Qi Zhao 1, Yimin Niu 1, Kinzo Matsumoto 1*, Koichi Tsuneyama 2, Ken Tanaka 3, Takeshi Miyata 4,5 and Takako Yokozawa 6

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1 Zho et l. BMC Complementry nd Alterntive Medicine 212, 12:188 RESEARCH ARTICLE Open Access Chotosn meliortes cognitive nd emotionl deficits in n niml model of type 2 dietes: possile involvement of cholinergic nd VEGF/PDGF mechnisms in the rin Qi Zho 1, Yimin Niu 1, Kinzo Mtsumoto 1*, Koichi Tsuneym 2, Ken Tnk 3, Tkeshi Miyt 4,5 nd Tkko Yokozw 6 Astrct Bckground: Dietes is one of the risk fctors for cognitive deficits such s Alzheimer s disese. To otin etter understnding of the nti-dementi effect of chotosn (CTS), Kmpo formul, we investigted its effects on cognitive nd emotionl deficits of type 2 dietic d/d mice nd puttive mechnism(s) underlying the effects. Methods: Seven-week-old d/d mice received dily dministrtion of CTS ( mg/kg, p.o.) nd the reference drug tcrine (THA: 2.5 mg/kg, i.p.) during n experimentl period of 7 weeks. From the ge of 9-week-old, the nimls underwent the novel oject recognition test, the modified Y-mze test, nd the wter mze test to elucidte cognitive performnce nd the elevted plus mze test to elucidte nxiety-relted ehvior. After completing ehviorl studies, Western lotting nd immunohistochemicl studies were conducted. Results: Compred with ge-mtched non-dietic control strin (m/m) mice, d/d mice exhiited impired cognitive performnce nd n incresed level of nxiety. CTS meliorted cognitive nd emotionl deficits of d/d mice, wheres THA improved only cognitive performnce. The phosphorylted levels of Akt nd PKCα in the hippocmpus were significntly lower nd higher, respectively, in d/d mice thn in m/m mice. Expression levels of the hippocmpl cholinergic mrker proteins nd the numer of the septl cholinergic neurons were lso reduced in d/d mice compred with those in m/m mice. Moreover, the d/d mice hd significntly reduced levels of vsculogenesis/ngiogenesis fctors, vsculr endothelil growth fctor (VEGF), VEGF receptor type 2, pltelet-derived growth fctor-b, nd PDGF receptor β, in the hippocmpus. CTS nd THA tretment reversed these neurochemicl nd histologicl ltertions cused y dietes. Conclusion: These results suggest tht CTS meliortes dietes-induced cognitive deficits y protecting centrl cholinergic nd VEGF/PDGF systems vi Akt signling pthwy nd tht CTS exhiits the nxiolytic effect vi neuronl mechnism(s) independent of cholinergic or VEGF/PDGF systems in d/d mice. Keywords: Chotosn, Dietes, Cognitive deficits, Cholinergic system, VEGF/PDGF systems * Correspondence: mkinzo@inm.u-toym.c.jp 1 Division of Medicinl Phrmcology, Institute of Nturl Medicine, University of Toym, 263 Sugitni, 263 Sugitni, Toym , Jpn Full list of uthor informtion is ville t the end of the rticle 212 Zho et l.; licensee BioMed Centrl Ltd. This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

2 Zho et l. BMC Complementry nd Alterntive Medicine 212, 12:188 Pge 2 of 18 Bckground Dietes is one of the most importnt risk fctors implicted in cognitive deficits such s Alzheimer s disese (AD) nd vsculr dementi (VD) nd is ssocited with impired cognitive function, including lerning, memory, nd processing speed [1,2]. In previous clinicl study, out 8% of AD ptients ppered to e dietic or to hve norml lood glucose levels nd defects in insulin signling tht were ssocited with ccumultion of the neurofirillry tngles (NFTs) nd senile plques of AD [3]. Similr lerning nd memory deficits hve een demonstrted in d/d mice, n niml model of type 2 dietes tht fils to respond to leptin, 16 kd protein hormone with key role in ppetite, metolism, nd regultion of energy intke nd energy expenditure [4,5]. This niml model exhiits not only hyperglycemi nd hyperinsulinemi ut lso impired cognitive performnce, long-term potentition, nd emotionl ehvior [6]. These deficits hve een reported to ecome evident in dulthood t 1 weeks old nd over. However, the mechnisms underlying cognitive dysfunction in dietes hve not een clerly understood [3]. Chotosn (CTS, or Gouteng Sn in trditionl Chinese medicine) is Kmpo (i.e. Chinese medicine) formul consisting of ten medicinl hers nd gypsum firosum. It hs long een used to tret chronic hedche, pinful tension of the shoulders nd cervicl muscles, vertigo, morning hedche, hevy feeling of the hed, flushing, tinnitus, nd insomni, prticulrly in middle-ged or older ptients with wek physicl constitutions [7]. Moreover, doule-lind nd plceo-controlled clinicl studies [7,8] demonstrted tht CTS is effective in the tretment of stroke ptients with cognitive impirments nd ptients with mild to moderte dementi of the Alzheimer type [9]. Consistent with these clinicl findings, it ws demonstrted tht dily dministrtion of CTS improves cererl flow nd exhiits n nti-hypertensive effect in spontneously hypertensive rts [1,11]. In ddition, we reported tht CTS meliortes cognitive deficits oserved in niml models of vsculr dementi [12,13] nd suggested tht the effects of CTS re medited y meliortion of dysfunction of centrl cholinergic systems, which ply n importnt role in lerning, memory, nd cognitive performnce. These clinicl nd neurophrmcologicl findings rise the possiility tht not only centrl cholinergic systems ut lso fctors/mechnism(s) involved in the lood circultion system my ccount for nti-dementi effects of CTS. Evidence indictes tht the ngiogenic growth fctors VEGF nd PDGF re involved in the dverse vsculr effects of hyperglycemi such s dietic nephropthy nd retinopthy [14,15]. However, retrdtion of ngiogenesis, prticulrly in the rins of ged nimls is severe enough to impir synptic plsticity, moleculr iologicl process importnt in lerning nd memory, nd requires long-lsting increses in metolic demnd supported y the genertion of new cpillries [16]. Indeed, recent findings hve indicted tht VEGF nd PDGF re importnt not only in ngiogenesis ut lso in neuroprotection nd neurogenesis in the rin [16] nd tht elevtion of the levels of these fctors improves cognitive nd emotionl performnce in n niml model of dementi [17-21]. Moreover, in the peripherl system, the protective effect of cholinergic drugs such s donepezil, n cetylcholinesterse inhiitor ginst AD, on ischemic cell dmge ppers to e medited y phosphtidyl inositol-3 phosphte kinse/akt phosphoryltion/vegf systems. We hve recently reported tht CTS dministrtion lso exhiits eneficil effect on cognitive deficits cused y ging, one of the risk fctors for Alzheimer disese (AD) nd cererovsculr disese-relted dementi [22] nd tht meliortion of VEGF/PDGF systems in the rin is likely involved in the effects of CTS [2]. These findings prompted us to investigte whether CTS cn meliorte dietes-relted neuropsychitric symptoms nd, if so, whether cholinergic nd VEGF/ PDGF systems re involved in the ction of CTS in the d/d mice. For this im, we used tcrine, n cetylcholinesterse inhiitor with nti-dementi ctivity, nd compred its effects on dietes-induced cognitive nd emotionl deficits nd neurochemicl ltertions with those of CTS. Methods Animls Mle 6-week-old C57BLKS/J-d/d mice nd their gemtched non-dietic m/m littermtes were purchsed from Jpn SLC Inc. (Hmmtsu, Jpn). The nimls were housed in groups of 6 9 mice/cge ( cm) in lortory niml room, which re mintined t 25 ± 1 C with 65 ± 5% humidity, on 12 h light/drk cycle (lights on: 7:3-19:3) for 1 week efore the strt of the experiments. The nimls were given food nd wter d liitum. The study ws conducted ccording to the experimentl schedule depicted in Figure 1. After 1 week of cclimtiztion, lood smples were collected from the til vein to mesure serum glucose level. The d/d mice were rndomly divided into 5 groups nd then received dily dministrtion of the test drugs, except for one group tht ws used for nxiolytic drug tretment. The drug dministrtion ws continued during the experimentl period. All niml reserch procedures used in the present study were in ccordnce with the Guiding Principles for the Cre nd Use of Animls (NIH Pulictions No. 8-23, revised in 1996). The present study ws lso pproved y the Institutionl Animl Use nd Cre Committee of the University of Toym (pprovl No.: S-29 INM-1).

3 Zho et l. BMC Complementry nd Alterntive Medicine 212, 12:188 Pge 3 of 18 Dily dministrtion of test drugs 7 weeks old Dy 9 weeks old 14 weeks old Biochemicl nd Histochemicl Dy14 Dy 49 nlyses ORT MYT WMT EPMT Blood smpling Behviorl experiments Blood smpling Figure 1 Schemtic drwing of experimentl schedule. After one week of cclimtiztion, the d/d mice were rndomly divided into 5 groups. Age-mtched m/m nd d/d mice were used s controls in the ehviorl nd neurochemicl experiments. Drug tretment Test drug dministrtion ws performed using feeding needle once dily t round 9.m. during the experimentl period. The m/m nd d/d vehicle (control) groups were given wter perorlly (p.o.), while the other groups were orlly dministered CTS dily t doses of 375 nd 75 mg/kg ody weight or injected intrperitonelly with THA (2.5 mg/kg) (Sigm-Aldrich Jpn, Tokyo, Jpn) dissolved in physiologicl sline, except when stted otherwise. Preprtion nd chemicl profiling of CTS extrct CTS used in this study ws purchsed from Tsumur Co. (Tokyo, Jpn) nd ws the sme lot (Lot #224-71) s used in previous studies [13,2]. CTS ws extrcted from mixture of 3. prts Uncrie Uncis cum Rmulus (hooks nd rnch of Uncri rhynchophyll MIQUEL), 3. prts Aurntii Noilis pericrpium (peel of Citrus unshiu MARKOVICH), 3. prts Pinellie tuer (tuer of Pinelli ternte BREITENBACH), 3. prts Ophiopogonis tuer (root of Ophiopogon jponicus KER-GAWLER), 3. prts Hoelen (sclerotium of Pori cocos WOLF), 2. prts Ginseng rdix (root of Pnx ginseng C.A. MEYER), 2. prts Sphoshnikovie rdix (root nd rhizome of Sposhnikovi divrict SCHISCHKIN), 2. prts Chrysnthemi flos (flower of Chrysnthemum morifolium RAMATULLE), 1. prt Glycyrrhize rdix (root of Glycyrrhiz urlensis FISHER), 1. prt Zingieris rhizome (rhizome of Zingier officinle ROSCOE), nd 5. prts Gypsum firosum (CSO 4 2H 2 O). The yield of CTS extrct ws 16.1%. To identify the chemicl constituents of CTS, 3 dimensionl high-performnce liquid chromtogrphy (3D-HPLC) nlysis ws conducted s previously descried [11,13]. Briefly,Chotosn(2.5g, Tsumur, Tokyo, Jpn) ws filtered nd then sujected to HPLC nlysis. HPLC equipment ws controlled with n SLC-1A (Shimdzu, Kyoto, Jpn) using TSKGELODS-8TS column ( mm), eluting with solvents (A).5 M AcONH 4 (ph 3.6) nd (B) CH 3 CN. A liner grdient of 1% A nd % B chnging over 6 min to % A nd 1% B ws used. The flow rte ws controlled with n LC-1 AD pump t 1. ml/min. The eluent from the column ws monitored nd ws processed using n SPD-M1A diode rry. For chemicl profiling of CTS, liquid chromtogrphymss spectrometry (LC-MS) nlysis ws performed with Shimdzu LC-IT-TOF mss spectrometer equipped with n electrospry ioniztion (ESI) interfce. The ESI prmeters were s follows: source voltge +4.5 kv, cpillry temperture 2 C, nd neulizer gs 1.5 l/min. The mss spectrometer ws operted in positive ion mode scnning from m/z 2 to 2. A Wters Atlntis T 3 column (2.1 mm i.d. 15 mm, 3 μm) ws used nd the column temperture ws mintined t 4 C. The moile phse ws inry eluent of (A) 5 mm mmonium cette solution nd (B) CH 3 CN under the following grdient conditions: -3 min liner grdient from 1% to 1% B, 3-4 min isocrtic t 1% B. The flow rte ws.15 ml/min. Mss spectrometry dt otined from the extrct hve een listed in MssBnk dtse [23] nd stored together with the phrmcologicl informtion on the extrct in the Wkn-Yku Dtse system (WknDB ID: LCMS: Chotosn/111 wknd.u-toym.c.jp/wiki/lcms:chotosn/111), Institute of Nturl Medicine, University of Toym. Voucher specimen (CTS: No ) otined from Tsumur Co. Ltd. hs een deposited t our institute. Mesurement of serum glucose Before nd fter completing the ehviorl studies, serum smples were collected from til vein of ech niml group under pentoritl (5 mg/kg, i.p.) nesthesi. The serum glucose levels were mesured using commercilly ville kit (Glucose CII-Test, Wko Pure Chem., Osk, Jpn) s previously descried [5]. Behviorl ssessment Novel oject recognition test (ORT) ORT ws conducted s previously descried [13,2,21]. The pprtus consisted of squre ren (5 5 4 cm) mde of polyvinyl chloride with gry wlls nd lck floor (Figure 2A). The ojects for recognition hd visul ptterns or visully different shpes to enle discrimintion. The ORT consisted of smple phse tril nd test

4 Zho et l. BMC Complementry nd Alterntive Medicine 212, 12:188 Pge 4 of 18 A) Oject recognition test Drugs dministrtion A1 A2 A1 3-6 min Smple trils 3 min 3 min A1 F Test trils N B) Smple phse Time exploring oject (s) m/m vehicle oject A1 oject A2 d/d vehicle (mg/kg/dy) Smple tril Test tril CTS THA C) Test tril Fmilir oject (F) New oject (N) D) Discrimintion index (DI=(N-F)/(N+F) Time exploring oject (s) m/m *** vehicle vehicle d/d 375 CTS 75 * ** 2.5 THA (mg/kg/dy) Discrimintion index m/m ** vehicle vehicle 375 d/d CTS * * THA (mg/kg/dy) Figure 2 Effects of CTS nd THA on oject discrimintion performnce of d/d mice in the ORT. The ORT ws conducted on dys fter strting drug dministrtion. Ech dtum represents the men ± S.D. (6 9 mice per group). (A) The dt from the smple trils of the ORT. The niml ws plced into the ren where two identicl smple ojects mde of glss (ojects A1 nd A2) were plced in two djcent corners of the ren nd ws llowed to explore for five minutes. (B) The dt from the test phse trils conducted ten minutes fter the smple phse trils. In the test phse trils, the time nimls spent exploring fmilir oject or new oject ws mesured during 5-minute oservtion period. ***P<.1 nd **P<.1 vs. the time spent exploring fmilir oject (pired t-test). (C) Discrimintion index (DI) in the ORT. DI ws clculted s descried in the text. **P < =.1 vs. vehicle-treted m/m group (t-test). *P <.5 vs. vehicle-treted d/d group Tukey test. phse tril. In the smple phse tril, ech mouse ws first plced in the oservtion ox where two identicl ojects, ojects O1 nd O2 (ech of which ws cm white cup), were plced seprtely, nd llowed to explore the ren freely for 1 min. The totl time tht the mouse spent exploring ech of the two ojects ws mesured nd then the mouse ws returned to the home cge. In the test phse tril performed 3 min fter the smple phse tril, one of the two ojects ws replced y n identicl copy (oject F) nd the other y novel oject (oject N). Performnce of the nimls in this test ws video-recorded for lter nlysis. In these trils, the explortion of n oject ws defined s directing the nose to the oject t distnce of <2 cm ccording to previous reports [13,21,24] nd the time spent exploring ech of the two ojects ws nlyzed using SMART W ver. 2.5 (PnL, S.L., Brcelon, Spin) with tri-wise module to detect the hed, center mss, nd setil. A discrimintion index (DI) ws clculted ccording to the following eqution [13,21,24]: DI = (T n T f )/(T n + T f ). Here, T n nd T f represent the times spent exploring new nd fmilir ojects, respectively. The ox ren nd ojects were clened using 75% ethnol etween trils to prevent uild-up of olfctory cues. Modified Y-mze test (MYM) A modified version of the Y-mze test [25] ws used s previously descried [26]. The pprtus used for the MYM test ws equivlent to the one used in the originl Y-mze test nd it consisted of lck polypropylene wlls with 3 rms ech 4 cm long, 12 cm wide t the top, 3 cm wide t the white ottom, nd 18 cm high. This test ws two-tril tsk with smple phse tril nd test phse tril tht were seprted y n inter-tril intervl (Figure 3A). In the smple phse tril, ech mouse ws individully plced in the mze with one of the 3 rms closed. The nimls were llowed to explore the other 2 rms freely for 1 min. Test phse tril ws conducted 3 min fter the smple phse tril. In the

5 Zho et l. BMC Complementry nd Alterntive Medicine 212, 12:188 Pge 5 of 18 A) Modified Y- mze test closed rm novel rm Drug dministrtion Smple trils Test trils 3 min 3-6 min 3 min Smple tril (1 min) Test tril (1 min) B) Time in novel rm C) Locomotor ctivity % Time in novel rm m/m ### d/d 33% veh veh (mg/kg/dy) CTS * * THA Totl distnce (cm/5 min) m/m # d/d veh veh (mg/kg/dy) Figure 3 Dietes-induced sptil working memory deficit in the MYM test nd its reversl y test drugs. A) Schemtic drwings of the Y-mze nd the experimentl procedure. The mze ws surrounded y different sptil cues. A) Experimentl protocol of the MYM test. The smple tril nd test trils were conducted for 5 min t 3-min intervl. (B) Ech test ws conducted 3 min (THA) or 6 min fter dily orl (CTS) or i.p. (THA) dministrtion of test drugs. % Time spent in novel rm (B) nd totl locomotion distnce during 5-min oservtion period were nlyzed. Ech dt column represents the men ± S.D. (n = 6 9). #P<.5 nd ###P<.1 compred with vehicle-treted m/m group. *P<.5 compred with vehicle-treted d/d group (Tukey test). CTS THA test phse tril, the niml ws gin plced in the mze with ll 3 rms opened, nd llowed to explore the rms freely. In the test phse tril performed 3 min fter the smple phse tril, the closed rm tht ws opened in the test phse tril ws defined s the new rm. The rms nd ottom were clened using 75% ethnol etween trils to prevent uild-up of olfctory cues. The niml ehvior ws video-recorded for lter nlysis. Percent time spent in the new rm nd numers of totl rm entries were nlyzed using the SMART W ver. 2.5 system (PnL, S.L., Brcelon, Spin). Morris wter mze test (MWM) The MWM test ws performed y slightly modifying the protocol dopted in previous studies [5]. Briefly, the MWM took plce during the first hlf of the light phse. The test ws performed in circulr pool (11 cm dimeter, height 3 cm) filled with wter mde opque with non-ft milk nd mintined t 26 ± 1 C nd circulr escpe pltform (11 cm dimeter) ws sumerged 1 cm elow the wter surfce. Cues were hung t four loctions t the north, west, south, nd est corners of the swimming pool wll. In the first dys, the nimls were sujected to visile tril (Visile 1) of the wter mze in which the pltform ws mde visile 1 cm ove the wter surfce. Trining trils were performed dily for 5 dys 1 dy fter the visile tril. Mice received 3 trils per dy s trining trils. Ech tril lsted until the niml found the pltform, or for mximum oservtion period of 12 sec for m/m nd d/d groups; nimls tht filed to find the pltform within the mximum oservtion period were guided there y the experimenter. In ech tril, they were plced into the pool, fcing the wll, with strt loctions vried pseudo-rndomly. One dy fter the lst cquisition trining session, nimls were tested in single 12-sec proe tril without the pltform. For ech tril, the ltency to rech the pltform (escpe ltency), moved distnce, nd men swim speed were recorded vi video cpture nd imge nlysis using the SMART W system (PnL, S.L., Brcelon, Spin). Elevted plus mze test (EPM) The EPM test ws conducted y slightly modifying the protocol descried in our previous reports [2,21]. Briefly, the EPM ws comprised of two open rms (22 8 cm) nd two rms enclosed y high wlls ( cm), with n open roof, the two rms of ech type eing positioned opposite to ech other s previously descried. The mze ws set 6 cm ove the floor. Ech mouse ws individully plced t the center of the mze fcing one of the enclosed rms nd llowed to explore the mze freely during 1-min oservtion period. In this experiment, dizepm (1 mg/kg) ws used s stndrd nxiolytic drug nd ws dministered intrperitonelly to the gemtched nïve d/d mice 3 min efore the test. Mze performnce ws video-recorded for lter nlysis. Time

6 Zho et l. BMC Complementry nd Alterntive Medicine 212, 12:188 Pge 6 of 18 spent in open rms nd totl locomotion distnce in the mze were nlyzed s indices of emotionl ehvior using the SMART W system (PnL, S.L., Brcelon, Spin). Western lot nlysis Western lotting ws performed y slightly modified version of method descried previously [5,2,21,26]. Briefly, hippocmpl tissues were dissected from the rin nd proteins were extrcted y homogeniztion in protein lysis uffer (5 mm Tris (ph 7.4), 15 mm NCl,.5% sodium deoxycholte, 1%(v/v) NP-4,.1% (v/v) sodium dodecyl sulfte (SDS), 15 mm NF, 8.12 μg/ml protinin, 2 mm sodium orthovndte, 1 μg/ml leupeptin, nd 2 mm phenylmethylsulfonyl fluoride using TissueLyser W (Qigen, Osk, Jpn). Lyste smples were centrifuged t 1, rpm (4 C) for 5 min, nd the superntnts were centrifuged for protein concentrtion ssy. The protein concentrtion ws determined using BCA protein ssy kit (Thermo Scientific, Rockford, IL, U.S.A.) nd microplte reder (Sunrise Clssic; TECAN Jpn, Kwski, Jpn). Totl protein (15 μg) prepred from ech smple ws electrophoresed on 7.5% SDS-polycrylmide gel nd then electro-lotted onto polyvinylidene difluoride memrne (Cler Blot Memrne-p; ATTO). The memrnes were incuted in 5% non-ft milk-contining wsh uffer (5 mm Tris (ph 7.5), 15 mm sodium chloride, nd.1% Tween 2) for 1 h t room temperture. They were then proed with specific ntiody of interest t 4 C for 24 h. After the memrnes were rinsed in TBS-T, the lots were incuted with ovine nti-got IgG secondry ntiodies linked with horserdish peroxidse (Snt Cruz Biotechnology, CA, USA) or nti-rit or nti-mouse secondry ntiodies linked with horserdish peroxidse (DkoCytomtion EnVision + System-HRPleled Polymer) (Dko Cytomtion, Inc., Crpinteri, CA, USA) ccording to the mnufcturer s instructions. The immune complexes were detected y the enhnced chemiluminescence method (Immoilon W Western Chemiluminescent HRP Sustrte) (Millipore, Temecul, CA, USA) nd imged using Lumino Imge Anlyzer LAS-4 (Fujifilm, Tokyo, Jpn). Bnd imges were nlyzed y VH-H1A5 softwre (Keyence, Osk, Jpn). The quntity of immunorective nds ws normlized y compring with their expression levels in tretment-nïve control mice. The dilution rtios nd commercil sources of the ntiodies used were s follows: nti-choline cetyltrnsferse (ChAT) got polyclonl ntiody (AB-144P; 1:3 dilution) (Millipore, CA, USA); nti-β-ctin mouse monoclonl ntiody (8226; 1:1, dilution) nd nti-pdgfr-α rit polyclonl ntiody (61219; 1:5 dilution) (Acm, Tokyo, Jpn); nti-akt (pn) (C67E7) rit monoclonl ntiody (#4691; 1:1 dilution), nti-pdgfr-β (28E1) rit monoclonl ntiody (#3169; 1:5 dilution), nti-phospho-akt (Ser473) rit polyclonl ntiody (#46; 1:5 dilution), nti-phospho- PKCα/βII (Thr638/641) rit polyclonl ntiody (#9375; 1:1 dilution), nti-pkcα (C67E7) rit polyclonl ntiody (#256; 1:1 dilution) (Cell Signling Technology, Dnvers, MA, USA); nti-muscrinic ACh receptor rit polyclonl ntiodies (1:5 dilution) (M1: sc-916; M3: sc- 918; M5: sc-916), nti-pdgf-a (E-1) mouse monoclonl ntiody (sc-9974; 1:1 dilution), nti-pdgf-b (H- 55) rit polyclonl ntiody (sc-7878; 1:1 dilution), nd nti-vegf (A-2) rit polyclonl ntiody (sc-152; 1:1 dilution), (Snt Cruz Biotechnology, Snt Cruz, CA, USA); nd nti-vegfr2 rit polyclonl ntiody (A-951; 1:5 dilution) (Signlwy Antiody, USA). Immunohistochemistry CTS dministrtion-induced chnges in expression levels of ChAT in the medil septum nd VEGF in the reti of m/m nd d/d mice were lso exmined y immunohistochemicl nlysis [5]. Mice were fixed y intrcrdic perfusion with 4% prformldehyde in phosphte-uffered sline (PBS) under nesthesi. Brin nd retinl tissues were post-fixed with 4% prformldehyde overnight t 4 C. A series of 5 μm sections were otined y using sliding microtome (IVS-41, SAKURA Finetek Jpn, Tokyo, Jpn). The prffinemedded specimens were deprffinized in xylene nd dehydrted with ethnol. Endogenous peroxidse ws locked with.1% hydrogen peroxide-methnol for 3 minutes t room temperture. Wshed with Trisuffered sline (TBS), the specimens were incuted in microwve oven (25 W, 4 s on nd 3 s off, MI-77, Azumy, Tokyo, Jpn) in trget retrievl solution (Dko, Denmrk) for 15 minutes nd then wshed with distilled wter nd TBS. Nonspecific inding ws locked y tretment with specil locking regent (Dko, Denmrk) for 15 minutes. For stining of ChAT positive cells in the medil septum, the specimens were chllenged with 1:4 dilution of got nti-chat polyclonl ntiody (AB-144P: Millipore, CA, USA) nd then incuted in moist ox t 4 C overnight. Wshed with TBS, the specimens were incuted with peroxidse-conjugted ovine nti-got IgG (Snt Cruz Biotechnology, Snt Cruz, CA, USA). After three wshes in TBS, rection product ws detected with 3,3 -diminoenzidine tetrhydrochloride (.25 mg/ml) nd hydrogen peroxide solution (.1%). Counter-stined with hemtoxylin, the sections were rinsed, dehydrted nd covered. Also included in ech stining run were negtive controls in which the primry ntiody ws omitted. The imges were cptured with microscope (AX-8, Olympus, Jpn). For stining of VEGF positive cells in the retin, the specimens were chllenged with

7 Zho et l. BMC Complementry nd Alterntive Medicine 212, 12:188 Pge 7 of 18 1:4 dilution of nti-rit VEGF (A-2) polyclonl ntiody (sc-152: Snt Cruz Biotechnology, Snt Cruz, CA, USA) in trget retrievl solution (Dko, Denmrk), incuted in microwve oven (25 W, 4 s on nd 3 s off, MI-77, Azumy, Tokyo, Jpn) for 15 minutes, nd then incuted in moist ox t 4 C overnight. After wshing the specimens with TBS, they were incuted with Alex Fluor W 488-conjugted got nti-rit IgG ntiodies (A118; 1:4 dilution, Invitrogen, Tokyo, Jpn) for 3 min t room temperture. Fluorescent imges were cptured using fluorescent microscope (AX-8, Olympus, Tokyo, Jpn). Sttisticl nlysis Sttisticl nlysis of the dt ws conducted ccording to Currn-Everett nd Benos [27]. All dt re expressed s the men ± S.D. The ehviorl dt were nlyzed using pired Student s t-test or one-wy repeted mesure ANOVA or two-wy repeted mesure ANOVA followed y the Tukey test for multiple comprisons mong different groups. The neurochemicl dt were compred using unpired Student s t-test or one-wy ANOVA followed y the Tukey test for multiple comprisons. Differences of P <.5 were considered significnt. Results Behviorl studies CTS- nd THA-induced meliortion of non-sptil cognitive deficits of d/d mice in ORT We first evluted the non-sptil cognitive performnce of d/d mice using the ORT, which ssesses short-term memory s indicted y explortion time. Anlysis of performnce in the smple phse tril of the ORT reveled no differences in totl time exploring two ojects etween ech group of m/m nd d/d mice [t =.951, df = 15, P=.356, t-test] [F(3, 23) = 1.914, P=.156, one-wy ANOVA] (Figure 2B). These results role out the possiility tht dietic stte cused deficits of motivtion nd sensory motor function. However, in the test phse tril conducted 3 min fter the smple phse tril, the m/m mice spent significntly longer time exploring new oject thn exploring fmilir oject [t = , df = 7, P=.1, pired t-test] nd exhiited cler preference for the novel oject, wheres d/d mice did not show significnt difference [t =.816, df = 8, P=.438, pired t-test]. On the other hnd, d/d mice treted with CTS (75 mg/kg per dy, p.o.) nd THA (2.5 mg/kg per dy, p.o) spent significntly longer exploring the novel oject thn exploring fmilir oject [CTS: t = , df = 5, P=.15, pired t-test nd THA: t = , df = 5, P=.2, pired t-test] (Figure 2C). These dt demonstrted tht vehicletreted d/d mice filed to discriminte novel oject (vs. m/m group: t = 2.96, df = 15, P =.1, t-test), wheres CTS- nd THA-treted d/d mice were clerly le to discriminte the two ojects. In fct, the post hoc nlysis following one-wy ANOVA [F(3,23) = 4.62, P=.19] reveled tht the CTS- nd THA-treted d/d groups hd DI significntly higher thn the vehicle-treted d/d group (Figure 2D). Effect of CTS on working memory performnce of d/d mice in the MYM test A MYM test ws used to evlute the effect of CTS on sptil working memory in d/d nimls. As shown in Figure 3, the rtio of the time tht vehicle-treted control mice spent visiting the novel rm to the time spent visiting the other two fmilir rms ws significntly higher thn the chnce level of 33.3%, indicting preference for the novel rm over the fmilir rms. The vehicle-treted d/d mice spent significntly shorter time exploring the novel rm thn the m/m mice [t = 4.175, df = 15, P=.1, t-test]. In contrst, the d/d mice treted with CTS nd THA spent significntly longer time exploring the novel rm thn the vehicle-treted group [drug tretment of d/d groups: F(3,23) = 4.224, P=.16; vehicle group vs. CTS 375 mg/kg per dy group, P=.58; vehicle group vs. CTS 75 mg/kg per dy group, P=.46; vehicle group vs. THA 2.5 mg/kg per dy group, P=.17, one-wy ANOVA] (Figure 3B). Locomotor ctivity of nimls mesured s totl distnce in the MYM test ws significntly shorter in the d/d mice thn in the m/m mice [t = 3.647, df = 15, P=.2, t-test], ut the ctivity of d/d mice ws not significntly ffected y CTS or THA dministrtion [F drug (3,23) = 2.275, P=.17, one-wy ANOVA] (Figure 3C). Effect of CTS on sptil lerning nd memory performnce of d/d mice in the MWM test Consistent with our previous report [5], d/d mice showed significntly impired sptil lerning nd memory performnce in the MWM test. In the visile tril conducted on the first dy, there ws no significnt difference in the time spent finding the pltform etween the two groups, eliminting the possiility of motivtionl or sensory motor deficits. The vehicle-treted m/m nd d/d groups could lern the loction of the sumerged pltform following repeted dily trining [F trining (4,6) = 17.72, P<.1, two-wy repeted mesure ANOVA] (Figure 4-A1), ut there ws significnt difference in lerning performnce etween two niml groups [F niml (1,15) = P<.1, two-wy repeted mesures ANOVA]. The escpe ltency of the control d/d group ws significntly greter thn tht of the control m/m group [F niml trining (4,6) = 5.385, P<.1, two-wy repeted mesure ANOVA]. Additionlly, the control d/d mice group displyed significntly longer swimming distnce to find the pltform

8 Zho et l. BMC Complementry nd Alterntive Medicine 212, 12:188 Pge 8 of 18 Escpe ltency (s) A) 1) Escpe ltency m/m + vehicle ### d/d + vehicle d/d + CTS (375) ** ** d/d + CTS (75) 12 d/d + THA (2.5) Visile Trining (Block of 3 trils/dy) Swimming speed (cm/s) ) Swimming speed visile ### m/m + vehicle d/d + vehicle Trining (Block of 3 trils/dy) % Time in trget qudrnt m/m Veh. Veh. 375 d/d CTS THA (mg/kg/dy) Figure 4 Effects of CTS nd THA on wter mze performnce of d/d mice. Lerning performnce of the nimls ws nlyzed in the trining test y ltency (A) nd swimming speed (B). Ech dt point indictes the men ± S.D. for 6 9 nimls in ech group. ### P<.1 vs. vehicle-treted m/m group, nd ** P<.1 vs. vehicle-treted d/d group (one-wy repeted mesures ANOVA). Memory retrievl performnce (D) elucidted in the proe test. Ech dtum represents the men of time spent in the trget qudrnt ± S.D. ### P<.1 vs. vehicle-treted m/m group (t-test). * P <.5 vs. respective vehicle-treted d/d group (one-wy repeted mesure ANOVA followed y Tukey test). B) # * * [F distnce (1,15) = 45.64, P<.1, two-wy repeted mesure ANOVA] (dt not shown) nd swimming speed to find the pltform [F niml (1,15) = , P<.1, two-wy repeted mesures ANOVA] in the trining trils (Figure 4-A2). These findings indicte tht d/d mice hd not only lerning nd memory dysfunction ut lso motor dysfunction. We lso exmined the effect of CTS nd THA on sptil cognitive performnce of d/d mice in the MWM test. The meliorting effects of THA (2.5 mg/kg per dy, i.p.) tretment significntly improved the reduced escpe ltency during the oservtion period in the d/d niml group [F drug (1,13) = P <.1, two-wy repeted mesure ANOVA] [F trining (4,52) = , P<.1, twowy repeted mesure ANOVA] [F drug trining (4,52) = 3.591, P=.12, two-wy repeted mesure ANOVA] nd swimming distnce to find the pltform [F drug (1,13) = , P<.1, two-wy repeted mesure ANOVA] in the trining tests (Figure 4-A1) without ffecting the swimming speed of this niml group [F drug (1,13) = 2.942, P=.11, two-wy repeted mesure ANOVA] (Figure 4-A2). Interestingly, dily tretment of d/d mice with 75 mg/kg CTS lso resulted in significnt decrese in escpe ltencies of these niml groups [F drug (1,13) = , P<.1, two-wy repeted mesure ANOVA](Figure 4-1), nd [F drug (4,52) = 8.73, P<.1, two-wy repeted mesure ANOVA] swimming distnce [F drug (1,13) = , P<.1, two-wy repeted mesure ANOVA] (dt not shown), while CTS dministrtion hd no effect on swimming speed of d/d niml groups [F drug (1,13) = 2.355, P=.149, two-wy repeted mesure ANOVA] (Figure 4-A2). In ddition, CTS 375 mg/kg per dy dministrtion led to no significnt chnges in the trining [F drug (1,13) = 1.31, P=.273, two-wy repeted mesure ANOVA] [F trining (4,52) = 5.711, P<.1, two-wy repeted mesure ANOVA] [F drug (1,13) =.132, P=.722, two-wy repeted mesure ANOVA], in the swimming distnce (dt not shown) nd swimming speed [F drug (1,13) =.599, P=.453, two-wy repeted mesure ANOVA] (Figure 4-A2). In the proe test conducted 1 dy fter the 5-dy trining period, swimming time of the d/d control mice in the trget qudrnt where the pltform hd een locted during trining ws significntly shorter thn tht of the m/m mice [t = 6.682, df = 15, P<.1, t-test]. The d/d groups treted with dily dministrtion of CTS ( mg/kg per dy nd THA (2.5 mg/kg per dy) spent significntly more time in the trget qudrnt during the oservtion period thn d/d mice treted with vehicle [F drug (3,23) =14.231, P<.1, one-wy ANOVA] (Figure 4C). Effect of CTS on emotionl performnce of d/d mice in EPM test The EPM test ws conducted to evlute the effect of CTS on nxiety-relted ehvior of d/d mice y using

9 Zho et l. BMC Complementry nd Alterntive Medicine 212, 12:188 Pge 9 of 18 dizepm s reference nxiolytic drug. The vehicletreted d/d mice showed significntly decresed percentge of time spent in open rms during 1-min oservtion period compred with the m/m controls (t = 5.82, df = 15, P<.1, t-test) (Figure 5A). In contrst, drug tretment of d/d mice significntly ltered EPM ehvior [F drug (3,23) = 3.32, P=.38, one-wy ANOVA]. In fct, the d/d mice tht cutely received single dose of 1. mg/kg dizepm efore the test spent significntly more time in the open rms thn the vehicle-treted d/d mice. The CTS-treted d/d mice lso spent slightly ut significntly longer in the open rms thn the vehicle-treted d/d group (Figure 5A), wheres THA tretment hd no effect on the emotionl performnce of d/d mice. The totl locomotion distnce in the mze ws significntly less in the vehicle-treted d/d mice thn in the vehicle-treted m/m mice [t = 9.119, df = 15, P=.1, t-test], ut the decrese of locomotor ctivity of the d/d mice ws not ffected y the drug dministrtion [F drug (3,23) =.2935, P=.83, one-wy ANOVA] (Figure 5C). Indeed, totl locomotion distnces nd swimming speeds of this dietic niml model in MYT nd WMT, respectively, were out 5% less thn those of the m/m. Biochemicl studies Effects of CTS nd THA on serum glucose in dietic d/d mice Body weights nd serum glucose levels of ech niml group were mesured efore nd fter completion of the ehviorl ssessments. As summrized in Tle 1, oth ody weights nd glucose levels were significntly higher in the d/d mice thn those in the ge-mtched m/m non-dietic control mice. THA (2.5 mg/kg per dy, i.p.) or CTS (375 7 mg/kg per dy, p.o.) hd no effect on these prmeters of d/d mice. Reduced AKT phosphoryltion nd incresed PKCα phosphoryltion in the d/d mice re reversed y CTS nd THA dministrtion To understnd the moleculr mechnism(s) underlying CTS-induced improvement of cognitive deficits in d/d mice, we first exmined the effects of CTS on type 2 dietes-relted AKT nd PKC signling y mesuring unphosphorylted nd phosphorylted levels of AKT nd PKCα in the hippocmpus (Figure 6). No differences in the hippocmpl AKT nd PKCα levels were found etween the vehicle-treted m/m nd d/d mice (Akt: t =.59, df = 8, P=.571; PKCα: t = -.92, df = 8, P=.394), wheres the expression levels of phosphorylted AKT nd PKCα in the control d/d group were significntly decresed (p-akt: t = 4.69, df = 8, P=.2) nd incresed (p-pkcα/βii: t = , df = 8, P<.1), respectively, compred with the levels in the vehicletreted m/m mice. Neither CTS nor THA tretment hd ny effect on the expression levels of these signling fctors in the d/d mice. On the other hnd, repeted dministrtion of CTS nd THA significntly incresed the level of p-akt [F drug (3,16) = , P<.1, one-wy ANOVA] nd decresed the level of phosphorylted A) B) % Time spent in open rms Veh. 8 # # Veh. * CTS (325) * CTS (75) THA (2.5) DZ* (1.) Totl distnce (cm/5 min) Veh. Veh. CTS (325) CTS (75) THA (2.5) DZ* (1. ) m/m d/d Figure 5 Effects of CTS dministrtion on EPM performnce of d/d mice. The nimls received n EPM test t round the ge of 13 weeks old. The 7-week-old d/d mice were orlly dministered wter (vehicle), mg/kg CTS, 2.5 mg/kg THA once dily during the experimentl period. The EMT ws conducted 1 hr fter CTS dministrtion. The ge-mtched nïve d/d mice received i.p. injection of 1 mg/kg dizepm 3 min efore the test. Ech dtum represents the men ± S.D. (6 9 nimls per group). The proportion of time spent in open rms (A) nd the totl locomotion distnce on the mze (B) were clculted. The dt re expressed s the men ± S.D. ###P<.1 vs. vehicletreted m/m group (Student s t-test). *P<.5, **P<.1 vs. vehicle-treted d/d group (one-wy ANOVA followed y Tukey test). m/m d/d

10 Zho et l. BMC Complementry nd Alterntive Medicine 212, 12:188 Pge 1 of 18 Tle 1 Effect of CTS nd THA tretment on ody weight nd serum glucose level in d/d mice 7 weeks old 14 weeks old m/m d/d m/m d/d Vehicle Vehicle CTS (375) CTS (75) THA (2.5) Body weight (g) 2.6 ± ± ± ± ± ± ±2.1 Serum glucose (mg/dl) ± ± ± ±17.4# 42.8 ± ± ± 67.1 Body weights nd serum glucose levels were first mesured t the ge of 7 weeks old just efore strting drug dministrtion. The d/d mice were rndomly seprted into 4 groups nd underwent dily dministrtion of vehicle, 375 mg/kg nd 75 mg/kg CTS, or 2.5 mg/kg THA. After completion of the ehviorl study, the ody weight nd serum glucose levels of ech niml were gin mesured t the ge of 14 weeks old. CTS nd THA represent chotosn extrct nd tcrine, respectively. Ech dtum represents the men ± S.D. (n = 6 9). ### P <.1 vs. respective 7w-m/m or 14w-m/m group (t-test). PKCα/βII [F drug (3,16) = 7.624, P<.2, one-wy ANOVA] in d/d mice (Figure 6). No significnt difference in β-ctin expression in the hippocmpus ws oserved etween vehicle-treted m/m nd d/d groups (t = 1.629, df = 8, P=.142). No effect of CTS or THA ws found in terms of the β-ctin level in the hippocmpus [F drug (3,16) =.68,P=.62, one-wy ANOVA]. Effects of CTS nd THA on the hippocmpl expression levels of VEGF nd PDGF nd their receptors in d/d mice Since the VEGF/PDGF systems hve een shown to ply ngiogenic nd neurotrophic roles in the centrl nervous system nd their function declines with ging [28-3], we evluted the effects of the CTS tretment on the hippocmpl VEGF/VEGFR2 nd PDGF/PDGFR systems in d/d mice. Western lotting nlysis (Figure 7) reveled tht, compred with the vehicle-treted m/m mice, the vehicle-treted d/d mice showed reduced levels of VEGF-A (t = 3.881, df = 8, P=.5, t-test), VEGFR2 (t = 3.233, df = 8, P=.12, t-test), PDGFR-α (t = 2.855, df = 8, P=.21, t-test), PDGF-B (t = 4.73, df = 1, P=.2, t-test), nd PDGFR-β (t = 3.541, df = 8, P=.8, t-test). The level of PDGF-A lso showed tendency to e lower in the d/d group thn in the m/m A) Akt 6 kd p-akt 6 kd PKCα 8 kd m/m control d/d control c d/d + CTS (375) p-pkcα β-ctin 8 kd 42 kd d d/d + CTS (75) e d/d + THA (2.5) B) 2 Akt c d e p-akt PKCα p-pkcα β-ctin Reltive expression level ( % of norml control) ## *** *** ### *** * c d e c d e c d e c d e c d e Figure 6 Effects of CTS nd THA tretment on Akt, p-akt, PKCα, p-pkcα/βii, nd β-ctin in the hippocmpi of d/d mice. After completing the ehviorl studies, the nimls were decpitted nd proteins were extrcted from the hippocmpi in ech niml group. A) Typicl photos indicting the expression levels of ech fctor in the hippocmpus of vehicle-treted m/m (lne ), nd vehicle (lne )-, CTS (325 mg/kg per dy: lne c; 75 mg/kg per dy: lne d)-, nd THA (2.5 mg/kg per dy; lne e)-treted d/d mice. B) Quntittive comprisons of test drug-induced chnges in Akt, p-akt, PKCα, p-pkcα/βii, nd β-ctin in the hippocmpi of d/d mice. The dt re expressed s the percentge of the vlue otined from nïve control m/m mice. Ech dt column represents the men ± S.D. otined from 5-6 rin smples. #P<.5 or ##P<.1 vs. vehicle-treted SAMR1 group (Student s t-test). ##P<.1, *P<.5, ***P<.1 vs. respective vehicle-treted d/d group (one-wy ANOVA followed y Tukey test).

11 Zho et l. BMC Complementry nd Alterntive Medicine 212, 12:188 Pge 11 of 18 A) VEGF-A VEGFR2 42 kd 18 kd PDGF-A 31 kd m/m control PDGFR-α PDGF-B 18 kd 43 kd c d d/d control d/d + CTS (375) d/d + CTS (75) PDGFR-β 19 kd e d/d + THA (2.5) B) Reltive expression level ( % of norml control) VEGF ## β-ctin ** ** c d e c d e VEGFR2 # * * c d e PDGF-A c d e 42 kd PDGFR-α # c d e PDGF-B ## * * c d e PDGFR-β ## *** * c d e Figure 7 Effects of CTS nd THA on VEGF/VEGFR2 nd PDGF/PDGFR expression in the hippocmpus of d/d mouse. A) Typicl photos indicting the expression levels of ech fctor in the hippocmpus of vehicle-treted m/m (lne ), nd vehicle (lne )-, CTS (325 mg/kg per dy: lne c; 75 mg/kg per dy: lne d)-, nd THA (2.5 mg/kg per dy; lne e)-treted d/d mice. B) Quntittive comprisons of ech fctor mong different niml groups were conducted s descried in the text. The dt re expressed s the percentge of the vlue otined from nïve control m/m mice. Ech dt column represents the men ± S.D. otined from 5 6 rin smples. #P <.5, ##P <.1 vs. vehicle-treted m/m group (Student s t-test). *P <.5, **P <.1, ***P <.1 vs. respective vehicle-treted d/d group (one-wy ANOVA followed y Tukey test). group ut the difference etween the two groups ws insignificnt (t = 1.793, df = 8, P=.111, t-test). The CTS nd THA dministrtions significntly up-regulted the expression levels of VEGF-A [F drug (3,16) = 9.998, P<.1, one-wy ANOVA], VEGFR2 [F drug (3,16) = 5.267, P=.1, one-wy ANOVA], PDGF-B [F drug (3,2) = 4.233, P=.18, one-wy ANOVA], nd PDGFRβ [F drug (3,16) = 7.898, P =.2, one-wy ANOVA] in the d/d group. These drug tretments filed to reverse the down-regulted expression of PDGFRα in the d/d mice [F drug (3,16) =.831, P=.496, one-wy ANOVA]. Immuno-histochemicl nlysis ws lso conducted to know whether CTS nd THA tretments lter the expression level of VEGF in the retinl tissue of d/d mice since VEGF hs een implicted s mjor contriutor to the development of dietic complictions such s dietic retinopthy [31,32]. As shown in Figure 8, VEGFpositive cells were clerly oserved in pigment epithelium nd inner segment lyers. Dietic d/d mice exhiited hyperplsi in inner nd outer nucler lyers, inner segments, nd outer plexiform lyers. They lso showed incresed expression of VEGF, prticulrly in inner segments nd outer plexiform lyers. However, no cler difference in the retinl morphology or the VEGF stining level ws oserved etween vehicle-treted groups nd CTS- or THA-treted d/d groups. Effects of CTS nd THA on downregulted expression of hippocmpl cholinergic mrker proteins in d/d mice Since the centrl cholinergic systems ply n importnt role in lerning nd memory function, we next evluted the effects of CTS nd THA tretment on the expression levels of cholinergic mrker proteins in the hippocmpi of d/d mice. Western lotting nlysis (Figure 9) reveled tht, compred with the control m/m mice, the vehicletreted dd group hd significntly reduced levels of ChAT (t = 5.74, df = 8, P<.1, t-test), M 1 AChR (t = 4.951, df = 8, P=.2, t-test), M 3 AChR (t = , df = 8, P<.1, t-test), nd M 5 AChR (t = 8.76, df = 8, P<.1, t-test). However, dministrtions of CT nd THA significntly up-regulted the expression levels of ChAT [F drug (3,16) = , P<.1, one-wy ANOVA], M 1 AChR [F drug (3,16) = 8.689, P=.1, one-wy ANOVA], M 3 AChR [F drug (3,16) = , P<.1, onewy ANOVA], nd M 5 AChR [F drug (3,16) = 8.887, P=.1, one-wy ANOVA] in d/d groups.

12 Zho et l. BMC Complementry nd Alterntive Medicine 212, 12:188 Pge 12 of 18 1) () () 2) (c) (d) 5µm 3) 4) Figure 8 Effects of CTS nd THA on VEGF expression in the retinl tissue in d/d mice. The retinl tissues were otined from the nimls perfused with prformldehyde s descried in the text. The rrows,, c, nd d represent pigment epithelium, outer segments, inner segments, nd outer plexiform lyers, respectively. VEGF-positive portions were identified y VEGF immunostining. Photos 1, 2, 3, nd 4 were from the retinl tissues of m/m control, vehicle-treted d/d, CTS (75 mg/kg per dy)-treted d/d, nd THA (2.5 mg/kg per dy)-treted d/d group, respectively. Arrows,, c, nd d represent pigment epithelium, outer segments, inner segments, nd outer plexiform lyer, respectively. Immunohistochemicl exmintion of effects of CTS nd THA on septl cholinergic cells in d/d mice Our previous report indicted tht 19-week-old d/d mice hd significntly lower numer of cholinergic cells in the medil septum nd sl forerin thn ge-mtched m/m mice nd 19-week-old d/d mice, suggesting dietes-induced degenertion of centrl cholinergic systems. To understnd the mechnism y which CTS tretment reverses dietes-induced decrese of cholinergic mrker proteins in the hippocmpus, we immunohistochemiclly nlyzed ChAT-immunopositive neurons in the medil septum tht minly project to the hippocmpl re. Consistent with previous findings [5], the vehicle-treted d/d mice showed smller numer of ChAT-immunopositive cells in the medil septum thn the vehicle-treted m/m mice (Figure 1). The d/d mice dministered with CTS nd THA for the experimentl period hd level of ChAT-immunopositive cells similr to tht in the control m/m mice. Discussion This study ws conducted to investigte if CTS hs n meliortive effect on cognitive deficits oserved in d/d mice s one of the dietes-relted neuropsychitric symptoms. The results demonstrte tht this type 2 dietes niml model exhiits severe cognitive deficits, nxiety-like emotionl ehvior, degenertion of the sl forerin cholinergic complexes, nd down-regultion of Akt signling nd VEGF/PDGF systems in the rin nd tht CTS is cple of ttenuting these ehviorl nd neuroiologicl deficits. Our findings suggest tht CTS exhiits eneficil effects on dietes-induced cognitive nd emotionl deficits nd tht its effects on cognitive deficits re medited y ttenuting dietes-induced dysfunction of centrl cholinergic systems, nd Akt signling nd VEGF/PDGF systems in the rin. Ameliortive effects of CTS on dietes-induced neuropsychitric symptoms In this study, three different types of ehviorl tsks, nmely, ORT, MYT, nd WMT, were used to elucidte non-sptil short-term memory [13,21], novelty-relted sptil working memory [25,26], nd hippocmpusdependent sptil lerning nd reference memory [12,33], respectively, in dietic d/d mice nd non-dietic m/m mice. The results reveled tht, compred with nondietic control mice, dietic mice exhiited clerly impired cognitive lerning nd memory performnce in these ehviorl tsks. Moreover, it ws oserved tht dietic mice showed reduced motor ctivity, tht is,

13 Zho et l. BMC Complementry nd Alterntive Medicine 212, 12:188 Pge 13 of 18 A) ChAT 68 kd M 1 ACh R 52 kd m/m control d/d control d/d + CTS (375) M 3 ACh R 75 kd c M 5 ACh R 5 kd d e d/d + CTS (75) d/d + THA (2.5) β-ctin 42 kd B) Reltive expression level ( % of norml control) c d e ChAT M 1 AChR M 3 AChR M 5 AChR ### ** *** ## *** ** ### *** *** ### *** ** c d e c d e c d e c d e Figure 9 Effects of CTS nd THA on ChAT, M 1,M 3, nd M 5 receptor protein expression in the hippocmpus of d/d mouse. Typicl photos indicting the expression levels of ech fctor in the hippocmpus of vehicle-treted m/m (lne ), nd vehicle (lne )-, CTS (325 mg/kg per dy: lne c; 75 mg/kg per dy: lne d)-, nd THA (2.5 mg/kg per dy; lne e)-treted d/d mice. B) Quntittive comprisons of ech fctor mong different niml groups were conducted s descried in the text. The dt re expressed s the percentge of the vlue otined from nïve control m/m mice. Ech dt column represents the men ± S.D. otined from 5-6 rin smples. ##P <.1, ###P <.1 vs. vehicletreted m/m group (Student s t-test). **P <.1, ***P <.1 vs. respective vehicle-treted d/d group (one-wy ANOVA followed y Tukey test). locomotor ctivity in the MYT nd swimming speed in the WMT. These findings re consistent with previous reports tht d/d mice re hypolocomotive [5,34] nd hve cognitive deficits [5,6,35]. Therefore, it is likely tht lerning nd memory deficits of d/d mice oserved in this study were cused y reduced motor ctivity relevnt to elevted ody weight of this niml group. However, this possiility seems to hve een ruled out y the dt otined in our present nd previous studies [5]. Indeed, our dt indicted tht dily dministrtions of THA nd Kmpo medicines meliorted cognitive lerning nd memory performnce of d/d mice without ffecting their locomotor nd swimming ility. Considering the fct tht CTS nd THA hd no effect on serum glucose levels or ody weights in d/d mice, these drugs likely meliorte nd/or prevent dietes-induced cognitive deficits vi mechnism independent of nti-hyperglycemi nd/or nti-oesity. The present study lso demonstrted tht d/d mice exhiited nxiety-like ehvior in the EPM test, suggesting tht they re more susceptile to phoi-driven nxiety thn the control m/m strin. This finding is in ccord with the work of Dinel et l. [6]. They reported tht d/d mice hd neuropsychitric symptoms such s cognitive deficits nd nxiety-like ehviors nd tht the symptoms were ssocited with incresed inflmmtory cytokines nd reduced expression of rin-derived neurotrophic fctor (BDNF) in the hippocmpus. Interestingly, in our present study, the nxiety-relted ehviors of d/d mice were significntly ttenuted in the d/d groups tht hd received n cute injection of the nxiolytic drug dizepm nd dily dministrtion of CTS. Moreover, it ws of interest to note tht, in contrst to CTS, THA hd no effect on the emotionl performnce of d/d mice. These results suggest tht CTS hs eneficil effect on neuropsychitric symptoms of dietic nimls nd llow us to speculte on possile involvement of GABAergic mechnisms in the nxiolytic-like ction of CTS in d/d mice. However, this possiility cn e ruled out for couple of resons. First, it is generlly recognized tht fcilittion of centrl GABAergic systems y drugs such s dizepm

14 Zho et l. BMC Complementry nd Alterntive Medicine 212, 12:188 Pge 14 of 18 1) 2) 3) 4) Figure 1 Effects of CTS nd THA tretment on dietes-induced cholinergic neuron degenertion in the medil septum. Cholinergic neurons were identified y ChAT immunostining in vehicle-treted m/m (1), nd vehicle (2)-, CTS (75 mg/kg per dy: 3)-, nd THA (2.5 mg/kg per dy; 4)-treted d/d mice. Cholinergic neurons were stined with nti-chat ntiody, showing tht predominnt ChAT expression occurred in the cell ody of cholinergic neurons. Scle r = 1 μm. impirs lerning nd memory performnce [36,37]. Secondly, in our previous study using SAMP8, senescenceccelerted mouse model, we demonstrted tht repeted dministrtion of CTS meliorted not only cognitive deficits ut lso reduced level of nxiety oserved in this niml model [38]. Thirdly, there is possiility tht the nxiolytic-like effect of CTS oserved in d/d mice is medited y interction of CTS with BNDF nd VEGF/ PDGF systems in the rin since these systems re reportedly implicted in ttenution of nxiety ehvior in rodents [17,39]. However, considering the present nd previous dt otined from THA-treted d/d nimls, this possiility seems unlikely. THA dministrtion filed to ffect nxiety ehvior of d/d nimls, ut it significntly revered the reduced levels of BDNF [5] nd VEGF/ PDGF in the rin. Therefore, the nxiolytic-like ction of CTS my e medited y neuronl mechnism(s) independent of centrl cholinergic nd VEGF/PDGF systems. Further investigtions re needed to clrify the mechnism(s) underlying the nxiolytic-like effect of CTS in d/ d mice. Effects of CTS nd THA on dietes-induced neuroiologicl dysfunctions in the rin We next elucidted the effects of CTS nd THA dministrtion on phosphoryltion of Akt nd PKCα s indices of impired signling nd ctivtion of PKC relted to hyperglycemi-induced complictions in d/d mice [4-42]. In this study, Akt phosphoryltion nd PKCα/ βii utophosphoryltion in the hippocmpi of d/d mice were significntly down-regulted nd up-regulted, respectively, compred with those in the control m/m mice nd these ltertions cused y dietes were reversed y CTS nd THA dministrtions. Evidence indictes tht the Akt ctivtion involves tyrosine kinse receptors, such s receptors for insulin, or certin growth fctors, such s vsculr endothelil growth fctor, nd tht it is triggered y Akt phosphoryltion vi phosphtidylinositol 3 kinse nd phosphoinositidedependent protein kinses. An Akt ctivtion mechnism is reduced in the CNS of dietes models including d/d mice [43,44], lthough there is report with conflicting findings [45]. Moreover, recently it ws reported