An Overview of the Histology of Aging Skin in Laboratory Models Tapan K. Bhattacharyya Contents Abstract This article is an overview of Introduction.......................................... 1 histomorphological changes documented dur- TheHumanScenario:SkinAgingHistology...... 2 ing various stages of life in many laboratory AginginRodentModels............................. 3 modelsutilizedinskinagingstudies.Abewil- deringvarietyincutaneousagingresponsehas MorphologicalChangeswithAginginOther Species............................................. 7 been observed in different strains. The com- monlyusedlaboratorymodelsliketheratand CalorieRestriction(CR)andSkinAging.......... 7 the mouse show dissimilar aging changes in Conclusion............................................ 7 skin. There are a few mouse models which References............................................ 10 seem to resemble the general trend of skin attrition related to advancing age observed in some human studies. Caloric restriction has beenobservedtomodulateskinagingchanges in the rat and the mouse. Despite the wide variation in observational studies related to agingchanges,somerodentmodelsareuseful toagingresearchandexperimentalresponseof agingskin. Introduction Human populations worldwide are living longer, andskinfromolderpeoplebecomesmoresuscep- tible to diseases and malformations apart from “Whatliesbeneaththisagingskin?Theuntolddestruction beingravagedbyenvironmentaltraumalikeultra- stealthycreeping,Thebonestheorgansthenerves,the violetradiation.Thescienceofclinicaldermatol- brain,Iambreathing,Iamfunctioning,AmIliving?” ogydocumentsnumerousdiseasesofhumanskin, JulieA.Crippin,2008 but scant information has been paid to basic T.K.Bhattacharyya(*) researchinthemicroanatomyofagingskin.This Otolaryngology-Head&NeckSurgery,Universityof isasubjectthatinvokesbiologicalandevolution- Illinois,Chicago,IL,USA ary interest about age-induced involution of the e-mail:[email protected] #Springer-VerlagBerlinHeidelberg2015 1 M.A.Farageetal.(eds.),TextbookofAgingSkin, DOI10.1007/978-3-642-27814-3_1-2 2 T.K.Bhattacharyya integumentary system. It is logical to introspect fewdecades.Skinatrophyismarkedonlyafterthe aboutthephenomenonofchronologicalagingof fifthdecadeofhumanlifeandshowsaplethoraof animal skin from other mammalian species. morphologicalchangesincludingepidermalthin- Despite many descriptive studies published in ning,flatteningofthedermal-epidermaljunction, early literature, there are still many gaps in our loss of melanocytes, and immunocompetent knowledgeaboutthepatternsofintrinsicagingin Langerhans cells; some physiological functions, various laboratory models. Although animal suchassurfacelipidproduction,andthermoregu- models may not fully corroborate biochemical lation are also affected by the aging process data of aging skin and may show inconsistent (reviewinRittieandFisher[3]). results, they are still useful tools to interpolate Therearealsodermalchangessuchasreduced the fundamental etiologies of skin aging and fibroblastpopulationandsebaceousglands.These antiagingeffects[1]. histopathologic events have been reviewed One reason for the lack of documentation in [4]. Morphometric measurement of collagen thisareaofresearchisscantavailabilityofanimal fibers from stained human skin biopsies further models of skin aging with clear documentation showedthatcollagenfiberdensitystarteddecreas- regardingageorthestagesofthelifecycle.Ani- ing from around 30–40 years, with thinner and mal husbandry for long-term maintenance of morespacedfibers[5].Otherinvestigatorsfound aginganimalsinadisease-freecolonyisacostly nodifferenceinepidermalordermalthicknessina project and is usually beyond the reach of an study of wound healing comparing skin from average laboratory. The aging human skin with young and elderly volunteers [6]. The biochemi- increasedroughnessandwrinklingissagging,and calprofileofcollagenmetabolismofhumanskin, inelastic,andshowsmanysignsofatrophy,such however, changes with age. A steady decline in as epidermal thinning or abnormal collagen and synthesis of hydroxyproline in human skin up to elastic fibers. It is logical to ask if similar or thefourthdecadehasbeendescribed[7]. differentconditionsareobservedwhenacompar- Reports of dermal elastic fiber changes with ativeanatomical analysis isextendedtodifferent age including abnormalities and disintegration models from the mammalian kingdom. This havebeenpublishedbysomeauthors.Asperthe review explores whether intrinsic aging also study of Vitellaro-Zuccarello et al. [5], elastic affects skin histology in laboratory models in a fiber distribution has a different aging pattern in comparablemanner.Thedataareverylimitedand men and women. While the density of elastic wereacquiredthroughpainstakinginvestigations fibersinthepapillarydermisisnotmodifiedasa by researchers dating back to several decades function of age, in the reticular dermis of both using classical histological techniques; the sexes, the fiber density increases in the first reported quantitative data were often decade, followed by a drop only in the female. uncorroboratedbystatisticalmethodology. Biochemically,elastinbiosynthesisisstableupto approximately the fourth decade of life [7]. Although reports by different investigators TheHuman Scenario: Skin Aging vary in detail due to different kinds of sampling Histology and histological methods employed, a general trend of attrition of the dermal connective tissue Morphologicalchangesofthehumanintegument fibersisnotedasacorrelateoftheagingprocess. due to intrinsic aging caused by time-induced Histopathologicchangesintheepidermisandder- physiological changes are understandable, but mis of human skin in sun-exposed and canbealteredduetopersonalandenvironmental sun-protected areas have been reviewed by factors,canvaryindifferentanatomicalsites,and Monteiro-Riviere[8]. are also linked to endocrine factors [2]. Despite theseproblems,severalaccountsofintrinsicaging of human skin have been published over the last AnOverviewoftheHistologyofAgingSkininLaboratoryModels 3 Aging inRodentModels detailed study in young and old C57B1/6NNia mice[13].Infact,theepidermisfromtheearand Someaccountsofhistologicalandcellularkinetic footpadshowedastatisticallysignificantincrease changesthroughoutthelifespanorrepresentative inthicknessandaugmentedcellsize.Theindexof stages of rodent life span are available. Most of labeling with tritiated thymidine showed no dif- thestudieswereconductedonthehairlessmouse, ference between young and old mice. Moreover, otherstrainsofmice(CBA,C57131/6NNia,Balb/ inC57BL/6Nmice,thenumberofepidermalcell c),andrats(Wistarrat,Fischer344).Itisdifficult layersandtheepidermalthicknessremainedcon- tomake ameaningful comparisonbetween older stantfrom1to22monthsofage[14]. studies related to skin aging histopathology, as Most of the reported studies on mouse skin different authors have reported data on both were mainly concerned with the aging effect on sexes of various mouse models, which were theepidermalcellsizeorcellkinetics,andscarce often based upon limited samplenumbers.Some attention has been devoted to the morphological of these important articles lack statistical evalua- changes of the dermal constituents in aging ani- tion. However, some noteworthy findings from mals. CBA mouse skin was investigated in three early literature on different rodent models are age groups (1, 6, and 27 months) from animals summarizedhere. procured from NIH colonies [15]. As the rate of The hairless mouse owes its hairlessness to a skin aging differs in different areas of the body homozygous recessive genetic condition, and [13], samples were studied from the dorsal, ven- structural changes of skin accompanying devel- tral, and pinna skin and the footpad of these opment of hairlessness in this animal were young,youngadult,andoldanimals.Anegative describedintheolderliterature.Age-relatedmod- linear effect of age on epidermal depth with a ificationsinepidermalcellkineticsinthisspecies significant reduction in cell count (cell/mm) and were described by Iverson and Schjoelberg pilosebaceousunitprofilesindorsalskinsamples [9]. Using autoradiography, it was shown that andfootpadwasobserved.Thesebaceousglands epidermal cell proliferation increased from birth appeared atrophied with pyknotic nuclei (Figs. 1 to approximately 20 weeks of age and remained and 2). No consistent change in depth of the steady. This detailed study could not confirm dermisorareafractionofcollagenasdetermined whether epidermal thickness or cell proliferation by histomorphometry was noticed. The dermal ratedecreasessystematicallywithincreasingage. elastic fibers in the dorsal skin and footpad Ontheotherhand,Haratakeetal.[10]presented showedproliferationinhigheragegroups inthis datashowingthatthethicknessoftheepidermisin mousemodel.Thiscanbecomparedwithastudy hairlessmicedecreaseswithintrinsicaging.There in the C57/B16 mice from NIH colonies [16] wasalsolessincorporationoftritiatedthymidine wheredecreaseddermalcellularityandthickness intheepidermisinoldermice. and decreased epidermal proliferation were The tritiated thymidinetechnique was usedin reported. miceupto19monthsofage,andthedatarevealed Further investigation of the CBA mice from anage-dependentdeclineinthecellproliferation NIH colonies as described in the previous para- rate[11].ThedatareportedfromBalb/cmice[12] graph [15] was made to compare epidermal mor- seemed to indicate epidermal atrophy with age. phometryandtheindexofproliferatingcellnuclear Theepidermiswasthinner,withsmallernuclei,in antigen (PCNA-I) by immunohistochemistry in 20-month-old animals compared to 2-month-old respect to the dorsal and pinna skin, ventral skin, animals,althoughtherewasnodecreaseinmitotic and the footpad fromother areas ofaging experi- activity and DNA labeling index. The loss of mental animals (Bhattacharyya, unpublished epidermal mass was related to a decrease in pro- observations).Therewasanattritionofepidermal teinandRNAcontentoftheepidermis.However, thicknessindorsalandventralskinandthefootpad similarepidermalchangeswerenotobservedina in relation to aging. PCNA-I showed a reduction 4 T.K.Bhattacharyya Fig.1 Histologicalpreparationofthedorsalskininaging shrinkageofsebaceousfollicles.Dermalelasticfiberscan CBA mice in young (a), young adult (b), and old (c) beseeninc.Verhoeff-vanGiesenstain animals,showingincreasingatrophyoftheepidermisand Fig.2 Errorbarchartof Three age groups: young, adult, old epidermalwidth measurementsinthree groupsofCBAmice m) 15.00 µ h ( pt 12.50 e d pi e D 10.00 S 1 ± n 7.50 a e M 5.00 1.00 2.00 3.00 Age across the ages only in the pinna skin and dorsal murinespecies.InanearlystudyofWistarInstitute surface(Table1;Figs.3a–c). rats,nosignificantage-relatedalterationwasnoted The aging skin in the rat shows nonuniform althoughtheauthordescribedmanyqualitativedif- patternsindifferentstrainswhenitcomestoepider- ferences in epidermal layers [17]. In Sprague mal and dermal thickness, and the age-associated Dawley rats, the foot epidermis was explored to changes seem to differ from the trend noted in determine age-related changes in cell kinetics AnOverviewoftheHistologyofAgingSkininLaboratoryModels 5 Table1 PCNA-IandepidermalthicknessdatafromagingCBAmice PCNA-I EPIwidth(μm) DS Y 31.9+5.0 16.3+0.8 AD 32.6+5.4 14.1+1.0 O 24.1+4.3 12.3+0.7 *F5.46,P0.01 *F32.43,P0.0005 PS Y 37.0+7.2 15.6+1.5 AD 26.8+5.3 14.1+1.3 O 31.2+5.9 13.3+3.2 *F4.01,P0.04 FP Y 40.8+12.2 88.7+11.8 AD 32.6+6.5 72.9+8.1 O 40.9+6.1 55.3+8.7 *F17.28P0.0001 VS Y 35.0+4.8 14.6+0.6 AD 34.8+2.7 12.1+0.9 O 32.1+3.1 9.8+0.9 *F54.91P0.0005 DSdorsalskin,PSpinnaskin,FPfootplate,VSventralskin,Yyoung,ADadult,Oold usingsingle-pulse[3H]-thymidinelabelingandthe increase is greatly diminished with aging. This is percentlabeledmitosistechnique[18]andledtothe duetoreducedcollagenbiosynthesisandincreased conclusionthatthereisaprogressivedeclineinthe degradation of macromolecules, but the balance ratesofcellproliferationassociatedwithage.How- betweensynthesisanddegradationremainspositive ever,thesedatawerepresentedfromratsonlyupto inrats. the age of 52 weeks. The authors of this chapter Aging changes in cells other than epidermal referred to five reports published earlier, which keratinocytes, such as melanocytes or cells of showed that rodent epidermal cell proliferation Langerhans,havealsobeendocumentedinsome decreasedinmiddleageandthenremainedconstant studies.Ultravioletradiationhasimportanthealth or increased in senile animals. Skin from aging consequences on the Langerhans cells of human Wistarratsuptotheageof34monthswasstudied skin.ThenumericaldensityofLangerhanscellsin using histomorphometric analysis by Voros and aging inbred mice was studied from epidermal Robert [19]. Average epidermal and dermalthick- sheets and showed reduction when compared to nessdidnotshowappreciablechangewithsenility thatinyounganimals,althoughcutaneousimmu- inthisspecies.InagingFischer344rats,epidermal noreactivity was not compromised [23]. thicknessremainedconstantfrom3monthsofage Age-relatedneurodegenerativechangesinperiph- onward[14].However,increasingvaluesinepider- eralnervesareawidespreadphenomenonofclin- maldepthandnuclearpopulationwerenotedinthe ical importance, and rat studies have attested to ventral and dorsal skin and footplate skin samples thisinhibitorypattern.Age-associatedlossinsize from young, 1-year-old, and 2-year-old Fischer ofMeissner’scorpuscles,withsmalleranddisor- 344rats[20,21].Earlier,Lapiere[22]commented ganized axonal processes, was reported in the thatinsteadofbecomingatrophic,ratskinincreases digital pads of mice aged to the maximum life in size, with more collagen, although the rate of expectancy[24]. 6 T.K.Bhattacharyya a Bar graph CBA mice pcna/epi width DSPCNAI 120.00 DSEPlum PSPCNAI PSEPIUM FPPCNAI 100.00 FPEPIUM VSPCNAI VSEPIUM 80.00 n ea 60.00 M 40.00 20.00 0.00 Young Adult Old AGEGROUP Error bars: +/- 1 SD b c Fig.3 (a)Acompositebardiagramshowingepidermal specimen shows PCNA staining in epidermis (EPI, width and PCNA-I (PCNA index) in skin samples from arrow). CT cartilage. (c) Pinna skin immunostained for dorsalandventralareas,footpad,andthepinnainthreeage PCNAinanoldspecimenshowsabsenceofreactivenuclei groups. (b). Pinna skin section from a CBA young inathinnerepidermis(EPI) Interpretation of morphological changes of changesinagingmice,rats,rabbits,andhamsters aging skin has many limitations, as discreet bio- havebeenpublished.Onlyafewpapersarecited chemicalchangesunderlyingsuchalterationscan- here[25,26].Oxidativedamagetothelipidsand notbevisualized.Incontrasttosparselyavailable DNA increases with age in Fischer 344 rats, accountsofdermalhistochemicalormorphologi- which was studied by measuring antioxidant cal transformations in relation to life history in enzymeactivity[27].Inthehairlessmouse,how- laboratory models, biochemical studies showing ever, skin aging was not accelerated due to quantitative changes in dermal glycosaminogly- decreasedantioxidantcapacity[28].Suchmolec- cans, hydroxyproline concentration, acid muco- ularchangesoftheskininintrinsicallyagedlab- polysaccharides, and skin collagen and elastin oratory animals can only be revealed by AnOverviewoftheHistologyofAgingSkininLaboratoryModels 7 immunohistochemistry as more suitable anti- dermalcollagen, elastic fibers, fibroblast density, bodiesbecomeavailableforresearch. capillaryprofiles,andstainingintensityofdermal glycosaminoglycans). The Fischer 344 rat showed many age-related skin changes, and Morphological Changes withAging thesewerepreventedordelayedbyCR,presum- inOtherSpecies ably due tometabolic alterations imposed by the dietaryregimen(Fig.4). Somesporadicaccountshavealsobeenpublished CR reduces cell proliferation in some tissues, relatingtomammalsotherthancommonlyavail- with inhibited pace of DNA replication, and this able laboratory rodent models. Age-induced makesthosetissueslesssusceptibletoDNAdam- reduction in sebaceous glands was described in age by carcinogens. Epidermal cell proliferation sheepbyWarrenetal.[29].Epidermalflattening, as quantified by immunohistochemistry was also fewer hair follicles and sebaceous glands, and a correlated to age-related changes in epidermal decreaseinmelanocyteswereage-relatedchanges thicknessinthesecolony-raisedFischer344rats. in the hairless dog [30]. Veterinary textbooks Just as CR somewhat inhibited the trend of describe aging changes in skin of domesticated increasing epidermal width in aging rats, the dogs and cats. Senile changes in the skin of old keratinocyte proliferation rate as measured by cats and dogs include alopecia, callus formation staining of proliferating cell nuclear antigen over pressure points, orthokeratotic hyperkerato- (PCNA)wascorrespondinglylowerinagingCR sis of the epidermis, and atrophied hair follicles rats.Thistrendwasobservedintheepitheliumof [31]. Due to certain structural similarities with thedorsalskinaswellasthefootplate(Figs.5and humanskin,thepigskinmodelhasbeenusedin 6)[34].InC57BL/6Jmice,epidermalcellprolif- many experiments for studying responses to sur- eration was reduced by CR and alternate-day gicalandphysiologicalmanipulations[32],butno fastingregimens[36].AstudyinSENCARmice account is available on aging morphological also shows that dietary calorie restriction may changesinthisspecies. inhibitgeneexpressioninskintissuesrelevantto cancerrisks[37].CRhasbeenreportedtoreduce theleveloffreeradicalsandpreventaccumulation CalorieRestriction(CR)andSkinAging of advanced glycation products and thus may be beneficialtoagedskin.Ontheotherhand,lossof Calorie restriction (CR) can reverse subdermal adipose tissue stores, with restricted age-associated alterations in many organs like feeding, may accentuate fine wrinkles in human the heart, the liver, and the brain [33] and has facial skin. CR can diminish subdermal adipose been shown to exert beneficial effects on many tissue [21]; therefore, its aesthetic effect on the skin disorders, although its morphological effect profile of the aging human face remains to be on the aging skin has not been thoroughly seen. explored. Whether CR can modify age-related histomorphological features of the skin in colony-raised rats was evaluated with morpho- Conclusion metric procedures in Fischer 344 rats. [20, 21, 34, 35]. Three age groups (young, adult, old) of Duetotheeaseofstudyingskinagingphenomena thisstrainbelongingtoadlibitumandCRfeeding and age-associated progressive changes in short- regimens were obtained from NIH colonies, and lived mammals, published studies were mostly skin samples from the dorsum, footpad, and confined to laboratory rodent models. Unfortu- abdominalskinwereanalyzedwithmorphometric nately,manyearlierdescriptionsofthehistopath- procedurestoevaluatevariousskincompartments ologic changes in aging skin in such models did (thicknessofstratumcorneum,epidermis,PCNA notconsideroldorsenilespecimensandtherefore index, dermis, fat layer, percentage fraction of lackaproperpopulationsampling.Thus,thestory 8 T.K.Bhattacharyya Dermis depth in abdominal skin in CR rats 700 600 500 s er 400 et Mean value m o sd cr 300 Mi 200 100 0 1 2 3 4 5 6 Mean value 402 585.3 622.1 418.8 375.36 429.73 sd 49.86 189 170.1 72.8 103.2 114.65 ALY ALAD ALO CRY CRAD CRO Fig.4 BargraphrepresentationofthedermisdepthfromFischer344ratsinCRstudy.Threeagegroupsofadlibitum (AL)andcalorie-restricted(CR)animalsarerepresented Fig.5 Representationof Title RAT CR Study FP EpiW vs PCNA epidermalwidthandPCNA indexfromfootplate(FP)in 175 FPEWidth ratsfromCRstudy.Three agegroupsfromALandCR FPPcnal animalsarerepresented 150 125 D S 100 1 – + n a e 75 M 50 25 0 Young Adult OLD Young Adult OLD AnOverviewoftheHistologyofAgingSkininLaboratoryModels 9 Fig.6 PCNAstaininginfootplateepidermisfromadult observed. (b) CR rat, old group. The footplate section (a)andold(b)CRrats.(a)CRrat,adultgroup,footplate showsregressionoftheepidermisandfewerPCNAposi- section.Abroadepidermiswithathickcornifiedlayerand tivecells brownstainedPCNAreactivecellsinthebasallayercanbe issomewhatincompletewhencomparedtogeron- underproperhusbandryconditionsandmaybea tologic cutaneous changes studied from skin more suitable animal for studying the process of biopsysamplesof90-year-oldhumanpatients!It senescenceoftheintegumentarysystem.Virtually shouldbenotedthatrodentlifespanisalsohighly nodataareavailableonhistologicalchangesfrom variable in different strains. Early literature has intrinsic aging in other animals like the dog, the recordedthatamonginbredmicestrainsusedfor domesticpig,ortherabbit,althoughsuchspecies aging research,ameanlifespancanvaryfroma are extensively used for biomedical and physio- lowof276daysintheAKR/Jstraintoamaximum logicalstudies. of 799 days in the LP/J strain [38]. The lack of Wrinkle formation is a vexing problem of availability of suitably aged rodent models is a aging human facial skin, and there is little infor- handicaptosuchresearch.Thisreview,however, mationaboutskinwrinklesinanyanimalmodelat showsthatthemousemodelmaybemoresuitable the senile state. Suitable models for studying the fordocumentingage-relatedhistologicalchanges histophysiologyofwrinklesortheirreversalhave that can be compared to human data, despite the not been reported, except for one study. Cross- caveat that comparing skin from laboratory- breedingbetweentheWistarandthewildratwas grown rodents with a short life span with the reportedtogeneratearatmodel(theIshibashirat) human skin assaulted by years of disease and in which skin aging, with wrinkles and furrows, environmental challenge is a rather risky appeared at 12 weeks. Wrinkle formation in this endeavor. Histological deficits in intrinsically new model was due to a reduction in elastin and aged human skin affect the epidermis, dermal collagencontentsoftheagingskin[40].Thiskind thickness, cellularity, and elastic fiber system. of experimental model would prove useful to Efforts should be made to search for analogous define the underlying anatomical correlates of chronologicalchanges inanimal modelsthat can skin wrinkleformation,tostudy its profilometry, beutilizedforstudiesaimedatrejuvenationofthe andtotesttheeffectoftopicalanti-wrinkleprod- aged skin. Similarities between human and uctsforitsamelioration[41]. murine skin have been reported in many studies, Inrecentyears,stemcellsinskinhaveattracted makingmouseskinasuitablematerialforstudy- alotofscientificintrospectionduetotheirpoten- ing inflammatory skin diseases. Apart from cer- tial clinical application in wound healing, burns, tain similarities in basic molecular and andalopecia[42],andthesecellscangothrough physiological processes between the mouse and self-renewalandterminaldifferentiationandmay the human [39], the mouse can be maintained regulate tissue aging [43]. It remains to be 10 T.K.Bhattacharyya determined whether stem cells in aging mouse HI,editors.Dermatoxicology.NewYork:CRCPress; skincanshowtemporalregression.Invitrostud- 2008.p.39–49. 9.IversenOH,SchjoelbergAR.Agerelatedchangesof ies have shown aged mouse epidermal epidermalcellkineticsinthehairlessmouse.Virchows keratinocytes can function comparably as those ArchBCellPatholInclMolPathol.1984;46:135–43. cells from young mice [44]. Keratin 15 (K15) 10.HaratakeA,UchidaY,MimuraK,etal.Intrinsically promoter activity, which is specific for stem aged epidermis displays diminished UVB-induced alterations in barrier function. 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