Experimental Gerontology 44 (2009) 183–189

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Experimental Gerontology

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Age-related bone loss in the LOU/c rat model of healthy ageing

Gustavo Duque a,b,*, Daniel Rivas b, Wei Li a,c, Ailian Li c, Janet E. Henderson c,Guylaine Ferland d, Pierrette Gaudreau e

a Aging Bone Research Program, Nepean Clinical School, University of Sydney, Level 5, South Block, Nepean Hospital, Penrith, NSW 2750, Australiab Lady Davis Institute for Medical Research, McGill University, Montreal, Que., Canada H3T 1E2c Department of Medicine, JTN Wong Laboratories for Mineralized Tissue Research, McGill University Health Centre, Montreal, Que., Canada H3G 1Y6d Institut Universitaire de Gériatrie de Montréal, Montreal, Que., Canada H3W 1W5e Laboratory of Neuroendocrinology of aging, Centre hospitalier de l’Université de Montréal Research Center (CRCHUM), Montreal, Que., Canada H1W 4A4

a r t i c l e i n f o a b s t r a c t

Article history:Received 5 June 2008Received in revised form 8 October 2008Accepted 10 October 2008Available online 21 October 2008

Keywords:AgingOsteoporosisAnimal modelsOsteoblastsAdipocytesBone turnover

0531-5565/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.exger.2008.10.004

* Corresponding author. Address: Aging Bone ReseaSchool, University of Sydney, Level 5, South Block, Ne2750, Australia. Tel.: +61 2 4734 4279; fax: +61 2 47

E-mail address: gduque@med.usyd.edu.au (G. Duq

Inbred albino Louvain (LOU) rats are considered a model of healthy aging due to their increased longevityin the absence of obesity and with a low incidence of common age-related diseases. In this study, wecharacterized the bone phenotype of male and female LOU rats at 4, 20 and 27 months of age using quan-titative micro computed tomographic (mCT) imaging, histology and biochemical analysis of circulatingbone biomarkers. Bone quality and morphometry of the distal femora, assessed by mCT, was similar inmale and female rats at 4 months of age and deteriorated over time. Histochemical staining of undecal-cified bone showed a significant reduction in cortical and trabecular bone by 20 months of age. Thereduction in mineralized tissue was accompanied by reduced numbers of osteoblasts and osteoclastsand a significant increase in marrow adiposity. Biochemical markers of bone turnover, C-telopeptideand osteocalcin, correlated with the age-related bone loss whereas the calciotropic hormones PTH andvitamin D remained unchanged over time. In summary, aged LOU rats exhibit low-turnover bone lossand marrow fat infiltration, which are the hallmarks of senile osteoporosis, and thus represent a novelmodel in which to study the molecular mechanisms leading to this disorder.

� 2008 Elsevier Inc. All rights reserved.

1. Introduction

Age-related bone loss is the primary underlying cause of frac-tures in older persons (Raisz, 2005). Cellular changes in the ageingskeleton include an increased rate of adipogenesis at the expenseof a reduction in osteoblastogenesis (Duque and Troen, 2008;Justesen et al., 2001). Fewer osteoblasts results in a reduction inbone formation, which fails to compensate for the bone lost duringresorption. Although the number and activity of osteoclasts is aug-mented by estrogen deprivation during menopause, there is evi-dence that bone turnover declines in elderly men and women(Khosla and Riggs, 2005; Seeman, 2002). From a structural per-spective, these changes result in decreased trabecular bone, signif-icant cortical thinning and expansion of the marrow cavity, whichis progressively infiltrated by fat (Duque and Troen, 2008; Seeman,2002).

At the molecular level, these changes are the consequence ofcomplex interactions between growth factors, hormones, matrixmolecules and the commitment of common precursor cells to

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rch Program, Nepean Clinicalpean Hospital, Penrith, NSW

34 1817.ue).

one lineage or another (Chan and Duque, 2002). Research intothe etiology and pathogenesis of osteoporosis has been advancedby the use of animal models to mimic these interactions (Priemelet al., 2002; Watanabe, 2008). Although osteopenia is a commonfeature in these models, the pathophysiology of their bone loss cor-relates poorly with that of age-related bone loss (Priemel et al.,2002). In fact, in an attempt to avoid the systemic changes to manyorgan systems that occur with aging, few studies have used purelyaged animals (Priemel et al., 2002; Watanabe and Hishiya, 2005).

The ovariectomized rodents are the most commonly used ani-mal model of osteopenia and osteoporosis (Watanabe and Hishiya,2005). Their bone phenotype is characterized by low bone massresulting from increased bone turnover associated with estrogendeficiency and elevated osteoclastic activity (Iwaniec et al.,2006). However, there is limited evidence that estrogen depriva-tion is actually a model of senile osteoporosis since, in contrastof what happens in the ovariectomized models, the ageing humanskeleton is characterized by low bone mass, enhanced bone fragil-ity and increased fracture risk arising from a reduction in bone for-mation, uncompensated bone resorption, and an increase in bonemarrow adipogenesis (Chan and Duque, 2002; Duque, 2008; Juste-sen et al., 2001; Seeman, 2002). In order to better understand thecellular and molecular mechanisms of senile osteoporosis, charac-terization of additional animal models are of great interest.

184 G. Duque et al. / Experimental Gerontology 44 (2009) 183–189

The objective of the current study was therefore to undertake acharacterization of bone phenotype in 4-, 20- and 27-month-oldmale and female LOU rats, a strain which has been described as amodel of healthy aging (Alliot et al., 2002). Inbred male and femaleLOU rats have an increased life expectancy when compared withthe Wistar strain from which they originated. They have been usedin numerous studies of nutrition in aging and have been shown tomaintain their lean body mass and avoid serious disorders such asspontaneous tumors and renal failure throughout their lifespan(Alliot et al., 2002). In this cross-sectional study, we have shownthat aged male and female LOU rats exhibit low-turnover bone lossin the presence of marrow adiposity, thus identifying them as analternative relevant model to study the molecular mechanisms ofage-related bone loss in humans.

Fig. 1. Age-related changes in body weight of 4-, 20- and 27-month-old male andfemale LOU rats. ap < 0.01 compared with 4-month-old males; bp < 0.001 males vs.females.

2. Materials and methods

2.1. Animals

Young mature 4-month-old rats, and old 20-, and 27-month-oldmale and female LOU/c rats were studied. They were obtained fromthe Aging LOU Rat Colony Infrastructure of the Quebec Network forResearch on Aging (RQRV; www.rqrv.com) (LOU/c/rqrv) (LOU). The4-month-old females were virgins. Their ancestors were obtainedat three months of age, from Professor Josette Alliot’s colony(LOU/c/jall; University Blaise Pascal, Clairmont-Ferrand, FR), inSeptember 2001. Breeding has been performed in Centre hospita-lier de l’Université de Montréal (CHUM) Research Center animalfacilities since then. Longevity characteristics of our LOU are simi-lar to those previously reported (Alliot et al., 2002). The RQRV LOUrat colony is fed the same chow regimen as Professor Alliot’s col-ony (SAFE, Augy, FR). After weaning at 21 days, rats are fed theR03 growing diet (6% fish protein, 20.2% vegetable protein, 4.6%vitamins and mineral mixture, 69.2% cereals) for three weeks andsubsequently with the R04 maintenance diet (4% fish protein, 8%vegetable protein, 4.1% vitamins and mineral mixture, 83.9% cere-als). They are kept in temperature (22 �C), humidity (65%) andlighting-controlled (12:12 light–dark cycle, light on at 07:00)rooms. They had free access to chow and water. The rats werekilled by rapid decapitation in block design fashion between09:00 and 13:00, after an overnight fast. Trunk blood was collectedand serum kept frozen at �80 �C for the various assays. Bones wererapidly dissected out and processed. All animal protocols were ap-proved by the Animal Care Committee of CHUM Research Center incompliance with guidelines of the Canadian Council for AnimalCare.

2.2. Quantitative radiologic imaging

Micro-CT was performed, using a modification of previouslypublished methods (Valverde-Franco et al., 2004), on the left femurafter removal of soft tissues and overnight fixation in 4% parafor-maldehyde. A Skyscan 1172 instrument (Skyscan, Antwerp, Bel-gium) equipped with a 1.3 Mp camera was used to capture 2Dserial cross-sections, which were used to reconstruct 3-dimen-sional images for the quantification of the volume of bone in thedistal metaphysis. Bone mineral density was estimated using aset of calibrated phantoms purchased from Skyscan.

2.3. Histology and histomorphometry

For marrow fat analysis, the right femur was cleaned of soft tis-sue, fixed for 36 h in 4% paraformaldehyde, rinsed thoroughly inPBS, decalcified in 10% EDTA and processed for paraffin embedding.Serial 4-lm sections cut on a modified Leica RM 2155 rotary

microtome (Leica Microsystems, Richmond Hill, Ontario, Canada)were deparaffinized in xylene and immersed in 2 changes of pro-pylene glycol before staining with Oil red O (Sigma–Aldrich,Canada Ltd., Oakville, ON) and counterstaining with haematoxylinto quantify adipocyte number and fat volume as described previ-ously (Duque et al., 2004). Fat volume was also quantified usingundecalcified, Von-Kossa stained sections (Richard et al., 2005).

For histomorphometric analyses the left femur was fixed over-night in 4% paraformaldehyde, rinsed in 3 changes of PBS andembedded in polymethylmethacrylate (MMA) or a mixture of50% MMA and 50% glycolmethacrylate (GMA). Serial 4- to 6-lmsections of MMA-embedded tissues were left unstained or stainedwith von Kossa, while 4-lm MMA–GMA sections were stained fortartrate resistance acid phosphatase (TRAP) and alkaline phospha-tase (ALP) activity as described previously (Valverde-Franco et al.,2004). Images were captured using a Leica DMR microscope (LeicaMicrosystems) equipped with a Retiga 1300 camera (Qimaging,Burnaby, British Columbia, Canada) and the primary histomorpho-metric data obtained using Bioquant Nova Prime image analysissoftware (Bioquant Image Analysis Corp., Nashville, Tennessee).Nomenclature and abbreviations conform to those recommendedby the American Society for Bone and Mineral Research (Parfittet al., 1987).

2.4. Serum biochemistry and bone biomarkers

Serum biochemistry (Ca, creatinine, PO4) was determined at theRodent Diagnostics Lab (McGill University, Montréal, Quebec, Can-ada) using routine automated techniques. Bone biomarkers, calcio-tropic and sex hormones were analyzed at the Centre for Bone andPeriodontal Research using commercial assays for serum C-termi-nal telopeptide of type I collagen (CTX) (RatLaps ELISA, Nordic Bio-science), 17b estradiol and testosterone (IBL Immuno-Biological,Hamburg, Germany), osteocalcin (ImmunoDiagnostic SystemsLtd., UK), PTH (Immunotopics Inc., San Clemente, CA, USA) and25(OH)-vitamin D (ImmunoDiagnostic Systems Ltd., UK). The interand intra-assay coefficient of variation (CV) for CTX was 7%, CVs for17b estradiol, osteocalcin and testosterone were 6%, and for PTHwas 10%.

2.5. Statistical analysis

Data are presented as the means ± SD. After normalization forgrowth, comparisons of body weight curves were done with one-way ANOVA for repeated measures. Comparisons between the dif-ferent age groups were made by ANOVA and Dunnett’s test todetermine statistical differences between 4-month-old and older

Fig. 2. MicroCT analysis (A–F) to evaluate distal femur structure and sections of undecalcified bone stained with von Kossa (A–F, right panels) (magnification 10�) to evaluatemineralized tissue (black) and fat volume (white). The figure shows three-dimensional images of the trabecular bone and cross-sectional images of the cortical bone from ratsaged 4, 20 and 27 months (A–F). Lower bone volume, trabecular bone and cortical thickness and higher cortical porosity was observed in aged rats (B, C, E and F) comparedwith young mature rats (A and B). Bone mineral density (BMD) (G) and bone volume/trabecular volume (BV/TV) (H) were significantly lower in males and females old ratscompared to young ones. ap < 0.01, bp < 0.001 compared with 4 months of age; cp < 0.01, dp < 0.001 males vs. females. Values are representative of four to six sections in eachgroup of animals.

1 For interpretation of color mentioned in this figure the reader is referred to theweb version of the article.

G. Duque et al. / Experimental Gerontology 44 (2009) 183–189 185

rats. Student’s t test was used to compare male and female ratsfrom a same age group. A p value of 60.05 was considered statis-tically significant.

3. Results

3.1. Body weights

Fig. 1 shows that male rats weighed significantly more(p < 0.001) than female rats at all time points. Male rats increasedtheir body weight by 62% ± 6% (p < 0.01) between 4 and 20 months,with little change thereafter, whereas female rats showed no signif-icant change in body weight between 4, 20 and 27 months of age.

3.2. Quantitative micro CT and histologic analyses of the distal femur

Fig. 2 shows a qualitative (A–F) and quantitative (G–H) declinein bone density and architecture over time in both male and femalerats. 3D reconstruction and 2D sagittal histological sections of thedistal femur show a significant loss in trabecular and cortical boneas well as higher levels of cortical porosity between 4 and 27months of age in male and female rats (Fig. 2). Bone mineral den-sity (BMD) increased by 30 ± 3% (p < 0.01), from 4 to 20 months ofage in male rats followed by a 42 ± 3% (p < 0.01) decline to 27months of age (Fig. 2G). In contrast, female rats showed a progres-sive 25 ± 3% (p < 0.01) decline in BMD after 4 months of age(Fig. 2G). The ratio of bone to tissue volume (BV/TV) declined overtime in both male and female rats and was significantly greater inmale compared with female rats at 4 and 20 months of age, but notat 27 months of age (Fig. 2H). Table 1 shows a small increase in tra-becular thickness (Tb.Th.) between 4 and 20 months in male ratsbut no significant changes in female rats. It is interesting to notethat trabecular separation (Tb.Sp) was increased in both maleand female aged rats, with the greatest increase (2.7 times) occur-ring in 27-month-old female rats (Table 1). A concomitant declinein trabecular number (Tr.N.) was seen, being highest (4 times) in

oldest females. Cortical BV/TV (Cor. BV/TV) was not different in4- and 20-month-old male rats, but was lower in the oldest group,whereas a decrease was observed in 20-month-old female rats,compared to young females.

3.3. Histochemical analyses of cellular activity

To determine the changes in bone cellularity in the differentgroups of rats, Fig. 3 shows sections of undecalcified bone stainedwith ALP to identify osteoblasts (brown A–F) and TRAP to identifyosteoclasts (red G–L).1 Quantification of cell numbers after normal-ization with bone surface revealed that osteoblast numbers werelower in both male and female aged rats compared to young rats (Ta-ble 1). Similar results were obtained for the number of osteoclasts,with the exception of higher values in 20-month-old females thanin young ones. Oldest male and female rats showed no differencein the number of distal femur osteoclasts.

3.4. Changes in bone marrow fat of aging LOU rats

Examination of bone marrow using two different methodolo-gies, von Kossa staining of undecalcified bone (Fig. 2) and oil redO staining of decalcified bone (Fig. 4), revealed that bone marrowfat content is higher in both aged male and female rats than inyoung rats. These data are in agreement with those on bone vol-ume which were lower in aged rats (Fig. 2, Table 1). At the threeages, male rats showed a significantly higher amount of fat contentthan female rats (p < 0.01).

3.5. Bone biomarker analysis

We next determined if the changes in bone parameters assessedby micro CT and by histologic analyses correlated with alterations

Fig. 3. Histological analysis of undecalcified distal femur from 4-, 20- and 27-month-old LOU Rats. Sections of femur fixed in 4% paraformaldehyde andembedded in plastic were stained for alkaline phosphatase (ALP)(A–F) activity toidentify osteoblasts or for tartrate resistance acid phosphatase (TRAP) (G–L) activityto identify osteoclasts. Staining patterns were similar in 4-month-old LOU rats (A, B,G and J). Both male and female showed a continuous decline in ALP staining at 20and 27 months of age (B, E, C and F). In contrast, whereas male rats show acontinuous and age related decline in TRAP staining (H, I), female rats showed anincrease in TRAP staining at 20 months of age (K). This increased was followed by adecrease in TRAP staining at 27 months of age (L). Magnification at source, leftpanels 10�. Micrographs are representative of those taken from five to sevensections in each group of animals.

Table 1Histological and morphometric parameters in distal femur of aging LOU rats.

Parameters Male Female

4 m 20 m 27 m 4 m 20 m 27 m

BV/TV (%) 29 ± 8c 18.1 ± 8.7 8.7 ± 2.2a 15.2 ± 2.7 11.8 ± 1.8a 8.4 ± 1.4a,b

Tb.Th. (lm) 89 ± 1 98 ± 1a 97 ± 4a 92 ± 9 90 ± 6 98 ± 1Tb.Sp. (lm) 403 ± 33 456 ± 12a 543 ± 49a,b 195 ± 12c 266 ± 36a,c 530 ± 74a,b

Tb.N (1/mm) 1.7 ± 0.2 1.2 ± 0.1a 0.8 ± 0.1a,b 3.2 ± 0.6c 1.9 ± 0.8a 0.8 ± 0.14a,b

Cor. BV/TV (%) 31 ± 3 32 ± 0.6 27 ± 0.1a 35 ± 2 29 ± 2a 27 ± 1a

OB (#) 286 ± 15 220 ± 12a 150 ± 20a,b 368 ± 42 215 ± 75a 120 ± 13a,b

OC (#) 76 ± 27 53 ± 3 44 ± 2a 74 ± 5 86 ± 2a,c 40 ± 7a,b

OB:OC 4:1 4:1 3:1 8:1c 2.5:1c 3:1

a p < 0.01 compared with 4 month-old rats.b p < 0.01 compared with 20 month-old rats.c p < 0.01 male vs. female at same age.

186 G. Duque et al. / Experimental Gerontology 44 (2009) 183–189

in circulating levels of calciotropic hormones and bone biomarkers,similar to those used in the clinical setting. Fig. 5A shows signifi-cantly higher values in serum C-telopeptide in female rats at 20months, corresponding to the perimenopause period and coincid-ing with the highest number of osteoclasts seen in distal femur.In 27-month-old females, a very low level of C-telopeptide was ob-served. In contrast, serum C-telopeptide was decreased in both 20and 27-month-old group compared to young rats, with the highestdecrease in the oldest group. C-telopeptide serum levels were notdifferent in oldest males and female rats. Serum osteocalcin(Fig. 5B) was significantly higher at 4 months of age in female thanmale rats. Level decreased with age and was not different in 27-month-old male vs. female rats. Serum 25(OH)-vitamin D

(Fig. 5C) declined over time in both male and female rats, whereasparathyroid hormone (Fig. 5D) remained constant over the timecourse. 17b-Estradiol (Fig. 5E) was significantly lower in old femalerats compared to young females. The low levels found in male ratswere not different in the three age groups. In contrast, serum tes-tosterone levels were significantly diminished in old male rats.They were consistently higher in male than in female rats (Fig. 5F).

4. Discussion

LOU rats are considered to be of Wistar origin. Their ancestorswere imported from an unknown place around 1937 probablyfrom a unique couple (Maisin, 1959). Alliot et al. (2002) have pro-posed the LOU rats as a model of healthy aging, based on a higherlongevity than Wistar rats and other common strains used in bio-medical research, the absence of obesity with age as well as severepathologies (nephropathy, pituitary tumors), which are commoncauses of death in aged rodents (Alliot et al., 2002; Dodane et al.,1991; McComb et al., 1984). In addition, LOU rats have been alsoreported to retain better neuroendocrine (Kappeler et al., 2004;Veyrat-Durebex et al., 2005) and neurological (Kollen et al.,2008) functions.

In this study, we were interested in determining whether, as inother systems, LOU rats showed a healthier bone phenotype thanaged male (Iida and Fukuda, 2002) and female (Fukuda and Iida,2004) Wistar rats. In agreement with previous reports (Alliotet al., 2002), body weights were higher in male than in femaleaging rats. Subsequently, we found that as in human aging, maleand female LOU rats showed a decrease in bone mineral densityand bone quality with age. This bone loss is associated with a sig-nificant reduction in bone volume/trabecular volume (BV/TV) andtrabecular number (Tb.N.) with an increase in trabecular separa-tion (Tb.Sp), indicating a significant and rapid decline in bone qual-ity. As previously reported in other animal models of age-relatedbone loss (Halloran et al., 2002; Wang et al., 2001), in our modelthe trabecular thickness (Tb.Th.) does not appear to decrease withage, suggesting that bone loss in this model is not a consequence oftrabecular thinning, at least in the distal femur. In contrast,changes in cortical structure are similar to those seen in the humanaging bone (Cooper et al., 2007) with a significant decline in corti-cal BV/TV and increased porosity with age.

Despite low serum levels of estrogens, female rats did not showthe accelerated decline in bone mass found in other rat strains andusually associated with either menopause (Banu et al., 2002; Prie-mel et al., 2002) or ovariectomy (Christopoulou et al., 2006). How-ever, higher levels of osteoclast number and serum markers ofbone resorption (C-telopeptides) were observed during the peri-menopause period in female LOU rats. Interestingly, this increasein osteoclast number was not seen at 27 months of age and wastherefore compatible with decreased bone turnover.

Fig. 4. Changes in bone marrow fat and fat volume/trabecular volume ratio of distal femur of aged LOU Rats. Sections of undecalcified bone were stained with oil red O andimages captured at original magnifications of 40� to evaluate fat volume (white areas, red arrows). Both, male and female rats, showed an age-related infiltration of bonemarrow by fat. This increase in marrow fat volume (FV) was correlated with trabecular volume (TV) using both oil red O and Von-Kossa staining and quantified using aBioquant Nova Prime image analysis software. Both aged groups showed a significant increase in the FV/TV ratio (E). In addition, male rats showed an earlier and significantlyhigher ratio at 4, 20 and 27 months of age than their females counterparts (E). Magnification at source was 40�. Micrographs are representative of four to six sections in eachgroup of animals. ap < 0.01 compared with 4 months; bp < 0.001 males vs. females at same age. (For interpretation of color mentioned in this figure the reader is referred to theweb version of the article.)

G. Duque et al. / Experimental Gerontology 44 (2009) 183–189 187

In addition, in spite their differences in body weight, BV/TV andhistomorphometrical parameters at early ages (4 and 20 month-old), male and female LOU rats showed no differences at 27months of age in their declined bone structure. In both cases, aphenotype compatible with age related bone loss at 27 monthsof age was found. From a cellular point of view, although therewas a significant increase in osteoclast number in female rats dur-ing their menopause, both male and female old rats showed a sig-nificant decrease in both osteoclast and osteoblast number at 27months of age. This reduction in cell numbers correlated withdecreasing serum levels of biochemical markers of bone formation(osteocalcin) and bone resorption (C-telopeptides) in 27 month-oldrats both male and female. Although the cellular changes in malerats rather correlate with those recently reported by Pietschmannet al. (2007), the mechanism by which decline in bone turnover isaccelerated after menopause in female rats remains to beelucidated.

We assessed whether these cellular and structural changes inbone were associated with serum levels of calciotropic hormones(vitamin D and PTH). Both aged male and female rats showed a sig-nificant decrease in their serum levels of 25(OH)-vitamin D. How-ever, this reduction in serum levels of vitamin D did not have asignificant effect on serum levels of PTH which remained stable. Fi-nally, in agreement with a previous study (Alliot et al., 2002), ser-um levels of creatinine and calcium remained unchanged in agedLOU rats (data no shown). These results suggest that the cellularand structural changes in bone of aged LOU rats are independentof serum levels of calciotropic hormones and/or renal function.

Apart from some difference in estimate BMD and bone architec-ture, it is important to mention that low levels of bone turnoverand high levels of bone marrow fat, are typical features of age-re-lated bone loss. A particularly interesting finding of this study wasthat while maintaining stable body weight, low adipose tissue

mass and low levels of serum leptin with aging (Veyrat-Durebexet al., 2005), old LOU rats showed significantly higher levels offat infiltration within the bone marrow than their younger coun-terparts. Although this infiltration was more prominent in agedmale rats, both males and females showed significantly higher lev-els of marrow fat. Increased bone marrow adipogenesis and fatinfiltration is a prevalent feature of age-related bone loss (Duque,2008; Justesen et al., 2001), thus LOU rats could become a particu-larly interesting model to study cross-talk of fat and bone cells inthe aging bone.

Taken together, our findings allow us to propose that LOU ratsare a useful model to study the mechanisms of age-related boneloss and low bone turnover osteoporosis. Although it is well ac-cepted that most of the cases of osteoporosis are due to high boneturnover (Raisz, 2005), most likely due to either estrogen defi-ciency or to prolonged steroid therapy, it is also accepted that thereis a particular type of osteoporosis due to low-turnover osteoporo-sis such as that seen in prolonged disuse or older subjects (Duqueand Troen, 2008; Kawaguchi et al., 1999; Khosla and Riggs, 2005;Seeman, 2002). Considering that the spatial and temporal varia-tions in mineral characteristics in individuals with low- andhigh-turnover osteoporosis are distinct and may contribute tothe reported differences in the bone mineral density, the risk ofmultiple fractures and the response to therapies, the identificationof an animal model to study low bone turnover osteoporosis ismandatory.

A rationale for proposing LOU rats as a new potential model tostudy the typical features of senile osteoporosis is given by com-paring them to other animal models of age-related bone loss. Con-trary to age-related bone changes seen in their proposed ancestors(Wistar rats) (Iida and Fukuda, 2002; Fukuda and Iida, 2004), agingLOU rats showed a faster and significantly higher decline in bonemass unrelated with their levels of calciotropic hormones. In addi-

Fig. 5. Biochemical analysis of (A) serum C-telopeptides, (B) Osteocalcin, (C) 25(OH)-vitamin D, (D) PTH, (E) 17b-estradiol, and (F) androgens. Female aging rats showed asignificant increase in C-telopeptide (bone resorption) during their perimenopausal period, followed by a significant decline at 27 months of age (A). In contrast, male ratsshowed a continuous decline in C-telopeptide throughout their lives. Although female rats showed higher levels of osteocalcin (bone formation) at 4 months of age, bothgroups showed a significant decrease in serum levels of osteocalcin reaching similar levels at 27 months of age (B). Both groups showed a significant decrease in their serumlevels of 25(OH)-vitamin D (C) without inducing an increase in serum levels of PTH (D). In addition, female rats showed a significant decrease in serum levels of17b-estradiolat both 20 and 27 months of age (E). Finally, male rats showed a progressive age-related decline in serum testosterone levels (F). Assays were performed in duplicate. Eachpoint is mean ± SE. ap < 0.01, bp < 0.001 compared with 4 months; cp < 0.001 males vs. females.

188 G. Duque et al. / Experimental Gerontology 44 (2009) 183–189

tion, in contrast with the early and significant infiltration of bonemarrow by fat in LOU rats, aging Wistar rats show stable levelsof marrow fat (Sottile et al., 2004).

Furthermore, in the case of the ovariectomized mouse and rat,this model mostly parallels the situation of humans after meno-pause where bone changes are associated with estrogen depriva-tion. Contrary to our proposed model, ovariectomized animalsshow high levels of bone turnover predominantly dependent onhigh levels of bone resorption (Iwaniec et al., 2006), which aremore suggestive of post-menopausal than senile osteoporosis.

A more recent approach to develop animal models for the studyof the aging skeleton has been either the use of premature agingphenotypes (Chen et al., 2004; Hishiya and Watanabe, 2004) oranimals deficient in specific genes required for bone cells function(Kawaguchi et al., 1999; Priemel et al., 2002; Watanabe and His-hiya, 2005; Watanabe, 2008). Although in some cases these animalmodels provide some of the features of age-related bone loss, mostof them show bone phenotypes that resemble the pathology of hu-man skeletal aging more than the physiological changes found inaging bone and their age-induced regulatory mechanisms.

In this study, we propose a rat model that meets the character-istics of low bone turnover and bone marrow fat infiltration, whichare common features seen in a considerable proportion of patientswith senile osteoporosis (Seeman, 2002). Although it is well ac-cepted that a single model cannot resemble the complete set offeatures and causes of osteoporosis in older persons, we consider

that this model could be used not only to assess some of the mech-anisms of osteoporosis in the older population but also to developnew potential therapeutic approaches. Another significant strengthof this model is the absence of either age-related diseases (i.e. renalfailure, pituitary tumors) or changes in calciotropic hormones thatcould affect the accuracy of the data obtained in other rat modelswhen looking at the mechanisms of the aging skeleton.

In summary, our data suggest that LOU rats show a similar pat-tern of bone changes as those seen in human aging bone, particu-larly in low bone turnover osteoporosis. However, the mechanismsthat explain these similarities remain unknown. We conclude thatthe elucidation of the mechanisms of age-related bone loss in LOUrats will also provide us with new evidence to understand some as-pects of age-related bone loss in humans.

Acknowledgements

This work was supported by a catalyst grant in aging researchfrom the Canadian Institutes of Health Research (Grant200703IAP) and a pilot project grant from the Quebec Networkfor Research on Aging (Nutrition Section). G Duque holds a Fellow-ship from the University of Sydney-Medical Research Foundationand was a Chercheur Boursier Junior 1 from Fonds de la Rechercheen Santé du Quebec. The authors are deeply grateful to ProfessorJosette Alliot (Université Blaise Pascal, Clermont-Ferrant, FR) forproviding the Quebec Network for Research on Aging with male

G. Duque et al. / Experimental Gerontology 44 (2009) 183–189 189

and female LOU/c/jall rats to start the network LOU rat colony. Wethank Mrs Miren Gratton and Julie Bédard for their technicalassistance.

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