This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights
Transcript
This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies areencouraged to visit:
We report here beneficial effects of life-long dietary restriction on the progression of age-associated cochleardegeneration in female Sprague–Dawley rats. Thirty-month old rats on a 70% dietary restriction were comparedto ad libitum fed age-matched rats, and three-month old adult rats. As expected, aged dietary restricted ratsdisplayed about 20% higher survival rate than age-matched rats fed ad libitum. This difference was reflectedalso in the auditory system. In the dietary restricted group, 73% of the subjects had preserved auditory reflexes(Preyer), and only modest degeneration of the stria vascularis of the inner ear was observed. In contrast, agedad libitum fed animals, of which only 15% had detectable Preyer reflexes, showed a marked thinning, cellulardegeneration and loss of cell processes in the stria vascularis. The extent of loss of sensory hair cells (~24%)was similar in both the aged groups, and neither group showed a significant reduction in the number of spiralganglion neurons across adult life-span. The observations thus demonstrate that dietary restriction delays age-related degradation of the auditory system. The results provide further insights into the mechanisms of strialpresbycusis.
A gradual loss of sensory perception is a prominent feature ofthe aging phenotype (Cowen and King, 2005). The effects of aging im-pact all sensory modalities but mostly those that rely on sophisticatedperipheral receptors, such as the cochlea of the inner ear.
Age-related hearing loss, presbycusis, is one of the most commoncauses of hearing loss (Frisina, 2009; Jonsson et al., 1998; Ohlemiller,2004; Schacht and Hawkins, 2005). In the developedworld, presbycusisaffects approximately 40% of the population at the age of 60, and thehearing loss progresses by approximately 1 dB per year thereafter.With an increasing elderly population, this becomes a problem for theindividual as well as for society (Ciorba et al., 2012). Environmentalfactors such as previous exposure to noise and ototoxic drug treatmentprobably affect the onset and severity of the auditory impairment butgenetic factors are likely to significantly influence the progression(Gates et al., 1999; Newman et al., 2012). Schuknecht (Schuknechtand Gacek, 1993) divided presbycusis into three different subtypesaccording to their suggested origin: 1) sensory presbycusis characterizedby high-frequency hearing loss, caused by the degeneration of the sen-sory hair cells; 2) neural presbycusis with an equal hearing loss all over
the frequency span and loss of word discrimination due to the degener-ation of spiral ganglion neurons (SGN) and/or components of the cen-tral auditory system; and 3) strial (metabolic) presbycusis reflecting ahearing loss with an equal drop over the entire frequency range, butwith relatively good speech discrimination. Histological examinationof human temporal bones shows atrophy of the metabolic tissue ofthe cochlea, the stria vascularis (SV) and the underlying spiral ligament(Schuknecht and Gacek, 1993). The classification scheme has been dis-puted since a combination of different subtypes has been observed(Ohlemiller, 2004).
Understanding the natural history and themechanisms of presbycusisrests in part on the use of animal models, in particular rodent models(Ohlemiller, 2004; Ohlemiller and Frisina, 2008). One cause of hearingloss in rodents is loss of sensory hair cells, primarily the outer hair cellsin the most apical and basal parts of the cochlea. Other age-relatedchanges described in mice, rats and Mongolian gerbils include degenera-tion of the hearing organ; the SV, the spiral ligament and loss of SGN(Buckiova et al., 2006; Hequembourg and Liberman, 2001; Keithleyet al., 1992, 2004; Ohlemiller and Gagnon, 2004; Spicer and Schulte,2002, 2005). Some strains, like the Fisher 344 rat and the DBA/2Jmouse, are known for their early onset of age-related hearing loss;whereas other strains like the Sprague–Dawley rat, the CBA mouse andthe BALB/c mouse show a later onset of this disability. Different celltypes are affected to a variable extent in different strains (Keithley et al.,2004) and there are also differences in the degenerative pattern alongthe length of the cochlea reflecting the frequency variation of the hearingloss (Ohlemiller and Gagnon, 2004; Spongr et al., 1997). The observed
Experimental Gerontology 48 (2013) 1173–1179
⁎ Corresponding author at: Dept. of Neuroscience, Retzius väg 8, B1:5, KarolinskaInstitutet, SE-171 77 Stockholm, Sweden. Tel.: +46 8 524 869 44; fax: +46 8 524 860 88.
strain differences in presbycusis highlight the importance of the geneticbackground.
Themost successfulmeans, yet known, to retard the negative impactof aging on the organism is to reduce dietary intake through caloric (CR)or dietary (DR) restriction. Although the precise mechanism by whichCR/DR enhances health-span and extends life-span remains unresolved,it apparently involves multiple adaptations of cellular metabolism andenergy production (Mair and Dillin, 2008; Masoro, 2003; Speakmanand Mitchell, 2011). Only few studies have addressed to what extentCR/DR counteracts the deteriorating effects of aging on hearing(Someya et al., 2010a). In 27-month old Fisher 344 rats, CRwas reportedto impede both the age-associated increase in hearing threshold and theconcomitant loss of hair cells observed in ad libitum (AL) fed animals(Seidman, 2000). Someya and coworkers reported that CR alsosuppressed apoptotic cell death of SGN and age-associated hearingloss in the C57Bl/6 mouse (Someya et al., 2007).
The aim of the present study was to examine in greater detail theend-point effects of DR compared to AL feeding (Altun et al., 2007)by unbiased stereological quantification of the total number of sen-sory hair cells and SGN, moreover, the volume and the fine structureof the SV, a fundamental metabolic structure of the inner ear. Theoutbred Sprague–Dawley albino rat strain was chosen as a model be-cause of its similarity to humans regarding diversity in life span,body weight, sensory and motor functions during aging (Altunet al., 2007).
2. Material and methods
2.1. Experimental design
A total of 53 female albino Sprague–Dawley rats, raised andmaintained in-house (colony founders delivered by Scanbur BK,Sollentuna, Sweden), that were alive at end-point time, were used forthe study. All animals were kept in the same holding unit on a 12/12 h day and night cycle, with a room temperature at 21 ± 0.2 °C andrelative humidity at 50 ± 10%. Post-weaning off springs were arrangedin sibling-groups and kept 3–5 in open type 4 cages (MakrolonTM, M4,Techniplast, Buguggiate (Va) Italy). Asp-woodchipwas used as beddingmaterial (Tapvei, Kortteinen, Finland) and as enrichment; the cageswere equipped with plastic-tubes and nesting material (paper). As theanimal aged and siblings died with advancing age the number ofanimals per cage decreased, but no animals were single-housed. Com-mercially available food-pellets (Lactamin R34, Lantmännen, Sweden)and water were served ad libitum. To avoid single-housing, the foodwas served once a day, a regime that allows all animals to fed and bringsdown the animal-to-animal body weight variation among cage-littermembers (Altun et al., 2007). Animalswereweighed at regular intervals(every other week or once a month). The chosen design for the studywas a cross-sectional two-point study comparing young adults (age:2–3 months) with the end-point age, i.e., 30 month old subjects. Tochallenge normal aging, a group of young females (n = 20; startingweight 50.4 +/– 5 g) derived from the same colony were post-weaning maintained on a life-long dietary restriction, correspondingto 70% of the food intake of the AL fed colony mates (n = 30; startingweight 48 +/− 9 g). It has previously been shown that, this restrictionextends life-span by about 20% (Altun et al., 2007). Thus, at sacrifice theDR group (65% survival, n = 13; end-point weight 311 +/− 13 g) hada better survival (Supplementary Fig. S1) than the AL fed animals(survival 43%, n = 13; end-point weight 445 +/− 19 g). Preyer'sreflex test was used to indicate auditory status of the animals. A qualita-tive non-blinded yes/no assessment of reflexes (ear contraction or bodymovement) was performed after presentation of a sound burst of 60 dBSPL above the cage. The test was repeated once if no reflexes wereobtained after the first presentation. All young animals (n = 10) hadpositive reflexes, while in the aging groups, 73% of the DR animals
(n = 11) and 15% of the AL animals (n = 13) displayed positivereflexes.
The animals were sacrificed and cochleae from the differentgroups were used for stereological estimation of total cell numberand volume as well as morphological evaluation using transmis-sion electron microscopy.
All experimental procedures followed Swedish regulations for thecare and use of laboratory animals (ethical permissions N122/06 andN394/09).
2.2. Estimation of cell numbers and volumes
Cochleae from young rats (n = 8), old AL rats (n = 9) and old DRrats (n = 9) were perfused with a fixative containing 2.5% glutaralde-hyde in phosphate buffer (pH 7.2) and then decalcified in 0.1 M EDTAuntil the bone became soft. The cochleaewere postfixedwith 1% osmiumtetroxide, dehydrated, infiltrated and randomly oriented before beingembedded in a 2-hydroxyethyl metacrylate-based resin (Technovit7100, Heraeus, Germany). Serial sections (24-μm thick) from the wholecochlea were stained with hematoxylin and eosin. A stereological designwas established for estimating the complete three-dimensional volumeof the SV, a structure extending from the base to the apex along the lateralwall of the cochlea. Total number of sensory hair cells as well as totalnumber and soma cell volume of SGN (Gundersen, 1988; Moller et al.,1990; Tandrup, 1993; Watanabe et al., 2010) were also estimated ineach cochlea using the Cavalieri principle (Gundersen et al., 1988b)and the optical fractionator technique (Gundersen et al., 1988a). Amicro-scope (Axioplan Zeiss) with a motorized stage and an electronicmicrocator (Prior ProScan II) with digital readout for measuringmovements in the z-direction was interfaced to a digital camera (Pixellink) and a personal computer running newCAST software program(Visiopharm, Denmark) that randomly placed counting grids over thelive image of the sections; for details see supplementary information. Inshort, volume estimation of stria vascularis was performed by placing acounting grid with test points over the section at 20× magnification. Alltest points that covered the SV were counted. To estimate the total num-ber of cells, a counting frame was randomly placed over the section at100× magnification and all inner and outer hair cells as well as bothtypes of SGN within the frame were counted. Type I neurons were atthe same time assessed for cell soma volume using the nucleator tech-nique, where the average distance from the most centrally placed nucle-olus to the cell membrane were calculated in four randomly placed testlines per neuron. Type I neuron was defined as having a large cell somawith one or several small scattered nucleoli in contrast to the smallertype II neuron, which has one large, centrally placed nucleolus.
2.3. Electron microscopy
The basal and apical parts of SV from three cochleae per group wereanalyzed at the cellular level using electron microscopy. The decalcifiedwhole cochleae were dehydrated, cleared with propylene oxide andembedded in Agar 100, (Agar 100 Resin kit, Agar Scientific Limited,England). Basal and apical turns from approximately the same place ofthe cochlea were remounted on a new block of Agar 100. The SV wasdissected out and 1-μm thick sections, stained with toluidine blue,were examined with a light microscope to control the orientation ofthe specimenbefore thin-sectioning. Thin-sectionswere put on formvarcoated copper grids and stained with uranyl acetate and lead citrate.Sections were examined with a transmission electron microscope(JEOL 1230, JEOL GmbH, Germany) and digital images were capturedat 2500× and 10,000× magnification.
2.4. Statistics
Data analysis was made using the SigmaPlot for Windows Version11.0 and results are presented asmean values with standard deviations
1174 P. Mannström et al. / Experimental Gerontology 48 (2013) 1173–1179
(SD). Comparisons of the stereological measurements were made withone-way ANOVA.When a statistically significant differencewas present(P b 0.05) post-hoc analysis was made using the Holm–Sidak multiplecomparison test. The total variation (CVtot = SD / mean)was calculatedfor each group of the stereological estimates. The total variation in theestimate depends both on error variance of the method (CVmethod)and the biological variation between animals. The relation can bedescribed with the following formula: CVtotal
2 = CEmethod2 + CVbiol
2 . Thestereological sampling design was determined so that the varianceerror of the method was below 0.5.
3. Results
3.1. Dietary restriction maintains stria vascularis integrity in aged animals
3.1.1. Preserved total volumeTomonitor possible degenerative changes in the SV, the total three-
dimensional volume was estimated from histological sections. Themean volume of SV in the aged AL animals was reduced by 27% com-pared to the young adult animals (Fig. 1 and Table 1; Holm–Sidak test;P b 0.001) while animals on DR showed only a 12% reduction of thevolume. The strial volume in aged DR animals was more preservedand significantly larger than in the AL animals (Holm–Sidak test;P = 0.011).
3.1.2. Modest cellular degenerationFurther examination of the SV and the adjacent spiral ligament with
transmission electron microscopy revealed signs of degeneration in allaged animals (Table 2). The degree of degeneration varied betweenindividuals but importantly it was much more severe in the aged ALanimals compared to the DR animals. The thickness, from the marginalcells to the basal cells, was greater in the basal part compared to theapical part in all cochleae, even from young animals. An age-relatedthinning and degeneration of the epithelium, both in the base butespecially in the apex, was more advanced in the AL group (Fig. 2A–F).Marginal cells in the SV of young animals showed numerous processesclose to the cell nuclei that continued further through the SV towardsthe intermediate cells and the basal cells (Fig. 3A and B). The spiralligament beneath the SV contained numerous fibrocytes (type I) andcollagen fibers (Fig. 3C).
The aged AL animals showed a remarkable reduction of the totalwidth from the marginal cells to the basal cells (Fig. 2C and F). Thethinning was more severe in the upper part of SV that connects to theReissner's membrane, while the lower part connecting to the spiralprominence was less affected. The cell membrane of the marginalcells, which borders the scala media, was usually retracted but hadintact microvilli protruding into scala media as well as tight junctionsbetween the cells (Fig. 4A). The fine processes of the marginal cellsthat intermingle with the underlying intermediate and basal cell layers
showed a variable degree of degeneration, being more prominentlycloser to themarginal cell nuclei (Fig. 4A), while appearing less affectedlycloser to the basal cells (Fig. 4B). Spherical lobules containing homoge-nous material were often seen in varying degrees and sizes in the placewhere the processes should have been (Fig. 4C). Some marginal cellswere completely disintegrated, displaying only remnants of cell materialor having undefined inclusions as a sign of a later stage of degeneration(Fig. 4D). In severe cases, the stria from the apical part of the cochleaconsisted of just a single flat cell-layer in the upper part adjacent to theReissner's membrane (Fig. 4E). The basal cells appeared less affectedand had normal gap junctions connecting to the intermediate and mar-ginal cells (Fig. 4B). The basementmembrane that surrounds the capillaryendothelial cells inside the SV were clearly thickened compared to theslender basement membranes seen in the younger animals, (Fig. 4F,compare to same-scale insert). In the spiral ligament, lateral to the SV,swelling of cells and nuclei was observed as well as empty intercellularspaces, mostly in the apical part of the cochlea (Fig. 4G). The basementmembranes surrounding the blood vessels in the spiral ligament werenot thickened as in the SV from the same animals.
The more modest degeneration of SV in DR animals affected mainlythe apical part of the cochlea while the SV in the basal part had a moreintact appearance (Fig. 2B and E). The total width of the SV was betterpreserved and the marginal cells appeared healthier with less affectedprocesses, both close to the cell nucleus and closer to the basal cells(Fig. 5A). The extensive degeneration seen in some of the AL animalswith only a single cell-layer was never found in any of the DR animals.However, the cell membranes of the marginal cells were usuallyretracted (Fig. 5B) and a few of the DR animals displayed degeneration
Young DR AL
SV
vol
ume
(mm
3 )
0.05
0.10
0.15
* ***
Fig. 1.DR significantly preserved the total volume of SV in old animals compared to old ALanimals (Holm–Sidak test; P = 0.011). The total SV volume was significantly reduced inAL rats compared to the young adults (Holm–Sidak test; P b 0.001).
Table 1Stereological estimation of cell volume and number in the inner ear.
Total number of SGN 19526 ± 2794 17248 ± 1114 17881 ± 1483Volume of SGN (μm3) 2918 ± 329 2992 ± 321 3300 ± 619
Mean ± standard deviation for each of the cell populations from the different animalgroups. DR = dietary restriction, AL = ad libitum, SV = stria vascularis, SGN = spiralganglion neurons.⁎⁎⁎ P b 0.001.⁎ P = 0.011.a Compared to old AL.b Compared to Young.
Table 2Observed changes at the cellular level in the SV and the spiral ligament.
1175P. Mannström et al. / Experimental Gerontology 48 (2013) 1173–1179
Author's personal copy
in the apical part of the stria with lobules as described above for theAL animals (Fig. 5C). The thickening of the basement membranesurrounding the blood vessels was also equally advanced as in the ALanimals (Fig. 5D). Inspection of the spiral ligament found that thedegeneration was less advanced in the DR animals (Fig. 5E), whereswelling and intercellular spaces were only occasionally observed.
3.2. Dietary restriction does not influence age-associated hair cell loss
Stereological estimation of the total number of sensory hair cells(Fig. 6 and Table 1) demonstrated a loss of 23–25% in both aged DRand AL animals compared to the young group (Holm–Sidak testP b 0.001). Microscopical investigations showed that hair cells weremainly missing in the very apical part and in the most basal part of
the aged cochleae. The losswas greater among the outer hair cells, com-pared to the inner hair cells, irrespective of feeding regime.
3.3. Spiral ganglion neuron number and volume do not change significantlywith age
The total number of SGN (types I and II counted together) tended todecrease somewhat (about 10%) in both aged AL and DR groups (Fig. 7Aand Table 1). However, there was no significant difference between thegroups (one-way ANOVA). The average volume of the SGN type I somadid not change significantly across adult life-span but varied to a greaterextent in the AL animals (Fig. 7B and Table 1). Microscopical observa-tions found cells with the smallest cell soma volume in the apical partwhereas the cells with the larger cell soma volume were mainly foundin the base of the cochlea. A frequency distribution analysis revealed
Fig. 2. Lowmagnification transmission electronmicrographs showing representative sections of the SV in the apical cochlear turn of (A) a young animal, (B) a DR animal, (C) an AL animal,and in the basal turn of (D) a young animal, (E) a DR animal and (F) an AL animal. The age-associated reduction in volume of the SV was less severe in the DR animals, and the SV wasthinner at the apical location compared to the basal in all groups. Arrows indicate the width of the SV for comparison. SL = spiral ligament, SV = stria vascularis, SM = scala media.Scale bar = 10 μm.
Fig. 3.Highmagnification transmission electronmicrographs of SV in young adult animals. (A)Marginal cells (MC)withmicrovilli (arrows) protruding into the scalamedia (SM) and tightjunctions (white arrowhead) shown as darker compartments between the cells. Processes (p) interdigit with the underlying cell layers. (B) Processes (p) originating frommarginal cellsconnect to the basal cells (BC) through gap junctions (arrow head). (C) Spiral ligament (SL) beside SVwith fibrocytes (F) and collagen fibers (cf). Scale bars A and B = 2 μm, C = 10 μm.
1176 P. Mannström et al. / Experimental Gerontology 48 (2013) 1173–1179
Author's personal copy
that the older animals had more cells with a larger volume than theyounger animals had (Supplementary Fig. S2).
4. Discussion
In the aging rat cochlea distinct morphological changes as degener-ation of the SV and loss of sensory hair cells, mainly the outer hair cells,in the most apical and basal parts of the cochlea were observed. Age-related hair cell loss in the Sprague–Dawley rat has previously beenshown in scanning electron microscopical observations of the surfaceof the hearing organ (Keithley and Feldman, 1982). Using stereology,we confirm this finding as well as the observation that the greatestloss occurred in the lower base and in the upper apex of the cochlea.Age-related hair cell loss both in lower base and/or in the apex hasbeen reported for other rat strains as well as other rodents (Fetoniet al., 2011; Keithley et al., 2004). The changes vary between speciesand strains regarding onset, severity and apical to basal gradients, andreflect accordingly the hearing status of the animals.
In contrast to the study by Keithley and Feldman (1979) we foundonly a small, not statistically significant, age-related loss of SGN inaged Sprague–Dawley rats. This discrepancy could be explained byother differences as gender (not specified in their paper), sub-strainvariations and different counting method.
Age-related degenerative findings of the SV and the spiral ligamenthave been extensively described in senescent Mongolian gerbils withstrial presbycusis (Spicer and Schulte, 2002, 2005; Thomopoulos et al.,1997). The observed changes on ultrastructural level included thinningof the epithelium, degeneration of the marginal cells and their process-es, and thickening of the basement membrane surrounding the bloodvessels; a degenerative pattern very similar to the one reported here
for the SD rat. In these previous studies, a concomitant decline in theendocochlear potential was also observed.
Aging and age-related diseases are very complex and depend on avariety of factors related to the genetic background as well as earlierlifetime exposures. The only established regime to slow down theaging process is by reducing the dietary intake during life (Speakmanand Mitchell, 2011). Animal studies have shown that a reduced dietaryintake prolongs longevity and reduces the occurrence of age-relateddiseases. Consistent with this, in Fisher 344 rats, DR was found to havea more robust effect on preserving hearing thresholds, and reducingouter hair cell loss than other protective treatments (Seidman, 2000).In the C57BL/6 mouse, DR has been shown to slow-down age-relatedhearing loss and in parallel reduces the occurrence of apoptotic celldeath of SGN (Someya et al., 2007). In agreementwith previous studies,we here demonstrate not only better survival rates but also maintainedPreyer reflexes with DR. The reflex test is commonly used as a measureof hearing status (Jero et al., 2001; Pharm andWillott, 1988), reflectingboth central and peripheral aspects of the auditory system. Histologically,the most beneficial effect of DR within the cochlea was seen at the levelof the SV. The total volume of the SVwas better maintained, and the pro-cesses of the marginal cells displayed significantly less degenerativechanges compared to animals fed AL. The degree of hair cell and SGNloss was however equally advanced for both the aging groups. The oldDR rats were more homogenous regarding cell number and volumecompared to the age-matched AL rats (where the inter-individual spreadwas greater). This observation is in agreement with earlier studies ofother body parameters in DR animals (Altun et al., 2007).
The extensive degeneration observed in the SV of the old AL animalsis a possible explanation of the functional decline of hearing reflexes.The cell types within SV maintain the electrical potential between thefluid-filled compartments of the cochlea by an active transport of K+
Fig. 4. High magnification transmission electron micrographs of SV in old AL animals displaying degenerative patterns. (A) Retracted cell borders of the marginal cells (MC) with normalmicrovilli (arrows) towards scalamedia (SM) andwith tight junctions (white arrowhead) between the cells. Processes (p) from themarginal cells displaying a different degree of degen-eration. (B) Less degenerated processes (p) frommarginal cells towards the basal cells (BC) and intact gap junctions (arrowhead). (C) Lobules (l) of different sizes in themarginal cell areacontaining homogenous material. (D) Cell debris (*) from amarginal cell, (E) single cell-layer (SC) in the apical part of the SV. (F) Blood vessel (BV)with thickened basement membrane(bm) compared to the slender basementmembrane surrounding the blood vessel from a young animal in same-scale insert (brackets for comparison). (G) Spiral ligament (SL)with emptyintercellular spaces (*) between the type I fibrocytes (F) and the collagen fibers (cf). Blood vessels (BV) with normal appearance. Scale bars A–F = 2 μm, G = 10 μm.
1177P. Mannström et al. / Experimental Gerontology 48 (2013) 1173–1179
to the sensory hair cells (Spicer and Schulte, 2005) and are thus essen-tial for normal auditory function. Imbalance of this complex systemreduces the electrical potential and results in hearing loss (Kerr et al.,1982). This is the mechanism which is believed to be the cause of strialpresbycusis (Spicer and Schulte, 2002, 2005). The DR animals displayedamore intact volume and better preservedmarginal cells and processeswithin SV. It is likely that the preserved SV integrity explains the betterauditory reflex responses in the DR animals.
In this study we have compared aged female AL and DR fedSprague–Dawley rats with young adult females and although DRprolongs health-span and life-span to a similar extent in both sexes(Altun et al., 2007), gender specific effects have been reported. Holehan
and Merry (1985a,b) showed that female Sprague–Dawley ratsmaintained on DR had lower peak serum levels of estrogen and folliclestimulating hormone, but that cycling and reproductionweremaintainedat a more advanced age than in AL fed rats. This is interesting since itwas previously reported that post-menopausal females are at greaterrisk than aged-matched males to develop hearing loss (Jonsson et al.,1998; Kilicdag et al., 2004; Kosus et al., 2012) and a protective role forestrogen was proposed. Further dissection of an estrogen protectivemechanism on hearing have utilized mice KO engineering of the cognateestrogen receptors (α− and β−) and linked β-receptor deficiency todeafness and loss of both hair cells and SGN (Meltser et al., 2008;Simonoska et al., 2009). Much less is known about the SV but evidencesuggest that the α- and possible also the β-receptor are expressed inthis anatomical region (Stenberg et al., 1999). Certainly this issuedeserves further attention but given that we did not find a significantdifference in the loss of hair cells between rats fed AL or on DR andthat the loss of SGN was minimal in both groups of aged rats, othermechanism by which DR protects cells and tissues should be considered.Recently, Someya and coworkers showed that a member of the sirtuinfamily; Sirt-3 (Someya et al., 2010b) is a critical mediator of DRs capacityto retard age-related hearing loss in C57BL/6J by reducing oxidative stressand apoptotic cell death in the cochlea (Someya et al., 2007). If thisapplies also for the SV, remains however to be shown.
We here present, using an unbiased stereological method, results onsensory hair cell and SGN number and size as well as volume and finestructure of the SV, both in young adult and old Sprague–Dawley ratsfed AL and on DR.We conclude that DR not only prolongs life andmain-tains auditory reflexes but also preserves the cellular integrity of the SV
Fig. 5.Highmagnification transmission electronmicrographs of SV in old DR animals. (A) Marginal cell (MC)with processes (p) projecting towards intermediate cells (IC) and basal cells(BC) appearing less degenerated than the processes found in the AL animals. (B)Marginal cell (MC) lining scala media (SM)with retracted cell borders, but intact microvilli (arrows) andtight junctions (white arrowheads) similar to the AL animals. (C) SV from apical part of the cochlea in a DR animal with a more degenerated appearance including smaller lobules ofhomogenous material (l). (D) Blood vessel (BV) with thickened basement membrane (bm and bracket) similar to the AL animals. (E) Intact fibrocytes (F) inside spiral ligament (SL).Scale bars A–D = 2 μm, E = 10 μm.
Young DR AL
Tot
al n
umbe
r of
hai
r ce
lls
2000
4000
6000
*** ***
Fig. 6. DR did not prevent the age-related hair cell loss in the aging animals. Aging itselfsignificantly reduced the total number of hair cells by 23–25% (post hoc Holm–Sidaktest; P b 0.001 for both comparisons).
1178 P. Mannström et al. / Experimental Gerontology 48 (2013) 1173–1179
in the inner ear. The results suggest that DR slows down the degenera-tive aging processes of the auditory system, a mechanism that possiblyalso could be activated by for example food supplementation.
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.exger.2013.07.004.
Conflict of interest
The authors have no conflicts of interests.
Acknowledgment
This work was supported by the Swedish Research Council (grantno. 2010-7209 to MU and grant no. 10820 to BU), Karolinska Institutet,the Tysta Skolan Foundation (MU), and in part by funding from theEuropean Union Seventh Framework Programme (FP7/2007-2013)under grant agreement no. 304925 (MU).
References
Altun, M., Bergman, E., Edstrom, E., Johnson, H., Ulfhake, B., 2007. Behavioral impairmentsof the aging rat. Physiol. Behav. 92, 911–923.
Buckiova, D., Popelar, J., Syka, J., 2006. Collagen changes in the cochlea of aged Fischer 344rats. Exp. Gerontol. 41, 296–302.
Ciorba, A., Bianchini, C., Pelucchi, S., Pastore, A., 2012. The impact of hearing loss on thequality of life of elderly adults. Clin. Interv. Aging 7, 159–163.
Cowen, T.U.B., King, R.H.M., 2005. Aging in the peripheral nervous system. Peripher.Neuropathy 483–507.
Fetoni, A.R., Picciotti, P.M., Paludetti, G., Troiani, D., 2011. Pathogenesis of presbycusis inanimal models: a review. Exp. Gerontol. 46, 413–425.
Frisina, R.D., 2009. Age-related hearing loss: ear and brain mechanisms. Ann. N. Y. Acad.Sci. 1170, 708–717.
Gates, G.A., Couropmitree, N.N., Myers, R.H., 1999. Genetic associations in age-relatedhearing thresholds. Arch. Otolaryngol Head Neck Surg. 125, 654–659.
Gundersen, H.J., 1988. The nucleator. J. Microsc. 151, 3–21.Gundersen, H.J., Bagger, P., Bendtsen, T.F., Evans, S.M., Korbo, L., Marcussen, N., Moller, A.,
Nielsen, K., Nyengaard, J.R., Pakkenberg, B., Sorensen, F.B., Vesterby, A., West, M.J.,1988a. The new stereological tools: disector, fractionator, nucleator and point sampledintercepts and their use in pathological research and diagnosis. APMIS 96, 857–881.
Gundersen, H.J., Bendtsen, T.F., Korbo, L., Marcussen, N., Moller, A., Nielsen, K., Nyengaard,J.R., Pakkenberg, B., Sorensen, F.B., Vesterby, A., 1988b. Some new, simple and efficientstereological methods and their use in pathological research and diagnosis. APMIS 96,379–394.
Hequembourg, S., Liberman, M.C., 2001. Spiral ligament pathology: a major aspect of age-related cochlear degeneration in C57BL/6 mice. J. Assoc. Res. Otolaryngol. 2, 118–129.
Holehan, A.M., Merry, B.J., 1985a. Lifetime breeding studies in fully fed and dietaryrestricted female CFY Sprague–Dawley rats. 1. Effect of age, housing conditions anddiet on fecundity. Mech. Ageing Dev. 33, 19–28.
Holehan, A.M., Merry, B.J., 1985b. Modification of the oestrous cycle hormonal profile bydietary restriction. Mech. Ageing Dev. 32, 63–76.
Jero, J., Coling, D.E., Lalwani, A.K., 2001. The use of Preyer's reflex in evaluation of hearingin mice. Acta Otolaryngol. 121, 585–589.
Jonsson, R., Rosenhall, U., Gause-Nilsson, I., Steen, B., 1998. Auditory function in 70- and75-year-olds of four age cohorts. A cross-sectional and time-lag study of presbyacusis.Scand. Audiol. 27, 81–93.
Keithley, E.M., Feldman, M.L., 1979. Spiral ganglion cell counts in an age-graded series ofrat cochleas. J. Comp. Neurol. 188, 429–442.
Keithley, E.M., Feldman, M.L., 1982. Hair cell counts in an age-graded series of rat cochleas.Hear. Res. 8, 249–262.
Keithley, E.M., Ryan, A.F., Feldman, M.L., 1992. Cochlear degeneration in aged rats of fourstrains. Hear. Res. 59, 171–178.
Keithley, E.M., Canto, C., Zheng, Q.Y., Fischel-Ghodsian, N., Johnson, K.R., 2004. Age-relatedhearing loss and the ahl locus in mice. Hear. Res. 188, 21–28.
Kerr, T.P., Ross, M.D., Ernst, S.A., 1982. Cellular localization of Na+, K+-ATPase in themammalian cochlear duct: significance for cochlear fluid balance. Am. J. Otolaryngol.3, 332–338.
Kilicdag, E.B., Yavuz, H., Bagis, T., Tarim, E., Erkan, A.N., Kazanci, F., 2004. Effects of estrogentherapy on hearing in postmenopausal women. Am. J. Obstet. Gynecol. 190, 77–82.
Kosus, N., Kosus, A., Turhan, N.O., Kurtaran, H., 2012. Hearing levels in menopausalwomen and the effect of tibolone on audiological functions. J. Obstet. Gynaecol. 32,294–297.
Mair, W., Dillin, A., 2008. Aging and survival: the genetics of life span extension by dietaryrestriction. Annu. Rev. Biochem. 77, 727–754.
Meltser, I., Tahera, Y., Simpson, E., Hultcrantz, M., Charitidi, K., Gustafsson, J.A., Canlon, B.,2008. Estrogen receptor beta protects against acoustic trauma in mice. J. Clin. Invest.118, 1563–1570.
Moller, A., Strange, P., Gundersen, H.J., 1990. Efficient estimation of cell volume andnumber using the nucleator and the disector. J. Microsc. 159, 61–71.
Newman, D.L., Fisher, L.M., Ohmen, J., Parody, R., Fong, C.T., Frisina, S.T., Mapes, F., Eddins,D.A., Frisina, D.R., Frisina, R.D., Friedman, R.A., 2012. GRM7 variants associated withage-related hearing loss based on auditory perception. Hear. Res. 294, 125–132.
Ohlemiller, K.K., 2004. Age-related hearing loss: the status of Schuknecht's typology. Curr.Opin. Otolaryngol Head Neck Surg. 12, 439–443.
Ohlemiller, K.K., Frisina, R.D., 2008. Clinical Characterization of Age-related Hearing Lossand Its Neural and Molecular Bases. In: Popper, A.N., Fay, R.R., Schacht, J. (Eds.).
Ohlemiller, K.K., Gagnon, P.M., 2004. Apical-to-basal gradients in age-related cochleardegeneration and their relationship to “primary” loss of cochlear neurons. J. Comp.Neurol. 479, 103–116.
Pharm, K., Willott, J.F., 1988. Acoustic startle response in young and aging C57BL/6J andCBA/J mice. Behav. Neurosci. 102, 881–886.
Schacht, J., Hawkins, J.E., 2005. Sketches of otohistory. Part 9: presby[a]cusis. Audiol.Neurootol. 10, 243–247.
Schuknecht, H.F., Gacek, M.R., 1993. Cochlear pathology in presbycusis. Ann. Otol. Rhinol.Laryngol. 102, 1–16.
Seidman, M.D., 2000. Effects of dietary restriction and antioxidants on presbyacusis.Laryngoscope 110, 727–738.
Simonoska, R., Stenberg, A.E., Duan, M., Yakimchuk, K., Fridberger, A., Sahlin, L., Gustafsson,J.A., Hultcrantz, M., 2009. Inner ear pathology and loss of hearing in estrogen receptor-beta deficient mice. J. Endocrinol. 201, 397–406.
Someya, S., Yamasoba, T., Weindruch, R., Prolla, T.A., Tanokura, M., 2007. Caloric restrictionsuppresses apoptotic cell death in the mammalian cochlea and leads to prevention ofpresbycusis. Neurobiol. Aging 28, 1613–1622.
Someya, S., Tanokura, M.,Weindruch, R., Prolla, T.A., Yamasoba, T., 2010a. Effects of caloricrestriction on age-related hearing loss in rodents and rhesus monkeys. Curr. AgingSci. 3, 20–25.
Someya, S., Yu, W., Hallows, W.C., Xu, J.Z., Vann, J.M., Leeuwenburgh, C., Tanokura, M.,Denu, J.M., Prolla, T.A., 2010b. Sirt3 mediates reduction of oxidative damage andprevention of age-related hearing loss under caloric restriction. Cell 143, 802–812.
172, 172–185.Spicer, S.S., Schulte, B.A., 2005. Pathologic changes of presbycusis begin in secondary
processes and spread to primary processes of strial marginal cells. Hear. Res. 205,225–240.
Spongr, V.P., Flood, D.G., Frisina, R.D., Salvi, R.J., 1997. Quantitative measures of hair cellloss in CBA and C57BL/6 mice throughout their life spans. J. Acoust. Soc. Am. 101,3546–3553.
Stenberg, A.E., Wang, H., Sahlin, L., Hultcrantz, M., 1999. Mapping of estrogen receptorsalpha and beta in the inner ear of mouse and rat. Hear. Res. 136, 29–34.
Tandrup, T., 1993. A method for unbiased and efficient estimation of number and meanvolume of specified neuron subtypes in rat dorsal-root ganglion. J. Comp. Neurol.329, 269–276.
Watanabe, F., Kirkegaard, M., Matsumoto, S., Gont, C., Mannstrom, P., Ulfendahl, M.,Fridberger, A., 2010. Signaling through erbB receptors is a critical functional regulatorin the mature cochlea. Eur. J. Neurosci. 32, 717–724.
A B
Fig. 7. No significant differences between any of the groups were detected, neither for (A) total number of SGN, nor for (B) averaged soma volume of the neurons.
1179P. Mannström et al. / Experimental Gerontology 48 (2013) 1173–1179
