osteoporosis is a metabolic bone disease characterized by loss of bone mass and disruption of bone microarchitecture , and consequently increases the risk of fracture3,5,11,14 ) . it is well known that estrogen plays a fundamental role in skeletal growth and bone homeostasis1 - 3,6 ) . this is of particular importance in postmenopausal women , whose estrogen levels are naturally lowered , and in whom the incidence of osteoporosis is high , because a rapid decrease in estrogens in this population is the predominant cause of the imbalance between bone formation and bone resorption12 ) . although no single animal model precisely mimics the human condition of osteoporosis , appropriate animal models can provide information on bone quality and structure not obtainable during clinical trials . ovariectomy ( ovx ) provides the most popular model for studying events associated with postmenopausal osteoporosis with estrogen deficiency3,5,12,16 ) , and it has been well established that ovariectomy elicits bone loss and increased bone turnover in rats3,5,7 - 9,11,12,16 ) . thus , ovx rat models of osteoporosis can mimic conditions in postmenopausal women , and are suitable for the evaluations of potential therapeutics designed to prevent or treat osteoporosis7 - 9,16 ) . in the present study , in an effort to devise a reliable and reproducible ovx rat model of osteoporosis , we performed histomorphometric analyses of the lumbar spine using micro - computed tomography ( ct ) scans and evaluated changes in serum bone turnover markers after ovariectomy in rats . all animal experiments were performed in accordance with the animal care guidelines issued by the national institute health , and were approved by the institutional animal care committee at kyungpook national university . twelve female sprague - dawley rats ( 11 weeks old , body weight 190 - 210 g ) were purchased from samtako bio inc . ( osan , korea ) and acclimated to conditions for one week before the experiment . animals were housed in an air - conditioned room ( relative humidity 45 - 65% ) under a 12-h light / dark cycle at 222 and given free access to food and tap water . acclimated rats were assigned to one of two groups , either a sham - operated group ( sham , controls ; n=6 ) or a surgically ovariectomized group ( ovx ; n=6 ) . at surgery , rats were initially anesthetized intraperitoneally with a mixture of xylazine ( 10 mg / kg ) and ketamine ( 60 mg / kg ) . a 2 cm skin incision was then made just medial to the most bulging part of the back . after pulling away periovarian fat with the ovaries , complete bilateral ovariectomy was performed , and then muscle , fascia , and skin were sutured using 4 - 0 silk . eight weeks after surgery , all animals were sacrificed and blood samples were collected by cardiac puncture for serum isolation . serum was separated by centrifugation ( at 1500g ) and then stored at -80 until required for bone metabolic marker assays . serum estrogen ( estradiol , e2 ) was determined using an estradiol elisa kit ( elisa , drg instruments gmbh , germany ) . in addition , 4th lumbar vertebrae were removed , fixed in a 3.7% formaldehyde in phosphate - buffered saline solution ( ph 7.4 ) for 16 h and then stored ( 4 ) in 80% ethanol for bone mass measurements . serum osteocalcin levels and alkaline phosphatase ( alp ) activities ( both sensitive biochemical markers of bone formation ) were determined using osteocalcin eia kits ( nordic bioscience diagnostics , herlev , denmark ) and quantichrome alp assay kits ( dalp-250 , bioassay systems , ca , usa ) , respectively . serum levels of c - terminal telopeptide fragment of type i collagen c - terminus ( ctx ) , which is generated by the osteoclast and is a marker of bone resorption , were determined using ratlaps elisa kits ( nordic bioscience diagnostics , herlev , denmark)3,6 ) . bone histomorphometric parameters and the microarchitectural properties of 4th lumbar vertebrae were determined using a micro - ct system ( explore locus sp , ge healthcare ) with x - ray energy settings of 80 kv and 80 a . samples were scanned over one entire 3600 rotation at an exposure time of 3000 ms / frame . an isotopic resolution of 15 - 40 m voxel size that displayed the microstructure of rat lumbar vertebra was selected , and the angle of increment around the sample was set to 0.4 , which resulted in the acquisition of 900 2d images . a modified feldkamp cone - beam algorithm was used for 3d reconstruction . for bone analysis , bone mineral contents ( bmc ) , bone mineral densities ( bmd ) , trabecular bone volume fractions ( bv / tv , % ) , trabecular thicknesses ( tb.th . ) , trabecular numbers ( tb.n . ) , trabecular separations ( tb.sp . ) , and cortical bone mineral densities ( cr.bmd ) were used for the quantitative analysis , which was performed using 2.0 + aba microview software provided with the micro - ct system13,14,17 ) . each bone was positioned on the two lower supports of the anvil of a universal testing machine ( instron 4202 ; instron , canton , ma , usa ) . load was applied to the midportion of the 4th lumbar vertebrae using a crosshead speed of 1.5 mm / min for all the tests . the load versus displacement data were recorded automatically by the instron software ( instron series ix automated materials tester , version 8.04.00 ) , which subsequently calculates the mechanical parameters from the load - displacement curves . repeated measure anova was used to compare body weights in the ovx and sham groups . the two - sample t test was used to identify significant differences between the groups , and p values of < 0.05 were considered significant . all animal experiments were performed in accordance with the animal care guidelines issued by the national institute health , and were approved by the institutional animal care committee at kyungpook national university . twelve female sprague - dawley rats ( 11 weeks old , body weight 190 - 210 g ) were purchased from samtako bio inc . ( osan , korea ) and acclimated to conditions for one week before the experiment . animals were housed in an air - conditioned room ( relative humidity 45 - 65% ) under a 12-h light / dark cycle at 222 and given free access to food and tap water . acclimated rats were assigned to one of two groups , either a sham - operated group ( sham , controls ; n=6 ) or a surgically ovariectomized group ( ovx ; n=6 ) . at surgery , rats were initially anesthetized intraperitoneally with a mixture of xylazine ( 10 mg / kg ) and ketamine ( 60 mg / kg ) . a 2 cm skin incision was then made just medial to the most bulging part of the back . after pulling away periovarian fat with the ovaries , complete bilateral ovariectomy was performed , and then muscle , fascia , and skin were sutured using 4 - 0 silk . eight weeks after surgery , all animals were sacrificed and blood samples were collected by cardiac puncture for serum isolation . serum was separated by centrifugation ( at 1500g ) and then stored at -80 until required for bone metabolic marker assays . serum estrogen ( estradiol , e2 ) was determined using an estradiol elisa kit ( elisa , drg instruments gmbh , germany ) . in addition , 4th lumbar vertebrae were removed , fixed in a 3.7% formaldehyde in phosphate - buffered saline solution ( ph 7.4 ) for 16 h and then stored ( 4 ) in 80% ethanol for bone mass measurements . serum osteocalcin levels and alkaline phosphatase ( alp ) activities ( both sensitive biochemical markers of bone formation ) were determined using osteocalcin eia kits ( nordic bioscience diagnostics , herlev , denmark ) and quantichrome alp assay kits ( dalp-250 , bioassay systems , ca , usa ) , respectively . serum levels of c - terminal telopeptide fragment of type i collagen c - terminus ( ctx ) , which is generated by the osteoclast and is a marker of bone resorption , were determined using ratlaps elisa kits ( nordic bioscience diagnostics , herlev , denmark)3,6 ) . bone histomorphometric parameters and the microarchitectural properties of 4th lumbar vertebrae were determined using a micro - ct system ( explore locus sp , ge healthcare ) with x - ray energy settings of 80 kv and 80 a . samples were scanned over one entire 3600 rotation at an exposure time of 3000 ms / frame . an isotopic resolution of 15 - 40 m voxel size that displayed the microstructure of rat lumbar vertebra was selected , and the angle of increment around the sample was set to 0.4 , which resulted in the acquisition of 900 2d images . a modified feldkamp cone - beam algorithm was used for 3d reconstruction . for bone analysis , bone mineral contents ( bmc ) , bone mineral densities ( bmd ) , trabecular bone volume fractions ( bv / tv , % ) , trabecular thicknesses ( tb.th . ) , trabecular numbers ( tb.n . ) , trabecular separations ( tb.sp . ) , and cortical bone mineral densities ( cr.bmd ) were used for the quantitative analysis , which was performed using 2.0 + aba microview software provided with the micro - ct system13,14,17 ) . each bone was positioned on the two lower supports of the anvil of a universal testing machine ( instron 4202 ; instron , canton , ma , usa ) . load was applied to the midportion of the 4th lumbar vertebrae using a crosshead speed of 1.5 mm / min for all the tests . the load versus displacement data were recorded automatically by the instron software ( instron series ix automated materials tester , version 8.04.00 ) , which subsequently calculates the mechanical parameters from the load - displacement curves . repeated measure anova was used to compare body weights in the ovx and sham groups . the two - sample t test was used to identify significant differences between the groups , and p values of < 0.05 were considered significant . 1 shows changes in mean body weights in the two groups with different time . at the start of the experiment , however , at 4 weeks after surgery , mean body weight was significantly greater in the ovx group compared with the sham group , and this significance was maintained throughout the period ( p=0.015 , 0.037 , and 0.011 at 4 , 6 , and 8 weeks , respectively ) . as shown in fig . 2 , 8 weeks after surgery , serum estradiol levels were significantly lower in the ovx group ( 1.610.62 pg / ml vs. 19.953.47 pg / ml ; p=0.001 ) . 3a , b summarize the serum levels of the biochemical markers 
 of bone formation , osteocalcin , and alp , and fig . the ovx group had on average a 75.4% higher osteocalcin level than the sham group , which was statistically significant ( p=0.002 ) . iu / l in the ovx group , they were not significantly different ( p=0.136 ) . on the other hand , serum ctx levels in the ovx group were 72.5% higher than in the sham group , which was significant ( p=0.018 ) . as shown in the representative micro - ct images , ovx rats had fewer trabecular bone structures than the sham controls ( fig . 4 ) . in the analyses of micro - ct scans of 4th lumbar vertebrae , the ovx group showed a significant 12% reduction in bmd , and a significant 9.1% reduction in trabecular bone volume fraction ( bv / tv ) ( p=0.005 and p=0.002 , respectively ) . furthermore , mean bmc in the ovx group was lower than in the sham group ( 22.384.12 mg vs. 25.524.37 mg , respectively ) , though this was not significantly different ( p=0.252 ) . however , tb.n . in the ovx group were lower than that of the sham group ( 4.20.6 in the sham group and 3.80.5 in the ovx group ) , which was not significantly different ( p=0.351 ) . in the histomorphometric analysis of cortical bone , the ovx group also showed a significant 6.2% reduction in cr.bmd versus the sham group ( p=0.021 ) ( fig . the three point bending test showed that the 4th lumbar vertebrae of ovx group have on average 39% lower maximal load value compared to that of the sham group ( p=0.001 ) ( fig . 1 shows changes in mean body weights in the two groups with different time . at the start of the experiment , however , at 4 weeks after surgery , mean body weight was significantly greater in the ovx group compared with the sham group , and this significance was maintained throughout the period ( p=0.015 , 0.037 , and 0.011 at 4 , 6 , and 8 weeks , respectively ) . as shown in fig . 2 , 8 weeks after surgery , serum estradiol levels were significantly lower in the ovx group ( 1.610.62 pg / ml vs. 19.953.47 pg / ml ; p=0.001 ) . fig . 3a , b summarize the serum levels of the biochemical markers 
 of bone formation , osteocalcin , and alp , and fig . the ovx group had on average a 75.4% higher osteocalcin level than the sham group , which was statistically significant ( p=0.002 ) . iu / l in the ovx group , they were not significantly different ( p=0.136 ) . on the other hand , serum ctx levels in the ovx group were 72.5% higher than in the sham group , which was significant ( p=0.018 ) . as shown in the representative micro - ct images , ovx rats had fewer trabecular bone structures than the sham controls ( fig . 4 ) . in the analyses of micro - ct scans of 4th lumbar vertebrae , the ovx group showed a significant 12% reduction in bmd , and a significant 9.1% reduction in trabecular bone volume fraction ( bv / tv ) ( p=0.005 and p=0.002 , respectively ) . furthermore , mean bmc in the ovx group was lower than in the sham group ( 22.384.12 mg vs. 25.524.37 mg , respectively ) , though this was not significantly different ( p=0.252 ) . however , tb.n . in the ovx group were lower than that of the sham group ( 4.20.6 in the sham group and 3.80.5 in the ovx group ) , which was not significantly different ( p=0.351 ) . in the histomorphometric analysis of cortical bone , the ovx group also showed a significant 6.2% reduction in cr.bmd versus the sham group ( p=0.021 ) ( fig . the three point bending test showed that the 4th lumbar vertebrae of ovx group have on average 39% lower maximal load value compared to that of the sham group ( p=0.001 ) ( fig . the causes of postmenopausal osteoporosis are accelerated bone resorption and systemic calcium imbalance due to menopause - induced estrogen deficiency3,5,11,12,14 ) . the animal models of osteoporosis are essential for anticipating treatment efficacy and safety2,10 ) , and of the various animal models used , rodents have numerous advantages , for example , they are inexpensive , easy to house , the rapid generation time , and their skeletons are sensitive to the loss of ovarian hormones18 ) . there is extensive literature studying ovx rat including the histomorphometric changes and the biochemical markers of bone turnover2 - 6,10,11,13,14,17 ) . in the present study , we investigated changes in bone turnover and bone loss after ovariectomy in spraque - dawley rats . at 8 weeks after ovariectomy , serum estradiol levels in the ovx group were significantly lower than in the sham group ( 1.640.62 pg / ml vs. 19.943.46 pg / ml ) , which supports the presence of ovarian deficiency in the ovx model . furthermore , as was expected , mean body weights were greater in the ovx group than in the sham group ( p=0.011 ) . omi et al.9 ) described that weight gain efficiency ( body weight gain / food intake ) in their ovx group was significantly greater than in the sham group . turner et al.18 ) also found that ovariectomy increases body weight more in ovx group than in sham group . accordingly , our results are comparable to those of previous studies that have used an ovx rat model3,7 - 9,12,16,18 ) . biochemical markers of bone turnover have been widely used as measures of the status of bone remodeling . the bone remodeling cycle involves the achievement of balance between the bone resorbing activities of osteoclasts and the bone forming activities of osteoblasts . after menopause , bone resorption markers increase due to the activation of bone resorption by estrogen deficiency , and bone formation increases to fill the higher number of resorption cavities , and as a result , increase in their serum levels . the currently accepted mechanism of skeletal bone loss in estrogen deficient rats is that of an imbalance in bone turnover , that is , where bone resorption exceeds bone formation1,16 ) . the biochemical markers of bone turnover used in clinical and experimental studies include osteocalcin and alp ( a sensitive marker of bone formation ) and ctx ( a sensitive marker of bone resorption ) . in a study of ovariectomized rats , kim et al.3 ) found higher plasma osteocalcin and ctx concentrations in their ovx group than in sham operated animals , and similarly , it has been reported that estrogen deficiency increases serum alp , osteocalcin , and ctx levels , which indicated the increased bone turnover2,9,12,15 ) . in the present study , ovariectomized animals were found to have higher osteocalcin , alp and ctx level than sham controls , indicating increased bone turnover due to menopause - induced estrogen deficiency , which is entirely consistent with previous studies2,9,12,15 ) . in ovariectomized animals , as in postmenopausal women , bone loss induced by ovarian deficiency mainly results from trabecular bone loss3 ) . although bmd measurements could be performed by dual - energy x - ray absorptiometry , micro - ct can also be used for monitoring bone microarchitecture and bmd in small animals . scanned micro - ct images can separate cancellous and cortical bone , and therefore , they can be used to produce realistic depictions of apparent and tissue bmds , which in turn reflect degrees of bone mineralization . in the present study , we analyzed bone histomorphometric parameters and microarchitectural properties , bmd , bv / tv , % , tb.th . lei et al.5 ) reported a significant decrease in femoral bmd in 8-week ovx rats as compared 8-week sham rats . in a recent study of histological observa - tions of bone resections of vertebrae and femurs , bone mass was found to be obviously reduced in ovariectomized rats than in sham group10 ) . similarly , in the present study , we also observed that bmd and bv / tv in 4th lumbar vertebrae and cortical bmd were significantly lower in the ovx group . these findings support the notion that our model successfully mimics the effects of osteoporosis . when considering animal models of osteoporosis , it is important that bone sites should be taken into account . it is well known that , in ovariectomized animals , cortical bone loss does not match that of trabecular bone2,9 ) . in a study of ovariectomized rats , ferretti et al.2 ) reported that bone mass started to decrease earlier and more extensively in trabecular than in cortical bone . in particular , mean bmd of 4th lumbar vertebra at 8 weeks after surgery , had reduced by 12.0% , but mean cortical bmd had only reduced by 6.2% . the present study demonstrates that ovariectomy in rats significantly increases bone turnover , and this was confirmed by elevated levels of bone turnover markers . furthermore , it shows a significant loss of trabecular bone in lumbar vertebrae 8 weeks after ovariectomy . finally , our study demonstrates that the ovariectomized rat model offers a reproducible and reliable model of osteoporosis .