an antioxidant dietary supplement improves brain derived neurotrophic factor levels in serum of aged...

Research ArticleAn Antioxidant Dietary Supplement ImprovesBrain-Derived Neurotrophic Factor Levels in Serum ofAged Dogs Preliminary Results

Sara Sechi1 Francesca Chiavolelli2 Nicoletta Spissu1 Alessandro Di Cerbo3

Sergio Canello2 Gianandrea Guidetti2 Filippo Fiore1 and Raffaella Cocco1

1Department of Veterinary Medicine Pathology and Veterinary Clinic Section Via Vienna 2 07100 Sassari Italy2SANYpet Spa Research and Development Department Via Austria 3 Bagnoli di Sopra 35023 Padua Italy3School of Specialization in Clinical Biochemistry ldquoG drsquoAnnunziordquo University Via dei Vestini 31 66100 Chieti Italy

Correspondence should be addressed to Raffaella Cocco rafcounissit

Received 23 March 2015 Revised 1 June 2015 Accepted 7 June 2015

Academic Editor Antonio Ortega-Pacheco

Copyright copy 2015 Sara Sechi et al This 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

Biological aging is characterized by a progressive accumulation of oxidative damage and decreased endogenous antioxidant defensemechanisms The production of oxidants by normal metabolism damages proteins lipids and nucleotides which may contributeto cognitive impairment In this study 36 dogs were randomly divided into four groups and fed croquettes of different compositionsfor 6 months We monitored derivatives of reactive oxygen metabolites (dROMs) and biological antioxidant potential (BAP) levelsin dogsrsquo plasma samples as well as brain-derived neurotrophic factor (BDNF) serum levels at the beginning and at the end of thedietary regime Our results showed that a dietary regime enriched with antioxidants induced a significant decrease of plasmalevels of dROMs (119901 lt 0005) and a significant increase in BDNF serum levels (119901 lt 0005) after six months Thus we hypothesizeda possible role of the diet in modulating pro- and antioxidant species as well as BDNF levels in plasma and serum respectively Inconclusion the proposed diet enrichedwith antioxidantsmight be considered a valid alternative and a valuable strategy to counteractaging-related cognitive decline in elderly dogs

1 Introduction

Biological aging is characterized by a progressive accumula-tion of oxidative damage and decreased endogenous antioxi-dant defense mechanisms [1] The production of oxidants bynormalmetabolism damages proteins lipids and nucleotideswhich may contribute to neurodegeneration and subse-quently cognitive impairment such as Alzheimerrsquos (AD) andParkinsonrsquos diseases (PD) [2 3] Although the body normallyhas sufficient protection producing endogenous antioxidantsenzymes such as catalase glutathione and superoxide dis-mutase an imbalance in the pro-oxidantantioxidant speciescould increase the risk for lipid peroxidation DNA and pro-tein damage Oxidative damage also affects neuron functionand may contribute to a cognition decline with age Theexcess of reactive oxygen species in neuronal level can leadto irreversible damage of cytoskeleton and the microtubular

network [4]Microtubules important dynamics polar forma-tions of the cytoskeleton are abundant in neurons wherethey provide a scaffold also for their dendrites Because ofoxidative stress conditions the peroxynitrite may react withtyrosine to form 3-nitro-L-tyrosine (3NT)This final productmay be selectively incorporated into the 120572-tubulin resultingin an irreversible blocking of the characteristic dynamicsof microtubules causing morphological changes neuronaldeath and consequent onset of neurodegenerative diseasesSeveral experimental evidences have shown the importanceof polyunsaturated fatty acids (PUFAs) in human and animalnutrition and development [5] Further an omega-3 andomega-6 deficiency likely correlates with the development ofbehavioral disorder [6]

Thus we hypothesized that a valid alternative and valu-able strategy to counteract aging-related cognitive declineand neurodegeneration in aged dogs might be a dietary

Hindawi Publishing CorporationJournal of Veterinary MedicineVolume 2015 Article ID 412501 9 pageshttpdxdoiorg1011552015412501

2 Journal of Veterinary Medicine

supplementation with antioxidants Recent studies of animalmodels of neurodegenerative diseases suggest that dietaryrestriction can increase neurons endurance to age-related anddisease-specific stresses [7] Neurotrophic factors includ-ing nerve growth factor (NGF) and brain-derived neu-rotrophic factor (BDNF) can protect neurons against deathas observed for in vivo and in vitro models of acute (stroketrauma and seizures) and chronic (Alzheimerrsquos and Parkin-sonrsquos diseases) neurodegenerative diseases [8]

Our interest in the neurological effects of age stems froma more general concern with the development of a caninemodel of human cognitive aging Dogs show age-relatedpathology similar to that observed in elderly humans likelearning and development impairments [9] Although theexact mechanisms have not been established so far recentevidences indicate that a combination of an antioxidant-enriched diet with essential fatty acids like omega-3 andomega-6 can be used to reduce age-dependent impairmentsand cognitive decline in aged dogs [10]We focused on BDNFdue to its role in supporting the survival and growth of manyneuronal subtypes and in mediating the synaptic efficacyneuronal connectivity and plasticity [11]

We hypothesized that BDNF serum levels might bemodulated by an antioxidant-enriched diet

In humans BDNF mRNA and protein are decreased inthe cortex and hippocampus in mild cognitive impairment(MCI) andAD [12] and are also decreased in cognitive decline[13] In animals decreased BDNF serum levels result in a LTPand memory deficit thus increasing BDNF availability in thebrain may be a viable strategy to counteract cognitive declinewith aging

The canine model provides us with an opportunity totest the relationship between cognitive impairment in agingrelated to a decrease in BDNF serum levels and the effects ofnon-pharmacological interventions

The aim of this study was to evaluate the effects of anovel dietary supplement endowed with antioxidant prop-erties as an adjuvant in the prevention of oxidative stressconditions and neurodegenerative disorders in aged dogsThe supplement consisted in a mixed formula of fishedor chicken proteins rice carbohydrates Grifola frondosaCurcuma longa Carica papaya Punica granatum Aloe veraPolygonum cuspidatum Solanum lycopersicum Vitis viniferaRosmarinus officinalis and an Omega 36 ratio of 1 08

Grifola Frondosa is a culinary-medicinal mushroom thatmay play an important role in the prevention of many age-associated neurological dysfunctions including Alzheimerrsquosand Parkinsonrsquos diseases The mushroom shows neuropro-tective antioxidant and anti-(neuro)inflammatory effects infact it reduces beta amyloid-induced neurotoxicity neuriteoutgrowth stimulation and nerve growth factor (NGF) syn-thesis [14] It can be considered a useful therapeutic agentin the management andor treatment of neurodegenerativediseases [15] It was suggested that there could be an overallimprovement in cognitive abilities of subjects when incorpo-rated in their daily diet [16]

Curcuma Longa is a naturally occurring phytochemicalcompound endowed with powerful free radical scavenging

activity [17] Farinacci et al observed that curcumin polyphe-nol derived from Curcuma longa reduced neutrophils adhe-sion and superoxide production in vitro [18] Moreoverit resulted beneficially in improving spatial attention andmotivation deficits associated with impaired cognition inaging and AD in both humans and dogs [19] Head et alobserved that 9 aged beagles provided with a medical foodcocktail containing 95 of curcuminoids for 3 months hadsignificantly lower error scores andweremore accurate acrossall distances suggesting an overall improvement in spatialattention [20] da Rocha et al demonstrated that C longahad neuroprotective effects in vitro and in vivo by targetingbiochemical pathways associated with neurodegenerativedisorders that include cognitive impairments energyfatiguemood and anxiety [21 22]

Carica papaya is a natural compound with a high phe-nolic and flavonoid content which explains its free radicalscavenging and antioxidant potential [22ndash24] Mehdipour etal demonstrated its antioxidant effect in vitro and reporteda significant decrease in blood lipid peroxidation while theblood total antioxidant power resulted significantly increased(119901 lt 0001) [24]

Punica granatum is a plant containing some speciesof flavonoids and anthocyanins for example delphinidincyaniding and pelargonidin which have been shown to havean antioxidant activity in vitro [25] and in vivo [26] Theantioxidant action of P granatum is related to its free radicalscavenging activity against superoxide ionsmainly due to thepresence of anthocyanins and to the ability to form metalchelates [25] Rehydration effects have been also reported[27]

Aloe vera and its extracts have medicinal propertiesattributed to the active components such as anthronechromone aloe verasin and hydroxylation [28 29] Sahu et aldemonstrated that Aloe vera had different degrees of antiox-idant activity [30] The antioxidant properties of this plantmay depend on the radical scavenging activity Moreoverlife-long dietary supplementation of A vera was shown tosuppress many age-related consequences in vivo Due to thepresence of phenolic acids polyphenols sterols fatty acidsand indoles A vera may result to be effective in relievingsymptoms associated with or preventing neurodegeneration[31]

Polygonum cuspidatum is an important natural sourceof resveratrol [32] Due to its numerous pharmacologicalactivities it has been used as an antioxidant [33] Antioxidantactivities of P cuspidatum have been reported both in vivoand in vitro study [34] Moreover three of its dimersparthenocissin A quadrangularing A and pallidol haveshown strong free radical quenching and selective singletoxygen scavenging activity [35] Trans-resveratrol has beenused as antidepressant in chronically stressed rats probablyby acting on themonoaminergic systems such as the seroton-ergic and noradrenergic [33] In fact chronic treatment withtrans-resveratrol was found to inhibit monoamine oxidase-A(MAO-A) activity in all the four brain regions particularly inthe frontal cortex and hippocampus [36]

Solanum lycopersicum is rich of vitamins C and Elycopene beta-carotene lutein and flavonoids such as

Journal of Veterinary Medicine 3

quercetin phenols ascorbic acid (AsA) and dehydroascorbicacid (DHA) It is considered an important plant able toprevent chronic diseases and improve energy balance andantioxidant activity [37] Both direct and indirect antioxidantactivity as indicated by reduced malondialdehyde (MDA)and nitric oxide (NO) production and increased glutathioneperoxidase (GPx) and superoxide dismutase (SOD) activitysupport the conclusion that tomatoes containing antho-cyanins can potentially provide better protection againstoxidative stress related chronic diseases [38]

Vitis vinifera can be considered as a potential sourceof natural antioxidants due to the presence of carotenoidssuch as lutein beta-carotene and polyphenols [39] Theantioxidant activity of grape extracts is due to their reducingpower [40]The grape seed flavanolprocyanidin compoundsmay act as similar as reductones by donating electrons andreaching with free radicals to convert them to more stableproducts and terminating the free radical chain reaction [40]Antioxidant activities ofV viniferawere also assessed by theircapacity to prevent Fe2+-induced lipid peroxidation inmicro-somes and their action onCu2+-induced lipid peroxidation inlow-density lipoproteins [41] Astringin a stilbenoid presentinV vinifera is endowedwith an important antioxidant effectand a higher radical scavenger activity [41]

Rosmarinus officinalis is known to exert an antiprolifer-ative antioxidant and antibacterial activity [42] The crudeextract also has shown antioxidant and anti-inflammatoryactivities inhibiting NO production and reducing proin-flammatory cytokines (IL-1120573) and enzymes (COX-2) mRNAexpression in LPS-activated cells thus highlighting itschemopreventive potential [43] Afonso et al showed thatphenolic compounds from R officinalis protected againsthypercholesterolemia-induced oxidative stress increasingthe activities of antioxidant enzymes [44] Several literaturereports have demonstrated that R officinalis exerted multiplebenefits for neuronal system and alleviated mood disorders[45] In particular its active compounds luteolin carnosicacid and rosmarinic acid exhibited neurotrophic effects byimproving cholinergic functions [46] and showed neuropro-tective properties by inhibiting amyloid precursor proteinsynthesis and higher brain-derived neurotrophic factor pro-duction in hypothalamus cells [45] Antidepressant-like effectof R officinalis may be mediated by an interaction with thedopaminergic system through the activation of dopamineD1and D2 receptors [47]

Omega 36 fatty acids a good balance of the Omega36 fatty acids ratio in the food is a basic requirementto improve the inflammatory and neurological background[48] More in detail n-3 polyunsaturated fatty acids usuallyfound in fish oil such as eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA) are known to both decrease theproduction of proinflammatorymediators and inhibit naturalkiller cell activity [49]Moreover preclinical studies suggestedthat low plasma omega-6 and omega-3 fatty acids levelswere associated with accelerated decline of peripheral nervefunction with aging [50] Intake of PUFAs mainly omega-3 and omega-6 was shown to increase BDNF productionin brain [50 51] Docking studies on PUFAs and their

Table 1 Dogsrsquo diet groups and their features

Group Pet food Sex Mean age plusmn SEM1 Organic chicken 2M 6F 9 plusmn 0082 Chicken + antioxidants 5M 3F 7 plusmn 0253 Fish 6M 2F 9 plusmn 0634 Fish + antioxidants 3M 5F 9 plusmn 094Antioxidants added in groups 2 and 4 are Grifola frondosa Curcuma longaCarica papayaPunica granatumAloe veraPolygonum cuspidatum Solanumlycopersicum Vitis vinifera and Rosmarinus officinalis an Omega 36 ratio of1 08

metabolites with BDNF revealed that PUFAs metabolitesmainly LXA 4 NPD1 and HDHA had more binding affinitytowards BDNF [51] These metabolites of PUFAs are alsoresponsible for modulation of BDNF activity [51]

2 Methods

21 Subjects Thirty-six dogs of different breeds were ran-domly and equally divided into four groups based on age anddiet (Table 1) First group made up of 8 dogs (three malesand five females age 9 plusmn 008 mean plusmn standard error of themean) was fed a control diet with organic chicken Secondgroup made up of 8 dogs (three males and five females age7 plusmn 025 mean plusmn standard error of the mean) was fed achicken-based food enriched with natural antioxidants Thethird group made up of 8 dogs (four males and four femalesage 9 plusmn 063 mean plusmn standard error of the mean) was fed afish-based meal and the fourth one was made up of 8 dogs(three males and five females age 9 plusmn 094 mean plusmn standarderror of the mean) and was fed a fished-based meal enrichedwith natural antioxidants

d-ROMs and BAP tests (Free Radical Analytical SystemFRAS 4 HampD srl Langhirano PR Italy) were performedbefore (T0) and at the end (T1) of the treatment in all animalsin order to determine the oxidative stress statusThe four dietswere administered for a six-month period

22 Sample Collection and Analysis Blood samples werecollected from each dog before the new dietary regime (T0)and at the end of the treatment (T1) after six months fromcephalic vein and stored in two tubes one with heparin andthe otherwithout anticoagulant Heparinized plasma samplesand serum samples were obtained by blood centrifugation at4000 gtimes 15min at 37∘C

The derivatives of reactive oxygen metabolites (dROMs)and the biological antioxidant potential (BAP) as indicatorsof oxidative stress were measured by portable spectropho-tometer (Free Radical Analytical System FRAS 4 HampD srlLanghirano PR Italy) on plasma samples In the dROMstest reactive oxygen metabolites (primarily hydroperoxides)of the sample in presence of iron released from plasmaproteins by an acidic buffer generate alkoxyl and peroxylradicals according to the Fenton reaction Such radicalsthen oxidize an alkyl-substituted aromatic amine (NN-dietylparaphenylendiamine) thus producing a pink-colored


derivative which is photometrically quantified at 505 nm[52] The dROMs concentration is directly proportional tothe colour intensity and is expressed as UCARR (CarratelliUnits) One UCARR corresponds to 08mgL hydrogenperoxide The reference values of dROMS are summarizedbelow

(i) reference value 50ndash90 UCARR(ii) threshold borderline 92ndash95 UCARR(iii) condition of mild oxidative stress 100ndash120 UCARR(iv) condition of oxidative stress 140ndash200 UCARR(v) condition of strong oxidative stress220ndash300UCARR(vi) strong oxidative stress over 300 UCARR

In the BAP test plasma samples were addedwith a coloredsolution obtained by mixing a ferric chloride solution with athiocyanate derivative solution which causes a discolorationwhose intensity is measured photometrically at 505 nm andit is proportional to the ability of the plasma to reduce ferricions The results are expressed as 120583MolL of reduced ferricions Both tests were validated for canine species [53] Rangevalues of BAP are listed below

(i) reference value 2000ndash4000120583MolL(ii) optimal values gt2200120583MolL(iii) threshold borderline 2200ndash2000120583MolL(iv) discrete deficiency state 2000ndash1800120583MolL(v) deficiency state 1800ndash1600 120583MolL(vi) strong deficiency state 1600ndash1400 120583MolL(vii) very strong deficiency state lt1400120583MolL

23 BDNF Analysis BDNF analysis was performed withBG BDNF ELISA kit (Blue Gene Biotech CO LTD Shang-hai China) designed for the quantitative determination ofcanine BDNF The ELISA test reaction was performed usingCrocodile mini Workstation (Totertek Berthold PforzheimGermany)The kit utilizes amonoclonal anti-BDNF antibodyand BDNF-HRP conjugate The assay sample and buffer areincubated together with BDNF-HRP conjugate in precoatedplate for one hour After the incubation period the wellsare washed five times The wells are then incubated witha substrate for HRP enzyme The product of the enzyme-substrate reaction forms a blue colored complex Finally astop solution is added to stop the reaction which will thenturn the solution yellow The intensity of color is measuredspectrophotometrically at 450 nm with a microplate readerThe intensity of the color is inversely proportional to theBDNF concentration since BDNF from samples and BDNF-HRP conjugate compete for the anti-BDNF antibody bindingsite Since the number of sites is limited as more sites areoccupied by BDNF from the sample fewer sites are led tobind BDNF-HRP conjugate A standard curve is then plottedrelating the intensity of the color (OD) to the concentrationof standards The BDNF concentration of each sample isinterpolated from this standard curve

24 Statistical Analysis All data are presented as the meanplusmn SEM An unpaired 2-sample Studentrsquos 119905-test was used tocompare the differences in plasma dROMs BAP and BDNFlevels between the four groups All statistical analyses wereperformed with GraphPad Prism 6 (GraphPad Software IncSan Diego CA USA) 119901 lt 005 was considered significant

3 Results

31 Oxidative Stress Status Evaluation We firstly analyzeddROMs and BAP levels as a measure of the oxidative stressstatus of dogs Analysis was made on dogs plasma in all ofthe four groups before starting the new dietary regime (T0)and at the end after six months (T1)

As shown in Figure 1 a significant decrease in plasmalevels of dROMs after sixmonths of feeding regime in groups2 and 4 (food supplemented with antioxidant) was observed(119901 lt 0005) dROMs levels remained unaltered in the first(control group) and third groups (fish-based meal withoutantioxidant addition)

These data indicate that a diet enriched with naturalantioxidant might be able to promote a decrease of reactivespecies in plasma of aged dogs

Antioxidant influence evaluated with BAP analysisremained unchanged in all groups of dogs after the diet(Figure 2)

Natural antioxidants seemed to modulate the balancebetween pro- and antioxidant species through the decreaseof dROMs without increasing natural antioxidant defense

32 BDNF Evaluation Literature reports have shown adecrease in BDNF serum levels to negatively correlate withcognitive decline and deficits in LTP and memory in dogs[13] BDNF is a neurotrophic factor that can protect neuronsagainst death supporting the survival and growth of manyneuronal subtypes [8] An increase in BDNF serum levels isone of the factors underlying improvements in learning andmemory [8 54] Increasing BDNF availability in the brain bydiet may be a viable strategy to counteract cognitive declinewith aging [7 55 56]

We reported a significant BDNF serum levels increase ingroups of dogs that received a diet enriched with antioxidant119901 lt 0005 (Figure 3) while in the other groups BDNF serumlevels remain unchanged

4 Discussion

The purpose of this study was to evaluate the possiblepotential ability of a long-term dietary antioxidant supple-mentation in controlling the oxidative stress and the generalhealth status of aged dogs Moreover we demonstrated apossible modulation of BDNF serum levels by a diet withantioxidant supplementation

Environmental stress and aging may induce psychologi-cal stress that possibly influences also the nutrition of pets Anunbalanced diet deficient in essential nutrientsmay representa risk factor of degenerative diseases Among the several


UCA

RR

T0 T1

Group 1 dROMs200

150

100

50

0

Time of analysis

Group 3 dROMs

Time of analysis

UCA

RR

T0 T1

200

150

100

50

0

Group 2 dROMs

UCA

RR

T0 T1

200

150

100

50

0

Time of analysis

lowastlowast

Group 4 dROMs

Time of analysis

UCA

RR

T0 T1

200

150

100

50

0

lowastlowast

Figure 1 Graphical representation of dROMs in plasma of aged dogs before and after the 6 months of the dietary regime A significantdecrease of dROMs levels was observed in dogs fed with antioxidant supplementation in the second group (lowastlowast119901 lt 0005) and in the fourthgroup (lowastlowast119901 lt 0005) respectively

mechanisms by which nutrients influence the health statusthe balance of oxidative stress has a relevant role Constantlyin the animal species metabolic oxidative reactions takeplace The goal of these reactions is to balance free radicalsproduction with antioxidants molecules

The inhibitory activity of antioxidant molecules wasobserved in in vitro studies using plants derivative com-pounds such as flavonoids anthocyanins and other poliphe-nols evaluating their effects on the converting activities of 120572-amylase 120572-glucosidase and angiotensin-converting enzyme(ACE) They all have shown an inhibitory activity on 120572-amylase 120572-glucosidase and ACE [57]

Furthermore a significant reduction in dROMs valuesin the experimental diet enriched with natural antioxidantswas observed In apparently healthy dogs serum levels ofthe dROMs ranged between 50 and 90 UCARR Thesevalues were in agreement with those reported by Pasquiniet al [53] The antioxidant supplementation significantlydecreased dROMs levels from 155 UCARR (T0) to 120UCARR (T1) in the second group and from 150UCARR (T0)to 95 UCARR (T1) in the forth group Differently dogs fed

the control diet deficient in antioxidant nutrients did notmodulate the oxidative stress status The antioxidant statusrevealed by BAP test was not affected by nutrition and valueswere at optimal levels throughout the observational periodProbably antioxidant supplements only affected dROMsspecies but not the endogenous antioxidant components ofdogs which remained in the initial optimal condition Thismight be related to the really efficacy of the experimental dietin modulating the oxidative stress

The antioxidant formulation employed in this experi-ment was based on Grifola frondosa Curcuma longa Caricapapaya Punica granatum Aloe vera Polygonum cuspidatumSolanum lycopersicum Vitis vinifera and Rosmarinus offici-nalis extracts

All these compounds contain anthocyanins and polyphe-nols with antioxidant effects [7]

With this study we showed a decrease in dROMs speciesfollowing the administration of the analyzed antioxidantformulation

Our study reporting the scavenger activity of an antiox-idant supplementation in dogs diet is in agreement with


Group 4 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)

Group 3 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)

T0 T1

Group 1 BAP3000

2000

1000

0

Time of analysis

(120583m

olL

)

Group 2 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)

Figure 2 Graphical representation of BAP in plasma of aged dogs before and after 6 months of the dietary regime BAP levels did not showsignificant modifications in all groups of dogs after the diet

other studies about antioxidant effects of singular activeprinciples included into an antioxidant supplementation [58]The anthocyanins and polyphenols have antioxidant effects insome pathological conditions such as metabolic disordersaging-related diseases cardiovascular diseases cancer andinflammatory-related disturbs as well as carotenoids andflavonoids [39 59] These compounds are inhibitors of lipidperoxidation probably by interfering with the glutathioneactivity [60]

Resveratrol (3541015840-trihydroxystilbene) is a polyphenolnaturally present in grapes berries peanuts and othervegetables [61] with therapeutic and neuroprotective func-tions [7] Moreover trans-resveratrol which is highly pre-sented in Polygonum cuspidatum could modulate BDNFlevels through the monoaminergic system activation [36]In our study we observed that a diet enriched with naturalantioxidants was able to increase BDNF serum levels from100 plusmn 05 pgmL to 180 plusmn 08 pgmL while in the othergroups remained unchanged As to Grifola frondosa animprovement in cognitive abilities in aged dogs was observed

when adding Curcuma longa and Aloe vera to a daily dietthrough a stimulation of the BDNF synthesis [16] Accordingto what was observed by Sasaki et al luteolin carnosic acidand rosmarinic acid from Rosmarinus officinalis exerteda neuroprotective activity probably modulating the neu-rotrophic metabolic pathway in the neuronal system [46]Finally Kumar et al observed that PUFAs like omega-3 andomega-6 along with their metabolites had more bindingaffinity towards BDNF [51] Further the serum level increaseof both fatty acids might be modulated by these metaboliteswhich in turn could regulate the BDNF production in thebrain Neurotrophic factors such as BDNF can protectneurons against death and be a preventive approach inneurodegenerative conditions [7 8]

By means of this new canine model of aging we showedthat providing antioxidants within a specific dietary supple-ment it was possible to restore the balance between pro-and antioxidants species possibly modulating also BDNFserum levels Future studies in both aged humans and dogswill be more effective if antioxidants combinations will be


Group 1 BDNF150

100

50

0

BDN

F le

vels

(pg

mL)

T0 T1Time of analysis

Group 3 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

150

100

50

0

Time of analysis

Group 2 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

300

200

100

0

Time of analysis

lowastlowast

Group 4 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

150

100

50

0

Time of analysis

lowastlowast

Figure 3 Graphical representation of BDNF in plasma of aged dogs before and after the 6 months of the dietary regime A significantincrease of BDNF levels was observed in dogs fed with antioxidant supplementation in the second group (lowastlowast119901 lt 0005) and in the fourthgroup (lowastlowast119901 lt 0005) respectively

evaluated along with additional lifestyle improvements suchas cognitive training and physical exercise

Conflict of Interests

None of the authors has any financial or personal relationshipthat could inappropriately influence or bias the content of thepaper

Acknowledgment

This paper was supported in part by grants from RegionalLaw 7 August 2007 n∘7 ldquoPromozione della Ricerca scientificae dellrsquoinnovazione tecnologica in Sardegnardquo

References

[1] T M Hagen D L Yowe J C Bartholomew et al ldquoMitochon-drial decay in hepatocytes from old rats membrane potentialdeclines heterogeneity and oxidants increaserdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 94 no 7 pp 3064ndash3069 1997

[2] M Tavakkoli R Miri A R Jassbi et al ldquoCarthamus Salvia andStachys species protect neuronal cells against oxidative stress-induced apoptosisrdquo Pharmaceutical Biology vol 52 no 12 pp1550ndash1557 2014

[3] X L Wang G H Xing B Hong et al ldquoGastrodin preventsmotor deficits and oxidative stress in the MPTP mouse modelof Parkinsonrsquos disease involvement of ERK12-Nrf2 signalingpathwayrdquo Life Sciences vol 114 no 2 pp 77ndash85 2014

[4] K Fukui A Masuda A Hosono et al ldquoChanges in micro-tubule-related proteins and autophagy in long-term vitamin E-deficient micerdquo Free Radical Research vol 48 no 6 pp 649ndash658 2014

[5] R Molteni R J Barnard Z Ying C K Roberts and F Gomez-Pinilla ldquoA high-fat refined sugar diet reduces hippocampalbrain-derived neurotrophic factor neuronal plasticity andlearningrdquo Neuroscience vol 112 no 4 pp 803ndash814 2002

[6] T Murphy G P Dias and S Thuret ldquoEffects of diet on brainplasticity in animal and human studies mind the gaprdquo NeuralPlasticity vol 2014 Article ID 563160 32 pages 2014

[7] W Duan J Lee Z Guo andM P Mattson ldquoDietary restrictionstimulates BDNF production in the brain and thereby protectsneurons against excitotoxic injuryrdquo Journal of Molecular Neuro-science vol 16 no 1 pp 1ndash12 2001


[8] M P Mattson S Maudsley and B Martin ldquoBDNF and 5-HT a dynamic duo in age-related neuronal plasticity andneurodegenerative disordersrdquo Trends in Neurosciences vol 27no 10 pp 589ndash594 2004

[9] A L S Dowling and EHead ldquoAntioxidants in the caninemodelof human agingrdquo Biochimica et Biophysica Acta vol 1822 no 5pp 685ndash689 2012

[10] R J Kearns M G Hayek J J Turek et al ldquoEffect of agebreed and dietary omega-6 (n-6) omega-3 (n-3) fatty acidratio on immune function eicosanoid production and lipidperoxidation in young and aged dogsrdquo Veterinary Immunologyand Immunopathology vol 69 no 2ndash4 pp 165ndash183 1999

[11] D K Binder andH E Scharfman ldquoBrain-derived neurotrophicfactorrdquo Growth Factors vol 22 no 3 pp 123ndash131 2004

[12] B Michalski and M Fahnestock ldquoPro-brain-derived neu-rotrophic factor is decreased in parietal cortex in AlzheimerrsquosdiseaserdquoMolecular Brain Research vol 111 no 1-2 pp 148ndash1542003

[13] S Peng J Wuu E J Mufson and M Fahnestock ldquoPrecursorform of brain-derived neurotrophic factor and mature brain-derived neurotrophic factor are decreased in the pre-clinicalstages of Alzheimerrsquos diseaserdquo Journal of Neurochemistry vol93 no 6 pp 1412ndash1421 2005

[14] S Ling-Sing Seow M Naidu P David K-H Wong and VSabaratnam ldquoPotentiation of neuritogenic activity of medicinalmushrooms in rat pheochromocytoma cellsrdquo BMC Comple-mentary and Alternative Medicine vol 13 article 157 2013

[15] C-W Phan P David M Naidu K-H Wong and V Sabarat-nam ldquoTherapeutic potential of culinary-medicinal mushroomsfor the management of neurodegenerative diseases diversitymetabolite and mechanismrdquo Critical Reviews in Biotechnology2014

[16] V SabaratnamW Kah-Hui M Naidu and P David ldquoNeuronalhealthmdashcan culinary and medicinal mushrooms helprdquo Journalof Traditional andComplementaryMedicine vol 3 no 1 pp 62ndash68 2013

[17] S Prasad S C Gupta A K Tyagi and B B AggarwalldquoCurcumin a component of golden spice from bedside tobench and backrdquo Biotechnology Advances vol 32 no 6 pp1053ndash1064 2014

[18] M Farinacci M Colitti and B Stefanon ldquoModulation of ovineneutrophil function and apoptosis by standardized extracts ofEchinacea angustifolia Butea frondosa and Curcuma longardquoVeterinary Immunology and Immunopathology vol 128 no 4pp 366ndash373 2009

[19] D Chin P Huebbe K Pallauf and G Rimbach ldquoNeuropro-tective properties of curcumin in Alzheimerrsquos Diseasemdashmeritsand limitationsrdquo Current Medicinal Chemistry vol 20 no 32pp 3955ndash3985 2013

[20] E Head H L Murphey A L S Dowling et al ldquoA combinationcocktail improves spatial attention in a canine model of humanaging and Alzheimerrsquos diseaserdquo Journal of Alzheimerrsquos Diseasevol 32 no 4 pp 1029ndash1042 2012

[21] M D da Rocha F P dias Viegas H C Campos et al ldquoTherole of natural products in the discovery of new drug candidatesfor the treatment of neurodegenerative disorders II AlzheimerrsquosdiseaserdquoCNS andNeurological DisordersmdashDrug Targets vol 10no 2 pp 251ndash270 2011

[22] G Srikanth S Manohar Babu C H N Kavitha M E BhanojiRao N Vyaykumar and C H Pradeep ldquoStudies on in-vitroantioxidant activities of Carica papaya aqueous leaf extractrdquo

Research Journal of Pharmaceutical Biological and ChemicalSciences vol 1 no 2 pp 59ndash65 2010

[23] K Imao H Wang M Komatsu and M Hiramatsu ldquoFreeradical scavenging activity of fermented papaya preparationand its effect on lipid peroxide level and superoxide dismutaseactivity in iron-induced epileptic foci of ratsrdquo Biochemistry andMolecular Biology International vol 45 no 1 pp 11ndash23 1998

[24] S Mehdipour N Yasa G Dehghan et al ldquoAntioxidant poten-tials of Iranian Carica papaya juice in vitro and in vivo arecomparable to120572-tocopherolrdquoPhytotherapyResearch vol 20 no7 pp 591ndash594 2006

[25] N P Seeram R N Schulman and D Heber PomegranatesAncient Roots to Modern Medicine Taylor amp Francis BocaRaton Fla USA 2006

[26] K N Chidambara Murthy G K Jayaprakasha and R P SinghldquoStudies on antioxidant activity of pomegranate (Punica grana-tum) peel extract using in vivo modelsrdquo Journal of Agriculturaland Food Chemistry vol 50 no 17 pp 4791ndash4795 2002

[27] P C Pande L Tiwari and H C Pande ldquoEthnoveterinaryplants of Uttaranchalmdasha reviewrdquo Indian Journal of TraditionalKnowledge vol 6 no 3 pp 444ndash458 2007

[28] M D Boudreau and F A Beland ldquoAn evaluation of the biolog-ical and toxicological properties of Aloe barbadensis (Miller)Aloe verardquo Journal of Environmental Science and HealthmdashPartC Environmental Carcinogenesis and Ecotoxicology Reviews vol24 no 1 pp 103ndash154 2006

[29] K Eshun and Q He ldquoAloe vera a valuable ingredient forthe food pharmaceutical and cosmetic industriesmdasha reviewrdquoCritical Reviews in Food Science and Nutrition vol 44 no 2pp 91ndash96 2004

[30] P K SahuDDGiri R Singh et al ldquoTherapeutic andmedicinaluses of Aloe vera a reviewrdquo Pharmacology amp Pharmacy vol 4no 8 pp 599ndash610 2013

[31] F Nejatzadeh-Barandozi ldquoAntibacterial activities and antioxi-dant capacity of Aloe verardquo Organic and Medicinal ChemistryLetters vol 3 article 5 2013

[32] L Chen Y Han F Yang and T Zhang ldquoHigh-speed counter-current chromatography separation and purification of resver-atrol and piceid from Polygonum cuspidatumrdquo Journal of Chro-matography A vol 907 no 1-2 pp 343ndash346 2001

[33] Y Xu Z Wang W You et al ldquoAntidepressant-like effect oftrans-resveratrol involvement of serotonin and noradrenalinesystemrdquo European Neuropsychopharmacology vol 20 no 6 pp405ndash413 2010

[34] X Q R Sheela and V A Raman ldquoIn-vitro antioxidant activityof Polygonium barbatum Leaf extractrdquoAsian Journal of Pharma-ceutical and Clinical Research vol 4 supplement 1 2011

[35] I Gulcin ldquoAntioxidant properties of resveratrol a structure-activity insightrdquo Innovative Food Science and Emerging Tech-nologies vol 11 no 1 pp 210ndash218 2010

[36] Y Yu R Wang C Chen et al ldquoAntidepressant-like effectof trans-resveratrol in chronic stress model behavioral andneurochemical evidencesrdquo Journal of Psychiatric Research vol47 no 3 pp 315ndash322 2013

[37] M Dorais D L Ehret and A P Papadopoulos ldquoTomato(Solanum lycopersicum) health components from the seed tothe consumerrdquo Phytochemistry Reviews vol 7 no 2 pp 231ndash250 2008

[38] H Li Z Deng R Liu S Loewen and R Tsao ldquoBioaccessibilityin vitro antioxidant activities and in vivo anti-inflammatoryactivities of a purple tomato (Solanum lycopersicum L)rdquo FoodChemistry vol 159 pp 353ndash360 2014


[39] C-I Bunea N Pop A C Babes C Matea F V Dulf and ABunea ldquoCarotenoids total polyphenols and antioxidant activityof grapes (Vitis vinifera) cultivated in organic and conventionalsystemsrdquo Chemistry Central Journal vol 6 article 66 2012

[40] G K Jayaprakasha R P Singh andK K Sakariah ldquoAntioxidantactivity of grape seed (Vitis vinifera) extracts on peroxidationmodels in vitrordquo Food Chemistry vol 73 no 3 pp 285ndash2902001

[41] B Fauconneau P Waffo-Teguo F Huguet L Barrier ADecendit and J-M Merillon ldquoComparative study of radicalscavenger and antioxidant properties of phenolic compoundsfromVitis vinifera cell cultures using in vitro testsrdquo Life Sciencesvol 61 no 21 pp 2103ndash2110 1997

[42] A I Hussain F Anwar S A Shahid Chatha A Jabbar SMahboob and P Singh Nigam ldquoRosmarinus officinalis essentialoil antiproliferative antioxidant and antibacterial activitiesrdquoBrazilian Journal of Microbiology vol 41 no 4 pp 1070ndash10782010

[43] S Cheung and J Tai ldquoAnti-proliferative and antioxidant proper-ties of rosemary Rosmarinus officinalisrdquo Oncology Reports vol17 no 6 pp 1525ndash1531 2007

[44] M S Afonso A M de O Silva E B Carvalho et al ldquoPhenoliccompounds from Rosemary (Rosmarinus officinalis L) atten-uate oxidative stress and reduce blood cholesterol concentra-tions in diet-induced hypercholesterolemic ratsrdquo Nutrition andMetabolism vol 10 article 19 2013

[45] C Ramachandran K-W Quirin E Escalon and S J Mel-nick ldquoImproved neuroprotective effects by combining Bacopamonnieri and Rosmarinus officinalis supercritical CO

2extractsrdquo

Journal of Evidence-Based Complementary and AlternativeMedicine vol 19 no 2 pp 119ndash127 2014

[46] K Sasaki A El Omri S Kondo J Han and H IsodaldquoRosmarinus officinalis polyphenols produce anti-depressantlike effect through monoaminergic and cholinergic functionsmodulationrdquo Behavioural Brain Research vol 238 no 1 pp 86ndash94 2013

[47] D G MacHado V B Neis G O Balen et al ldquoAntidepressant-like effect of ursolic acid isolated from Rosmarinus officinalisL in mice evidence for the involvement of the dopaminergicsystemrdquo Pharmacology Biochemistry and Behavior vol 103 no2 pp 204ndash211 2012

[48] A P Simopoulos ldquoThe importance of the ratio of omega-6omega-3 essential fatty acidsrdquo Biomedicine and Pharma-cotherapy vol 56 no 8 pp 365ndash379 2002

[49] R Verlengia R Gorjao C C Kanunfre et al ldquoEffects ofEPA and DHA on proliferation cytokine production and geneexpression in Raji cellsrdquo Lipids vol 39 no 9 pp 857ndash864 2004

[50] F Lauretani S Bandinelli B Bartali et al ldquoOmega-6 andomega-3 fatty acids predict accelerated decline of peripheralnerve function in older personsrdquoEuropean Journal ofNeurologyvol 14 no 7 pp 801ndash808 2007

[51] Y P Kumar G S Srinivas Y Mitravinda E L Malla andA A Rao ldquoAgonistic approach of omega-3 omega-6 and itsmetabolites with BDNF an In-silico studyrdquo Bioinformation vol9 no 18 pp 908ndash911 2013

[52] A Pasquini E Luchetti V Marchetti G Cardini and E LIorio ldquoAnalytical performances of d-ROMs test and BAP testin canine plasma Definition of the normal range in healthyLabrador dogsrdquo Veterinary Research Communications vol 32no 2 pp 137ndash143 2008

[53] A Pasquini E Luchetti and G Cardini ldquoEvaluation ofoxidative stress in hunting dogs during exerciserdquo Research inVeterinary Science vol 89 no 1 pp 120ndash123 2010

[54] M F Egan M Kojima J H Callicott et al ldquoThe BDNFval66met polymorphism affects activity-dependent secretion ofBDNF and human memory and hippocampal functionrdquo Cellvol 112 no 2 pp 257ndash269 2003

[55] A Wu Z Ying and F Gomez-Pinilla ldquoDietary omega-3 fattyacids normalize BDNF levels reduce oxidative damage andcounteract learning disability after traumatic brain injury inratsrdquo Journal of Neurotrauma vol 21 no 10 pp 1457ndash1467 2004


[57] G Oboh A O Ademosun A O Ademiluyi O S OmojokunE E Nwanna and K O Longe ldquoIn vitro studies on the antiox-idant property and inhibition of 120572-amylase 120572-glucosidase andangiotensin I-converting enzyme by polyphenol-rich extractsfrom cocoa (Theobroma cacao) beanrdquo Pathology Research Inter-national vol 2014 Article ID 549287 6 pages 2014

[58] D K Gessner A Fiesel E Most et al ldquoSupplementation of agrape seed and grape marc meal extract decreases activities ofthe oxidative stress-responsive transcription factorsNF-kappaBand Nrf2 in the duodenal mucosa of pigsrdquo Acta VeterinariaScandinavica vol 55 article 18 2013

[59] K B Pandey and S I Rizvi ldquoPlant polyphenols as dietaryantioxidants in human health and diseaserdquo Oxidative Medicineand Cellular Longevity vol 2 no 5 pp 270ndash278 2009

[60] J Oslash Moskaug H Carlsen M C W Myhrstad and RBlomhoff ldquoPolyphenols and glutathione synthesis regulationrdquoThe American Journal of Clinical Nutrition vol 81 no 1 pp277Sndash283S 2005

[61] W Yu Y-C Fu and W Wang ldquoCellular and moleculareffects of resveratrol in health and diseaserdquo Journal of CellularBiochemistry vol 113 no 3 pp 752ndash759 2012

Submit your manuscripts athttpwwwhindawicom

Veterinary MedicineJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Veterinary Medicine International



International Journal of

Microbiology


AnimalsJournal of

EcologyInternational Journal of


PsycheHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Evolutionary BiologyInternational Journal of


Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

Biotechnology Research International


Agronomy




Journal of Parasitology Research

Hindawi Publishing Corporation httpwwwhindawicom


Volume 2014

Zoology

GenomicsInternational Journal of


InsectsJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


VirusesJournal of

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Cell BiologyInternational Journal of



Case Reports in Veterinary Medicine


supplementation with antioxidants Recent studies of animalmodels of neurodegenerative diseases suggest that dietaryrestriction can increase neurons endurance to age-related anddisease-specific stresses [7] Neurotrophic factors includ-ing nerve growth factor (NGF) and brain-derived neu-rotrophic factor (BDNF) can protect neurons against deathas observed for in vivo and in vitro models of acute (stroketrauma and seizures) and chronic (Alzheimerrsquos and Parkin-sonrsquos diseases) neurodegenerative diseases [8]

Our interest in the neurological effects of age stems froma more general concern with the development of a caninemodel of human cognitive aging Dogs show age-relatedpathology similar to that observed in elderly humans likelearning and development impairments [9] Although theexact mechanisms have not been established so far recentevidences indicate that a combination of an antioxidant-enriched diet with essential fatty acids like omega-3 andomega-6 can be used to reduce age-dependent impairmentsand cognitive decline in aged dogs [10]We focused on BDNFdue to its role in supporting the survival and growth of manyneuronal subtypes and in mediating the synaptic efficacyneuronal connectivity and plasticity [11]

We hypothesized that BDNF serum levels might bemodulated by an antioxidant-enriched diet

In humans BDNF mRNA and protein are decreased inthe cortex and hippocampus in mild cognitive impairment(MCI) andAD [12] and are also decreased in cognitive decline[13] In animals decreased BDNF serum levels result in a LTPand memory deficit thus increasing BDNF availability in thebrain may be a viable strategy to counteract cognitive declinewith aging

The canine model provides us with an opportunity totest the relationship between cognitive impairment in agingrelated to a decrease in BDNF serum levels and the effects ofnon-pharmacological interventions

The aim of this study was to evaluate the effects of anovel dietary supplement endowed with antioxidant prop-erties as an adjuvant in the prevention of oxidative stressconditions and neurodegenerative disorders in aged dogsThe supplement consisted in a mixed formula of fishedor chicken proteins rice carbohydrates Grifola frondosaCurcuma longa Carica papaya Punica granatum Aloe veraPolygonum cuspidatum Solanum lycopersicum Vitis viniferaRosmarinus officinalis and an Omega 36 ratio of 1 08

Grifola Frondosa is a culinary-medicinal mushroom thatmay play an important role in the prevention of many age-associated neurological dysfunctions including Alzheimerrsquosand Parkinsonrsquos diseases The mushroom shows neuropro-tective antioxidant and anti-(neuro)inflammatory effects infact it reduces beta amyloid-induced neurotoxicity neuriteoutgrowth stimulation and nerve growth factor (NGF) syn-thesis [14] It can be considered a useful therapeutic agentin the management andor treatment of neurodegenerativediseases [15] It was suggested that there could be an overallimprovement in cognitive abilities of subjects when incorpo-rated in their daily diet [16]

Curcuma Longa is a naturally occurring phytochemicalcompound endowed with powerful free radical scavenging

activity [17] Farinacci et al observed that curcumin polyphe-nol derived from Curcuma longa reduced neutrophils adhe-sion and superoxide production in vitro [18] Moreoverit resulted beneficially in improving spatial attention andmotivation deficits associated with impaired cognition inaging and AD in both humans and dogs [19] Head et alobserved that 9 aged beagles provided with a medical foodcocktail containing 95 of curcuminoids for 3 months hadsignificantly lower error scores andweremore accurate acrossall distances suggesting an overall improvement in spatialattention [20] da Rocha et al demonstrated that C longahad neuroprotective effects in vitro and in vivo by targetingbiochemical pathways associated with neurodegenerativedisorders that include cognitive impairments energyfatiguemood and anxiety [21 22]

Carica papaya is a natural compound with a high phe-nolic and flavonoid content which explains its free radicalscavenging and antioxidant potential [22ndash24] Mehdipour etal demonstrated its antioxidant effect in vitro and reporteda significant decrease in blood lipid peroxidation while theblood total antioxidant power resulted significantly increased(119901 lt 0001) [24]

Punica granatum is a plant containing some speciesof flavonoids and anthocyanins for example delphinidincyaniding and pelargonidin which have been shown to havean antioxidant activity in vitro [25] and in vivo [26] Theantioxidant action of P granatum is related to its free radicalscavenging activity against superoxide ionsmainly due to thepresence of anthocyanins and to the ability to form metalchelates [25] Rehydration effects have been also reported[27]

Aloe vera and its extracts have medicinal propertiesattributed to the active components such as anthronechromone aloe verasin and hydroxylation [28 29] Sahu et aldemonstrated that Aloe vera had different degrees of antiox-idant activity [30] The antioxidant properties of this plantmay depend on the radical scavenging activity Moreoverlife-long dietary supplementation of A vera was shown tosuppress many age-related consequences in vivo Due to thepresence of phenolic acids polyphenols sterols fatty acidsand indoles A vera may result to be effective in relievingsymptoms associated with or preventing neurodegeneration[31]

Polygonum cuspidatum is an important natural sourceof resveratrol [32] Due to its numerous pharmacologicalactivities it has been used as an antioxidant [33] Antioxidantactivities of P cuspidatum have been reported both in vivoand in vitro study [34] Moreover three of its dimersparthenocissin A quadrangularing A and pallidol haveshown strong free radical quenching and selective singletoxygen scavenging activity [35] Trans-resveratrol has beenused as antidepressant in chronically stressed rats probablyby acting on themonoaminergic systems such as the seroton-ergic and noradrenergic [33] In fact chronic treatment withtrans-resveratrol was found to inhibit monoamine oxidase-A(MAO-A) activity in all the four brain regions particularly inthe frontal cortex and hippocampus [36]

Solanum lycopersicum is rich of vitamins C and Elycopene beta-carotene lutein and flavonoids such as









2 Methods












3 Results








4 Discussion




UCA

RR

T0 T1

Group 1 dROMs200

150

100

50

0

Time of analysis

Group 3 dROMs

Time of analysis

UCA

RR

T0 T1

200

150

100

50

0

Group 2 dROMs

UCA

RR

T0 T1

200

150

100

50

0

Time of analysis

lowastlowast

Group 4 dROMs

Time of analysis

UCA

RR

T0 T1

200

150

100

50

0

lowastlowast











Group 4 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)

Group 3 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)

T0 T1

Group 1 BAP3000

2000

1000

0

Time of analysis

(120583m

olL

)

Group 2 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)







Group 1 BDNF150

100

50

0

BDN

F le

vels

(pg

mL)


Group 3 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

150

100

50

0

Time of analysis

Group 2 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

300

200

100

0

Time of analysis

lowastlowast

Group 4 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

150

100

50

0

Time of analysis

lowastlowast





Acknowledgment


References

















































2extractsrdquo

























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of














2 Methods












3 Results








4 Discussion




UCA

RR

T0 T1

Group 1 dROMs200

150

100

50

0

Time of analysis

Group 3 dROMs

Time of analysis

UCA

RR

T0 T1

200

150

100

50

0

Group 2 dROMs

UCA

RR

T0 T1

200

150

100

50

0

Time of analysis

lowastlowast

Group 4 dROMs

Time of analysis

UCA

RR

T0 T1

200

150

100

50

0

lowastlowast











Group 4 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)

Group 3 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)

T0 T1

Group 1 BAP3000

2000

1000

0

Time of analysis

(120583m

olL

)

Group 2 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)







Group 1 BDNF150

100

50

0

BDN

F le

vels

(pg

mL)


Group 3 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

150

100

50

0

Time of analysis

Group 2 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

300

200

100

0

Time of analysis

lowastlowast

Group 4 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

150

100

50

0

Time of analysis

lowastlowast





Acknowledgment


References

















































2extractsrdquo

























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of













3 Results








4 Discussion




UCA

RR

T0 T1

Group 1 dROMs200

150

100

50

0

Time of analysis

Group 3 dROMs

Time of analysis

UCA

RR

T0 T1

200

150

100

50

0

Group 2 dROMs

UCA

RR

T0 T1

200

150

100

50

0

Time of analysis

lowastlowast

Group 4 dROMs

Time of analysis

UCA

RR

T0 T1

200

150

100

50

0

lowastlowast











Group 4 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)

Group 3 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)

T0 T1

Group 1 BAP3000

2000

1000

0

Time of analysis

(120583m

olL

)

Group 2 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)







Group 1 BDNF150

100

50

0

BDN

F le

vels

(pg

mL)


Group 3 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

150

100

50

0

Time of analysis

Group 2 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

300

200

100

0

Time of analysis

lowastlowast

Group 4 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

150

100

50

0

Time of analysis

lowastlowast





Acknowledgment


References

















































2extractsrdquo

























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







UCA

RR

T0 T1

Group 1 dROMs200

150

100

50

0

Time of analysis

Group 3 dROMs

Time of analysis

UCA

RR

T0 T1

200

150

100

50

0

Group 2 dROMs

UCA

RR

T0 T1

200

150

100

50

0

Time of analysis

lowastlowast

Group 4 dROMs

Time of analysis

UCA

RR

T0 T1

200

150

100

50

0

lowastlowast











Group 4 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)

Group 3 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)

T0 T1

Group 1 BAP3000

2000

1000

0

Time of analysis

(120583m

olL

)

Group 2 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)







Group 1 BDNF150

100

50

0

BDN

F le

vels

(pg

mL)


Group 3 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

150

100

50

0

Time of analysis

Group 2 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

300

200

100

0

Time of analysis

lowastlowast

Group 4 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

150

100

50

0

Time of analysis

lowastlowast





Acknowledgment


References

















































2extractsrdquo

























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







Group 4 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)

Group 3 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)

T0 T1

Group 1 BAP3000

2000

1000

0

Time of analysis

(120583m

olL

)

Group 2 BAP

T0 T1

3000

2000

1000

0

Time of analysis

(120583m

olL

)







Group 1 BDNF150

100

50

0

BDN

F le

vels

(pg

mL)


Group 3 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

150

100

50

0

Time of analysis

Group 2 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

300

200

100

0

Time of analysis

lowastlowast

Group 4 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

150

100

50

0

Time of analysis

lowastlowast





Acknowledgment


References

















































2extractsrdquo

























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of







Group 1 BDNF150

100

50

0

BDN

F le

vels

(pg

mL)


Group 3 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

150

100

50

0

Time of analysis

Group 2 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

300

200

100

0

Time of analysis

lowastlowast

Group 4 BDNF

BDN

F le

vels

(pg

mL)

T0 T1

150

100

50

0

Time of analysis

lowastlowast





Acknowledgment


References

















































2extractsrdquo

























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of















































2extractsrdquo

























Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of













Microbiology


AnimalsJournal of








Volume 2014



Agronomy







Volume 2014

Zoology



InsectsJournal of




VirusesJournal of






an antioxidant dietary supplement improves brain derived neurotrophic factor levels in serum of aged...

Health & Medicine

bdnf levels

serum of aged dogs

pro tein damage

antioxidant dietary

dogs plasma samples

elderly dogs

cognitive impairment

dietary regime