintained in the 3timesTg-AD mice at 5 months of age (119865

319=

3046 119875 lt 0001 Figure 4(a)) which was declined at 13months (119865

327= 251 119875 = 008 Figure 4(a)) However the

MP diet restored the spatial learning ability in the 3timesTg-ADmice (119865

323= 428119875 lt 005 Figure 4(a)) For spatial learning

acquisition there was a significant difference between groups(119865217= 473 119875 lt 005 Figure 4(b)) According to post

hoc LSDmultiple comparison the spatial learning acquisitionwas significantly declined at 13 months of age as comparedwith 5 months of age (119875 lt 005 Figure 4(b)) However theMP diet had no effect in terms of attenuating the deficit inspatial learning acquisition (119875 = 011 Figure 4(b))

Two hours after the last testing trial the retrieval of short-term memory was characterized and a significant differencewas identified among the 3timesTg-AD mice (119865

217= 6390

119875 lt 0001 Figure 4(c)) Post hoc multiple comparisonfurther showed that the retrieval of short-term memory wassignificantly impaired at 13 months of age as compared with5 months of age (119875 lt 0001 Figure 4(c)) and the MPdiet significantly rescued the deficit in short-term memoryof the 13-month-old mice (119875 lt 0001 Figure 4(c)) Forty-eight hours after the last testing trial the retrieval of long-term memory was assessed and significant differences wereobserved between groups (119865

217= 4996 119875 lt 0001

Figure 4(d)) From post hoc multiple comparison we alsofound that the retrieval of long-term memory was signifi-cantly impaired in the 13-month-old mice as compared withthe 5-month-old mice (119875 lt 0001 Figure 4(d)) and the MPdiet significantly decreased the deficit in long-term memoryas compared with the regular diet in the 13-month-old mice(119875 lt 005 Figure 4(d))

In addition the swimming velocity was significantlydecreased in the 13-month-old mice as compared with the5-month-old mice (119875 lt 005 Figure 4(e)) However theMP diet did not change the swimming velocity (119875 = 083Figure 4(e)) ANCOVA analysis was performed to determinethe diet effect on swimming velocity as a cofactor in theMWM task We found that swimming velocity had noinfluence on the MP diet to attenuate the impairment inspatial memory retrieval Furthermore both the body weightand blood glucose did not differ between the 3timesTg-AD miceadministered the MP diet and the regular diet (data notshown) Therefore these results indicated that the 3timesTg-AD mice exhibited cognitive dysfunction and the MP dietattenuated the retrieval impairment of spatial memory at 13months of age

Table 3 The results of immunostaining in 3timesTg-AD mice afterdietary supplementation

Target Regular diet MP dietNeuN 716 plusmn 2869 1037 plusmn 7830lowastlowastlowast

Calbindin 20 plusmn 371 91 plusmn 991lowastlowast

BDNF 19 plusmn 101 27 plusmn 067lowastlowastlowast

ChAT 42 plusmn 500 62 plusmn 580lowast

TH 57 plusmn 466 111 plusmn 564lowastlowastlowast

5-HT 36 plusmn 283 67 plusmn 541lowastlowastlowast

APP 165 plusmn 407 159 plusmn 662A12057340

613 plusmn 2939 656 plusmn 2008A12057342

685 plusmn 1472 604 plusmn 2301

Tau pSer202 37 plusmn 222 19 plusmn 244

Each value represents the mean plusmn SEM (119899 = 3ndash5 for each group)All values were compared with the regular diet grouplowastIncreased (119875 lt 005) lowastlowastincreased (119875 lt 001) lowastlowastlowastincreased (119875 lt 0001)Decreased (119875 lt 005) decreased (119875 lt 001) decreased (119875 lt 0001)

331 MP Supplementary Diet Promoted the Neuroprotectionin 3timesTg-AD Mice For 3timesTg-AD mice the MP supplemen-tary diet significantly protected the hippocampal neurons(119875 lt 001 Figure 5(a) and Table 3) and increased calciumbinding protein level in the dentate gyrus (DG) of thehippocampus (119875 lt 001 Figure 5(b) and Table 3) and anincreased BDNF level in the hippocampus (119875 lt 0001Figure 5(c) andTable 3) In addition the effects of theMPdieton cholinergic (choline acetyltransferase immunoreactiveChAT-ir) noradrenergic (tyrosine hydroxylase immunore-active TH-ir) and serotonergic (serotonin immunoreactive5-HT-ir) neurons were also examined in the mouse brain(Figure 5 and Table 3) MP diet significantly prevented theloss of cholinergic neurons in the medial septum (MS) verti-cal diagonal band of Broca (VDB) and horizontal diagonalband of Broca (HDB) regions of the 3timesTg-AD mice (119875 lt005 Figure 5(d) and Table 3) The MP diet also significantlyreduced the loss of noradrenergic neurons in the locuscoeruleus (LC) region (119875 lt 0001 Figure 5(e) and Table 3)and serotonergic neurons in the raphe nucleus (119875 lt 0001Figure 5(f) and Table 3) in the 3timesTg-AD mice Thereforethese findings showed that theMP diet increased the calciumbinding protein and BDNF levels associated with protectingcholinergic noradrenergic serotonergic and hippocampalneurons in the 3timesTg-AD mice These results suggested thattheMP diet might exert neuroprotection via increasing levelsof calbindin and BDNF in the 3timesTg-AD mice

332 MP Supplementary Diet Reduced the Deposition ofA120573 and p-Tau (S202S262) and Increased the Level of theNR2ANR2B Ratio in 3timesTg-AD Mice There was no signif-icant difference between regular and MP diet in the levelsof APP (119875 = 054 Figure 6(a) and Table 3) and A120573

40(119875 =

029 Figure 6(b) and Table 3) HoweverMP diet significantlydecreased the levels of A120573

42(119875 lt 005 Figure 6(c) and

Table 3) and BACE1 (119875 lt 005 Figure 6(d)) in the hippocam-pus as compared with regular diet We further found thatthe level of the NR2ANR2B ratio in the hippocampus was


Training day

Late

ncie

s (s)

0

10

20

30

40

50

60

TG-regular dietTG-MP diet

TG-5 months

1 2 3 4

lowast

(a)

Treatment

Late

ncie

s (s)

0

10

20

30

40

50

60

5 months Regular diet MP diet13 months

lowast

(b)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60



lowastlowastlowast lowastlowastlowast

(c)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60



lowastlowastlowastlowast

(d)

Swim

min

g ve

loci

ty (c

ms

)

0

10

20

30

Treatment


lowast

(e)

Figure 4 MP diet attenuated the impairment in spatial memory retrieval in 3timesTg-AD mice (a) The spatial learning ability was measuredin the 3timesTg-AD mice from training days 1sim4 The 3timesTg-AD mice showed a poor spatial learning ability at 13 months of age but the MPdiet attenuated the impairment in spatial learning ability (b) The spatial learning acquisition was measured in the 3timesTg-AD mice The poorspatial learning acquisition of 13-month-old 3timesTg-AD mice could not be rescued by the MP diet (c) The retrieval of short-term memory inthe 3timesTg-ADmice The reduced time spent in the target quadrant of the 13-month-old 3timesTg-ADmice was significantly increased by the MPdiet (d)The retrieval of long-termmemory in the 3timesTg-ADmiceTheMP diet significantly increased the time spent in the target quadrant ofthe 13-month-old 3timesTg-ADmice (e)The swimming velocity of the 3timesTg-ADmice TheMP diet had no influence on the reduced velocity ofthe 13-month-old 3timesTg-AD mice Data are expressed as means plusmn SEM 119899 = 16-17group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 comparedwith the 13-month-old mice treated with a regular diet


Neu

NN

euN


CA1

DG

CA1

DG

100120583m

(A) (B)

(C) (D)

(a)

Calb

indi

n

DG DG

50120583m

(A) (B)

(b)

BDN

FBD

NF

DGDG

CA1 CA1

50120583m

5120583m

(A) (B)

(C) (D)

(c)

ChAT

MS

VDB

HDB

MS

VDB

HDB

(A) (B)

(d)

Figure 5 Continued


THLC

LC

(A) (B)


(e)

5-H

T

Raphe Raphe

500120583m

(A) (B)

(f)

Figure 5 Neuroprotective effects of the MP diet in 3timesTg-AD mice Immunostaining images of neurons in the hippocampus (a) calbindinlevels in the hippocampus (b) BDNF in the hippocampus (c) ChAT in the MSDB region (d) TH in the LC region (e) and 5-HT in theraphe nucleus (f) Scale bars are 100 120583m in panel (a) 50 120583m in panels (b) and (c) and 500 120583m in panels (d)sim(f) Arrowheads indicate positivestaining signals 119899 = 3ndash5group

significantly increased in the mice administered the MP dietas compared with a regular diet (119875 lt 005 Figure 6(e))In addition the phosphorylated levels of p-tau (S202) (119875 lt0001 Figure 6(f) and Table 3) and p-tau (S262) (119875 lt 005Figure 6(g)) were also reduced after administration of theMP diet However there were no significant differencesidentified in the other related molecules including the totalA120573 level total tau level inactive GSK3120573 (pS9) CDK5 andseveral signaling kinases (ERK JNK and Akt) (data notshown) Therefore these results indicated that the MP dietlargely reduced the amyloidal deposition and p-tau level(S202S262) and increased the level of the NR2A2B ratio inthe hippocampus of the 3timesTg-AD mice

333 MP Supplementary Diet Decreased Oxidative Stress andInflammatory Responses in 3timesTg-AD Mice We also foundthat the MP diet significantly increased the serum GSH anddecreased the serum IL-6 concentration as comparedwith theregular diet (119875 lt 005 Figures 7(a) and 7(b)) Moreover aseries of inflammatory-related signaling pathways involvedin pathogenesis of AD animal models and patients [19ndash21]were also characterized in this study Among these pathwayswe further found that both the levels of phosphorylatedp38 MAPK (119875 lt 001 Figure 7(c)) and COX2 (119875 lt005 Figure 7(d)) were decreased in the 3timesTg-AD miceadministered anMP diet From the immunostaining analysisof the mouse hippocampus we observed that the MP diet

significantly decreased the activation of astrocytes (GFAPpositive staining) and microglia (Iba1 positive staining withround or amoeboid cells) as compared with the regular diet(119875 lt 005 and 119875 lt 0001 resp Figures 7(e)-7(f) andTable 3) These results showed that the MP diet exerted anti-inflammatory and antioxidative activities in the 3timesTg-ADmice

4 Discussion

In this study the neuroprotective property of MP treatmentwas first evaluated using an OHSC platform Furthermorethe effects and molecular mechanisms of the long-term MPsupplementary diet were elucidated in both B6 and 3timesTg-AD male mice We demonstrated that (1) MP treatmentexhibited neuroprotective activity via increasing the BDNFlevel in hippocampal slices (2) the MP diet attenuated thecognitive impairment associated with an increasing BDNFlevel increased anti-inflammation and decreased p-tau level(S202) in older B6 male mice and (3) the MP diet alsoattenuated the deficit in spatial memory retrieval associ-ated with increases in antioxidation anti-inflammation theNR2ANR2B ratio neurotransmitter neurons hippocam-pal neurons calcium binding protein and BDNF leveland decreased A120573

42 BACE1 activated glia cells and p-tau

(S202S262) in 3timesTg-AD male mice This was the first study



APP

CA1 CA1

(A) (B)

(a)

CA1

DG

CA1

DG

A12057340

A12057340

(A) (B)

(C) (D)

(b)

CA1

DG

CA1

DG

A12057342

A12057342

(A) (B)

(C) (D)

(c)

Regular MP

TreatmentRegular MP

BACE

1ac

tin

00

02

04

06

08

BACE

1Ac

tin

lowast

(d)

Figure 6 Continued


TreatmentRegular MP

Regular MP

NR2

AN

R2B

00

05

10

15

20N

R2A

NR2

BAc

tin

lowast

(e)

CA1 CA1


Tau

pSer

202

100 120583m

(A) (B)

(f)

TreatmentRegular MP

000

015

030

045

060Regular MP

Actin

lowast

Tau

pSer

262a

ctin

Tau

pSer

262

(g)

Figure 6 Effects of the MP diet on amyloid deposition p-tau (S202S262) and NR2ANR2B in 3timesTg-AD mice Immunostaining images ofAPP (a) A120573

40(b) and A120573

42(c) in the hippocampus of the mice The levels of BACE1 (d) and NR2ANR2B ratio (e) in the hippocampus

identified by western blot The MP diet significantly decreased the level of BACE1 and increased the level of the NR2ANR2B ratioImmunostaining images of p-tau (S202) (f) in the hippocampus of the mice (g) The level of p-tau (S262) measured in the hippocampusby western blotTheMP diet greatly decreased the level of p-tau (S262) Scale bar = 100 120583m and arrowheads indicate positive staining signalsData are expressed as means plusmn SEM 119899 = 3ndash5group lowast119875 lt 005 comparison between the regular and MP diet groups

to demonstrate themultifunctional properties ofMP in termsof attenuating the cognitive dysfunction of AD

At first the polyphenolic xanthone-enriched MP sig-nificantly reduced the cell death level associated with anincreasing BDNF level in hippocampal slices This result isconsistent with previous evidence showing that the naturalproduct possesses a potential neuroprotective activity for thetreatment of neurodegenerative diseases [22 23] In an in vivostudy we found that the older B6 mice had cognitive dys-function associated with increasing systematic IL-6 and p-taulevels (S202) and a decreasing BDNF level and MP dietary

supplementation attenuated these impairments significantlyAccumulating evidence indicates that BDNF is critical forthe survival and guidance of neurons to influence the long-term potentiation neuroplasticity learning and memory[24 25] Evidence suggests that low BDNF is correlatedwith high IL-6 in the cognitive dysfunction of multiplesclerosis patients [26] Recent study has also shown that achronic inflammatory state resulted from increased secretionof proinflammatory cytokines and mediators in the elderly[27] Previous study has shown that a systemic immunechallenge in wild-type mice might play an important role


Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)

Figure 7The antioxidative and anti-inflammatory activities of MP in 3timesTg-ADmice (a)The systemic GSH level was determined by ELISAwith mouse serum MP diet significantly increased the GSH level in the mouse serum (b) The systemic IL-6 level was determined by ELISAwithmouse serumMP diet significantly decreased the IL-6 level in themouse serum (c)The level of pp38 wasmeasured in the hippocampusof the mice by western blot MP diet significantly decreased the level of pp38 (d)The level of COX2 was measured in the hippocampus of themice by western blot MP diet significantly decreased the level of COX2 Immunostaining images of activated astrocytes (e) and microglia (f)in the hippocampus Scale bar = 100 120583mand arrowheads indicate positive staining signals Data are expressed asmeans plusmn SEM 119899 = 3ndash5grouplowast119875 lt 005 and lowastlowast119875 lt 001 comparison between the regular and MP diet groups

in inducing tau protein phosphorylation to develop an AD-like neuropathology during the course of aging [28] Recentevidence further indicated that inflammatorymediators suchas IL-6 could modulate tau phosphorylation independent ofthe A120573 levels in amousemodel [29]Therefore these findingssuggest that the cognitive dysfunction shown in the olderB6 mice is associated with p-tau (S202) inflammation andreduced BDNFMPdietary supplementation could effectivelyattenuate these impairments in mice

In this study we observed that 3timesTg-AD mice showedcognitive dysfunction at 13 months of age as comparedwith 5 months of age The MP diet alleviated the cognitiveimpairment associated with increases in serum GSH BDNFthe NR2ANR2B ratio calbindin neurotransmitter neuronsand hippocampal neurons and decreases in serum IL-6 levelactivated glia pp38 COX2 p-tau (S202S262) A120573

42 and

BACE1 in the hippocampus of 3timesTg-AD mice However nosignificant alterations were observed in CDK5 GSK3120573 JNKERK total tau and total A120573 in the hippocampus of the 3timesTg-AD mice (data not shown) Evidence has also revealed thatthe impairment of social recognition in 3timesTg-AD mice wasnot associated with increasing total tau and A120573 deposition[30] MP dietary supplementation attenuated the cognitivedysfunction associated with an increasing BDNF level anddecreasing inflammatory-related signals (IL-6 pp38 COX2and activated glia cells) and p-tau (S202S262) The sameresults were also observed in the older B6 mice except forpp38 COX2 and p-tau (S262) Previous study has shown thatboth pp38 and COX-2 are upregulated in AD transgenicmice[31]

In addition MP dietary supplementation also decreasedoxidative stress and the deposition of A120573

42associated with

a reducing BACE1 level Evidence also shows that BACE1inhibitor impacted amyloid deposition [32] Previous studyfurther showed that the methanol extract of MP attenuates

A12057342-induced ROS in SK-N-SH cells [33] GSH is the most

abundant intracellular antioxidant that protects cells againstoxidative damage caused by ROS [34 35] Therefore theseresults revealed that theMP diet attenuated the cognitive dys-function associated with antioxidative anti-inflammatoryand neurotrophic activity through decreasing the depositionof A120573

42and tau protein phosphorylation in the AD mice

Impaired synaptic function has been linked with the ADpathological process [36] NMDARs are known to maintainthe synaptic plasticity and contribute to memory formation[37] The bioactivity of NMDARs regulates synaptic functionandneurotransmission to sustain normal long-termpotential(LTP) and memory formation [38 39] LTP requires activa-tion of the NR2A subunit but not the NR2B subunit [40]Evidence also suggests that a synaptic plasticity alterationwas associated with a decrease in the NR2ANR2B ratioin both a neurotoxic and transgenic model of Parkinsonrsquosdisease [41] Previous study further suggested that a highNR2ANR2B ratio would be required for LTP induction[42] In this study the MP diet restored the spatial memoryretrieval associated with increasing levels of the NR2ANR2Bratio cholinergic neurons in MSDB serotonergic neuronsin the Raphe nucleus noradrenergic neurons in the LCregion and calcium-binding protein calbindin D28K in theDG subregion of the hippocampus of 3timesTg-AD mice Ourprevious studies also showed that the NR2ANR2B ratiocalbindin and neurons (cholinergic serotonergic and nora-drenergic) involved in neurotransmission play pathogenicroles in memory loss in AD [43] Evidence also shows thatsymptomatic drug treatment for AD might be beneficiallydirected toward amelioratingmultiple neurotransmitter defi-ciencies [44] Recent evidence further indicated that cal-bindin depletion might be an important contributor to thepathogenesis of AD [45] Therefore these results showedthat the MP diet attenuated the spatial memory impairment


associated with the protection of cognitive-related signalsthe NR2ANR2B ratio the neurotransmitter neurons andcalcium-binding protein in 3timesTg-AD mice In conclusionour results demonstrated that the therapeutic strategy of MPdietary supplementation attenuated cognitive dysfunction viamultifunctional properties Therefore the multifunctionalstrategy might be a potential therapy against multifactor-mediated AD

Conflict of Interests

The authors have declared that no competing interests exist

Authorsrsquo Contribution

Hei-Jen Huang andWei-Lin Chen contributed equally to thiswork

Acknowledgments

The authors thank Yu-Xuan Lin and Shi-Yun Huang fortheir assistance in animal care This work was supported inpart by research grants from the National Science Council(NSC 102-2325-B-003-001 and NSC 102-2321-B-003-004)Ministry of Science and Technology (MOST 103-2325-B-003-003MOST 103-2321-B-003-003 andMOST 103-2320-B-436-001) National Taiwan Normal University (103T3040B07)and Mackay Junior College of Medicine Nursing and Man-agement (MKC102R06) The authorsrsquo gratitude is extendedto the Molecular Imaging Core Facility of National TaiwanNormal University under the auspices of the Ministry ofScience and Technology Their gratitude also goes to theAcademic Paper Editing Clinic NTNU

References

[1] H W Querfurth and F M LaFerla ldquoAlzheimerrsquos diseaserdquo TheNew England Journal of Medicine vol 362 no 4 pp 329ndash3442010

[2] D J Selkoe ldquoAlzheimerrsquos disease genes proteins and therapyrdquoPhysiological Reviews vol 81 no 2 pp 741ndash766 2001

[3] S E Lesne M A Sherman M Grant et al ldquoBrain amyloid-120573oligomers in ageing andAlzheimerrsquos diseaserdquoBrain vol 136 no5 pp 1383ndash1398 2013

[4] M Carmo Carreiras E Mendes M Jesus Perry A P Fran-cisco and J Marco-Contelles ldquoThe multifactorial nature ofAlzheimerrsquos disease for developing potential therapeuticsrdquo Cur-rent Topics inMedicinal Chemistry vol 13 no 15 pp 1745ndash17702013

[5] B Lin ldquoPolyphenols and neuroprotection against ischemia andneurodegenerationrdquoMini-Reviews in Medicinal Chemistry vol11 no 14 pp 1222ndash1238 2011

[6] J Hou J Xue M Lee J Yu and C Sung ldquoLong-termadministration of ginsenosideRh1 enhances learning andmem-ory by promoting cell survival in the mouse hippocampusrdquoInternational Journal of Molecular Medicine vol 33 no 1 pp234ndash240 2014

[7] R M Ortega ldquoImportance of functional foods in the Mediter-ranean dietrdquo Public Health Nutrition vol 9 no 8 pp 1136ndash11402006

[8] F Gutierrez-Orozco and M L Failla ldquoBiological activities andbioavailability of mangosteen xanthones a critical review of thecurrent evidencerdquo Nutrients vol 5 no 8 pp 3163ndash3183 2013

[9] L-G Chen L-L Yang and C-C Wang ldquoAnti-inflammatoryactivity of mangostins from Garcinia mangostanardquo Food andChemical Toxicology vol 46 no 2 pp 688ndash693 2008

[10] H-A Jung B-N Su W J Keller R G Mehta and AD Kinghorn ldquoAntioxidant xanthones from the pericarp ofGarcinia mangostana (Mangosteen)rdquo Journal of Agriculturaland Food Chemistry vol 54 no 6 pp 2077ndash2082 2006

[11] J J Wang B J S Sanderson and W Zhang ldquoCytotoxic effectof xanthones from pericarp of the tropical fruit mangosteen(Garcinia mangostana Linn) on human melanoma cellsrdquo Foodand Chemical Toxicology vol 49 no 9 pp 2385ndash2391 2011

[12] S M Al-Massarani A A El Gamal N M Al-Musayeib et alldquoPhytochemical antimicrobial and antiprotozoal evaluationof Garcinia Mangostana pericarp and 120572-mangostin its majorxanthone derivativerdquoMolecules vol 18 no 9 pp 10599ndash106082013

[13] B Marquez-Valadez P D Maldonado S Galvan-Arzate etal ldquoAlpha-mangostin induces changes in glutathione levelsassociated with glutathione peroxidase activity in rat brainsynaptosomesrdquo Nutritional Neuroscience vol 15 no 5 pp 13ndash19 2012

[14] J Wang W Bi A Cheng et al ldquoTargeting multiple pathogenicmechanisms with polyphenols for the treatment of Alzheimerrsquosdisease-experimental approach and therapeutic implicationsrdquoFrontiers in Aging Neuroscience vol 6 article 42 2014

[15] C Chitchumroonchokchai K M Riedl S Suksumrarn SK Clinton A D Kinghorn and M L Failla ldquoXanthones inmangosteen juice are absorbed and partially conjugated byhealthy adultsrdquoThe Journal of Nutrition vol 142 no 4 pp 675ndash680 2012

[16] T Su B Paradiso Y-S Long W-P Liao and M SimonatoldquoEvaluation of cell damage in organotypic hippocampal sliceculture from adult mouse a potential model system to studyneuroprotectionrdquo Brain Research vol 1385 pp 68ndash76 2011

[17] H Doi M-A Shibata E Shibata et al ldquoPanaxanthone isolatedfrom pericarp of Garcinia mangostana L suppresses tumorgrowth and metastasis of a mouse model of mammary cancerrdquoAnticancer Research vol 29 no 7 pp 2485ndash2495 2009

[18] H-J Huang Y-H Chen K-C Liang et al ldquoExendin-4 pro-tected against cognitive dysfunction in hyperglycemic micereceiving an intrahippocampal lipopolysaccharide injectionrdquoPLoS ONE vol 7 no 7 Article ID e39656 2012

[19] M H Cobb ldquoMAP kinase pathwaysrdquo Progress in Biophysics andMolecular Biology vol 71 no 3-4 pp 479ndash500 1999

[20] I Ferrer R Blanco M Carmona and B Puig ldquoPhosphory-lated mitogen-activated protein kinase (MAPKERK-P) pro-tein kinase of 38kDa (p38-P) stress-activated protein kinase(SAPKJNK-P) and calciumcalmodulin-dependent kinase II(CaM kinase II) are differentially expressed in tau depositsin neurons and glial cells in tauopathiesrdquo Journal of NeuralTransmission vol 108 no 12 pp 1397ndash1415 2001

[21] J F Schindler J B Monahan and W G Smith ldquoP38 pathwaykinases as anti-inflammatory drug targetsrdquo Journal of DentalResearch vol 86 no 9 pp 800ndash811 2007

[22] NWang LWu Y Cao YWang and Y Zhang ldquoThe protectiveactivity of imperatorin in cultured neural cells exposed tohypoxia re-oxygenation injury via anti-apoptosisrdquo Fitoterapiavol 90 pp 38ndash43 2013


[23] H Wei G Wu J Chen et al ldquo(2S)-5 21015840 51015840-trihydroxy-7-methoxyflavanone a natural product from abacopteris penan-giana presents neuroprotective effects in vitro and in vivordquoNeurochemical Research vol 38 no 8 pp 1686ndash1694 2013

[24] M Blurton-Jones M Kitazawa H Martinez-Coria et alldquoNeural stem cells improve cognition via BDNF in a trans-genic model of Alzheimer diseaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 106 no32 pp 13594ndash13599 2009

[25] S Vaynman Z Ying and F Gomez-Pinilla ldquoHippocampalBDNF mediates the efficacy of exercise on synaptic plasticityand cognitionrdquo European Journal of Neuroscience vol 20 no10 pp 2580ndash2590 2004

[26] A K Patanella M Zinno D Quaranta et al ldquoCorrelationsbetween peripheral blood mononuclear cell production ofBDNF TNF-alpha IL-6 IL-10 and cognitive performances inmultiple sclerosis patientsrdquo Journal of Neuroscience Researchvol 88 no 5 pp 1106ndash1112 2010

[27] J Park T Miyakawa A Shiokawa H Nakajima-Adachi MTanokura and S Hachimura ldquoSplenic stromal cells from agedmice produce higher levels of IL-6 compared to young micerdquoMediators of Inflammation vol 2014 Article ID 826987 9 pages2014

[28] D Krstic A Madhusudan J Doehner et al ldquoSystemic immunechallenges trigger and drive Alzheimer-like neuropathology inmicerdquo Journal of Neuroinflammation vol 9 article 151 2012

[29] A M Birch L Katsouri and M Sastre ldquoModulation ofinflammation in transgenic models of Alzheimerrsquos diseaserdquoJournal of Neuroinflammation vol 11 article 25 2014

[30] D Arsenault A Dal-Pan C Tremblay et al ldquoPAK inactivationimpairs social recognition in 3xTG-ADmicewithout increasingbrain deposition of tau and A120573rdquo Journal of Neuroscience vol 33no 26 pp 10729ndash10740 2013

[31] A Sanchez D Tripathy X Yin et al ldquop38 MAPK a mediatorof hypoxia-induced cerebrovascular inflammationrdquo Journal ofAlzheimerrsquos Disease vol 32 no 3 pp 587ndash597 2012

[32] S Eketjall J Janson F Jeppsson et al ldquoAZ-4217 a high potencyBACE inhibitor displaying acute central efficacy in different invivo models and reduced amyloid deposition in Tg2576 micerdquoJournal of Neuroscience vol 33 no 24 pp 10075ndash10084 2013

[33] P Moongkarndi C Srisawat P Saetun et al ldquoProtective effectof mangosteen extract against 120573-amyloid-induced cytotoxicityoxidative stress and altered proteome in SK-N-SH cellsrdquo Journalof Proteome Research vol 9 no 5 pp 2076ndash2086 2010

[34] J I Chuang T Y Chang and H S Liu ldquoGlutathione depletion-induced apoptosis of Ha-ras-transformed NIH3T3 cells can beprevented bymelatoninrdquoOncogene vol 22 no 9 pp 1349ndash13572003

[35] P Guha A Dey R Sen M Chatterjee S Chattopadhyay andS K Bandyopadhyay ldquoIntracellular GSH depletion triggeredmitochondrial bax translocation to accomplish resveratrol-induced apoptosis in the U937 cell linerdquo Journal of Pharmacol-ogy and Experimental Therapeutics vol 336 no 1 pp 206ndash2142011

[36] P N Lacor M C Buniel P W Furlow et al ldquoA120573 oligomer-induced aberrations in synapse composition shape and densityprovide amolecular basis for loss of connectivity in Alzheimerrsquosdiseaserdquo Journal of Neuroscience vol 27 no 4 pp 796ndash8072007

[37] A H Rezvani ldquoInvolvement of the NMDA system in learningand memoryrdquo in Animal Models of Cognitive Impairment E DLevin and J J Buccafusco Eds Boca Raton Fla USA 2006

[38] Z Cui R Feng S Jacobs et al ldquoIncreased NR2ANR2B ratiocompresses long-term depression range and constrains long-term memoryrdquo Scientific Reports vol 3 article 1036 2013

[39] P Paoletti C Bellone and Q Zhou ldquoNMDA receptor subunitdiversity impact on receptor properties synaptic plasticity anddiseaserdquo Nature Reviews Neuroscience vol 14 no 6 pp 383ndash400 2013

[40] P V Massey B E Johnson P R Moult et al ldquoDifferential rolesof NR2A and NR2B-containing NMDA receptors in corticallong-term potentiation and long-term depressionrdquo Journal ofNeuroscience vol 24 no 36 pp 7821ndash7828 2004

[41] C Costa C Sgobio S Siliquini et al ldquoMechanisms underlyingthe impairment of hippocampal long-term potentiation andmemory in experimental Parkinsonrsquos diseaserdquo Brain vol 135no 6 pp 1884ndash1899 2012

[42] K Yashiro and B D Philpot ldquoRegulation of NMDA receptorsubunit expression and its implications for LTD LTP andmetaplasticityrdquoNeuropharmacology vol 55 no 7 pp 1081ndash10942008

[43] H-J Huang K-C Liang H-C Ke Y-Y Chang and H MHsieh-Li ldquoLong-term social isolation exacerbates the impair-ment of spatial working memory in APPPS1 transgenic micerdquoBrain Research vol 1371 pp 150ndash160 2011

[44] S A Lyness C Zarow and H C Chui ldquoNeuron loss in keycholinergic and aminergic nuclei in Alzheimer disease a meta-analysisrdquo Neurobiology of Aging vol 24 no 1 pp 1ndash23 2003

[45] S-Y KookH JeongM J Kang et al ldquoCrucial role of calbindin-D28119896

in the pathogenesis of Alzheimerrsquos disease mouse modelrdquoCell Death and Differentiation vol 21 pp 1575ndash1587 2014


individual polyphenols [14] These putative health claimsare based on in vitro observations and anecdotal reports ofthe top-selling botanical supplement containing mangosteenfruit in the United States [15] However the proposed healthbenefits of MP have only received very limited attention interms of cognitive dysfunction in neurodegenerative diseasessuch as AD Therefore in the present study we evaluatedthe effects of MP by in vitro organotypic hippocampal sliceculture (OHSC) and by using in vivo B6 and AD mousesystems

2 Materials and Methods

21 Mice B6 and 3timesTg-AD (harboring PS1M146V APPSweand TauP30IL transgenes) mice were purchased from theNational Breeding Center for Laboratory Animals Taiwanand the Jackson Laboratory (004807) respectively B6 malemice (5-week-old) were used to measure the neuroprotec-tion of MP in the organotypic hippocampal slice cultureIn addition the supplement of regular diet and MP dietwas administrated in the B6 male (aged 3 weeks and 5months) and 3timesTg-AD (aged 5 months) mice The micewere housed in individual-ventilation cages and maintainedon a 12 h lightdark cycle at a controlled room temperatureand humidity in accordance with standard use protocols andanimal welfare regulations The mice consumed food andwater ad libitum All experiments were performed duringthe light phase between 7 am and 7 pm All study protocolswere also approved by the Institutional Animal Care and UseCommittee of National Taiwan Normal University TaipeiTaiwan

22 Organotypic Hippocampal Slice Culture (OHSC) OHSCswere conducted in a slightly modified manner from theprevious report [16] Young adult male B6 mice (5-week-old) were anesthetized by Avertin (04 gkg body weightSigma St LouisMOUSA) and decapitatedThe hippocampiwere rapidly dissected in ice-cold artificial cerebrospinalfluid (aCSF) consisting of the following (in mM) NaCl 118KCl 25 MgSO

43 NaH

2PO411 NaHCO

326 CaCl

21

and glucose 11 (all reagents from Sigma) bubbled with 95O25CO

2 Subsequently coronal slices (350 120583m thick) were

cut with a vibratome (VT1200S Leica Wetzlar Germany)The sliced hippocampi were transferred onto sterile 04 120583mporousmembrane confetti (Millicell-PCFMillipore Ireland)and cultured with medium consisting of 50 MEM 25horse serum 18 HBSS 4mM L-glutamine 12mM glucose45mM NaHCO

3 20mM sucrose 100UmL penicillin and

100mgmL streptomycin (all reagents from Gibco or Sigma)in a humidified 5 CO

2atmosphere at 37∘C The medium

was changed three times a week On day in vitro (DIV) 5slices were treated with MP (10 120583M) or vehicle (DMSO) for12 hr The cell death level of the slices was then examined byPI staining Furthermore the BDNF level was examined toevaluate the neuroprotective effect of MP treatment on thehippocampal slices

23 PIUptakeAssay inOHSC PI uptake is an effective assess-ment of cell damage At the stated time point slices were

incubated with 5120583gmL PI (Sigma) in culture medium for30minThe stained slices were observed using a fluorescencemicroscope (Leica Wetzlar Germany) and the fluorescentintensity was quantified using AxioVision software (CarlZeiss Jena Germany)

24 Diets The male B6 (aged 3 weeks and 5 months) and3timesTg-AD (aged 5 months) mice were randomly divided intotwo groups with 15sim17 animals in each group (i) regular diet(LabDiet 5010 calories provided by protein 275 fat 135and carbohydrate 59) and (ii) regular diet with 5000 ppmof MP (Lord Duke Biotechnology Company containing3560mgg 120572-mangostin 063mgg 120573-mangostin 146mgg3-isomangostin 142mgg 8-deoxygartanin 155mgg gar-tanin and 132mgg 9-hydroxycalabaxanthone) supplementas in previous studies [15 17] Baseline behavioral assessmentwas conducted in themale 3timesTg-ADmice at 5 months of ageMice were maintained on MP dietary supplementation for 8months

25 Morris Water Maze Task (MWM) The Morris watermaze task was performed as previously described [18] Inbrief it consists of pretraining training testing and probingAll trials lasted for a maximum of 60 sec On the day priorto spatial training all mice underwent pretraining in orderto assess their swimming ability and to acclimatize them tothe pool In the three 60 sec pretraining trials the mousewas released into the water facing the wall of the poolfrom semirandomly chosen cardinal compass points Afterthree trials of acclimatization each mouse was placed onthe invisible platform located at the center of the targetquadrant and allowed to stay there for 20 sec The mice weregiven a 4-day training session consisting of four 60-secondtraining trials (intertrial interval 20ndash30min) per day Thehidden platformwas always placed at the same location of thepool (northeast quadrant as the target quadrant) throughoutthe training period During each trial from semirandomlychosen cardinal compass points the mouse was releasedinto the water facing the pool wall After climbing onto theplatform the mouse was allowed to rest on it for 20 sec Ifthe mouse failed to swim to the platform within 60 sec itwould be placed on the platform to stay on it for 20 sec by anexperimenter Twenty-four hours after the last training trialall mice were given three testing trials to assess the time takento climb onto the hidden platform Two and forty-eight hoursafter the last testing trial all mice were given two probe trialsto evaluate the retrieval of the short- and long-term spatialmemory of the platform

26 ELISA Analyses Blood samples were collected from theretroorbital plexus of anesthetized mice and centrifuged at2000timesg for 15min at 4∘C after the MWM (119899 = 3sim5 for eachgroup) Sera were collected and stored at minus80∘C until useThe levels of glutathione (GSH) and IL-6 in the serum weremeasured using a Glutathione assay kit (Cayman ChemicalMI USA) and an IL-6 ELISA kit (RampD Systems MN USA)respectively For the BDNF assay hippocampal slices werehomogenized in 8 times volume of lysis buffer containing 50mM










3 Results



359= 488




























142= 861 119875 lt 001 Figure 2(c))


142= 613


MPDMSO

500120583m

(a)

PI u

ptak

e (

)

0

25

50

75

100

MPDMSOTreatment

lowastlowast

(b)

BDN

F le

vel (

ngm

g)

0

200

400

600

800

TreatmentMPDMSO

lowastlowast

(c)



142= 732 119875 lt 001 Figure 2(c)) Post






140=



1 2 3 4 1 2 3 4

Late

ncie

s (s)

0

10

20

30

40

50

60


YoungerDay

Older

(a)

Younger Older

Late

ncie

s (s)

0

10

20

30

40

50

60lowastlowast

(b)

Younger Older

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60


lowast

(c)

Younger Older

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60



(d)

Younger Older

Swim

min

g ve

loci

ty (c

ms

)

0

6

12

18

24

30

Regular dietMP diet

lowast

(e)




BDN

FBD

NF

CA1

DG


(A) (B) (C) (D)

(E) (F) (G) (H)

50120583m

5120583m

(a)

CA1

DG

pTau

Ser

202

pTau

Ser

202

(A) (B) (C) (D)

(E) (F) (G) (H)

50120583m

(b)

GFA

P

DG

(A) (B) (C) (D)

50120583m

(c)

Iba-1

(A) (B) (C) (D)

50120583m

5120583m

(d)

Figure 3 Continued


IL-6

leve

l in

seru

m (p

gm

L)0

350

700

1050

1400

Younger Older

Regular dietMP diet

lowast

(e)











127= 5408 119875 lt




127=


125= 3136 119875 lt 0001 Figure 3(b)


111=





119= 1747










319=








217= 6390


217= 4996 119875 lt 0001











613 plusmn 2939 656 plusmn 2008A12057342

685 plusmn 1472 604 plusmn 2301





40(119875 =


42(119875 lt 005 Figure 6(c) and



Training day

Late

ncie

s (s)

0

10

20

30

40

50

60


TG-5 months

1 2 3 4

lowast

(a)

Treatment

Late

ncie

s (s)

0

10

20

30

40

50

60


lowast

(b)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(c)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(d)

Swim

min

g ve

loci

ty (c

ms

)

0

10

20

30

Treatment


lowast

(e)



Neu

NN

euN


CA1

DG

CA1

DG

100120583m

(A) (B)

(C) (D)

(a)

Calb

indi

n

DG DG

50120583m

(A) (B)

(b)

BDN

FBD

NF

DGDG

CA1 CA1

50120583m

5120583m

(A) (B)

(C) (D)

(c)

ChAT

MS

VDB

HDB

MS

VDB

HDB

(A) (B)

(d)

Figure 5 Continued


THLC

LC

(A) (B)


(e)

5-H

T

Raphe Raphe

500120583m

(A) (B)

(f)





4 Discussion






APP

CA1 CA1

(A) (B)

(a)

CA1

DG

CA1

DG

A12057340

A12057340

(A) (B)

(C) (D)

(b)

CA1

DG

CA1

DG

A12057342

A12057342

(A) (B)

(C) (D)

(c)

Regular MP

TreatmentRegular MP

BACE

1ac

tin

00

02

04

06

08

BACE

1Ac

tin

lowast

(d)

Figure 6 Continued


TreatmentRegular MP

Regular MP

NR2

AN

R2B

00

05

10

15

20N

R2A

NR2

BAc

tin

lowast

(e)

CA1 CA1


Tau

pSer

202

100 120583m

(A) (B)

(f)

TreatmentRegular MP

000

015

030

045

060Regular MP

Actin

lowast

Tau

pSer

262a

ctin

Tau

pSer

262

(g)


40(b) and A120573







Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References

























































3 Results



359= 488




























142= 861 119875 lt 001 Figure 2(c))


142= 613


MPDMSO

500120583m

(a)

PI u

ptak

e (

)

0

25

50

75

100

MPDMSOTreatment

lowastlowast

(b)

BDN

F le

vel (

ngm

g)

0

200

400

600

800

TreatmentMPDMSO

lowastlowast

(c)



142= 732 119875 lt 001 Figure 2(c)) Post






140=



1 2 3 4 1 2 3 4

Late

ncie

s (s)

0

10

20

30

40

50

60


YoungerDay

Older

(a)

Younger Older

Late

ncie

s (s)

0

10

20

30

40

50

60lowastlowast

(b)

Younger Older

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60


lowast

(c)

Younger Older

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60



(d)

Younger Older

Swim

min

g ve

loci

ty (c

ms

)

0

6

12

18

24

30

Regular dietMP diet

lowast

(e)




BDN

FBD

NF

CA1

DG


(A) (B) (C) (D)

(E) (F) (G) (H)

50120583m

5120583m

(a)

CA1

DG

pTau

Ser

202

pTau

Ser

202

(A) (B) (C) (D)

(E) (F) (G) (H)

50120583m

(b)

GFA

P

DG

(A) (B) (C) (D)

50120583m

(c)

Iba-1

(A) (B) (C) (D)

50120583m

5120583m

(d)

Figure 3 Continued


IL-6

leve

l in

seru

m (p

gm

L)0

350

700

1050

1400

Younger Older

Regular dietMP diet

lowast

(e)











127= 5408 119875 lt




127=


125= 3136 119875 lt 0001 Figure 3(b)


111=





119= 1747










319=








217= 6390


217= 4996 119875 lt 0001











613 plusmn 2939 656 plusmn 2008A12057342

685 plusmn 1472 604 plusmn 2301





40(119875 =


42(119875 lt 005 Figure 6(c) and



Training day

Late

ncie

s (s)

0

10

20

30

40

50

60


TG-5 months

1 2 3 4

lowast

(a)

Treatment

Late

ncie

s (s)

0

10

20

30

40

50

60


lowast

(b)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(c)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(d)

Swim

min

g ve

loci

ty (c

ms

)

0

10

20

30

Treatment


lowast

(e)



Neu

NN

euN


CA1

DG

CA1

DG

100120583m

(A) (B)

(C) (D)

(a)

Calb

indi

n

DG DG

50120583m

(A) (B)

(b)

BDN

FBD

NF

DGDG

CA1 CA1

50120583m

5120583m

(A) (B)

(C) (D)

(c)

ChAT

MS

VDB

HDB

MS

VDB

HDB

(A) (B)

(d)

Figure 5 Continued


THLC

LC

(A) (B)


(e)

5-H

T

Raphe Raphe

500120583m

(A) (B)

(f)





4 Discussion






APP

CA1 CA1

(A) (B)

(a)

CA1

DG

CA1

DG

A12057340

A12057340

(A) (B)

(C) (D)

(b)

CA1

DG

CA1

DG

A12057342

A12057342

(A) (B)

(C) (D)

(c)

Regular MP

TreatmentRegular MP

BACE

1ac

tin

00

02

04

06

08

BACE

1Ac

tin

lowast

(d)

Figure 6 Continued


TreatmentRegular MP

Regular MP

NR2

AN

R2B

00

05

10

15

20N

R2A

NR2

BAc

tin

lowast

(e)

CA1 CA1


Tau

pSer

202

100 120583m

(A) (B)

(f)

TreatmentRegular MP

000

015

030

045

060Regular MP

Actin

lowast

Tau

pSer

262a

ctin

Tau

pSer

262

(g)


40(b) and A120573







Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References








































































142= 861 119875 lt 001 Figure 2(c))


142= 613


MPDMSO

500120583m

(a)

PI u

ptak

e (

)

0

25

50

75

100

MPDMSOTreatment

lowastlowast

(b)

BDN

F le

vel (

ngm

g)

0

200

400

600

800

TreatmentMPDMSO

lowastlowast

(c)



142= 732 119875 lt 001 Figure 2(c)) Post






140=



1 2 3 4 1 2 3 4

Late

ncie

s (s)

0

10

20

30

40

50

60


YoungerDay

Older

(a)

Younger Older

Late

ncie

s (s)

0

10

20

30

40

50

60lowastlowast

(b)

Younger Older

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60


lowast

(c)

Younger Older

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60



(d)

Younger Older

Swim

min

g ve

loci

ty (c

ms

)

0

6

12

18

24

30

Regular dietMP diet

lowast

(e)




BDN

FBD

NF

CA1

DG


(A) (B) (C) (D)

(E) (F) (G) (H)

50120583m

5120583m

(a)

CA1

DG

pTau

Ser

202

pTau

Ser

202

(A) (B) (C) (D)

(E) (F) (G) (H)

50120583m

(b)

GFA

P

DG

(A) (B) (C) (D)

50120583m

(c)

Iba-1

(A) (B) (C) (D)

50120583m

5120583m

(d)

Figure 3 Continued


IL-6

leve

l in

seru

m (p

gm

L)0

350

700

1050

1400

Younger Older

Regular dietMP diet

lowast

(e)











127= 5408 119875 lt




127=


125= 3136 119875 lt 0001 Figure 3(b)


111=





119= 1747










319=








217= 6390


217= 4996 119875 lt 0001











613 plusmn 2939 656 plusmn 2008A12057342

685 plusmn 1472 604 plusmn 2301





40(119875 =


42(119875 lt 005 Figure 6(c) and



Training day

Late

ncie

s (s)

0

10

20

30

40

50

60


TG-5 months

1 2 3 4

lowast

(a)

Treatment

Late

ncie

s (s)

0

10

20

30

40

50

60


lowast

(b)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(c)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(d)

Swim

min

g ve

loci

ty (c

ms

)

0

10

20

30

Treatment


lowast

(e)



Neu

NN

euN


CA1

DG

CA1

DG

100120583m

(A) (B)

(C) (D)

(a)

Calb

indi

n

DG DG

50120583m

(A) (B)

(b)

BDN

FBD

NF

DGDG

CA1 CA1

50120583m

5120583m

(A) (B)

(C) (D)

(c)

ChAT

MS

VDB

HDB

MS

VDB

HDB

(A) (B)

(d)

Figure 5 Continued


THLC

LC

(A) (B)


(e)

5-H

T

Raphe Raphe

500120583m

(A) (B)

(f)





4 Discussion






APP

CA1 CA1

(A) (B)

(a)

CA1

DG

CA1

DG

A12057340

A12057340

(A) (B)

(C) (D)

(b)

CA1

DG

CA1

DG

A12057342

A12057342

(A) (B)

(C) (D)

(c)

Regular MP

TreatmentRegular MP

BACE

1ac

tin

00

02

04

06

08

BACE

1Ac

tin

lowast

(d)

Figure 6 Continued


TreatmentRegular MP

Regular MP

NR2

AN

R2B

00

05

10

15

20N

R2A

NR2

BAc

tin

lowast

(e)

CA1 CA1


Tau

pSer

202

100 120583m

(A) (B)

(f)

TreatmentRegular MP

000

015

030

045

060Regular MP

Actin

lowast

Tau

pSer

262a

ctin

Tau

pSer

262

(g)


40(b) and A120573







Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References

















































MPDMSO

500120583m

(a)

PI u

ptak

e (

)

0

25

50

75

100

MPDMSOTreatment

lowastlowast

(b)

BDN

F le

vel (

ngm

g)

0

200

400

600

800

TreatmentMPDMSO

lowastlowast

(c)



142= 732 119875 lt 001 Figure 2(c)) Post






140=



1 2 3 4 1 2 3 4

Late

ncie

s (s)

0

10

20

30

40

50

60


YoungerDay

Older

(a)

Younger Older

Late

ncie

s (s)

0

10

20

30

40

50

60lowastlowast

(b)

Younger Older

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60


lowast

(c)

Younger Older

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60



(d)

Younger Older

Swim

min

g ve

loci

ty (c

ms

)

0

6

12

18

24

30

Regular dietMP diet

lowast

(e)




BDN

FBD

NF

CA1

DG


(A) (B) (C) (D)

(E) (F) (G) (H)

50120583m

5120583m

(a)

CA1

DG

pTau

Ser

202

pTau

Ser

202

(A) (B) (C) (D)

(E) (F) (G) (H)

50120583m

(b)

GFA

P

DG

(A) (B) (C) (D)

50120583m

(c)

Iba-1

(A) (B) (C) (D)

50120583m

5120583m

(d)

Figure 3 Continued


IL-6

leve

l in

seru

m (p

gm

L)0

350

700

1050

1400

Younger Older

Regular dietMP diet

lowast

(e)











127= 5408 119875 lt




127=


125= 3136 119875 lt 0001 Figure 3(b)


111=





119= 1747










319=








217= 6390


217= 4996 119875 lt 0001











613 plusmn 2939 656 plusmn 2008A12057342

685 plusmn 1472 604 plusmn 2301





40(119875 =


42(119875 lt 005 Figure 6(c) and



Training day

Late

ncie

s (s)

0

10

20

30

40

50

60


TG-5 months

1 2 3 4

lowast

(a)

Treatment

Late

ncie

s (s)

0

10

20

30

40

50

60


lowast

(b)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(c)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(d)

Swim

min

g ve

loci

ty (c

ms

)

0

10

20

30

Treatment


lowast

(e)



Neu

NN

euN


CA1

DG

CA1

DG

100120583m

(A) (B)

(C) (D)

(a)

Calb

indi

n

DG DG

50120583m

(A) (B)

(b)

BDN

FBD

NF

DGDG

CA1 CA1

50120583m

5120583m

(A) (B)

(C) (D)

(c)

ChAT

MS

VDB

HDB

MS

VDB

HDB

(A) (B)

(d)

Figure 5 Continued


THLC

LC

(A) (B)


(e)

5-H

T

Raphe Raphe

500120583m

(A) (B)

(f)





4 Discussion






APP

CA1 CA1

(A) (B)

(a)

CA1

DG

CA1

DG

A12057340

A12057340

(A) (B)

(C) (D)

(b)

CA1

DG

CA1

DG

A12057342

A12057342

(A) (B)

(C) (D)

(c)

Regular MP

TreatmentRegular MP

BACE

1ac

tin

00

02

04

06

08

BACE

1Ac

tin

lowast

(d)

Figure 6 Continued


TreatmentRegular MP

Regular MP

NR2

AN

R2B

00

05

10

15

20N

R2A

NR2

BAc

tin

lowast

(e)

CA1 CA1


Tau

pSer

202

100 120583m

(A) (B)

(f)

TreatmentRegular MP

000

015

030

045

060Regular MP

Actin

lowast

Tau

pSer

262a

ctin

Tau

pSer

262

(g)


40(b) and A120573







Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References

















































1 2 3 4 1 2 3 4

Late

ncie

s (s)

0

10

20

30

40

50

60


YoungerDay

Older

(a)

Younger Older

Late

ncie

s (s)

0

10

20

30

40

50

60lowastlowast

(b)

Younger Older

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60


lowast

(c)

Younger Older

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60



(d)

Younger Older

Swim

min

g ve

loci

ty (c

ms

)

0

6

12

18

24

30

Regular dietMP diet

lowast

(e)




BDN

FBD

NF

CA1

DG


(A) (B) (C) (D)

(E) (F) (G) (H)

50120583m

5120583m

(a)

CA1

DG

pTau

Ser

202

pTau

Ser

202

(A) (B) (C) (D)

(E) (F) (G) (H)

50120583m

(b)

GFA

P

DG

(A) (B) (C) (D)

50120583m

(c)

Iba-1

(A) (B) (C) (D)

50120583m

5120583m

(d)

Figure 3 Continued


IL-6

leve

l in

seru

m (p

gm

L)0

350

700

1050

1400

Younger Older

Regular dietMP diet

lowast

(e)











127= 5408 119875 lt




127=


125= 3136 119875 lt 0001 Figure 3(b)


111=





119= 1747










319=








217= 6390


217= 4996 119875 lt 0001











613 plusmn 2939 656 plusmn 2008A12057342

685 plusmn 1472 604 plusmn 2301





40(119875 =


42(119875 lt 005 Figure 6(c) and



Training day

Late

ncie

s (s)

0

10

20

30

40

50

60


TG-5 months

1 2 3 4

lowast

(a)

Treatment

Late

ncie

s (s)

0

10

20

30

40

50

60


lowast

(b)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(c)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(d)

Swim

min

g ve

loci

ty (c

ms

)

0

10

20

30

Treatment


lowast

(e)



Neu

NN

euN


CA1

DG

CA1

DG

100120583m

(A) (B)

(C) (D)

(a)

Calb

indi

n

DG DG

50120583m

(A) (B)

(b)

BDN

FBD

NF

DGDG

CA1 CA1

50120583m

5120583m

(A) (B)

(C) (D)

(c)

ChAT

MS

VDB

HDB

MS

VDB

HDB

(A) (B)

(d)

Figure 5 Continued


THLC

LC

(A) (B)


(e)

5-H

T

Raphe Raphe

500120583m

(A) (B)

(f)





4 Discussion






APP

CA1 CA1

(A) (B)

(a)

CA1

DG

CA1

DG

A12057340

A12057340

(A) (B)

(C) (D)

(b)

CA1

DG

CA1

DG

A12057342

A12057342

(A) (B)

(C) (D)

(c)

Regular MP

TreatmentRegular MP

BACE

1ac

tin

00

02

04

06

08

BACE

1Ac

tin

lowast

(d)

Figure 6 Continued


TreatmentRegular MP

Regular MP

NR2

AN

R2B

00

05

10

15

20N

R2A

NR2

BAc

tin

lowast

(e)

CA1 CA1


Tau

pSer

202

100 120583m

(A) (B)

(f)

TreatmentRegular MP

000

015

030

045

060Regular MP

Actin

lowast

Tau

pSer

262a

ctin

Tau

pSer

262

(g)


40(b) and A120573







Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References

















































BDN

FBD

NF

CA1

DG


(A) (B) (C) (D)

(E) (F) (G) (H)

50120583m

5120583m

(a)

CA1

DG

pTau

Ser

202

pTau

Ser

202

(A) (B) (C) (D)

(E) (F) (G) (H)

50120583m

(b)

GFA

P

DG

(A) (B) (C) (D)

50120583m

(c)

Iba-1

(A) (B) (C) (D)

50120583m

5120583m

(d)

Figure 3 Continued


IL-6

leve

l in

seru

m (p

gm

L)0

350

700

1050

1400

Younger Older

Regular dietMP diet

lowast

(e)











127= 5408 119875 lt




127=


125= 3136 119875 lt 0001 Figure 3(b)


111=





119= 1747










319=








217= 6390


217= 4996 119875 lt 0001











613 plusmn 2939 656 plusmn 2008A12057342

685 plusmn 1472 604 plusmn 2301





40(119875 =


42(119875 lt 005 Figure 6(c) and



Training day

Late

ncie

s (s)

0

10

20

30

40

50

60


TG-5 months

1 2 3 4

lowast

(a)

Treatment

Late

ncie

s (s)

0

10

20

30

40

50

60


lowast

(b)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(c)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(d)

Swim

min

g ve

loci

ty (c

ms

)

0

10

20

30

Treatment


lowast

(e)



Neu

NN

euN


CA1

DG

CA1

DG

100120583m

(A) (B)

(C) (D)

(a)

Calb

indi

n

DG DG

50120583m

(A) (B)

(b)

BDN

FBD

NF

DGDG

CA1 CA1

50120583m

5120583m

(A) (B)

(C) (D)

(c)

ChAT

MS

VDB

HDB

MS

VDB

HDB

(A) (B)

(d)

Figure 5 Continued


THLC

LC

(A) (B)


(e)

5-H

T

Raphe Raphe

500120583m

(A) (B)

(f)





4 Discussion






APP

CA1 CA1

(A) (B)

(a)

CA1

DG

CA1

DG

A12057340

A12057340

(A) (B)

(C) (D)

(b)

CA1

DG

CA1

DG

A12057342

A12057342

(A) (B)

(C) (D)

(c)

Regular MP

TreatmentRegular MP

BACE

1ac

tin

00

02

04

06

08

BACE

1Ac

tin

lowast

(d)

Figure 6 Continued


TreatmentRegular MP

Regular MP

NR2

AN

R2B

00

05

10

15

20N

R2A

NR2

BAc

tin

lowast

(e)

CA1 CA1


Tau

pSer

202

100 120583m

(A) (B)

(f)

TreatmentRegular MP

000

015

030

045

060Regular MP

Actin

lowast

Tau

pSer

262a

ctin

Tau

pSer

262

(g)


40(b) and A120573







Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References

















































IL-6

leve

l in

seru

m (p

gm

L)0

350

700

1050

1400

Younger Older

Regular dietMP diet

lowast

(e)











127= 5408 119875 lt




127=


125= 3136 119875 lt 0001 Figure 3(b)


111=





119= 1747










319=








217= 6390


217= 4996 119875 lt 0001











613 plusmn 2939 656 plusmn 2008A12057342

685 plusmn 1472 604 plusmn 2301





40(119875 =


42(119875 lt 005 Figure 6(c) and



Training day

Late

ncie

s (s)

0

10

20

30

40

50

60


TG-5 months

1 2 3 4

lowast

(a)

Treatment

Late

ncie

s (s)

0

10

20

30

40

50

60


lowast

(b)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(c)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(d)

Swim

min

g ve

loci

ty (c

ms

)

0

10

20

30

Treatment


lowast

(e)



Neu

NN

euN


CA1

DG

CA1

DG

100120583m

(A) (B)

(C) (D)

(a)

Calb

indi

n

DG DG

50120583m

(A) (B)

(b)

BDN

FBD

NF

DGDG

CA1 CA1

50120583m

5120583m

(A) (B)

(C) (D)

(c)

ChAT

MS

VDB

HDB

MS

VDB

HDB

(A) (B)

(d)

Figure 5 Continued


THLC

LC

(A) (B)


(e)

5-H

T

Raphe Raphe

500120583m

(A) (B)

(f)





4 Discussion






APP

CA1 CA1

(A) (B)

(a)

CA1

DG

CA1

DG

A12057340

A12057340

(A) (B)

(C) (D)

(b)

CA1

DG

CA1

DG

A12057342

A12057342

(A) (B)

(C) (D)

(c)

Regular MP

TreatmentRegular MP

BACE

1ac

tin

00

02

04

06

08

BACE

1Ac

tin

lowast

(d)

Figure 6 Continued


TreatmentRegular MP

Regular MP

NR2

AN

R2B

00

05

10

15

20N

R2A

NR2

BAc

tin

lowast

(e)

CA1 CA1


Tau

pSer

202

100 120583m

(A) (B)

(f)

TreatmentRegular MP

000

015

030

045

060Regular MP

Actin

lowast

Tau

pSer

262a

ctin

Tau

pSer

262

(g)


40(b) and A120573







Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References





















































319=








217= 6390


217= 4996 119875 lt 0001











613 plusmn 2939 656 plusmn 2008A12057342

685 plusmn 1472 604 plusmn 2301





40(119875 =


42(119875 lt 005 Figure 6(c) and



Training day

Late

ncie

s (s)

0

10

20

30

40

50

60


TG-5 months

1 2 3 4

lowast

(a)

Treatment

Late

ncie

s (s)

0

10

20

30

40

50

60


lowast

(b)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(c)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(d)

Swim

min

g ve

loci

ty (c

ms

)

0

10

20

30

Treatment


lowast

(e)



Neu

NN

euN


CA1

DG

CA1

DG

100120583m

(A) (B)

(C) (D)

(a)

Calb

indi

n

DG DG

50120583m

(A) (B)

(b)

BDN

FBD

NF

DGDG

CA1 CA1

50120583m

5120583m

(A) (B)

(C) (D)

(c)

ChAT

MS

VDB

HDB

MS

VDB

HDB

(A) (B)

(d)

Figure 5 Continued


THLC

LC

(A) (B)


(e)

5-H

T

Raphe Raphe

500120583m

(A) (B)

(f)





4 Discussion






APP

CA1 CA1

(A) (B)

(a)

CA1

DG

CA1

DG

A12057340

A12057340

(A) (B)

(C) (D)

(b)

CA1

DG

CA1

DG

A12057342

A12057342

(A) (B)

(C) (D)

(c)

Regular MP

TreatmentRegular MP

BACE

1ac

tin

00

02

04

06

08

BACE

1Ac

tin

lowast

(d)

Figure 6 Continued


TreatmentRegular MP

Regular MP

NR2

AN

R2B

00

05

10

15

20N

R2A

NR2

BAc

tin

lowast

(e)

CA1 CA1


Tau

pSer

202

100 120583m

(A) (B)

(f)

TreatmentRegular MP

000

015

030

045

060Regular MP

Actin

lowast

Tau

pSer

262a

ctin

Tau

pSer

262

(g)


40(b) and A120573







Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References

















































Training day

Late

ncie

s (s)

0

10

20

30

40

50

60


TG-5 months

1 2 3 4

lowast

(a)

Treatment

Late

ncie

s (s)

0

10

20

30

40

50

60


lowast

(b)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(c)

Dur

atio

n in

targ

et re

gion

(s)

0

10

20

30

40

50

60




(d)

Swim

min

g ve

loci

ty (c

ms

)

0

10

20

30

Treatment


lowast

(e)



Neu

NN

euN


CA1

DG

CA1

DG

100120583m

(A) (B)

(C) (D)

(a)

Calb

indi

n

DG DG

50120583m

(A) (B)

(b)

BDN

FBD

NF

DGDG

CA1 CA1

50120583m

5120583m

(A) (B)

(C) (D)

(c)

ChAT

MS

VDB

HDB

MS

VDB

HDB

(A) (B)

(d)

Figure 5 Continued


THLC

LC

(A) (B)


(e)

5-H

T

Raphe Raphe

500120583m

(A) (B)

(f)





4 Discussion






APP

CA1 CA1

(A) (B)

(a)

CA1

DG

CA1

DG

A12057340

A12057340

(A) (B)

(C) (D)

(b)

CA1

DG

CA1

DG

A12057342

A12057342

(A) (B)

(C) (D)

(c)

Regular MP

TreatmentRegular MP

BACE

1ac

tin

00

02

04

06

08

BACE

1Ac

tin

lowast

(d)

Figure 6 Continued


TreatmentRegular MP

Regular MP

NR2

AN

R2B

00

05

10

15

20N

R2A

NR2

BAc

tin

lowast

(e)

CA1 CA1


Tau

pSer

202

100 120583m

(A) (B)

(f)

TreatmentRegular MP

000

015

030

045

060Regular MP

Actin

lowast

Tau

pSer

262a

ctin

Tau

pSer

262

(g)


40(b) and A120573







Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References

















































Neu

NN

euN


CA1

DG

CA1

DG

100120583m

(A) (B)

(C) (D)

(a)

Calb

indi

n

DG DG

50120583m

(A) (B)

(b)

BDN

FBD

NF

DGDG

CA1 CA1

50120583m

5120583m

(A) (B)

(C) (D)

(c)

ChAT

MS

VDB

HDB

MS

VDB

HDB

(A) (B)

(d)

Figure 5 Continued


THLC

LC

(A) (B)


(e)

5-H

T

Raphe Raphe

500120583m

(A) (B)

(f)





4 Discussion






APP

CA1 CA1

(A) (B)

(a)

CA1

DG

CA1

DG

A12057340

A12057340

(A) (B)

(C) (D)

(b)

CA1

DG

CA1

DG

A12057342

A12057342

(A) (B)

(C) (D)

(c)

Regular MP

TreatmentRegular MP

BACE

1ac

tin

00

02

04

06

08

BACE

1Ac

tin

lowast

(d)

Figure 6 Continued


TreatmentRegular MP

Regular MP

NR2

AN

R2B

00

05

10

15

20N

R2A

NR2

BAc

tin

lowast

(e)

CA1 CA1


Tau

pSer

202

100 120583m

(A) (B)

(f)

TreatmentRegular MP

000

015

030

045

060Regular MP

Actin

lowast

Tau

pSer

262a

ctin

Tau

pSer

262

(g)


40(b) and A120573







Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References

















































THLC

LC

(A) (B)


(e)

5-H

T

Raphe Raphe

500120583m

(A) (B)

(f)





4 Discussion






APP

CA1 CA1

(A) (B)

(a)

CA1

DG

CA1

DG

A12057340

A12057340

(A) (B)

(C) (D)

(b)

CA1

DG

CA1

DG

A12057342

A12057342

(A) (B)

(C) (D)

(c)

Regular MP

TreatmentRegular MP

BACE

1ac

tin

00

02

04

06

08

BACE

1Ac

tin

lowast

(d)

Figure 6 Continued


TreatmentRegular MP

Regular MP

NR2

AN

R2B

00

05

10

15

20N

R2A

NR2

BAc

tin

lowast

(e)

CA1 CA1


Tau

pSer

202

100 120583m

(A) (B)

(f)

TreatmentRegular MP

000

015

030

045

060Regular MP

Actin

lowast

Tau

pSer

262a

ctin

Tau

pSer

262

(g)


40(b) and A120573







Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References


















































APP

CA1 CA1

(A) (B)

(a)

CA1

DG

CA1

DG

A12057340

A12057340

(A) (B)

(C) (D)

(b)

CA1

DG

CA1

DG

A12057342

A12057342

(A) (B)

(C) (D)

(c)

Regular MP

TreatmentRegular MP

BACE

1ac

tin

00

02

04

06

08

BACE

1Ac

tin

lowast

(d)

Figure 6 Continued


TreatmentRegular MP

Regular MP

NR2

AN

R2B

00

05

10

15

20N

R2A

NR2

BAc

tin

lowast

(e)

CA1 CA1


Tau

pSer

202

100 120583m

(A) (B)

(f)

TreatmentRegular MP

000

015

030

045

060Regular MP

Actin

lowast

Tau

pSer

262a

ctin

Tau

pSer

262

(g)


40(b) and A120573







Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References

















































TreatmentRegular MP

Regular MP

NR2

AN

R2B

00

05

10

15

20N

R2A

NR2

BAc

tin

lowast

(e)

CA1 CA1


Tau

pSer

202

100 120583m

(A) (B)

(f)

TreatmentRegular MP

000

015

030

045

060Regular MP

Actin

lowast

Tau

pSer

262a

ctin

Tau

pSer

262

(g)


40(b) and A120573







Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References

















































Regular MP0

1

2

3

4

Treatment

GSH

in se

rum

(120583M

)lowast

(a)

Regular MP

IL-6

in se

rum

(pg

mL)

0

10

20

30

40

Treatment

lowast

(b)

TreatmentRegular MP

Regular MPpp

38p

38

00

02

04

06

08

p38

pp38

Actin

lowastlowast

(c)

COX2

Actin

TreatmentRegular MP

Regular MP

COX2

act

in

000

004

008

012

016

lowast

(d)


GFA

P

(A) (B)

(e)

Figure 7 Continued


Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References

















































Iba-

1

100120583m

(A) (B)


(f)




42 and



42associated with












Acknowledgments


References






















































Acknowledgments


References









































































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ECAM2014-813672

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