The effect of different fatty acid sources on wound healing in rats assessed by matrix-assisted-laser-desorption-ionization mass-spectroscopy-imaging

The effect of different fatty acid sources on wound healing in rats assessed by matrix-assisted-laser-desorption-ionization mass-spectroscopy-imaging Tomáš Komprda1, Zbyšek Sládek2, Veronika Švehlová1, Zuzana Lacková3,4, RomanGuráň3,4, Tomáš Do3, Jan Wijacki2, Marcela Buchtová5, Jana Neuwirthová6, Břetislav Gál6,VojtěchAdam3,4,OndřejZítka3,4 1MendelUniversityinBrno,DepartmentofFoodTechnology,Brno,CzechRepublic 2MendelUniversityinBrno,DepartmentofAnimalMorphology,PhysiologyandGenetics,Brno,CzechRepublic 3MendelUniversityinBrno,DepartmentofChemistryandBiochemistry,Brno,CzechRepublic 4BrnoUniversityofTechnology,CentralEuropeanInstituteofTechnology,Brno,CzechRepublic 5CzechAcademyofSciences,InstituteofAnimalPhysiologyandGenetics,v.v.i.,Brno,CzechRepublic 6MasarykUniversity,St.Anne’sHospitalandFacultyofMedicine,DepartmentofOtorhinolaryngologyandHead andNeckSurgery,Brno,CzechRepublic Received March 15, 2019 Accepted October 29, 2019 Abstract The objective of the present study was to compare the effects of dietary oils containing polyunsaturatedfattyacids(PUFA)n-3andn-6,respectively,oncutaneouswoundhealingin rats,andtodemonstratetheusefulnessofthematrix-assisted-laser-desorption-ionizationmass spectroscopy-imaging(MALDIMSI)methodinthistypeofexperiment.SuperiorityofPUFA n-3inthiscontextwasthetestedhypothesis.FourgroupsofmaleWistarratsbytwelveanimals eachwerefedadietwithadded5%ofpalmoil(P;control),fishoil(F),Schizochytriummicroalga oil(Sch)andsaffloweroil(S),respectively,foreightweeks.Consequently,dorsalfull-thickness cutaneous excisions were performed, and selected markers of wound healing were evaluated 18dayspostexcision.Themedianofsignalintensitycorrespondingtoanamountofcollagen α-1 (III) fragment, quantified using MALDI MSI, decreased in a sequence P > F > Sch > S (P < 0.001). Using haematoxylin-eosin staining of the histological preparations, semiquantitativelyassessedepitheliumheighttendedtodecreaseintheorderofP>S>Sch>F; thewoundextentinthesequenceofP>Sch>S>F;andthesequenceoftheprogressofneoangiogenesiswasassessedasS>P>F≈Sch.Itwasconcludedthatthetestedhypothesiswas confirmedonlypartially:PUFAn-3showedbetterresultsregardingthewoundextent,butwere inferiorintermsofepitheliumheightandprogressofneo-angiogenesis.Thiswasthefirsttime MALDIMSIwassuccessfullyemployedforevaluatingskinwoundhealinginaratmodel. Docosahexaenoic acid, eicosapentaenoic acid, Schizochytrium oil, reepithelialization, MALDI MSI, collagen alpha-1(III) Woundhealingisadynamicsequentialprocessconsistingofthreeoverlappingstages: inflammation,cellproliferationandtissueremodelling(Caetanoetal.2016).Theeffects ofdietaryfattyacids,especiallylong-chainpolyunsaturatedfattyacids(LC-PUFAn-3) eicosapentaenoicacid(EPA;20:5n-3)anddocosahexaenoicacid(DHA;22:6n-3),known inflammation modulators, on wound healing in animal models are very inconsistent (Komprda2018):positiveeffects(Chenetal.2012;Olsonetal.2013)arecontradicted bynegativeones(Drzymala-Czyzetal.2012; dos Santos Rosaetal.2014),with betterresultsbeingoftenobtainedwithoilscontainingPUFAn-6(Otrantoetal.2010). The main objective of the present study was to evaluate the effect of fish oil (high EPAcontent)andaSchizochytriumsp.microalgaextract(richinDHA),respectively,in comparisonwithsaffloweroil(PUFAn-6linoleicacid)onselectedmarkersofthehealing ofacutaneousexcisioninrats.Asmallerextentofthewoundatthegiventimeinterval, better reepithelialization and neo-vascularization score by dietary oils containing LCPUFAn-3wasselectedasthetestedhypothesis.Oneoftheobjectiveswasalsotouse ACTA VET. BRNO 2019, 88: 443–449; https://doi.org/10.2754/avb201988040443 Address for correspondence: Tomáš Komprda DepartmentofFoodTechnology MendelUniversityinBrno Zemedelska1,61300Brno,CzechRepublic Phone:+420545133261 E-mail:[email protected] http://actavet.vfu.cz/ forthefirsttimethecutting-edge method of matrix-assisted-laser-desorption-ionization mass-spectroscopyimaging(MALDIMSI;Angeletal.2018)formonitoringthespatial distributionofaselectedcollagenfragmentincutaneousexcisionsinrats. Materials and Methods Animalsanddietaryinterventions Forty-eightadult malerats ofthe laboratorystrainWistarAlbino(BioTest,Konárovice,Czech Republic) attheageof8weeks(themeanliveweightof230±24.0g)wereused.Theratswerehousedinplasticboxes (53.5×32.5×30.5cm)offouranimalseachinaroommaintainedat23±1°C,humidityof60%and12/12h oflight/darkcycle(maximumintensityof200lx).TheexperimentwasperformedincompliancewiththeCzech NationalCouncilActNo.246/1992Coll.ontheprotectionofanimalsagainstcrueltyandtheamendedActNo. 162/1993Coll.,andwasapprovedbytheCommitteefortheProtectionofAnimalsagainstCrueltyoftheMendel UniversityinBrno(StatementNo.16252-MZE-17214). Theratsweredividedintofourgroupsby12animalseachandfedeightweekswitheitherthecontroldiet consistingofbasicfeedmixture(BFM;pelletizedcompletechowformiceandrats;Biokron,Blučina,Czech Republic)enrichedwith5%ofpalmoil(P),oroneofthethreeexperimentaldiets:BFMsupplementedwith5% offishoil(F),5%ofoilextractedfromtheSchizochytriumsp.microalga(Sch),and5%ofsaffloweroil(S;high contentoflinoleicacid),respectively.ThereasonforusingthePdietasacontrolinsteadofBFMalonewasthe requirementtokeepthedietsnotonlyisocaloric,butalsoisolipidic.Fattyacidcompositionofthefourdietsis showninTable1. Thedietswereprepared(atthelaboratory)asfollows:pelletizedchowwasgrinded,homogenizedwithan appropriateamountoftheparticularoilandcakesweighingapproximately200gwerepreparedbyhand.The animalswerefeddailyad libitumandhadfreeaccesstodrinkingwater.Feedconsumptionwasmeasureddaily pergivenbox;dailyfeedconsumptionperratwascalculatedasaone-fourthofthetotalintakepercage(dueto the ad libitumaccesstothefeed,majordifferencesinconsumptionbetweenanimalsinaboxwerenotpresumed). Theanimalswereweighedatweeklyintervals. Excisionsandsamplecollection After 56 days of fattening, all animals were anaesthetized by intramuscular administration of zolazepam (Virbac,Carros,France),andtwofull-thickness(epidermis,dermis)cutaneousexcisionsonthedorsumwere performedinthesagittalplaneusingacircularpuncherwithaninternaldiameterof8mm(StiefelLaboratories, Brentford,UK).Fortechnicalreasons,thetotalnumberofanimalswasrestrictedto48;considering12animals thenecessarynumberforsufficientcharacterizationofthedietarygroupinthehealingtimeperiod,wegaveup theideaofassessingseveralsubsequenthealingphasesand(basedonapreliminaryexperiment)optedforone 18-dayinterval.Aftertheexcision,theratswerehousedindividually:twoanimalswereplacedinthesameplastic boxasmentionedabove,partitionedwithacrossbarintotwoparts.Thewoundswerecheckeddailyforinfections andsincetherewerenone,noadditionalwoundtreatmentwasperformed.After18daysofcontinuationonthe particulardiet(P,F,Sch,S),theanimalsweresacrificedbyisofluraneoverdosingandthefollowingtissueswere taken:twosamplesofthewoundedskinfortheproductionofhistologicalpreparations(includingMALDIMSI) anda5galiquotoftheliverfordeterminationoffattyaciddeposition. 444 Table1.Fattyacidcontentintheratdietssupplementedwith5%ofpalmoil(P–control),5%ofsaffloweroil(S), 5%offishoil(F)and5%ofSchizochytriumsp.microalgaextract(Sch),respectively. Diet  P S F Sch Totallipids(%)  6.8 6.5 6.4 6.5  16:0 33.0 0.1 0.5 16.2  18:0 4.7 3.3 5.0 3.8 Fattyacidcontent(%oftheƩ 18:1n-9 36.6 17.1 39.7 22.0 ofalldeterminedfattyacids) 18:2n-6 21.1 73.3 24.3 21.8  18:3n-3 0.1 0.1 0.4 0.2  20:5n-3 0.2 0.5 6.3 1.0  22:6n-3 0.2 0.1 9.4 21.9 PUFAn-6/PUFAn-3  53.9 87.6 1.7 1.1 Fattyacidsdetermination Thecontentoffattyacidsinthedietsandintheliverwasdeterminedasperourpreviousstudy(Komprdaet al.2017). Histologicalpreparations Skinwoundsamplesincludingca2mmofskinsurroundingthewoundwerefixedin10%bufferedformalin, dehydratedbyagradualalcoholseries,clearedinxylene,embeddedinparaffinblocksandsectionedintothe sizeof5–8µmusingarotarymicrotomeIntracoMicromodelMT5505(IntracoMicroLtd.,Tachlovice,Czech Republic).Sectionsweresubsequentlystainedwithhaematoxylin-eosin(H&E).Thehistologicalpreparations werethenevaluatedusingalightmicroscopeOlympusBHTSPM1(OlympusAmericaInc.,USA).Morphometry andevaluationofthequalitativechangeswerecarriedoutineachsamplemanuallyusingtheocularmicrometer at×5(woundextent)and×200(reepithelializationextent),magnifications.Atleastthreesectionswereprepared fromeachtissuesample.Morphometrywasthendeterminedafterdigitalizationofthemicroscopicpreparations bymeansofthesoftwareLucia(LaboratoryImagingLtd.,Prague,CzechRepublic). MALDIMSIexperiment Formalin-fixedandparaffin-embeddedtissuesectionswith8µmthickness,mountedontoindium-tinoxide (ITO)glassslides,werepreparedaccordingtoHegeretal.(2016). The mass spectrometry imaging was performed on a MALDI-TOF/TOF mass spectrometer Bruker ultrafleXtreme(BrukerDaltonikGmbH,Bremen,Germany)usingaprotocolaccordingtoHegeretal.(2016). A total sample set spanned 8 ITO glass slides containing 24 tissue sections.As MALDI matrix, 30 mg/ml 2,5-dihydroxybenzoicacid(DHB)inmethanol/water(50:50,v/v)with1%trifluoroaceticacid(TFA)wasused. Therasterspotdiameterwas30μm.MALDIMSIofpeptideswasperformedinareflectorpositivemodeinthe m/zrangeof0–5kDa.Atotalof500spectraweresummedforeachspotusingtheRandomWalkrasterpattern, withnoevaluationcriteria.Them/zimagesweregeneratedandvisualizedusingtheSCiLSLab2014bsoftware (SCiLS–BrukerDaltonikGmbH,Bremen,Germany).FinalMALDIMSIimageswerepreparedintheGIMP 2.8software. For on-tissue digestion collagenaseA from Clostridium histolyticum was used.Approximately 300 μl of collagenaseAsolution(1mg/mlinwater)peroneslidewasappliedbyImagePrep(BrukerDaltonikGmbH, Bremen,Germany),theslidewaskeptinsideasmallboxwithhumidatmosphereat37°Cfor24handtheDHB matrixsolutionwasapplied.PriortoMSIanalysistheslidewasdriedinavacuumdesiccatorfor15min. Fromdifferenttypesofcollagen,collagenIII(togetherwithcollagenI)aretypicalfortheskin;therefore, collagenalpha-1(III)chainfragmentGAPGFRGPAGPNGIPGEK(accordingtoOktayetal.2018)wasused foranalysis. Statisticalanalysis TheMSIdatawereevaluatedbyAnderson-DarlingnormalitytestandKruskal-WallistestwithSCiLSLab softwareaccordingtoGuranetal.(2017).Histologicalpreparationswereonlysemi-quantitativelyassessed,and thereforewerenotstatisticallyevaluated.Regardingsupplementarymeasurements(weightcharacteristics,fatty aciddepositionintheliver),thedifferencesbetweendietarygroupswereevaluatedbyone-wayANOVAwithpost hocTukey’stestusingtheSTATISTICA12package(StatSoft,Tulsa,OK,USA). Results Weightcharacteristicsandfattyaciddepositionintheliver Liveweightofthecontrol(P-),S-,F-andSch-ratsafter56daysoffattening(finallive weightbeforeexcisions)was536.8±13.7,507.5±7.7,519.1±9.8and521.7±6.2g, respectively, and did not differ between dietary groups (P > 0.05). Daily weight gains (DWG;withintheperioduntiltheexcision,56days)oftheP-,S-,F-andSch-ratsare presented in Fig. 1 which shows that, in comparison with control, only safflower oil decreased (P < 0.05) DWG of rats. Deposition of EPA+DHA in the liver is shown in Fig.2.Thecontentofthebiologicallyactivesubstancestestedinthismainmetabolically activeorganindicatestheiravailability fortheprocess ofwoundhealing, amongother things. Histologicalexamination Semi-quantitativelyassessed(H&Estaining)epitheliumheightdecreasedintheorder ofP>S>Sch>F(PlateIII,Fig.3A).Thetotalextentofthewounddecreasedinthe sequenceofP>Sch>S>F(PlateIII,Fig.3B).Finally,thesequenceoftheextent ofneo-angiogenesiswasassessedasS>P>F≈Sch.Collagenfibreswereoriented 445 inparallelinthebasalpartofthegranulationtissueofthecontrolsamples(PlateIII, Fig.3Ba);theS-preparations(PlateIII,Fig.3Bb)didnotdifferinthisregard.Horizontally arranged collagen fibres switched to an all-direction orientation in the greater depth oftheF-preparations(Fig.III3Bc).Thecollagenfibresorientedinparallelthatwere accumulatedattheboundarybetweenthegranulationtissueandthecorium,switched (similarastheF-samples)toanall-directionorientationintheSch-preparations(Plate III,Fig.3Bd). 446 Fig.1.Dailyweightgainofratsfed56daysadietsupplementedwith5%ofpalmoil(P;control),5%ofsafflower oil(S),5%offishoil(F)and5%ofSchizochytrium sp. microalgaextract(Sch),respectively;a,b–meanswith differentlettersdifferatP<0.05,one-wayANOVAwithpost hoc Tukey’stest Fig.2.Depositionofeicosapentaenoicacid(EPA)anddocosahexaenoicacid(DHA)intheliverofratsfedadiet supplementedwith5%ofpalmoil(P;control),5%ofsaffloweroil(S),5%offishoil(F)and5%ofSchizochytriummicroalgaextract(Sch),respectively,for74days(56daysbefore+18daysafterexcision);A,B–means withdifferentlettersdifferatP<0.05(notindividualfattyacidsbutsumsofEPA+DHAarecompared);one-way ANOVAwithpost hocTukey’stest. Dietaryfishoilshowedmoreadvancedarrangementofcollagenfibresandtendedto decreasethewoundextent18daysafterexcisionincomparisonwithcontrol(P)which wasapositivefinding,butsimultaneouslytendedtoworsentheoutcomeintermsofthe epitheliumheightandaprogressofneo-angiogenesis. MALDIMSIofacollagenfragment The spatial distribution of collagen alpha-1 (III) chain fragment GAPGFRGPAGPNGIPGEK, m/z 1679.873 ± 0.100 Da, is shown in Fig. 4 (Plate III, Fig.4).Theregionswiththehighestintensitiesofthisfragmentwerefoundintheskin tissuesectionofcontrolgroupP;theintensitiestendedtodecreaseinarowP>F>S>Sch. Moreover,thedatashowninFig.4(yellowlines)confirmedthatthehealingprocessstarted onthesidesoftheincisionandcontinuedtowardsthecentre.Basedontheintensitybox plots(PlateIV,Fig.5),themedianofintensitywasdecreasing(P <0.001)inasequence P>F>Sch>S.Therefore,theoutputoftheMALDIMSImeasurementsdifferedsomewhat fromthehistologydata. Discussion Theeffectofdietaryoilontheweightcharacteristicsofrats ThereportedeffectsofPUFAn-3onDWGofratsarecontradictory:noeffectofdietary fishoil(Campiolietal.2012);atendencytoincreaseDWGintheF-ratsincomparison with safflower oil (this study; Fig. 1); a significant DWG decrease in the Sch-fed rats incomparisonwithsaffloweroil(Komprdaetal.2016).Thecontradictioncorroborates the conflicting epidemiological evidence of a presumed inverse association between consumptionofLC-PUFAn-3andobesity(Howeetal.2014). Histologicalevaluation Gerceketal.(2007)reportedsignificantlysmallermeanwounddepthbutlowermean scoresforepidermalanddermalregeneration,granulationtissuethicknessandangiogenesis in rats treated with parenteral fish oil emulsion 18 days after the skin wounding in comparisonwiththecontrol,whichwasverysimilartoourresults. Ontheotherhand,thepercentageofwoundclosuresevendaysaftertheexcisionon the back of rats was higher in control than in the fish oil-fed group in an experiment byOtrantoetal.(2010).Thepercentageofre-epithelializedwoundarea18dayspost excisionwasnotdifferentbetweenthesunfloweroilandfishoilgroup,butwaslowerin thefishoilgroupthaninthecontrol.Thesedatapartiallycorrespondwiththeresultsofthe presentexperiment,wheretheheightoftheepitheliumintheF-ratstendedtobelowerin comparisonwiththecontrolgroup. Otrantoet al. (2010)also reported delayedangiogenesis and impairedformationof the granulation tissue in the fish oil group and in the sunflower oil-fed rats; results of the present experiment are partially in accordance with the quoted study: we can also inferabetterscoreofdietarysaffloweroilthanfishoilasfarasre-epithelializationand vascularizationisconcerned. MappingofcollagendistributionbyMALDIMSI ThemostrecentandactuallytheonlystudyinwhichMALDIMSIwasusedinwound healingevaluatedthedistributionoflipidsinvolvedintheskinbarrierrepair/woundhealing processusinganin vitrolivingskinequivalentmodel(Lewisetal.2018).However,the animal(rat)modelusedinthepresentstudyismoresuitablethanin vitromodelsregarding therelevanceofgainedinformationtonaturalbiochemicalprocessesoccurringinaliving organism. 447 Theuse of formalin-fixedandparaffin-embedded (FFPE) tissuesections for MALDI MSIhasitslimitationsregardingtheprocessesofdeparaffinizationandantigenretrieval, duringwhichtheanalytesofinterestcanbedilutedandmoredelocalizedinthetissue. Also,theefficiencyoftheirionizationisusuallyworsethaninthecaseofcryosections (Pietrowskaetal.2016). Itcanbeconcludedthatthehypothesistestedinthepresentstudywasonlypartially confirmed: the results show ambiguous effects of oils containing PUFA n-3 on the histologicalmarkers,includingtheirinconclusiveeffectontheweightcharacteristicsof rats.Ontheotherhand,thepresentexperimentconfirmedthepoweroftheMALDI-TOF MSItechnique,whichwasappliedforthefirsttimeintheratskinhealingexperiment.In ordertoincreaseeffectivityofPUFAn-3inwoundhealing,theusefulnessoftheirtopical application in a combination with nanoparticles would be worthy of testing in future experiments. Acknowledgements TheexperimentwasfinanciallysupportedbytheInternalGrantAgencyofMendelUniversityinBrno(project no.TP3/2017)andbyCEITEC2020(LQ1601). 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Reepithelialization (A) and wound extend (B) 18 days post excision in rats fed a diet supplemented with 5% of palm oil (P; a), 5% of safflower oil (S; b), 5% of fish oil (F; c) and 5% of Schizochytrium microalga extract (Sch; d), respectively for 74 days (56 days before + 18 days after excision). Haematoxylin& eosin staining. Magnification was ×200 for (A) and ×5 for (B). The epithelium and its width is highlighted by yellow dashed lines and arrows Fig. 4. Haematoxylin-eosin images combined with matrix-assisted-laser-desorption-ionization-time-offlight mass spectroscopy imaging of the m/z 1679.873 ± 0.100 Da collagen alpha-1(III) chain fragment GAPGFRGPAGPNGIPGEK, in the dermis 18 days post-excision in rats fed (56 days before + 18 days after excision) a diet supplemented with 5% of palm oil (P – control; a), 5% of safflower oil (S; b), 5% of fish oil (F; c) and 5% of Schizochytrium microalga extract (Sch; d), respectively. The regions with the highest intensity of m/z 1679.873 ± 0.100 Da fragment are highlighted by red ovals Plate III Komprda T. et al.: The effect of different ... pp. 443-449 Fig. 5. Intensity box plot for the m/z 1679.873 ± 0.100 Da collagen α-1(III) chain fragment GAPGFRGPAGPNGIPGEK in the tissue sections of the dermis 18 days post excision in rats fed (56 days before + 18 days after excision) a diet supplemented with 5% of palm oil (P – control), 5% of safflower oil (S), 5% of fish oil (F) and 5% of Schizochytrium sp. microalga extract (Sch), respectively; each dietary group is represented by six tissue sections, several tens of thousands of the mass spectra were measured within each section, and intensity of each mass spectrum is depicted by a spot within the plot; the intensity decreased in the order of P > F > Sch > S (P< 0.001; Kruskal-Wallis test) Plate IV