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Erect bipedal stance exercise partially prevents orchidectomy-induced bone loss in the lumbar vertebrae of rats
Bone Vol. 27, No. 5 November 2000:667– 675
Erect Bipedal Stance Exercise Partially Prevents Orchidectomy-Induced Bone Loss in the Lumbar Vertebrae of Rats W. YAO,1 W. S. S. JEE,1 J. CHEN,1 C. S. TAM,2 R. B. SETTERBERG,1 and H. M. FROST1,3 1
Radiobiology Division, University of Utah, Salt Lake City, UT, USA Faculty of Science, University of Toronto, and Gen.Sci Regeneration Sciences Inc., ON, Canada 3 Department of Orthopedic Surgery, Southern Colorado Clinic, Pueblo, CO, USA 2
androgenic effects on the development or maintenance of the male skeleton.10,11,30,33 Bone loss induced by castration can be prevented by hormone replacement therapy with androgen,26,31 testosterone,28 estrogen,3,29 selective estrogen receptor modulator (SERM),15 parathyroid hormone,12 and prostaglandin E2 (PGE2).17 Treadmill exercise was reported to partially prevent orchidectomy-induced bone loss in the rat femur,13,27 increase cancellous bone mass in skeletally immature animals,14,18 and increase periosteal bone formation without significantly affecting cancellous bone mass in skeletally mature rats.35,36 Recently, we used a raised-cage model to “exercise” rats, which forced them to rise to a bipedal stance for feeding.34 This model increased cortical bone mass and partially prevented orchidectomy-induced tibial bone loss in 6-month-old male rats. However, how it affects the vertebrae remains unknown. In addition, although the effects of androgen deficiency have been well documented in rat tibia and femur,10,11,33 few studies have evaluated its effects on vertebral bodies. Therefore, we conducted this study to characterize cancellous and cortical bone histomorphometric changes of both the sagittal and transverse sections of lumbar vertebral bodies after orchidectomy and/or housing in a raised cage in 6-month-old male rats.
This study investigates the responses of the fourth and fifth lumbar vertebral bodies of 6-month-old male Sprague-Dawley (SD) rats to orchidectomy (orx) and to erect bipedal stance for feeding for 12 weeks in specially designed raised cages (RC) for which the heights were raised from 20 cm to 35.5 cm. A total of 30 rats were divided into groups of: baseline; sham ⴙ housed in normal height cage (NC); orx ⴙ NC; sham ⴙ RC; and orx ⴙ RC. Bone histomorphometry was performed on the triple-labeled undecalcified fourth sagittal (LVL-4) and fifth transverse (LVX-5) sections. We found that orchidectomy induced high-turnover trabecular and cortical bone loss in the lumbar vertebrae. Forcing the rats to rise to erect stance for feeding reduced trabecular and cortical bone loss caused by orx. Apparently, depressing the elevated bone resorption next to the marrow induced by orx, and stimulating bone formation at the ventral periosteal surfaces, caused these effects. Orchidectomy and raised cage had similar effects on the two vertebrae except that the percentage of trabecular bone loss was greater in the LVL-4 than in LVX-5, and that bipedal stance exercise increased the total tissue area and mineral apposition rates (0 – 80 day interval) of ventral periosteal and dorsal endocortical surfaces of LVX-5 to a greater extent than it did in LVL-4. Such findings suggest that forcing rats to rise to an erect bipedal stance for feeding helps prevent loss of trabecular and cortical bone “mass,” and presumably bone strength, in orchidectomized rats. This method also provides an inexpensive, noninvasive, reliable model to increase in vivo vertebral loading in rats that is similar in humans. (Bone 27:667– 675; 2000) © 2000 by Elsevier Science Inc. All rights reserved.
Materials and Methods Animals and Experimental Protocol Thirty 1-month-old male Sprague-Dawley rats (Simonsen Laboratories, Gilroy, CA) were acclimated to and aged in local vivarium conditions. They were housed six per cage (58 cm ⫻ 36 cm ⫻ 20 cm) with the room temperature maintained at 72°F and 12:12 hour light/dark cycles. The rats were allowed free access to water and pelleted commercial natural diet (Teklad Rodent Laboratory Chow #8604, Harlan-Teklad, Madison, WI), which contains 1.46% calcium, 0.99% phosphorus, and 4.96 IU/g of vitamin D3. At 6 months of age, the rats were divided randomly into the following five body-weight (b.w.)-matched groups with six rats per group: (1) baseline control (baseline); (2) shamorchidectomized ⫹ housed in the normal height cage (sham ⫹ NC); (3) bilaterally orchidectomized ⫹ housed in normal height cage (orx ⫹ NC); (4) sham-orchidectomized ⫹ housed in the raised cage (sham ⫹ RC); and (5) orchidectomized ⫹ housed in raised cage (orx ⫹ RC). The study lasted 12 weeks, so the rats were 6 months old when the experiment began, and 9 months old when it ended.
Key Words: Orchidectomy; Bipedal stance exercise; Lumbar vertebrae; Bone histomorphometry; Bone strength. Introduction Androgen deficiency, reduced physical activity, and loss of body weight are believed to contribute to the increased incidence of vertebral fracture in men after their sixth decade.1,22 Orchidectomized (orx) rats have been used as an animal model to study Address for correspondence and reprints: Dr. Wei Yao, Radiobiology Division, University of Utah, 729 Arapeen Drive, Suite 2334, Salt Lake City, UT 84108-1218. E-mail: [email protected] © 2000 by Elsevier Science Inc. All rights reserved.
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W. Yao et al. Effects of making rats rise to bipedal stance on lumbar vertebrae
Bone Vol. 27, No. 5 November 2000:667– 675
Table 1. Effects of orchidectomy and raised cage on body weights in 6-month-old male Sprague-Dawley rats Groups
Baseline
Sham ⫹ NC
orx ⫹ NC
Sham ⫹ RC
Orx ⫹ RC
Initial (g) Final (g)
392 ⫾ 32
384 ⫾ 26 387 ⫾ 69
388 ⫾ 34 387 ⫾ 19
394 ⫾ 29 437 ⫾ 38a,b
391 ⫾ 32 437 ⫾ 23a–c
KEY: NC, normal height cage; orx, orchidectomy; RC, raised cage. a Vs. baseline, p ⬍ 0.05. b Vs. sham ⫹ NC, p ⬍ 0.05. c Vs. orx ⫹ NC, p ⬍ 0.05.
At the beginning of the study, the rats in the sham ⫹ RC and orx ⫹ RC groups were housed in the specially designed cages with an initial height of 28 cm (58 cm ⫻ 36 cm ⫻ 28 cm) for 1 week. The heights of the cages were then raised 2.5 cm every third day until they reached the final height of 35.5 cm. At this height, the rats needed to stand upright on their hind limbs and reach upward for food and water. This height was maintained for 10 weeks. The body weights were recorded weekly, except for the first 2 weeks when they were weighed daily. The rats were observed to verify that they were able to stand upright and reach the food and water. The baseline rats received 20 mg tetracycline/kg b.w. (Sigma Chemical Co., St. Louis, MO) and 10 mg calcein/kg b.w. (Sigma) by subcutaneous injection at day 70 and day 80 of the study. All other rats received 90 mg xylenol orange/kg b.w. (Sigma) at the beginning of the study (day 0), and 20 mg tetracycline/kg b.w. and 10 mg calcein/kg b.w. subcutaneously at day 70 and day 80 of the study. Before autopsy, the rats were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg) and killed by cardiac puncture.
(BFR/TV), bone formation rate per unit of bone surface (BFR/ BS), and activation frequency (Ac.f), according to Parfitt et al.23,24 Results are presented as mean ⫾ SD. The statistical analyses were performed using SPSS version 10 for WINDOWS (SPSS Inc., Chicago, IL). The Kruskal–Wallis test was selected to analyze the differences between groups. Univariate analysis of variance (ANOVA) was used to observe the interaction between raisedcage and orchidectomy using body weights as a covariate. p ⬍ 0.05 was considered significant. Results Body Weight Changes No age-related changes occurred in body weights between baseline and sham ⫹ NC rats. Orchidectomy did not induce significant changes in body weights. In the sham ⫹ RC and orx ⫹ RC groups, body weights were higher by 21% as compared with sham ⫹ NC animals34 (Table 1). Bone Histomorphometric Measurements
Bone Histomorphometry The fourth and fifth lumbar vertebrae were stained with Villanueva bone stain, dehydrated in graded concentrations of ethanol, defatted in acetone, and embedded in methylmethacrylate (Fisher Scientific, Fairlawn, NJ). Serial transverse sections of the fifth LV were cut from the cranial end to the caudal end to 230 m thickness using a low-speed metallurgic saw. The first transverse section with an intact cortical ring of each fifth LV (LVX-5), and the midsagittal sections of the fourth LV (LVL-4), were selected and ground to 20 m for histomorphometric measurement. Histomorphometry was done using a semiautomatic image analysis system (OSTEOMEASURE, OsteoMeasure, Inc., Atlanta, GA) linked to a microscope equipped for transmitted and fluorescent light studies. We studied the entire transverse section of the fifth lumbar vertebral bodies and the sagittal sections of the fourth lumbar vertebral bodies, but omitted 0.5 mm from the growth plates. Static measurements included total cross-sectional area (T.Ar), trabecular bone area (B.Ar) and perimeter (B.Pm), and dorsal and ventral average cortical bone width (Ct.Wi, sampled once every 50 m). Dynamic measurements included single-labeled (sL.Pm) and double-labeled perimeter (dL.Pm), eroded perimeter (E.Pm), and interlabel width (L.Wi, at the 0 – 80 day and 70 – 80 day intervals) of the trabecular surface, and periosteal and endocortical surfaces of the dorsal and ventral cortices. These data were used to calculate trabecular volume (BV/TV), cortical thickness (Ct.Th), mineralized surface (MS/BS), eroded surface (ES/BS), mineral apposition rate (MAR at the 0 – 80 day and 70 – 80 day intervals), bone formation rate per unit of bone volume (BFR/BV), bone formation rate per unit of tissue volume
Histomorphometric profiles of the LVX-5 are shown in Tables 2 and 3. Compared with sham ⫹ NC animals, orchidectomy alone (orx ⫹ NC) induced 37%, 11%, and 12% losses of trabecular volume and ventral and dorsal cortical thickness. Bone formation increased on all the bone surfaces and endocortical eroded surface increased. Raised cages in sham-operated animals (sham ⫹ RC) increased the total cross-sectional area of the vertebral body by 11% due to increased bone formation at the ventral periosteal surface over the entire study period (0 – 80 days), and tended to decrease bone resorption at the endosteal surface. In orchidectomized animals in raised-cage conditions (orx ⫹ RC), the ventral and dorsal cortical thicknesses were higher by 29% and 25%, respectively, when compared with orx ⫹ NC animals, and these values were comparable to those of the sham control animals. These changes were accompanied by increased total mineral apposition rate at 0 – 80 days at the ventral periosteal surface and suppressed eroded surface at the endocortical surfaces (Figure 1). Histomorphometric profiles of the LVL-4 are shown in Tables 4 and 5. The changes induced by orchidectomy and/or raised cages were similar to that of LVX-5, except that LVL-4 showed more decreases in both cancellous and cortical bone loss after orx, but fewer increases in cortical bone thickness and total bone formation after RC (Figure 1). Discussion This study demonstrates that orchidectomy of rats at 6 months of age induced high-turnover cancellous and cortical bone loss next to marrow in lumbar vertebral bodies. Making orchidectomized rats rise to an erect stance for feeding partially prevented these
18.1 ⫾ 3.1 0.5 ⫾ 0.05 35.8 ⫾ 9.4 96.8 ⫾ 36.5 33.5 ⫾ 17.5 4.0 ⫾ 1.7 48.2 ⫾ 15.9 n.a. 0.7 ⫾ 0.0 34.7 ⫾ 11.4
Trabecular dynamics Mineralized surface (%) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day * 100) Bone formation rate/bone volume (%/yr) Bone formation rate/trabecular volume (%/yr) Eroded surface (%)
Ventral periosteal dynamics Mineralized surface (%) Mineral apposition rate (0–80 days, m/day) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day * 100) 52.8 ⫾ 10.5 0.8 ⫾ 0.1 0.6 ⫾ 0.1 35.5 ⫾ 13.8
21.6 ⫾ 5.8 0.5 ⫾ 0.1 43.2 ⫾ 14.7 103.7 ⫾ 38.3 31.6 ⫾ 11.1 3.3 ⫾ 0.5
9.4 ⫾ 0.3 25.0 ⫾ 0.4 3.7 ⫾ 0.1 181.3 ⫾ 9.8 276.3 ⫾ 33.2 230.2 ⫾ 11.3
Sham ⫹ NC
58.0 ⫾ 13.3 n.d. 1.1 ⫾ 0.1a,b 64.7 ⫾ 25.1
29.7 ⫾ 1.3a,b 0.7 ⫾ 0.1a,b 78.6 ⫾ 6.1a,b 215.0 ⫾ 26.6a,b 50.0 ⫾ 19.1 6.7 ⫾ 1.3b
9.3 ⫾ 0.6 15.8 ⫾ 2.1a,b 2.7 ⫾ 0.5a,b 297.1 ⫾ 70.6a,b 214.2 ⫾ 16.4a,b 196.6 ⫾ 14.6a,b
Orx ⫹ NC
61.0 ⫾ 12.8 1.2 ⫾ 0.1b 0.8 ⫾ 0.2 48.5 ⫾ 9.6
22.5 ⫾ 5.0 0.6 ⫾ 0.1 48.1 ⫾ 15.2 117.0 ⫾ 48.5 38.4 ⫾ 10.2 2.4 ⫾ 0.8
10.4 ⫾ 0.5a,b 27.4 ⫾ 6.0 4.0 ⫾ 0.5 164.4 ⫾ 45.1 299.1 ⫾ 21.5 255.2 ⫾ 12.2
Sham ⫹ RC
63.8 ⫾ 10.8 1.0 ⫾ 0.1c 1.0 ⫾ 0.1a,b 63.8 ⫾ 18.7a
32.0 ⫾ 9.6a 0.7 ⫾ 0.04a,b,d 77.8 ⫾ 21.7a,d 188.0 ⫾ 40.9a,b 49.1 ⫾ 9.1 2.9 ⫾ 1.4c
9.5 ⫾ 0.6 20.8 ⫾ 4.0c 3.2 ⫾ 0.5c 232.9 ⫾ 49.8c 275.9 ⫾ 15.2c 245.9 ⫾ 17.4c
orx ⫹ RC
Orx
n.s. n.s. 0.003 0.002
0.005 0.000 0.000 0.000 n.s. 0.012
n.s. 0.001 0.013 0.002 0.005 0.005
KEY: ANOVA, analysis of variance; n.a., not available; NC, normal height cage; n.d., not detectable; n.s., nonsignificant change; orx, orchidectomy; RC, raised cage. a Vs. baseline, p ⬍ 0.05. b Vs. sham ⫹ NC, p ⬍ 0.05. c Vs. orx ⫹ NC, p ⬍ 0.05. d Vs. sham ⫹ RC, p ⬍ 0.05.
9.4 ⫾ 0.4 26.5 ⫾ 5.2 4.4 ⫾ 1.2 158.5 ⫾ 54.0 271.9 ⫾ 32.2 222.9 ⫾ 35.1
Baseline
Static changes Total cross-sectional area (mm2) Trabecular volume (%) Trabecular number (#) Trabecular separation (m) Ventral cortical thickness (m) Dorsal cortical thickness (m)
Parameters
Table 2. Histomorphometric changes of the fifth lumbar vertebral body: transverse sections
n.s. 0.000 0.000 n.s.
n.s. n.s. n.s. n.s. n.s. 0.001
0.042 n.s. n.s. n.s. 0.020 n.s.
RC
n.s. 0.001 0.001 0.020
n.s. n.s. n.s. n.s. n.s. 0.050
n.s. n.s. n.s. n.s. n.s. n.s.
Orx * RC
Univariate ANOVA
Bone Vol. 27, No. 5 November 2000:667– 675 W. Yao et al. Effects of making rats rise to bipedal stance on lumbar vertebrae 669
29.3 ⫾ 5.6 0.5 ⫾ 0.05 14.5 ⫾ 2.8 16.9 ⫾ 9.3 n.a. 0.6 ⫾ 0.1 10.3 ⫾ 6.4 2.7 ⫾ 1.6
Dorsal periosteal dynamics Mineralized surface (%) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day)
Dorsal endocortical dynamic changes Mineralized surface (%) Mineral apposition rate (0–80 days, m/day) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day) Eroded surface (%) 24.8 ⫾ 2.4 n.d. 0.5 ⫾ 0.04 12.4 ⫾ 1.2 3.4 ⫾ 2.1
25.4 ⫾ 3.8 0.6 ⫾ 0.1 15.4 ⫾ 6.7
21.8 ⫾ 5.9 0.5 ⫾ 0.0 10.9 ⫾ 2.9 15.4 ⫾ 5.1
23.2 ⫾ 15.4 n.d. 0.8 ⫾ 0.1b 19.2 ⫾ 9.7b 10.8 ⫾ 0.7a,b
19.7 ⫾ 12.1 0.5 ⫾ 0.04 9.9 ⫾ 6.0
17.7 ⫾ 6.9 0.9 ⫾ 0.2a,b 15.1 ⫾ 2.8a,b 21.8 ⫾ 5.9a,b
Orx ⫹ NC
19.5 ⫾ 5.3 0.5 ⫾ 0.0b 0.6 ⫾ 0.1 11.9 ⫾ 6.4 1.8 ⫾ 0.7
22.8 ⫾ 12.5 0.5 ⫾ 0.04 12.8 ⫾ 9.2
15.3 ⫾ 3.0 0.7 ⫾ 0.1b 10.7 ⫾ 3.0b 11.6 ⫾ 3.0
Sham ⫹ RC
33.2 ⫾ 17.2 0.5 ⫾ 0.0b,c 0.8 ⫾ 0.3c 30.4 ⫾ 26.1 2.0 ⫾ 0.7c
34.8 ⫾ 15.5 0.5 ⫾ 0.1 21.4 ⫾ 12.1
18.7 ⫾ 3.0 1.0 ⫾ 0.5a,b 18.8 ⫾ 11.2a,b 18.8 ⫾ 5.8
orx ⫹ RC
n.s. n.s. 0.002 0.014 0.002
n.s. n.s. n.s.
n.s. 0.005 0.006 0.023
Orx
KEY: ANOVA, analysis of variance; n.a., not available; NC, normal height cage; n.d., not detectable; n.s., nonsignificant change; orx, orchidectomy; RC, raised cage. a Vs. baseline, p ⬍ 0.05. b Vs. sham ⫹ NC, p ⬍ 0.05. c Vs. orx ⫹ NC, p ⬍ 0.05. d Vs. sham ⫹ RC, p ⬍ 0.05.
22.0 ⫾ 4.0 0.5 ⫾ 0.0 11.5 ⫾ 2.4 15.1 ⫾ 4.4
Baseline
Ventral endocortical dynamics Mineralized surface (%) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day) Eroded surface (%)
Parameters
Sham ⫹ NC
Table 3. Histomorphometric changes of the fifth lumbar vertebral body: transverse sections
n.s. 0.000 n.s. n.s. 0.002
n.s. n.s. n.s.
n.s. n.s. n.s. n.s.
RC
n.s. n.s. n.s. n.s. 0.003
n.s. n.s. n.s.
n.s. n.s. n.s. n.s.
Orx ⴱ RC
Univariate ANOVA
670 W. Yao et al. Effects of making rats rise to bipedal stance on lumbar vertebrae Bone Vol. 27, No. 5 November 2000:667– 675
Bone Vol. 27, No. 5 November 2000:667– 675
W. Yao et al. Effects of making rats rise to bipedal stance on lumbar vertebrae
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Figure 1. Dorsal (A,C,E,G) and ventral (B,D,F,H) cortices from the fifth lumbar vertebral bodies of sham ⫹ NC (A,B), orx ⫹ NC (C,D), sham ⫹ RC (E,F), and orx ⫹ RC (G,H) animals. Note the thinner dorsal and ventral cortices, and increased erosion at the endocortical surface (arrowhead) in the orx ⫹ NC animal (C,D); new bone (bone formed started at the xylenol orange label [arrows] toward the bone surface) was added at the dorsal endocortical and the ventral periosteal surfaces in the raised-cage animals (E–H). Er, eroded surface; XO, xylenol orange label. Original magnification ⫻12.5; 20-m-thick Villanueva bone stain sections.
changes. Apparently these effects were due to suppression of the high bone turnover induced by orx on trabecular and endocortical surfaces, and stimulation of bone formation on the ventral periosteal surface. These findings suggest that making rats rise to an erect bipedal stance for feeding helps to prevent loss of both trabecular and cortical bone mass in the lumbar vertebrae of orchidectomized rats. Previous experimental animal studies have reported that orchidectomy induced cancellous bone loss of proximal tibial metaphyses in rats by increasing bone resorption accompanied by a suppression of cortical periosteal bone formation and an increase in cancellous bone turnover in young/growing rats.10,31 In our study, we found that orchidectomy-induced bone loss was associated with increased bone formation and resorption at bone surfaces (proximal tibial metaphyses and tibial shafts) next to the marrow and did not affect periosteal bone formation in cortical bone (tibial shafts).31 The current study found that, in 6-monthold Sprague-Dawley rats, 12 weeks of androgen withdrawal resulted in a 40% loss of cancellous bone in the lumbar vertebral bodies. Dynamic histomorphometry indicated that bone loss occurred next to the marrow and was accompanied by high turnover, as suggested by increases of eroded surface, mineralized surface, bone formation rate (BFR/TV), and bone turnover (BFR/BV). Orchidectomy also induced an approximately 10% reduction of cortical thickness due to increases of bone resorption on ventral and dorsal endocortical surfaces. There are few reports that have assessed lumbar vertebral histomorphometric profiles after orchidectomy. Erben et al. found that, at 3 months postorchidectomy, lumbar vertebrae lost about 25% of cancellous bone in 13-month-old Fisher rats, associated with increases in cancellous bone formation and resorption.5 But no cortical bone data were reported in their study. Our results, together with those of Erben and coworkers, suggest that, like estrogen deficiency-induced bone loss, orchidectomy caused high-bone-turnover osteopenia in the lumbar vertebral bodies. To our knowledge, this is the first report of bone histomorphometric data of the lumbar vertebral bodies that has forced rats to rise to an erect stance as opposed to running on all four limbs on a treadmill, wheel, or swimming and jumping. In growing rats
(1–3 months), treadmill exercise was found to increase the lumbar vertebral trabecular bone peak bone mass and bone strength,14,18 whereas, in adult rats (14 months), treadmill exercise did not affect trabecular bone, but cortical bone formation was found to increase.36 A recent report in tibiae found that bipedal resistance exercise dramatically increased cancellous bone but not cortical bone in 5-month-old male rats,32 but no data on the lumbar vertebral body were provided in that report. Similar to the effects of treadmill exercise on adult rats, we found that raised-cage treatment did not affect cancellous bone mass of the lumbar vertebral body, but cortical bone formation of the ventral periosteal surface increased over the entire study period and increased the total vertebral body cross-section area by 11%; bone resorption at the endosteal surface tended to decrease. In human vertebrae, smaller cross-sectional areas of vertebrae in women explained their lower maximum compressive loads when compared with men.4,19 In rats, the increase of vertebral total cross-sectional area by raised cages may be more important for increasing vertebral bone strength than the increase of trabecular bone mass. After 12 weeks of housing in raised cages, the orx rats lost only about 15% of cancellous bone compared with the 40% loss in those rats housed in normal height cages, and there was no net cortical bone loss. Dynamic histomorphometry showed that raised cages reduced orx-related increased bone resorption at the trabecular and dorsal endocortical surfaces. Moreover, animals in raised cages added new bone (increased mineral apposition rate at the 0 – 80 day interval) on the ventral periosteal and dorsal endocortical surfaces. Taken together, the prevention effects of raised cages on the lumbar cortical bone were attained mainly by stimulating bone formation at the ventral cortices and depressing endocortical resorption at the endocortical dorsal cortices. These observations suggest that, unlike exercise by treadmill running or swimming, the rats in raised cages had to exert considerable extra muscle force by using their spinal extensor muscles to become erect and reach upward. This exertion might increase mechanical forces on the ventral and dorsal cortices. Also, such forces could exert large enough strains to depress remodeling-dependent bone loss and to turn on modeling-dependent bone gain.6 –9 The same
17.3 ⫾ 4.5 3.5 ⫾ 0.7
19.2 ⫾ 4.7 3.7 ⫾ 0.6 67.3 ⫾ 7.1 n.a. 0.7 ⫾ 0.0 47.7 ⫾ 8.3
Ventral periosteal dynamics Mineralizing surface (%) Mineral apposition rate (0–80 days, m/day) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day) 88.8 ⫾ 12.7a 0.2 ⫾ 0.4 1.0 ⫾ 0.1a,b 91.8 ⫾ 18.2a
16.3 ⫾ 3.9 10.2 ⫾ 3.4a,b
23.9 ⫾ 2.7 0.7 ⫾ 0.1a,b 195.7 ⫾ 41.8a,b 17.2 ⫾ 1.6a,b
10.0 ⫾ 1.3 9.3 ⫾ 0.9a,b 1.6 ⫾ 0.3a,b 547.4 ⫾ 95.1a,b 255.4 ⫾ 11.2a,b 191.1 ⫾ 11.5a,b
Orx ⫹ NC
67.3 ⫾ 19.4 0.7 ⫾ 0.1 0.8 ⫾ 0.1 67.3 ⫾ 19.4
15.3 ⫾ 4.7 3.6 ⫾ 1.1
17.0 ⫾ 3.0 0.6 ⫾ 0.1 93.0 ⫾ 29.1 9.9 ⫾ 2.5
10.9 ⫾ 2.4 17.0 ⫾ 4.3 2.7 ⫾ 0.4 299.7 ⫾ 67.9 336.5 ⫾ 16.6 222.2 ⫾ 19.2
Sham ⫹ RC
77.9 ⫾ 20.4a 0.7 ⫾ 0.2c 0.9 ⫾ 0.1a,b 73.5 ⫾ 15.9a
23.1 ⫾ 5.4d 3.1 ⫾ 0.2c
23.7 ⫾ 3.8d 0.7 ⫾ 0.1a–c 174.4 ⫾ 48.8a,b,d 16.1 ⫾ 3.7a,b,d
10.9 ⫾ 0.7 13.5 ⫾ 2.1c 2.5 ⫾ 0.3c 333.7 ⫾ 65.2c 303.8 ⫾ 35.3c 212.6 ⫾ 10.4c
Orx ⫹ RC
n.s. n.s. 0.001 0.002
n.s. 0.000
0.001 0.000 0.000 0.000
n.s. 0.000 0.000 0.000 0.005 0.035
Orx
KEY: ANOVA, analysis of variance; n.a., not available; NC, normal height cage; n.d., not detectable; n.s., nonsignificant change; orx, orchidectomy; RC, raised cage. a Vs. baseline, p ⬍ 0.05. b Vs. sham ⫹ NC, p ⬍ 0.05. c Vs. orx ⫹ NC, p ⬍ 0.05. d Vs. sham ⫹ RC, p ⬍ 0.05.
82.2 ⫾ 8.8a 0.2 ⫾ 0.4 0.7 ⫾ 0.1 59.0 ⫾ 10.0
18.4 ⫾ 4.6 0.5 ⫾ 0.03 104.6 ⫾ 36.3 10.3 ⫾ 2.2
9.8 ⫾ 0.6 17.3 ⫾ 4.6 2.9 ⫾ 0.4 273.3 ⫾ 61.4 298.4 ⫾ 30.6 215.1 ⫾ 24.6
Sham ⫹ NC
18.1 ⫾ 3.6 0.5 ⫾ 0.1 111.1 ⫾ 28.0 10.2 ⫾ 2.2
9.1 ⫾ 0.5 17.5 ⫾ 1.2 3.2 ⫾ 0.3 241.9 ⫾ 30.7 296.9 ⫾ 25.7 209.1 ⫾ 20.8
Baseline
Trabecular dynamics Mineralizing surface (%) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone volume (%/yr) Bone formation rate/bone surface (m/day * 100) Bone formation rate/trabecular volume (%/yr) Eroded surface (%)
Static changes Total tissue area (mm2) Trabecular volume (%) Trabecular number (#) Trabecular separation (m) Ventral cortical thickness (m) Dorsal cortical thickness (m)
Parameters
Table 4. Histomorphometric changes of the fourth lumbar vertebral body: sagittal sections
n.s. 0.005 n.s. n.s.
n.s. 0.001
n.s. n.s. n.s. n.s.
n.s. n.s. n.s. n.s. 0.044 n.s.
RC
n.s. n.s. 0.004 0.018
n.s. 0.000
n.s. n.s. n.s. n.s.
n.s. n.s. 0.001 0.000 n.s. n.s.
Orx ⴱ RC
Univariate ANOVA
672 W. Yao et al. Effects of making rats rise to bipedal stance on lumbar vertebrae Bone Vol. 27, No. 5 November 2000:667– 675
51.8 ⫾ 2.0 0.5 ⫾ 0.03 28.1 ⫾ 2.3 32.2 ⫾ 11.3 n.a. 0.7 ⫾ 0.2 22.8 ⫾ 11.9 3.0 ⫾ 1.1
Dorsal periosteal dynamics Mineralizing surface (%) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day)
Dorsal endocortical dynamic changes Mineralizing surface (%) Mineral apposition rate (0–80 days, m/day) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day) Eroded surface (%) 22.4 ⫾ 1.8 n.d. 0.7 ⫾ 0.1 16.1 ⫾ 4.4 2.9 ⫾ 0.1
46.2 ⫾ 10.9 0.5 ⫾ 0.05 24.4 ⫾ 8.0
21.8 ⫾ 5.9 0.5 ⫾ 0.1 13.5 ⫾ 5.4 10.8 ⫾ 0.8
Sham ⫹ NC
22.8 ⫾ 3.6 n.d. 0.6 ⫾ 0.1 14.4 ⫾ 3.9 7.4 ⫾ 0.8a,b
31.6 ⫾ 11.5 0.5 ⫾ 0.04 17.3 ⫾ 7.5
17.7 ⫾ 6.9 0.5 ⫾ 0.01 11.1 ⫾ 1.4 14.8 ⫾ 1.6a,b
Orx ⫹ NC
25.5 ⫾ 4.6 0.3 ⫾ 0.01b 0.5 ⫾ 0.0 13.2 ⫾ 2.5 2.6 ⫾ 1.3
35.7 ⫾ 15.5 0.5 ⫾ 0.1 21.5 ⫾ 15.6
15.3 ⫾ 3.0 0.5 ⫾ 0.01 8.6 ⫾ 2.9a 10.3 ⫾ 3.0
Sham ⫹ RC
29.6 ⫾ 6.8 n.d. 0.6 ⫾ 0.1 17.8 ⫾ 3.5 4.2 ⫾ 1.0c
36.2 ⫾ 5.7 0.6 ⫾ 0.1 21.1 ⫾ 5.4
18.7 ⫾ 3.0 0.5 ⫾ 0.05 12.5 ⫾ 3.3 11.5 ⫾ 2.7
Orx ⫹ RC
n.s. n.s. n.s. n.s. 0.000
n.s. n.s. n.s.
n.s. n.s. n.s. 0.027
Orx
KEY: ANOVA, analysis of variance; n.a., not available; NC, normal height cage; n.d., not detectable; n.s., nonsignificant change; orx, orchidectomy; RC, raised cage. a Vs. baseline, p ⬍ 0.05. b Vs. sham ⫹ NC, p ⬍ 0.05. c Vs. orx ⫹ NC, p ⬍ 0.05. d Vs. sham ⫹ RC, p ⬍ 0.05.
22.0 ⫾ 4.0 0.5 ⫾ 0.1 15.2 ⫾ 4.4 10.2 ⫾ 4.4
Baseline
Ventral endocortical dynamics Mineralizing surface (%) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day) Eroded surface (%)
Parameters
Table 5. Histomorphometric changes of the fourth lumbar vertebral body: sagittal sections
n.s. n.s. n.s. n.s. 0.001
n.s. n.s. n.s.
n.s. n.s. n.s. n.s.
RC
n.s. n.s. n.s. n.s. 0.005
n.s. n.s. n.s.
n.s. n.s. 0.028 n.s.
Orx * RC
Univariate ANOVA
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observations were also seen in the long bones of in vivo loading models, in which the same resorption-to-formation drifts were observed, with enhanced periosteal bone formation and suppressed endocortical bone resorption.20,25 In addition, we found that periosteal bone formation occurred in the ventral cortices but not in the dorsal cortices. This could thicken and strengthen lumbar vertebrae in ways that would not put the spinal cord at risk. Moreover, the bipedal-stance exercise prevented the detrimental effects of androgen depletion on trabecular architecture (Tb.N and Tb.Sp), which are among the predictors of the presence of vertebral fracture in male osteoporosis.16 As this wholebody standing and squatting movement in rats is somewhat similar to the “squat” exercise in humans, we believe that exercise involving both concentric and eccentric muscle contraction against the spine may be useful in increasing spinal bone strength and preventing the occurrence of vertebral fracture. The current study included the histomorphometric profiles of both sagittal sections (LVL-4) and transverse sections (LVX-5) of rat vertebrae. In general, LVX-5 contained more trabecular bone and had thicker dorsal, but thinner ventral, cortices. The latter may be due to the fact that it was reported as the average of the entire ventral cortex, whereas that of LVL-4 was sectioned in the midbody where the thickest ventral cortex lies. Our data demonstrate that LVX-5 had higher trabecular bone turnover, but lower mineralized surfaces and bone formation rates at the periosteal and endocortical surfaces of the ventral and dorsal cortices. Orchidectomy and raised cage had similar effects on these two vertebrae except that the percentage of trabecular bone loss was greater in LVL-4 than in LVX-5, and the raised cage increased the total tissue area and mineral apposition rate (0 – 80 day interval) of ventral periosteal and dorsal endocortical surfaces of LVX-5 to a greater extent than it did in LVL-4. The changes might represent “true” differences between the two vertebrae or might be due to different sectioning methods. We believe the transverse sections may be more meaningful when studying the loading effects on the vertebrae, because they show the total amount of bone supporting the total compression loads on the vertebral body. The raised cages increased the body weights of the rats. It has been proposed and seems true that muscle force is the main determinant of postnatal and whole-bone strength and bone “mass.” We found that the increased body weight by use of the raised cage was accompanied by increases in hind-limb muscle mass and bone mass.34 Unfortunately, we did not assess the details of the relationship between bone formation and muscle weight in the lumbar vertebrae, because we did not obtain spinal muscle data in the current study. In summary, orchidectomy induced high bone turnover and trabecular and cortical bone loss in rat lumbar vertebral bodies. Forcing the rats to rise to an erect stance partially prevented those decreases. Such findings suggest that making orchidectomized rats rise to an erect bipedal stance for feeding helps to prevent loss of trabecular and cortical bone “mass,” and presumably bone strength. This method also provides us with an inexpensive, noninvasive, reliable model to increase in vivo vertebral loading in rats that is similar in humans. References 1. Baillie, S. P., Davison, C. E., Johnson, F. J., and Francis, R. M. Pathogenesis of vertebral body crush fracture in man. Age Aging 21:139 –141; 1992. 2. Bourrin, S., Ghaemmaghami, F., Vico, L., Chappard, D., Gharib, C., and Alexandre, C. Effect of a five-week swimming program on rat bone: A histomorphometric study. Calcif Tissue Int 51:137–142; 1992. 3. Coxam, V., Robins, S., Pastoureau, P., Gaumet, N., Lebecque, P., Davicco, M. J., Giry, J., and Barlet, J. P. Estrogen is more effective than dihydrotest-
Bone Vol. 27, No. 5 November 2000:667– 675
4.
5.
6. 7. 8. 9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19. 20.
21.
22. 23.
24.
25.
26.
27.
osterone to prevent bone loss in young castrated male rats. J Bone Miner Res 10(Suppl.):S311; 1995. Ebbesen, E. N., Thomsen, J. S., Beck-Nielsen, H., Nepper-Rasmussen, H. J., and Mosekilde, L. Age and gender-related differences in vertebral bone mass, density, and strength. J Bone Miner Res 14:1394 –1403; 1999. Erben, R. G., Eberle, J., Stahr, K., and Goldberg, M. Androgen deficiency induces high turnover osteopenia in aged male rats. A sequential histomorphometric study. J Bone Miner Res 15:1085–1098; 2000. Frost, H. M. Bone “mass” and the “mechanostat”: A proposal. Anat Rec 219:1–9; 1987. Frost, H. M. Perspective: On our age-related bone loss: Insights from a new paradigm. J Bone Miner Res 12:1539 –1546; 1997. Frost, H. M. Perspectives: The role of changes in mechanical usage set points in the pathogenesis of osteoporosis. J Bone Miner Res 7:253–261; 1992. Frost, H. M., Ferretti, J. L., and Jee, W. S. S. Perspective: Some roles of mechanical usage, muscle strength, and the mechanical in skeleton physiology, disease, and research. Calcif Tissue Int 62:1–7; 1998. Gunness, M. and Orwoll, E. Early induction of alteration in cancellous and cortical bones histology after orchidectomy in mature rats. J Bone Miner Res 10:1735–1743; 1995. Gurkan, L., Ekeland, A., Gautvik, K. M., Langerland, N., Ronningen, H., and Silheim, L. F. Bone changes after castration in rats: A model of osteoporosis. Acta Orthop Scand 57:67–70; 1986. Hock, J. M., Gera, I., Fonseca, J., and Raisz, L. G. Human parathyroid hormone-(1–34) increases bone mass in ovariectomized and orchidectomized rats. Endocrinology 122:2899 –2904; 1988. Horcajada-Molteni, M. N., Davicco, M. J., Collignon, H., Lebecque, P., Coxam, V., and Barlet, J. P. Does endurance running before orchidectomy prevent osteopenia in rats? Eur J Appl Physiol 80:344 –352; 1999. Iwamoto, J., Yeh, J. K., and Aloria, J. F. Different effect of treadmill exercise on three cancellous bone sites in the young growing rats. Bone 24:163–169; 1999. Ke, H. Z., Qi, H., Crawford, D. T., Chidsey-Frink, K. L., Simmons, H. A., and Thompson, D. D. Lasofoxifene (CP-336,156), a selective estrogen receptor modulator, prevents bone loss induced by aging and orchidectomy in the adult rat. Endocrinology 141:1338 –1344; 2000. Legrand, E., Chappard, D., Pascaretti, C., Duquenne, M., Krebs, S., Rohmer, V., Basle, M. F., and Audran, M. Trabecular bone microarchitecture, bone mineral density, and vertebral fracture in male osteoporosis. J Bone Miner Res 15:13–18; 2000. Li, M., Jee, W. S. S., Ke, H. Z., Tang, L. Y., Ma, Y. F., Liang, X. G., and Setterberg, R. B. Prostaglandin E2 administration prevents bone loss induced by orchidectomy in rats. J Bone Miner Res 10:66 –73; 1995. Mosekilde, L., Danielsen, C. C., Søgaard, C. H., and Thorling, E. The effect of long-term exercise on vertebral and femoral bone mass, dimensions, and strength-assessed in a rat model. Bone 15:292–301; 1994. Mosekilde, L. The effects of modeling and remodeling on human vertebral body architecture. Technol Health Care 6:287–297; 1998. Mosley, J. R. and Lanyon, L. E. Strain rate as a controlling influence on adaptive modeling in response to dynamic loading of the ulna in growing male rats. Bone 23:313–318; 1998. Newhall, K. M., Rodnick, K. J., Meulen, M. C., Carter, D. R., and Marcus, R. Effects of voluntary exercise on bone mineral content in rats. J Bone Miner Res 6:289 –296; 1991. Nguyen, T. V., Eisman, J. A., Kelly, P. J., and Sambrook, P. N. Risk factors for osteoporotic fractures in elderly men. Am J Epidemiol 144:255–263; 1996. Parfitt, A. M., Drezner, M. K., Glorieux, F. H., Janis, J. A., Malluche, H., Meunier, P. J., Ott, S. M., and Recker, R. R. Bone histomorphometry: Standardization of nomenclature, symbols and units. Report of the ASBMR Histomorphometry Committee. J Bone Miner Res 2:595– 610; 1987. Parfitt, A. M., Mathews, C. H. E., Villanueva, A. R., Kleerekoper, M., Frame, B., and Rao, D. S. Relationships between surface, area, and thickness of iliac trabecular bone in aging and in osteoporosis. J Clin Invest 72:1396 –1409; 1983. Turner, C. H., Forwood, M. R., Rho, J. Y., and Yoshikawa, T. Mechanical loading thresholds for lamellar and woven bone formation. J Bone Miner Res 9:87–97; 1994. Turner, R. T., Wakley, G. K., and Hannon, K. S. Different effects of androgens on cortical bone histomorphometry in gonadectomized male and female rats. J Orthop Res 8:612– 617; 1990. Tuukanen, J., Peng, Z., and Vaananen, H. K. Effect of running exercise on the bone mass induced by orchidectomy in the rats. Calcif Tissue Int 55:33–37; 1994.
Bone Vol. 27, No. 5 November 2000:667– 675 28. Vanderschueren, D., Van Herck, E., De Coster, R., and Bouillon, R. Aromatization of androgen is important for skeleton maintenance of aged male rats. Calcif Tissue Int 59:179 –183; 1996. 29. Vanderschueren, D., Van Herck, E., Nijs, J., Ederveen, A. G. H., De Coster, R., and Bouillon, R. Estrogen prevents the effect of aromatase inhibition on the skeleton of aged male rats. J Bone Miner Res 12(Suppl.):T483; 1997. 30. Verhas, M., Schoutens, M. A., L’Lermit-Baleriaux, M., Dourow, N., Verschaeren, A., Mone, M., and Heilponorn, A. The effect of orchidectomy on bone metabolism in aging rats. Calcif Tissue Int 39:74 –77; 1986. 31. Wakley, G. K., Schutte, H. D., Jr, Hannon, K. S., and Turner, R. T. Androgen treatment prevents loss of cancellous bone in the orchidectomized rats. J Bone Miner Res 6:325–330; 1991. 32. Westerlind, K., Fluckey, J. D., Gordon, S. E., Kraemer, W. J., Farrell, P. A., and Turner, R. T. Effects of resistance exercise training on cortical and cancellous bone in mature male rats. J Appl Physiol 84:459 – 464; 1998. 33. Wink, C. S. and Felts, W. J. L. Effects of castration on bone structure of male rats. Calcif Tissue Int 32:77– 82; 1980.
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34. Yao, W., Jee, W. S. S., Chen, J. L., Liu, H. Y., Tam, C. S., Cui, L., Zhou, H., Setterberg, R. B., and Frost, H. M. Making rats rise to erect bipedal stance for feeding partially prevented orchidectomy-induced bone loss and added bone to intact rats. J Bone Miner Res 15:1158 –1168; 2000. 35. Yeh, J. K., Aloia, J. F., and Barilla, M.-L. Effects of 17-estradiol replacement and treadmill exercise on vertebral and femoral bones of the ovariectomized rats. Bone Miner 24:223–234; 1994. 36. Yeh, J. K., Aloia, J. F., and Chen, M. M. Growth hormone administration potentiates the effect of treadmill exercise on long bone formation but not on the vertebrae in middle-aged rats. Calcif Tissue Int 54:38 – 43; 1994.
Date Received: April 4, 2000 Date Revised: July 25, 2000 Date Accepted: July 28, 2000
Erect Bipedal Stance Exercise Partially Prevents Orchidectomy-Induced Bone Loss in the Lumbar Vertebrae of Rats W. YAO,1 W. S. S. JEE,1 J. CHEN,1 C. S. TAM,2 R. B. SETTERBERG,1 and H. M. FROST1,3 1
Radiobiology Division, University of Utah, Salt Lake City, UT, USA Faculty of Science, University of Toronto, and Gen.Sci Regeneration Sciences Inc., ON, Canada 3 Department of Orthopedic Surgery, Southern Colorado Clinic, Pueblo, CO, USA 2
androgenic effects on the development or maintenance of the male skeleton.10,11,30,33 Bone loss induced by castration can be prevented by hormone replacement therapy with androgen,26,31 testosterone,28 estrogen,3,29 selective estrogen receptor modulator (SERM),15 parathyroid hormone,12 and prostaglandin E2 (PGE2).17 Treadmill exercise was reported to partially prevent orchidectomy-induced bone loss in the rat femur,13,27 increase cancellous bone mass in skeletally immature animals,14,18 and increase periosteal bone formation without significantly affecting cancellous bone mass in skeletally mature rats.35,36 Recently, we used a raised-cage model to “exercise” rats, which forced them to rise to a bipedal stance for feeding.34 This model increased cortical bone mass and partially prevented orchidectomy-induced tibial bone loss in 6-month-old male rats. However, how it affects the vertebrae remains unknown. In addition, although the effects of androgen deficiency have been well documented in rat tibia and femur,10,11,33 few studies have evaluated its effects on vertebral bodies. Therefore, we conducted this study to characterize cancellous and cortical bone histomorphometric changes of both the sagittal and transverse sections of lumbar vertebral bodies after orchidectomy and/or housing in a raised cage in 6-month-old male rats.
This study investigates the responses of the fourth and fifth lumbar vertebral bodies of 6-month-old male Sprague-Dawley (SD) rats to orchidectomy (orx) and to erect bipedal stance for feeding for 12 weeks in specially designed raised cages (RC) for which the heights were raised from 20 cm to 35.5 cm. A total of 30 rats were divided into groups of: baseline; sham ⴙ housed in normal height cage (NC); orx ⴙ NC; sham ⴙ RC; and orx ⴙ RC. Bone histomorphometry was performed on the triple-labeled undecalcified fourth sagittal (LVL-4) and fifth transverse (LVX-5) sections. We found that orchidectomy induced high-turnover trabecular and cortical bone loss in the lumbar vertebrae. Forcing the rats to rise to erect stance for feeding reduced trabecular and cortical bone loss caused by orx. Apparently, depressing the elevated bone resorption next to the marrow induced by orx, and stimulating bone formation at the ventral periosteal surfaces, caused these effects. Orchidectomy and raised cage had similar effects on the two vertebrae except that the percentage of trabecular bone loss was greater in the LVL-4 than in LVX-5, and that bipedal stance exercise increased the total tissue area and mineral apposition rates (0 – 80 day interval) of ventral periosteal and dorsal endocortical surfaces of LVX-5 to a greater extent than it did in LVL-4. Such findings suggest that forcing rats to rise to an erect bipedal stance for feeding helps prevent loss of trabecular and cortical bone “mass,” and presumably bone strength, in orchidectomized rats. This method also provides an inexpensive, noninvasive, reliable model to increase in vivo vertebral loading in rats that is similar in humans. (Bone 27:667– 675; 2000) © 2000 by Elsevier Science Inc. All rights reserved.
Materials and Methods Animals and Experimental Protocol Thirty 1-month-old male Sprague-Dawley rats (Simonsen Laboratories, Gilroy, CA) were acclimated to and aged in local vivarium conditions. They were housed six per cage (58 cm ⫻ 36 cm ⫻ 20 cm) with the room temperature maintained at 72°F and 12:12 hour light/dark cycles. The rats were allowed free access to water and pelleted commercial natural diet (Teklad Rodent Laboratory Chow #8604, Harlan-Teklad, Madison, WI), which contains 1.46% calcium, 0.99% phosphorus, and 4.96 IU/g of vitamin D3. At 6 months of age, the rats were divided randomly into the following five body-weight (b.w.)-matched groups with six rats per group: (1) baseline control (baseline); (2) shamorchidectomized ⫹ housed in the normal height cage (sham ⫹ NC); (3) bilaterally orchidectomized ⫹ housed in normal height cage (orx ⫹ NC); (4) sham-orchidectomized ⫹ housed in the raised cage (sham ⫹ RC); and (5) orchidectomized ⫹ housed in raised cage (orx ⫹ RC). The study lasted 12 weeks, so the rats were 6 months old when the experiment began, and 9 months old when it ended.
Key Words: Orchidectomy; Bipedal stance exercise; Lumbar vertebrae; Bone histomorphometry; Bone strength. Introduction Androgen deficiency, reduced physical activity, and loss of body weight are believed to contribute to the increased incidence of vertebral fracture in men after their sixth decade.1,22 Orchidectomized (orx) rats have been used as an animal model to study Address for correspondence and reprints: Dr. Wei Yao, Radiobiology Division, University of Utah, 729 Arapeen Drive, Suite 2334, Salt Lake City, UT 84108-1218. E-mail: [email protected] © 2000 by Elsevier Science Inc. All rights reserved.
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8756-3282/00/$20.00 PII S8756-3282(00)00377-X
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Table 1. Effects of orchidectomy and raised cage on body weights in 6-month-old male Sprague-Dawley rats Groups
Baseline
Sham ⫹ NC
orx ⫹ NC
Sham ⫹ RC
Orx ⫹ RC
Initial (g) Final (g)
392 ⫾ 32
384 ⫾ 26 387 ⫾ 69
388 ⫾ 34 387 ⫾ 19
394 ⫾ 29 437 ⫾ 38a,b
391 ⫾ 32 437 ⫾ 23a–c
KEY: NC, normal height cage; orx, orchidectomy; RC, raised cage. a Vs. baseline, p ⬍ 0.05. b Vs. sham ⫹ NC, p ⬍ 0.05. c Vs. orx ⫹ NC, p ⬍ 0.05.
At the beginning of the study, the rats in the sham ⫹ RC and orx ⫹ RC groups were housed in the specially designed cages with an initial height of 28 cm (58 cm ⫻ 36 cm ⫻ 28 cm) for 1 week. The heights of the cages were then raised 2.5 cm every third day until they reached the final height of 35.5 cm. At this height, the rats needed to stand upright on their hind limbs and reach upward for food and water. This height was maintained for 10 weeks. The body weights were recorded weekly, except for the first 2 weeks when they were weighed daily. The rats were observed to verify that they were able to stand upright and reach the food and water. The baseline rats received 20 mg tetracycline/kg b.w. (Sigma Chemical Co., St. Louis, MO) and 10 mg calcein/kg b.w. (Sigma) by subcutaneous injection at day 70 and day 80 of the study. All other rats received 90 mg xylenol orange/kg b.w. (Sigma) at the beginning of the study (day 0), and 20 mg tetracycline/kg b.w. and 10 mg calcein/kg b.w. subcutaneously at day 70 and day 80 of the study. Before autopsy, the rats were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg) and killed by cardiac puncture.
(BFR/TV), bone formation rate per unit of bone surface (BFR/ BS), and activation frequency (Ac.f), according to Parfitt et al.23,24 Results are presented as mean ⫾ SD. The statistical analyses were performed using SPSS version 10 for WINDOWS (SPSS Inc., Chicago, IL). The Kruskal–Wallis test was selected to analyze the differences between groups. Univariate analysis of variance (ANOVA) was used to observe the interaction between raisedcage and orchidectomy using body weights as a covariate. p ⬍ 0.05 was considered significant. Results Body Weight Changes No age-related changes occurred in body weights between baseline and sham ⫹ NC rats. Orchidectomy did not induce significant changes in body weights. In the sham ⫹ RC and orx ⫹ RC groups, body weights were higher by 21% as compared with sham ⫹ NC animals34 (Table 1). Bone Histomorphometric Measurements
Bone Histomorphometry The fourth and fifth lumbar vertebrae were stained with Villanueva bone stain, dehydrated in graded concentrations of ethanol, defatted in acetone, and embedded in methylmethacrylate (Fisher Scientific, Fairlawn, NJ). Serial transverse sections of the fifth LV were cut from the cranial end to the caudal end to 230 m thickness using a low-speed metallurgic saw. The first transverse section with an intact cortical ring of each fifth LV (LVX-5), and the midsagittal sections of the fourth LV (LVL-4), were selected and ground to 20 m for histomorphometric measurement. Histomorphometry was done using a semiautomatic image analysis system (OSTEOMEASURE, OsteoMeasure, Inc., Atlanta, GA) linked to a microscope equipped for transmitted and fluorescent light studies. We studied the entire transverse section of the fifth lumbar vertebral bodies and the sagittal sections of the fourth lumbar vertebral bodies, but omitted 0.5 mm from the growth plates. Static measurements included total cross-sectional area (T.Ar), trabecular bone area (B.Ar) and perimeter (B.Pm), and dorsal and ventral average cortical bone width (Ct.Wi, sampled once every 50 m). Dynamic measurements included single-labeled (sL.Pm) and double-labeled perimeter (dL.Pm), eroded perimeter (E.Pm), and interlabel width (L.Wi, at the 0 – 80 day and 70 – 80 day intervals) of the trabecular surface, and periosteal and endocortical surfaces of the dorsal and ventral cortices. These data were used to calculate trabecular volume (BV/TV), cortical thickness (Ct.Th), mineralized surface (MS/BS), eroded surface (ES/BS), mineral apposition rate (MAR at the 0 – 80 day and 70 – 80 day intervals), bone formation rate per unit of bone volume (BFR/BV), bone formation rate per unit of tissue volume
Histomorphometric profiles of the LVX-5 are shown in Tables 2 and 3. Compared with sham ⫹ NC animals, orchidectomy alone (orx ⫹ NC) induced 37%, 11%, and 12% losses of trabecular volume and ventral and dorsal cortical thickness. Bone formation increased on all the bone surfaces and endocortical eroded surface increased. Raised cages in sham-operated animals (sham ⫹ RC) increased the total cross-sectional area of the vertebral body by 11% due to increased bone formation at the ventral periosteal surface over the entire study period (0 – 80 days), and tended to decrease bone resorption at the endosteal surface. In orchidectomized animals in raised-cage conditions (orx ⫹ RC), the ventral and dorsal cortical thicknesses were higher by 29% and 25%, respectively, when compared with orx ⫹ NC animals, and these values were comparable to those of the sham control animals. These changes were accompanied by increased total mineral apposition rate at 0 – 80 days at the ventral periosteal surface and suppressed eroded surface at the endocortical surfaces (Figure 1). Histomorphometric profiles of the LVL-4 are shown in Tables 4 and 5. The changes induced by orchidectomy and/or raised cages were similar to that of LVX-5, except that LVL-4 showed more decreases in both cancellous and cortical bone loss after orx, but fewer increases in cortical bone thickness and total bone formation after RC (Figure 1). Discussion This study demonstrates that orchidectomy of rats at 6 months of age induced high-turnover cancellous and cortical bone loss next to marrow in lumbar vertebral bodies. Making orchidectomized rats rise to an erect stance for feeding partially prevented these
18.1 ⫾ 3.1 0.5 ⫾ 0.05 35.8 ⫾ 9.4 96.8 ⫾ 36.5 33.5 ⫾ 17.5 4.0 ⫾ 1.7 48.2 ⫾ 15.9 n.a. 0.7 ⫾ 0.0 34.7 ⫾ 11.4
Trabecular dynamics Mineralized surface (%) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day * 100) Bone formation rate/bone volume (%/yr) Bone formation rate/trabecular volume (%/yr) Eroded surface (%)
Ventral periosteal dynamics Mineralized surface (%) Mineral apposition rate (0–80 days, m/day) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day * 100) 52.8 ⫾ 10.5 0.8 ⫾ 0.1 0.6 ⫾ 0.1 35.5 ⫾ 13.8
21.6 ⫾ 5.8 0.5 ⫾ 0.1 43.2 ⫾ 14.7 103.7 ⫾ 38.3 31.6 ⫾ 11.1 3.3 ⫾ 0.5
9.4 ⫾ 0.3 25.0 ⫾ 0.4 3.7 ⫾ 0.1 181.3 ⫾ 9.8 276.3 ⫾ 33.2 230.2 ⫾ 11.3
Sham ⫹ NC
58.0 ⫾ 13.3 n.d. 1.1 ⫾ 0.1a,b 64.7 ⫾ 25.1
29.7 ⫾ 1.3a,b 0.7 ⫾ 0.1a,b 78.6 ⫾ 6.1a,b 215.0 ⫾ 26.6a,b 50.0 ⫾ 19.1 6.7 ⫾ 1.3b
9.3 ⫾ 0.6 15.8 ⫾ 2.1a,b 2.7 ⫾ 0.5a,b 297.1 ⫾ 70.6a,b 214.2 ⫾ 16.4a,b 196.6 ⫾ 14.6a,b
Orx ⫹ NC
61.0 ⫾ 12.8 1.2 ⫾ 0.1b 0.8 ⫾ 0.2 48.5 ⫾ 9.6
22.5 ⫾ 5.0 0.6 ⫾ 0.1 48.1 ⫾ 15.2 117.0 ⫾ 48.5 38.4 ⫾ 10.2 2.4 ⫾ 0.8
10.4 ⫾ 0.5a,b 27.4 ⫾ 6.0 4.0 ⫾ 0.5 164.4 ⫾ 45.1 299.1 ⫾ 21.5 255.2 ⫾ 12.2
Sham ⫹ RC
63.8 ⫾ 10.8 1.0 ⫾ 0.1c 1.0 ⫾ 0.1a,b 63.8 ⫾ 18.7a
32.0 ⫾ 9.6a 0.7 ⫾ 0.04a,b,d 77.8 ⫾ 21.7a,d 188.0 ⫾ 40.9a,b 49.1 ⫾ 9.1 2.9 ⫾ 1.4c
9.5 ⫾ 0.6 20.8 ⫾ 4.0c 3.2 ⫾ 0.5c 232.9 ⫾ 49.8c 275.9 ⫾ 15.2c 245.9 ⫾ 17.4c
orx ⫹ RC
Orx
n.s. n.s. 0.003 0.002
0.005 0.000 0.000 0.000 n.s. 0.012
n.s. 0.001 0.013 0.002 0.005 0.005
KEY: ANOVA, analysis of variance; n.a., not available; NC, normal height cage; n.d., not detectable; n.s., nonsignificant change; orx, orchidectomy; RC, raised cage. a Vs. baseline, p ⬍ 0.05. b Vs. sham ⫹ NC, p ⬍ 0.05. c Vs. orx ⫹ NC, p ⬍ 0.05. d Vs. sham ⫹ RC, p ⬍ 0.05.
9.4 ⫾ 0.4 26.5 ⫾ 5.2 4.4 ⫾ 1.2 158.5 ⫾ 54.0 271.9 ⫾ 32.2 222.9 ⫾ 35.1
Baseline
Static changes Total cross-sectional area (mm2) Trabecular volume (%) Trabecular number (#) Trabecular separation (m) Ventral cortical thickness (m) Dorsal cortical thickness (m)
Parameters
Table 2. Histomorphometric changes of the fifth lumbar vertebral body: transverse sections
n.s. 0.000 0.000 n.s.
n.s. n.s. n.s. n.s. n.s. 0.001
0.042 n.s. n.s. n.s. 0.020 n.s.
RC
n.s. 0.001 0.001 0.020
n.s. n.s. n.s. n.s. n.s. 0.050
n.s. n.s. n.s. n.s. n.s. n.s.
Orx * RC
Univariate ANOVA
Bone Vol. 27, No. 5 November 2000:667– 675 W. Yao et al. Effects of making rats rise to bipedal stance on lumbar vertebrae 669
29.3 ⫾ 5.6 0.5 ⫾ 0.05 14.5 ⫾ 2.8 16.9 ⫾ 9.3 n.a. 0.6 ⫾ 0.1 10.3 ⫾ 6.4 2.7 ⫾ 1.6
Dorsal periosteal dynamics Mineralized surface (%) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day)
Dorsal endocortical dynamic changes Mineralized surface (%) Mineral apposition rate (0–80 days, m/day) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day) Eroded surface (%) 24.8 ⫾ 2.4 n.d. 0.5 ⫾ 0.04 12.4 ⫾ 1.2 3.4 ⫾ 2.1
25.4 ⫾ 3.8 0.6 ⫾ 0.1 15.4 ⫾ 6.7
21.8 ⫾ 5.9 0.5 ⫾ 0.0 10.9 ⫾ 2.9 15.4 ⫾ 5.1
23.2 ⫾ 15.4 n.d. 0.8 ⫾ 0.1b 19.2 ⫾ 9.7b 10.8 ⫾ 0.7a,b
19.7 ⫾ 12.1 0.5 ⫾ 0.04 9.9 ⫾ 6.0
17.7 ⫾ 6.9 0.9 ⫾ 0.2a,b 15.1 ⫾ 2.8a,b 21.8 ⫾ 5.9a,b
Orx ⫹ NC
19.5 ⫾ 5.3 0.5 ⫾ 0.0b 0.6 ⫾ 0.1 11.9 ⫾ 6.4 1.8 ⫾ 0.7
22.8 ⫾ 12.5 0.5 ⫾ 0.04 12.8 ⫾ 9.2
15.3 ⫾ 3.0 0.7 ⫾ 0.1b 10.7 ⫾ 3.0b 11.6 ⫾ 3.0
Sham ⫹ RC
33.2 ⫾ 17.2 0.5 ⫾ 0.0b,c 0.8 ⫾ 0.3c 30.4 ⫾ 26.1 2.0 ⫾ 0.7c
34.8 ⫾ 15.5 0.5 ⫾ 0.1 21.4 ⫾ 12.1
18.7 ⫾ 3.0 1.0 ⫾ 0.5a,b 18.8 ⫾ 11.2a,b 18.8 ⫾ 5.8
orx ⫹ RC
n.s. n.s. 0.002 0.014 0.002
n.s. n.s. n.s.
n.s. 0.005 0.006 0.023
Orx
KEY: ANOVA, analysis of variance; n.a., not available; NC, normal height cage; n.d., not detectable; n.s., nonsignificant change; orx, orchidectomy; RC, raised cage. a Vs. baseline, p ⬍ 0.05. b Vs. sham ⫹ NC, p ⬍ 0.05. c Vs. orx ⫹ NC, p ⬍ 0.05. d Vs. sham ⫹ RC, p ⬍ 0.05.
22.0 ⫾ 4.0 0.5 ⫾ 0.0 11.5 ⫾ 2.4 15.1 ⫾ 4.4
Baseline
Ventral endocortical dynamics Mineralized surface (%) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day) Eroded surface (%)
Parameters
Sham ⫹ NC
Table 3. Histomorphometric changes of the fifth lumbar vertebral body: transverse sections
n.s. 0.000 n.s. n.s. 0.002
n.s. n.s. n.s.
n.s. n.s. n.s. n.s.
RC
n.s. n.s. n.s. n.s. 0.003
n.s. n.s. n.s.
n.s. n.s. n.s. n.s.
Orx ⴱ RC
Univariate ANOVA
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W. Yao et al. Effects of making rats rise to bipedal stance on lumbar vertebrae
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Figure 1. Dorsal (A,C,E,G) and ventral (B,D,F,H) cortices from the fifth lumbar vertebral bodies of sham ⫹ NC (A,B), orx ⫹ NC (C,D), sham ⫹ RC (E,F), and orx ⫹ RC (G,H) animals. Note the thinner dorsal and ventral cortices, and increased erosion at the endocortical surface (arrowhead) in the orx ⫹ NC animal (C,D); new bone (bone formed started at the xylenol orange label [arrows] toward the bone surface) was added at the dorsal endocortical and the ventral periosteal surfaces in the raised-cage animals (E–H). Er, eroded surface; XO, xylenol orange label. Original magnification ⫻12.5; 20-m-thick Villanueva bone stain sections.
changes. Apparently these effects were due to suppression of the high bone turnover induced by orx on trabecular and endocortical surfaces, and stimulation of bone formation on the ventral periosteal surface. These findings suggest that making rats rise to an erect bipedal stance for feeding helps to prevent loss of both trabecular and cortical bone mass in the lumbar vertebrae of orchidectomized rats. Previous experimental animal studies have reported that orchidectomy induced cancellous bone loss of proximal tibial metaphyses in rats by increasing bone resorption accompanied by a suppression of cortical periosteal bone formation and an increase in cancellous bone turnover in young/growing rats.10,31 In our study, we found that orchidectomy-induced bone loss was associated with increased bone formation and resorption at bone surfaces (proximal tibial metaphyses and tibial shafts) next to the marrow and did not affect periosteal bone formation in cortical bone (tibial shafts).31 The current study found that, in 6-monthold Sprague-Dawley rats, 12 weeks of androgen withdrawal resulted in a 40% loss of cancellous bone in the lumbar vertebral bodies. Dynamic histomorphometry indicated that bone loss occurred next to the marrow and was accompanied by high turnover, as suggested by increases of eroded surface, mineralized surface, bone formation rate (BFR/TV), and bone turnover (BFR/BV). Orchidectomy also induced an approximately 10% reduction of cortical thickness due to increases of bone resorption on ventral and dorsal endocortical surfaces. There are few reports that have assessed lumbar vertebral histomorphometric profiles after orchidectomy. Erben et al. found that, at 3 months postorchidectomy, lumbar vertebrae lost about 25% of cancellous bone in 13-month-old Fisher rats, associated with increases in cancellous bone formation and resorption.5 But no cortical bone data were reported in their study. Our results, together with those of Erben and coworkers, suggest that, like estrogen deficiency-induced bone loss, orchidectomy caused high-bone-turnover osteopenia in the lumbar vertebral bodies. To our knowledge, this is the first report of bone histomorphometric data of the lumbar vertebral bodies that has forced rats to rise to an erect stance as opposed to running on all four limbs on a treadmill, wheel, or swimming and jumping. In growing rats
(1–3 months), treadmill exercise was found to increase the lumbar vertebral trabecular bone peak bone mass and bone strength,14,18 whereas, in adult rats (14 months), treadmill exercise did not affect trabecular bone, but cortical bone formation was found to increase.36 A recent report in tibiae found that bipedal resistance exercise dramatically increased cancellous bone but not cortical bone in 5-month-old male rats,32 but no data on the lumbar vertebral body were provided in that report. Similar to the effects of treadmill exercise on adult rats, we found that raised-cage treatment did not affect cancellous bone mass of the lumbar vertebral body, but cortical bone formation of the ventral periosteal surface increased over the entire study period and increased the total vertebral body cross-section area by 11%; bone resorption at the endosteal surface tended to decrease. In human vertebrae, smaller cross-sectional areas of vertebrae in women explained their lower maximum compressive loads when compared with men.4,19 In rats, the increase of vertebral total cross-sectional area by raised cages may be more important for increasing vertebral bone strength than the increase of trabecular bone mass. After 12 weeks of housing in raised cages, the orx rats lost only about 15% of cancellous bone compared with the 40% loss in those rats housed in normal height cages, and there was no net cortical bone loss. Dynamic histomorphometry showed that raised cages reduced orx-related increased bone resorption at the trabecular and dorsal endocortical surfaces. Moreover, animals in raised cages added new bone (increased mineral apposition rate at the 0 – 80 day interval) on the ventral periosteal and dorsal endocortical surfaces. Taken together, the prevention effects of raised cages on the lumbar cortical bone were attained mainly by stimulating bone formation at the ventral cortices and depressing endocortical resorption at the endocortical dorsal cortices. These observations suggest that, unlike exercise by treadmill running or swimming, the rats in raised cages had to exert considerable extra muscle force by using their spinal extensor muscles to become erect and reach upward. This exertion might increase mechanical forces on the ventral and dorsal cortices. Also, such forces could exert large enough strains to depress remodeling-dependent bone loss and to turn on modeling-dependent bone gain.6 –9 The same
17.3 ⫾ 4.5 3.5 ⫾ 0.7
19.2 ⫾ 4.7 3.7 ⫾ 0.6 67.3 ⫾ 7.1 n.a. 0.7 ⫾ 0.0 47.7 ⫾ 8.3
Ventral periosteal dynamics Mineralizing surface (%) Mineral apposition rate (0–80 days, m/day) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day) 88.8 ⫾ 12.7a 0.2 ⫾ 0.4 1.0 ⫾ 0.1a,b 91.8 ⫾ 18.2a
16.3 ⫾ 3.9 10.2 ⫾ 3.4a,b
23.9 ⫾ 2.7 0.7 ⫾ 0.1a,b 195.7 ⫾ 41.8a,b 17.2 ⫾ 1.6a,b
10.0 ⫾ 1.3 9.3 ⫾ 0.9a,b 1.6 ⫾ 0.3a,b 547.4 ⫾ 95.1a,b 255.4 ⫾ 11.2a,b 191.1 ⫾ 11.5a,b
Orx ⫹ NC
67.3 ⫾ 19.4 0.7 ⫾ 0.1 0.8 ⫾ 0.1 67.3 ⫾ 19.4
15.3 ⫾ 4.7 3.6 ⫾ 1.1
17.0 ⫾ 3.0 0.6 ⫾ 0.1 93.0 ⫾ 29.1 9.9 ⫾ 2.5
10.9 ⫾ 2.4 17.0 ⫾ 4.3 2.7 ⫾ 0.4 299.7 ⫾ 67.9 336.5 ⫾ 16.6 222.2 ⫾ 19.2
Sham ⫹ RC
77.9 ⫾ 20.4a 0.7 ⫾ 0.2c 0.9 ⫾ 0.1a,b 73.5 ⫾ 15.9a
23.1 ⫾ 5.4d 3.1 ⫾ 0.2c
23.7 ⫾ 3.8d 0.7 ⫾ 0.1a–c 174.4 ⫾ 48.8a,b,d 16.1 ⫾ 3.7a,b,d
10.9 ⫾ 0.7 13.5 ⫾ 2.1c 2.5 ⫾ 0.3c 333.7 ⫾ 65.2c 303.8 ⫾ 35.3c 212.6 ⫾ 10.4c
Orx ⫹ RC
n.s. n.s. 0.001 0.002
n.s. 0.000
0.001 0.000 0.000 0.000
n.s. 0.000 0.000 0.000 0.005 0.035
Orx
KEY: ANOVA, analysis of variance; n.a., not available; NC, normal height cage; n.d., not detectable; n.s., nonsignificant change; orx, orchidectomy; RC, raised cage. a Vs. baseline, p ⬍ 0.05. b Vs. sham ⫹ NC, p ⬍ 0.05. c Vs. orx ⫹ NC, p ⬍ 0.05. d Vs. sham ⫹ RC, p ⬍ 0.05.
82.2 ⫾ 8.8a 0.2 ⫾ 0.4 0.7 ⫾ 0.1 59.0 ⫾ 10.0
18.4 ⫾ 4.6 0.5 ⫾ 0.03 104.6 ⫾ 36.3 10.3 ⫾ 2.2
9.8 ⫾ 0.6 17.3 ⫾ 4.6 2.9 ⫾ 0.4 273.3 ⫾ 61.4 298.4 ⫾ 30.6 215.1 ⫾ 24.6
Sham ⫹ NC
18.1 ⫾ 3.6 0.5 ⫾ 0.1 111.1 ⫾ 28.0 10.2 ⫾ 2.2
9.1 ⫾ 0.5 17.5 ⫾ 1.2 3.2 ⫾ 0.3 241.9 ⫾ 30.7 296.9 ⫾ 25.7 209.1 ⫾ 20.8
Baseline
Trabecular dynamics Mineralizing surface (%) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone volume (%/yr) Bone formation rate/bone surface (m/day * 100) Bone formation rate/trabecular volume (%/yr) Eroded surface (%)
Static changes Total tissue area (mm2) Trabecular volume (%) Trabecular number (#) Trabecular separation (m) Ventral cortical thickness (m) Dorsal cortical thickness (m)
Parameters
Table 4. Histomorphometric changes of the fourth lumbar vertebral body: sagittal sections
n.s. 0.005 n.s. n.s.
n.s. 0.001
n.s. n.s. n.s. n.s.
n.s. n.s. n.s. n.s. 0.044 n.s.
RC
n.s. n.s. 0.004 0.018
n.s. 0.000
n.s. n.s. n.s. n.s.
n.s. n.s. 0.001 0.000 n.s. n.s.
Orx ⴱ RC
Univariate ANOVA
672 W. Yao et al. Effects of making rats rise to bipedal stance on lumbar vertebrae Bone Vol. 27, No. 5 November 2000:667– 675
51.8 ⫾ 2.0 0.5 ⫾ 0.03 28.1 ⫾ 2.3 32.2 ⫾ 11.3 n.a. 0.7 ⫾ 0.2 22.8 ⫾ 11.9 3.0 ⫾ 1.1
Dorsal periosteal dynamics Mineralizing surface (%) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day)
Dorsal endocortical dynamic changes Mineralizing surface (%) Mineral apposition rate (0–80 days, m/day) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day) Eroded surface (%) 22.4 ⫾ 1.8 n.d. 0.7 ⫾ 0.1 16.1 ⫾ 4.4 2.9 ⫾ 0.1
46.2 ⫾ 10.9 0.5 ⫾ 0.05 24.4 ⫾ 8.0
21.8 ⫾ 5.9 0.5 ⫾ 0.1 13.5 ⫾ 5.4 10.8 ⫾ 0.8
Sham ⫹ NC
22.8 ⫾ 3.6 n.d. 0.6 ⫾ 0.1 14.4 ⫾ 3.9 7.4 ⫾ 0.8a,b
31.6 ⫾ 11.5 0.5 ⫾ 0.04 17.3 ⫾ 7.5
17.7 ⫾ 6.9 0.5 ⫾ 0.01 11.1 ⫾ 1.4 14.8 ⫾ 1.6a,b
Orx ⫹ NC
25.5 ⫾ 4.6 0.3 ⫾ 0.01b 0.5 ⫾ 0.0 13.2 ⫾ 2.5 2.6 ⫾ 1.3
35.7 ⫾ 15.5 0.5 ⫾ 0.1 21.5 ⫾ 15.6
15.3 ⫾ 3.0 0.5 ⫾ 0.01 8.6 ⫾ 2.9a 10.3 ⫾ 3.0
Sham ⫹ RC
29.6 ⫾ 6.8 n.d. 0.6 ⫾ 0.1 17.8 ⫾ 3.5 4.2 ⫾ 1.0c
36.2 ⫾ 5.7 0.6 ⫾ 0.1 21.1 ⫾ 5.4
18.7 ⫾ 3.0 0.5 ⫾ 0.05 12.5 ⫾ 3.3 11.5 ⫾ 2.7
Orx ⫹ RC
n.s. n.s. n.s. n.s. 0.000
n.s. n.s. n.s.
n.s. n.s. n.s. 0.027
Orx
KEY: ANOVA, analysis of variance; n.a., not available; NC, normal height cage; n.d., not detectable; n.s., nonsignificant change; orx, orchidectomy; RC, raised cage. a Vs. baseline, p ⬍ 0.05. b Vs. sham ⫹ NC, p ⬍ 0.05. c Vs. orx ⫹ NC, p ⬍ 0.05. d Vs. sham ⫹ RC, p ⬍ 0.05.
22.0 ⫾ 4.0 0.5 ⫾ 0.1 15.2 ⫾ 4.4 10.2 ⫾ 4.4
Baseline
Ventral endocortical dynamics Mineralizing surface (%) Mineral apposition rate (70–80 days, m/day) Bone formation rate/bone surface (m/day) Eroded surface (%)
Parameters
Table 5. Histomorphometric changes of the fourth lumbar vertebral body: sagittal sections
n.s. n.s. n.s. n.s. 0.001
n.s. n.s. n.s.
n.s. n.s. n.s. n.s.
RC
n.s. n.s. n.s. n.s. 0.005
n.s. n.s. n.s.
n.s. n.s. 0.028 n.s.
Orx * RC
Univariate ANOVA
Bone Vol. 27, No. 5 November 2000:667– 675 W. Yao et al. Effects of making rats rise to bipedal stance on lumbar vertebrae 673
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W. Yao et al. Effects of making rats rise to bipedal stance on lumbar vertebrae
observations were also seen in the long bones of in vivo loading models, in which the same resorption-to-formation drifts were observed, with enhanced periosteal bone formation and suppressed endocortical bone resorption.20,25 In addition, we found that periosteal bone formation occurred in the ventral cortices but not in the dorsal cortices. This could thicken and strengthen lumbar vertebrae in ways that would not put the spinal cord at risk. Moreover, the bipedal-stance exercise prevented the detrimental effects of androgen depletion on trabecular architecture (Tb.N and Tb.Sp), which are among the predictors of the presence of vertebral fracture in male osteoporosis.16 As this wholebody standing and squatting movement in rats is somewhat similar to the “squat” exercise in humans, we believe that exercise involving both concentric and eccentric muscle contraction against the spine may be useful in increasing spinal bone strength and preventing the occurrence of vertebral fracture. The current study included the histomorphometric profiles of both sagittal sections (LVL-4) and transverse sections (LVX-5) of rat vertebrae. In general, LVX-5 contained more trabecular bone and had thicker dorsal, but thinner ventral, cortices. The latter may be due to the fact that it was reported as the average of the entire ventral cortex, whereas that of LVL-4 was sectioned in the midbody where the thickest ventral cortex lies. Our data demonstrate that LVX-5 had higher trabecular bone turnover, but lower mineralized surfaces and bone formation rates at the periosteal and endocortical surfaces of the ventral and dorsal cortices. Orchidectomy and raised cage had similar effects on these two vertebrae except that the percentage of trabecular bone loss was greater in LVL-4 than in LVX-5, and the raised cage increased the total tissue area and mineral apposition rate (0 – 80 day interval) of ventral periosteal and dorsal endocortical surfaces of LVX-5 to a greater extent than it did in LVL-4. The changes might represent “true” differences between the two vertebrae or might be due to different sectioning methods. We believe the transverse sections may be more meaningful when studying the loading effects on the vertebrae, because they show the total amount of bone supporting the total compression loads on the vertebral body. The raised cages increased the body weights of the rats. It has been proposed and seems true that muscle force is the main determinant of postnatal and whole-bone strength and bone “mass.” We found that the increased body weight by use of the raised cage was accompanied by increases in hind-limb muscle mass and bone mass.34 Unfortunately, we did not assess the details of the relationship between bone formation and muscle weight in the lumbar vertebrae, because we did not obtain spinal muscle data in the current study. In summary, orchidectomy induced high bone turnover and trabecular and cortical bone loss in rat lumbar vertebral bodies. Forcing the rats to rise to an erect stance partially prevented those decreases. Such findings suggest that making orchidectomized rats rise to an erect bipedal stance for feeding helps to prevent loss of trabecular and cortical bone “mass,” and presumably bone strength. This method also provides us with an inexpensive, noninvasive, reliable model to increase in vivo vertebral loading in rats that is similar in humans. References 1. Baillie, S. P., Davison, C. E., Johnson, F. J., and Francis, R. M. Pathogenesis of vertebral body crush fracture in man. Age Aging 21:139 –141; 1992. 2. Bourrin, S., Ghaemmaghami, F., Vico, L., Chappard, D., Gharib, C., and Alexandre, C. Effect of a five-week swimming program on rat bone: A histomorphometric study. Calcif Tissue Int 51:137–142; 1992. 3. Coxam, V., Robins, S., Pastoureau, P., Gaumet, N., Lebecque, P., Davicco, M. J., Giry, J., and Barlet, J. P. Estrogen is more effective than dihydrotest-
Bone Vol. 27, No. 5 November 2000:667– 675
4.
5.
6. 7. 8. 9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19. 20.
21.
22. 23.
24.
25.
26.
27.
osterone to prevent bone loss in young castrated male rats. J Bone Miner Res 10(Suppl.):S311; 1995. Ebbesen, E. N., Thomsen, J. S., Beck-Nielsen, H., Nepper-Rasmussen, H. J., and Mosekilde, L. Age and gender-related differences in vertebral bone mass, density, and strength. J Bone Miner Res 14:1394 –1403; 1999. Erben, R. G., Eberle, J., Stahr, K., and Goldberg, M. Androgen deficiency induces high turnover osteopenia in aged male rats. A sequential histomorphometric study. J Bone Miner Res 15:1085–1098; 2000. Frost, H. M. Bone “mass” and the “mechanostat”: A proposal. Anat Rec 219:1–9; 1987. Frost, H. M. Perspective: On our age-related bone loss: Insights from a new paradigm. J Bone Miner Res 12:1539 –1546; 1997. Frost, H. M. Perspectives: The role of changes in mechanical usage set points in the pathogenesis of osteoporosis. J Bone Miner Res 7:253–261; 1992. Frost, H. M., Ferretti, J. L., and Jee, W. S. S. Perspective: Some roles of mechanical usage, muscle strength, and the mechanical in skeleton physiology, disease, and research. Calcif Tissue Int 62:1–7; 1998. Gunness, M. and Orwoll, E. Early induction of alteration in cancellous and cortical bones histology after orchidectomy in mature rats. J Bone Miner Res 10:1735–1743; 1995. Gurkan, L., Ekeland, A., Gautvik, K. M., Langerland, N., Ronningen, H., and Silheim, L. F. Bone changes after castration in rats: A model of osteoporosis. Acta Orthop Scand 57:67–70; 1986. Hock, J. M., Gera, I., Fonseca, J., and Raisz, L. G. Human parathyroid hormone-(1–34) increases bone mass in ovariectomized and orchidectomized rats. Endocrinology 122:2899 –2904; 1988. Horcajada-Molteni, M. N., Davicco, M. J., Collignon, H., Lebecque, P., Coxam, V., and Barlet, J. P. Does endurance running before orchidectomy prevent osteopenia in rats? Eur J Appl Physiol 80:344 –352; 1999. Iwamoto, J., Yeh, J. K., and Aloria, J. F. Different effect of treadmill exercise on three cancellous bone sites in the young growing rats. Bone 24:163–169; 1999. Ke, H. Z., Qi, H., Crawford, D. T., Chidsey-Frink, K. L., Simmons, H. A., and Thompson, D. D. Lasofoxifene (CP-336,156), a selective estrogen receptor modulator, prevents bone loss induced by aging and orchidectomy in the adult rat. Endocrinology 141:1338 –1344; 2000. Legrand, E., Chappard, D., Pascaretti, C., Duquenne, M., Krebs, S., Rohmer, V., Basle, M. F., and Audran, M. Trabecular bone microarchitecture, bone mineral density, and vertebral fracture in male osteoporosis. J Bone Miner Res 15:13–18; 2000. Li, M., Jee, W. S. S., Ke, H. Z., Tang, L. Y., Ma, Y. F., Liang, X. G., and Setterberg, R. B. Prostaglandin E2 administration prevents bone loss induced by orchidectomy in rats. J Bone Miner Res 10:66 –73; 1995. Mosekilde, L., Danielsen, C. C., Søgaard, C. H., and Thorling, E. The effect of long-term exercise on vertebral and femoral bone mass, dimensions, and strength-assessed in a rat model. Bone 15:292–301; 1994. Mosekilde, L. The effects of modeling and remodeling on human vertebral body architecture. Technol Health Care 6:287–297; 1998. Mosley, J. R. and Lanyon, L. E. Strain rate as a controlling influence on adaptive modeling in response to dynamic loading of the ulna in growing male rats. Bone 23:313–318; 1998. Newhall, K. M., Rodnick, K. J., Meulen, M. C., Carter, D. R., and Marcus, R. Effects of voluntary exercise on bone mineral content in rats. J Bone Miner Res 6:289 –296; 1991. Nguyen, T. V., Eisman, J. A., Kelly, P. J., and Sambrook, P. N. Risk factors for osteoporotic fractures in elderly men. Am J Epidemiol 144:255–263; 1996. Parfitt, A. M., Drezner, M. K., Glorieux, F. H., Janis, J. A., Malluche, H., Meunier, P. J., Ott, S. M., and Recker, R. R. Bone histomorphometry: Standardization of nomenclature, symbols and units. Report of the ASBMR Histomorphometry Committee. J Bone Miner Res 2:595– 610; 1987. Parfitt, A. M., Mathews, C. H. E., Villanueva, A. R., Kleerekoper, M., Frame, B., and Rao, D. S. Relationships between surface, area, and thickness of iliac trabecular bone in aging and in osteoporosis. J Clin Invest 72:1396 –1409; 1983. Turner, C. H., Forwood, M. R., Rho, J. Y., and Yoshikawa, T. Mechanical loading thresholds for lamellar and woven bone formation. J Bone Miner Res 9:87–97; 1994. Turner, R. T., Wakley, G. K., and Hannon, K. S. Different effects of androgens on cortical bone histomorphometry in gonadectomized male and female rats. J Orthop Res 8:612– 617; 1990. Tuukanen, J., Peng, Z., and Vaananen, H. K. Effect of running exercise on the bone mass induced by orchidectomy in the rats. Calcif Tissue Int 55:33–37; 1994.
Bone Vol. 27, No. 5 November 2000:667– 675 28. Vanderschueren, D., Van Herck, E., De Coster, R., and Bouillon, R. Aromatization of androgen is important for skeleton maintenance of aged male rats. Calcif Tissue Int 59:179 –183; 1996. 29. Vanderschueren, D., Van Herck, E., Nijs, J., Ederveen, A. G. H., De Coster, R., and Bouillon, R. Estrogen prevents the effect of aromatase inhibition on the skeleton of aged male rats. J Bone Miner Res 12(Suppl.):T483; 1997. 30. Verhas, M., Schoutens, M. A., L’Lermit-Baleriaux, M., Dourow, N., Verschaeren, A., Mone, M., and Heilponorn, A. The effect of orchidectomy on bone metabolism in aging rats. Calcif Tissue Int 39:74 –77; 1986. 31. Wakley, G. K., Schutte, H. D., Jr, Hannon, K. S., and Turner, R. T. Androgen treatment prevents loss of cancellous bone in the orchidectomized rats. J Bone Miner Res 6:325–330; 1991. 32. Westerlind, K., Fluckey, J. D., Gordon, S. E., Kraemer, W. J., Farrell, P. A., and Turner, R. T. Effects of resistance exercise training on cortical and cancellous bone in mature male rats. J Appl Physiol 84:459 – 464; 1998. 33. Wink, C. S. and Felts, W. J. L. Effects of castration on bone structure of male rats. Calcif Tissue Int 32:77– 82; 1980.
W. Yao et al. Effects of making rats rise to bipedal stance on lumbar vertebrae
675
34. Yao, W., Jee, W. S. S., Chen, J. L., Liu, H. Y., Tam, C. S., Cui, L., Zhou, H., Setterberg, R. B., and Frost, H. M. Making rats rise to erect bipedal stance for feeding partially prevented orchidectomy-induced bone loss and added bone to intact rats. J Bone Miner Res 15:1158 –1168; 2000. 35. Yeh, J. K., Aloia, J. F., and Barilla, M.-L. Effects of 17-estradiol replacement and treadmill exercise on vertebral and femoral bones of the ovariectomized rats. Bone Miner 24:223–234; 1994. 36. Yeh, J. K., Aloia, J. F., and Chen, M. M. Growth hormone administration potentiates the effect of treadmill exercise on long bone formation but not on the vertebrae in middle-aged rats. Calcif Tissue Int 54:38 – 43; 1994.
Date Received: April 4, 2000 Date Revised: July 25, 2000 Date Accepted: July 28, 2000