To determine both the preventive and recovery effects of tower climbing exercise on mass, strength, and local turnover of bone in ovariectomized (OVX) rats, we carried out two experiments. In experiment I, 60 Sprague-Dawley rats, 12 months of age, were assigned to four groups: a Baseline Control, Sham-Operated Sedentary, OVX-Sedentary and OVX-Exercise rats. Rats voluntarily climbed a 200-cm tower to drink water from a bottle set at the top. At 3 months, OVX elevated both the femoral cortex and lumbar trabecular turnover, leading to a reduction in bone mass and strength. However, in OVX-Exercise rats, those values were maintained at the same level as in the Sham-Sedentary rats. Thus, the climbing exercise, started after 3 days of OVX, prevented OVX-induced cortical and trabecular bone loss by depressing turnover elevation. After confirming the preventive effect, we evaluated the recovery effect of exercise. In experiment II, 90 Sprague-Dawley rats, 12 months of age, were assigned to six groups: a Baseline control, two groups of Sham-Operated Sedentary and OVX-Sedentary, and OVX-Exercise rats. The exercise started 3 months after the OVX operation. At 3 months, OVX increased the trabecular bone formation rate and osteoclast surface, leading to a decrease in compressive strength. In the midfemur, the cross-sectional area, moment of inertia, and bending load values decreased. At 6 months, in the OVX-Exercise rats, the parameters of breaking load in both the lumbar and midfemur, lumbar bone mass, and the total cross-sectional area recovered to the same levels as those in the Sham-Sedentary rats. However, the cortical bone area did not recover. Periosteal bone formation increased, while endosteal bone formation decreased. These results showed that the climbing exercise had both a preventive and recovery effect on bone strength in OVX rats. In the mid-femur, effects on bone formation were site-specific, and the cross-sectional morphology was improved without an increase in cortical bone area, supporting cortical drift by mechanical stimulation.
OSTEOPOROSIS IS A SERIOUS problem in elderly women and is characterized by bone loss, leading to fractures and high turnover of bone.(1) Many studies suggest that the effects of exercise have a beneficial effect on bone in humans(2, 3) and animals.(4, 5) Because exercise is also effective in maintaining bone mineral density (BMD) in early postmenopausal women, it has been proposed as a long-term solution to prevent osteoporosis.(3)
To examine the preventive or recovery effect of exercise with both bone mass and strength as the endpoints of an experiment, animal exercise models are used. Animal studies using voluntary wheel running,(6) jumping,(7) treadmill running,(8) or voluntary climbing(4) have demonstrated the beneficial effect of increased loading on bone mass and mechanical properties. It has been suggested that treadmill exercise prevented ovariectomy-induced bone loss by improving mechanical stress in young rats.(8) In mature and aged animals with estrogen deficiency, there are conflicting reports of positive effects(8, 9) or no significant effects.(10) These discrepancies may be because of the difference in age or the type of exercise. Resistance exercise such as jumping(11) or bipedal stance(12) have been examined in mature OVX rats and prevented the OVX-induced bone loss in the tibia. However, these studies did not investigate all the effects of exercise on bone mass, strength, and local turnover in detail. In the past, most studies focused on the preventive effects of exercise,(8,11,12) whereas there are few studies that investigated whether exercise recovered OVX-induced bone loss.(9) No studies have evaluated whether or not exercise has both a preventive and recovery effect in OVX rats.
We previously demonstrated that tower climbing exercise increased bone mass and strength mainly by increasing bone formation in intact rats.(4) In this experiment, we first confirmed the preventive effects of tower climbing exercise and then evaluated the influence of this exercise, started 3 months after ovariectomy, on trabecular and cortical bone in aged osteopenic rats. The purpose of this study was to discover the preventive and recovery effects of voluntary resistance exercise on mass, strength, and local turnover of bone in ovariectomized rats.
MATERIALS AND METHODS
Female Sprague-Dawley rats, 6 months of age, were purchased from Japan CLEA Inc. (Tokyo, Japan) and acclimatized for 6 months under standard laboratory conditions (22 ± 2°C, 60% humidity). The light/dark cycle was 12 h with lights on from 6:00 a.m. to 6:00 p.m. All rats were housed in metal cages. Drinking water was available at all times. All rats were fed commercial rat chow (Japan CLEA Inc.; calcium: 1200 mg/100 g, phosphorus; 1080 mg/100 g). The body weight of each rat was measured weekly. The protocol was approved by The University of Tsukuba's Institutional Animal Care and Use Committee.
The purpose of this experiment was to confirm the preventive effect of climbing exercise in mature OVX rats. Sixty rats, 12 months of age, were randomized to four groups of 15 animals each. Group BC was the Baseline Control. Thirty rats were ovariectomized (OVX) and the other 15 were sham-operated under anesthesia with sodium-Nembutal administered intraperitoneally. All the animals recovered well after surgery. The exercise was started 3 days after ovariectomy. Group SS was a Sham-Operated Sedentary control group killed after 3 months of the experiment. Groups OS and OE were OVX-Sedentary and OVX-Exercise groups, killed after 3 months of the experiment. To preclude any excess food intake induced by OVX, the daily consumption of each group was measured, and each group was given the mean amount of chow consumed by the SS group on the previous day. During the course of the experiment, four rats (1 SS, 2 OS, and 1 OE) died because of tumors or unknown causes, and they were not subjected to analyses. The other rats remained healthy.
The purpose of this experiment was to find out the recovery effect of exercise in aged osteopenic rats. Ninety rats, 12 months of age, were randomized to six groups of 15 animals each. Group 0BC was the Baseline Control. Forty-five rats were ovariectomized and the other 30 were sham-operated under anesthesia with sodium-Nembutal administered intraperitoneally. All the animals recovered well after surgery. The exercise started 3 months after ovariectomy, at the age of 15 months. Groups 3SS and 6SS were Sham-Operated Sedentary control groups killed after 3 and 6 months of the experiment, respectively. Groups 3OS and 6OS were OVX-Sedentary groups, killed after 3 and 6 months of OVX, respectively. Group 6OE was an OVX-Exercise group, killed after 6 months of OVX. To preclude any excess food intake induced because of ovariectomy, the daily consumption of each group was measured and each was given the mean amount of chow consumed by the SS group on the previous day. During the course of the experiment, 13 rats (1, 5, 3, and 4 from groups 3OS, 6SS, 6OS, and 6OE, respectively) died because of tumors or unknown causes, and they were not subjected to analyses. The other rats remained healthy.
At the end of both experiments, rats were killed by exsanguination under ether anesthesia. Soon after death, the hindlimb muscles (gastrocnemius, plantaris, soleus, tibia, and extensor digitorum longus) and abdominal fats (parametrial fat, perigastric fat, and mesenteric fat) were isolated. The combined weight of the hindlimb muscles and abdominal fats were measured. The third, fourth, and fifth lumbar vertebra (L3, L4, and L5) and bilateral femora were harvested. The L3, L4, and right femur (RF) were immediately fixed with 4% paraformaldehyde in a 0.1 M phosphate buffer containing 2% sucrose and stored at 4°C. L5 and the left femur (LF) were stored at −80°C until the mechanical tests. Bone labeling of rats with a subcutaneous injection of calcein (8 mg/kg body weight) was performed 14 and 4 days before death.
The groups of exercise rats were housed in metal cages with a wire mesh tower that had two water bottles set at the top.(4) There were no bottles in the bottoms of the cages. At the beginning, the bottles were set at a height of 20 cm. The set point of the drink bottles was gradually elevated to 200 cm over 1 week. The rats were monitored 24 h/day every 3 weeks during the experimental period, using a charge-coupled device (CCD) video camera (CCD-TRV95; Sony, Tokyo, Japan). The daily distances and time periods of climbing activity were obtained from the monitoring records. In 10–20% of exercise activity, the rats climbed the tower without drinking.
Bone mineral measurement
BMD (mg/cm2) and bone mineral content (BMC; mg) were measured on the LF using dual-energy X-ray absorptiometry (DXA) (DCS-3000; Aloka, Tokyo, Japan). The L5 body was prepared by removing the posterior segment, and bone mineral measurements were performed. The mineralization profiles of the specimens were stored with monitoring images, and the BMD and BMC values for the lumbar body and the femur mid-diaphyseal region (12 mm in length) were obtained.
A three-point bending test was performed as previously described(4, 13) using a load tester (Tensilon UTA-1T; Orientec, Tokyo, Japan). Each LF specimen was placed on a holding device with supports located at a distance of 12 mm, with the lesser trochanter proximal to, and in contact with, the proximal transverse bar. The midpoint served as the anterior (upper) loading point. A bending force was applied by the crosshead at a speed of 10 mm/minute until fracture occurred. The breaking load (N) and structural stiffness (N/mm) were obtained directly from the load-deformation curves that were recorded continually in a computerized monitor linked to the load tester.
Lumbar vertebral body
The L5 body specimen was fixed with a clamp at the bases of the transverse processes in the holder of a diamond band saw (Exakt, Norderstedt, Germany). By removing the cranial and caudal ends of the specimens, the plano-parallel ends at a height of 3.5 mm were obtained.(4, 13) Cylinder samples were placed centrally on the smooth surface of a steel disk attached to the load tester. A cranio-caudal compression force was applied to the specimen using a steel disk at a nominal deformation rate of 2 mm/minute. The breaking load (N) and structural stiffness (N/mm) were obtained, as were those of the femur specimens.
Lumbar vertebral body
Each L4 specimen was embedded in methylmethacrylate (MMA) after Villanueva's bone staining. From the middle portion of the specimen, 10-μm-thick undecalcified sagittal sections were cut on a microtome (Supercut 2050; Reichert-Jung, Heidelberg, Germany). L3 specimens were embedded in a mixture of MMA, hydroxyglycol methacrylate, and 2-hydroxyethylacrylate polymerized at 4°C. Six-micrometer-thick L3 specimens was obtained as described for L4. The L3 sections were then stained for tartrate-resistant acid phosphatase (TRAP).(14) Histomorphometry of L3 and L4 was performed with a semiautomatic image-analyzing system linked to a light microscope (Cosmozone 1S; Nikon, Tokyo, Japan). For each section, the area of the secondary spongiosa was measured, but the regions within 1.0 mm of the growth plate/metaphyseal junction and one cortical shell-width of the endocortical surface were not measured, so as to exclude the primary spongiosa.
For the structural parameters, the trabecular bone volume (BV; μm2), trabecular tissue volume (TV; μm2), and trabecular bone surface (BS; μm) were measured. The trabecular thickness (Tb.Th; μm), trabecular number (Tb.N; 1/mm), and trabecular bone separation (Tb.Sp; μm) were calculated by a parallel plate model assuming constant geometry.(15, 16) For the bone formation parameters of L4, the single-labeled surface (sLS; μm), double-labeled surface (dLS; μm), and BS (μm) were measured. The value of dLS/sLS ratio was obtained. The mineral apposition rate (MAR; μm/day) was calculated as the distance between double labels divided by the labeling interval and multiplied by π/4. The mineralizing surface per bone surface (MS/BS; %) was obtained by adding the values of the dLS/BS and one-half of the sLS/BS value. The surface referent bone formation rate (BFR/BS; μm3/μm2/day) was calculated by multiplying the MS/BS value by the MAR.(15–17) For the bone resorption parameters of L3, the osteoclast surface (Oc.S; μm) and BS (μm) were measured.(15–17) TRAP-positive cells that formed resorption lacunae at the surface of the trabeculae and contained one or more nuclei were identified as osteoclasts.(18)
An undecalcified section was obtained from the site of the mid-diaphysis of the RF. The specimen was embedded in MMA without staining to yield a 40-μm-thick crosscut ground section. Measurements were made on a cathode-ray tube monitor with a CCD camera (CCD High Gain Camera-1600A; Flovel Co., Tokyo, Japan) setting on the microscope using a semiautomatic image-analyzing program (Mac Scope; Mitani Corp., Fukui, Japan) on a computer.(4, 13) The total cross-sectional area (mm2), cortical bone area (mm2), and bone marrow area (mm2) were obtained. The endocortical surface was approximated as a regular, continuous line. The moment of inertia (mm4) of the cortical bone area for the medial-lateral axis was calculated directly by an image-analyzing computer.(4, 13) Dynamic parameters such as dLS/BS, sLS/BS, MS/BS, MAR, and BFR/BS were measured in the periosteal and endocortical envelopes. The eroded surface (ES/BS; %) was also measured in the endocortical perimeter.
All values are expressed as mean ± SEM. Data were assessed by two-way analysis of variance (ANOVA) for the time and treatment. When the treatment was found to have a significant overall effect, the difference between the experimental groups was assessed by Tukey's honestly significant difference (HSD) for each time period or Student's t-test at 15 months of age in experiment II.(4, 7) One-way ANOVA with repeated measurements was used to examine the significance of changes in the climbing distances and time. Statistical significance was set at less than 0.05. All analyses were performed with a commercially available statistical package (SPSS Version 10.0; SPSS, Tokyo, Japan).
This study demonstrated that climbing exercise enabled recovery of mechanical strength of both the femur and lumbar vertebrae in aged osteopenic rats and prevented cortical and trabecular bone loss in mature OVX rats. In the first experiment, the BMD and mechanical load values of the femur and lumbar vertebrae, reduced by ovariectomy, were maintained, depressing turnover elevation. Second, the values of mechanical load, total cross-sectional area, and moment of inertia recovered 3 months after starting the climbing exercise. Surprisingly, even in aged OVX rats, the exercise increased periosteal bone formation, whereas it decreased endosteal bone formation, indicating that cortical drift had occurred after 3 months of exercise.
Previous studies reported that OVX increased cancellous bone turnover in rats, whereas treadmill exercise mainly depressed bone resorption.(19) This bone formation was not stimulated, but rather decreased, in OVX rats. In experiment I, the climbing exercise significantly decreased both bone formation and resorption, suggesting that it could prevent elevated cancellous bone turnover in OVX rats. However, in experiment II, the exercise increased bone formation, while decreasing bone resorption, after transient elevation of cancellous bone turnover by OVX. These results were consistent with our previous reports in intact rats, using both climbing(4) and jumping exercise.(7) Why exercise decreases the cancellous bone formation in gonadal hormone deficiency rats is not yet understood. According to the bone remodeling concept,(1) the deactivation of osteoclasts leads to a decrease in osteoblasts. We observed that the resistance exercise decreased both the number and surface area of osteoclasts.(4, 7) The exercise may have decreased the osteoclasts in the early stage, leading to a decrease in osteoblast activities. This hypothesis is supported previous data that treadmill exercise mainly affects bone resorption in estrogen-deficient rats.(12, 19)
In the femoral cortex, OVX increased bone formation on both the periosteal and endocortical surfaces as in previous studies,(20) while the climbing exercise prevented elevation of those parameters in experiment I. However, in experiment II, exercise increased periosteal bone formation but decreased endosteal bone formation after transient turnover elevation by OVX. These results were consistent with our previous studies of intact rats,(4, 7) suggesting that exercise leads to an acceleration of cortical drift in aged OVX rats. Thus, the climbing exercise would be effective to restore bone strength in estrogen deficiency.
The femoral morphology parameters, except for bone marrow area, were decreased by OVX, leading to mechanical strength loss of the midfemur. We did not observe a significant increase in the eroded surface, which had an increased tendency compared with the sham group after 3 months of OVX. However, the cortical bone area was decreased, although both periosteal and endosteal bone formation were increased, by OVX. The imbalance of bone formation between the periosteal and endosteal surfaces is one possible reason for the decrease in bone mass after OVX. The climbing exercise maintained femoral cross-sectional morphology and femoral bone strength. Interestingly, in experiment II, although the cortical bone area was decreased by OVX, exercise recovered the cross-sectional area and bending load. As with the observation of cortical bone turnover in this study, these results also suggested that a cortical drift occurred in aged OVX rats. To support these findings, we also obtained the normalization of the femoral breaking load by the moment of inertia and midfemoral BMD. Those normalized breaking loads were both recovered, and the breaking load/BMD was further improved compared with that in the Sham-Sedentary group. These data strongly suggested that the exercise improved not only deteriorated structure, but also strengthened the bone material properties in OVX rats.
The BMD and breaking load of lumbar vertebrae seemed to vary with changes in the trabecular structure. The exercise-induced structural alterations were different between prevention and recovery. In experiment I, BV/TV, Tb.Th, and Tb.Sp were changed by the exercise, whereas in experiment II, Tb.Th was increased and the other three parameters did not recover. Resistance exercise mainly affects the lumbar vertebrae and tibia Tb.Th in intact rats.(4, 7) Trabecular thickness is increased by direct apposition in the unilateral hindlimb experiments in OVX rats.(12) Our data were consistent with these results. Because the exercise increased the values of dLS/sLS ratio in experiment II, these findings strongly indicate that disturbed osteoblast function at each trabecular surface site is improved by exercise. From these results, exercise improved both the structure of cancellous and cortical bone in aged OVX rats as in intact rats.(4, 7) This suggested that the bone structure in OVX rats was relatively more responsive to loading than that normal animals. This could be explained at least partly by the increased bone turnover rate, which is caused by estrogen deficiency.
In this study, the daily climbing times were 18–21 minutes/day in experiment I and 16–19 minutes/day in experiment II. These times were about two-thirds of those in growing rats.(4) However, the climbing exercise had a preventive and recovery effect on OVX-induced bone loss. We do not have a good explanation why ovariectomized and/or aged bone was more sensitive to exercise. With regard to the exercise distance, the climbing distance in experiment I was about 1.5 times that in experiment II. The reason for this difference was unclear. The difference in climbing distance suggested that the voluntary climbing style and/or learning style changed age-dependently after ovariectomy. In our behavioral observation, the rats preferred to wander, cling, and stay at the top of tower in experiment II more than in experiment I. Therefore, the exercise times in experiment II were similar to those in experiment I, although the exercise distances were different between the two experiments. However, this climbing exercise prevented deterioration and improved the frail bone strength induced by ovariectomy. Daily climbing time seemed to be more important as a valuation parameter than distance in this study. Recently, Robling et al. (21) showed that short but distributed loading bouts were osteogenic, and that rest periods inserted between loading bouts enhanced the osteogenic potential by restoring mechanosensitivity.(22) The climbing style, which had sufficient rest periods between voluntary climbing sessions and distributed climbing sessions throughout the day, conformed to those results. These findings also supported our suggestion.
The body weight increase in OVX rats was higher than that in sham animals. In each experiment, rats took an equal amount of food. This weight increase was also observed in pair-fed rats,(20) indicating that it is not only the amount of food that determines the difference. The increase in body weight seemed to be entirely the result of excessive fat deposition, because muscle mass was not increased by OVX. In present and previous studies, OVX rats had decreased bone mass and strength without a concomitant decrease in body mass,(9, 11) although the increased loading from body mass, mainly because of increased adipose tissue, increased the strain in the remaining bone. One reason for this is the increased voluntary load by fat apposition did not overcome the increased osteoclast activity by estrogen deficiency. Another reason could be that leptin inhibited bone formation through a hypothalamic relay.(23) Increased adipose tissue augmented the secretion of leptin.(24, 25) Because this bone loss mechanism is associated with various factors such as hormonal, dietary, biochemical, and other factors, these mechanisms remain unclear. However, in this study, exercise had the potential to decrease the fat mass and normalize bone strength. There were no differences in the hindlimb muscles when the bone strength was increased by exercise. To resolve this problem, we analyzed the relationship between hindlimb muscle mass and bending load. However, no relationships in either experiment I or II were found. Previously, the climbing exercise increased the bending load without significantly increasing hindlimb muscle after 8 weeks of exercise.(4) These data suggested, in this exercise, that the bone strength is not only caused by the load from muscle mass, but also mainly to exercise-induced direct load.
As far as studies of postmenopausal osteoporosis women and resistance exercise are concerned, the exercise periods varied, and some researchers reported that exercise increased and maintained the BMD.(26, 27) In postmenopausal women with osteoporosis receiving calcium and vitamin D3 supplements, 1- and 2-year resistance exercise increased the lumbar BMD, whereas only the supplement without exercise did not alter the BMD.(28) Further, in postmenopausal women with calcium supplements, longer than 2 years of resistance exercise increased the BMD of total and intertrochanter hip sites.(29) Based on these human studies, there is evidence that resistance exercise recovers and maintains bone mass in early and older postmenopausal women. Extrapolation of the present results to humans should be undertaken with caution, because this exercise pattern of quadrupedal rats is different from various human exercise patterns, although our data indicate that exercise could prevent and recover osteopenia and osteoporosis in postmenopausal women.
In conclusion, voluntary climbing exercise maintained the strength of both the cortical and trabecular bone reduced by OVX and further recovered these parameters. The trabecular thickness was markedly compensated while the other structural parameters did not fully recover. The effect of exercise on cortical and cancellous bone turnover was mainly the stimulus of bone formation after a transient increase in OVX-induced turnover. The climbing exercise prevented negative changes in cortical bone structure and, in aged osteopenic rats, accelerated cortical drift by increasing periosteal bone formation and decreasing endosteal bone formation.