In vivo rat assay: bone remodeling and steroid effects on juvenile bone by pQCT quantification in 7 days

Nansie A. McHugh, Haydee M. Vercesi, Robert W. Egan, and John A. Hey

Allergy, Schering-Plough Research Institute, Kenilworth, New Jersey 07033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Anesthetized Sprague-Dawley weanling rats were scanned for bone mineral density (BMD) values after 7 days of treatment to determine whether resorption/growth at the proximal tibia can be quantified by peripheral quantitative computed tomography scanning techniques. Because the weanling rat is in a rapid growth stage, all groups showed significant increases in change from baseline values of BMD. Bisphosphonate treatment produced significant dose-related changes in BMD with average increases of 195 and 241% (10 and 20 µg/kg) vs. 86% in control rats. We further characterized this model to determine effects of steroids on growing bone. Graded doses of glucocorticoid (3.5, 7.0, 10.5, 14.0, 28.0, and 42.0 mg · kg-1 · wk-1) caused no significant differences in trabecular BMD in 7 days between control and treated rats. Significant decreases in growth (weights) and increases in cortical bone area were observed, indicating that this model may be useful in comparing effects of nonsteroid, anti-inflammatory alternatives on juvenile bone. Although the relevance of this model to adult disease remains to be elucidated, it also provides a tool for mechanistic evaluation of therapeutic modalities or efficacy assessment for dose selection for longerterm models.

osteoporosis; bone mineral density; glucocorticoid; computed tomography; animal model


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ANIMAL MODELS that simulate osteoporosis, such as ovariectomized rodents (postmenopausal) and glucocorticoid-induced and senescence-related osteopenia, are typically conducted in aged animals over long periods of time. In the case of senescence-induced osteoporosis, decrements in bone density may not be observed for 3 to 6 mo. The cost of boarding and large amounts of drugs that are needed to sustain these chronic models are thus often prohibitive. Moreover, this protracted study time is rate limiting to evaluation and development of novel therapeutic agents.

Normal bone growth involves both bone resorption and bone formation in a well-controlled balance. It is proposed that administration of an anti-resorptive agent to the 21-day-old growing rat may disrupt the normal equilibrium at this time of fast growth, leading to a significantly higher bone mass in treated rats compared with age-matched untreated rats. In early studies, Schenk and colleagues [Muhlbauer et al. (16) and Schenk et al. (21)] utilized this 21-day-old model to screen bisphosphonate candidates. These studies showed that 7-day treatment with bisphosphonates yielded bones that demonstrated large increases in metaphysial density as measured by histology. The purpose of our study was to determine whether increases or decreases in apparent bone density can be quantified by peripheral quantitative computed tomography (pQCT) at an early time period.

The 21-day-old rat represents a juvenile model of bone growth. The juvenile bone is particularly sensitive to corticosteroids. Corticosteroids are prescribed for severe asthma, juvenile rheumatoid arthritis, or dermatologic disease and are known to reduce bone turnover, stunt bone growth, and decrease bone mineral density (BMD) (5, 6, 9, 15, 23). An average daily dose of 5 mg · kg-1 · day-1 will cause loss of BMD in the prepubertal child, and monitoring of bone density is recommended even for adults taking 7.5 mg · kg-1 · day-1 for >= 6 mo (17). In their retrospective review of 212 patients, Hougardy et al. (10) reported that the median daily dose was 10 mg prednisolone equivalent and the median duration of oral corticosteroid treatment was 50 wk, which is well above the dosage capable of eliciting bone loss. Moreover, at an average dose of 0.67 mg · m-2 · day-1 of inhaled steroids, there is a reduction in the acquisition of bone mineral in prepubertal children that compromises their peak bone mass and predisposes them to osteoporosis and a higher fracture risk as an adult (2). Therefore, we also wanted to determine whether we could quantify by pQCT with this model negative effects of corticosteroids on juvenile bone. Although pQCT scanning cannot replace the information obtained from histostaining, immunostaining, or histomorphometric analysis, it allows for the rapid and noninvasive evaluation of potential therapeutic anti-resorptive compounds in a shorter amount of time.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These studies were conducted under a protocol approved by the Schering-Plough Research Institute Animal Care and Use Committee. All of the studies employed male Sprague-Dawley weanling (21-day-old) rats weighing 44 ± 5 g that had been purchased from Charles River Laboratories (Wilmington, MA). The rats were given free access to water and a standard rat chow (Harlan Teklad Labdiet, Madison, WI).

Pilot study of two doses of bisphosphonate. This pilot study was conducted to determine whether changes in trabecular BMD could be measured in the growing rat by pQCT scanning techniques. Two doses were delivered, because we did not know how sensitive the CT scanner would be at detecting quantifiable changes in trabecular bone over 7 days. Fifteen rats were randomly assigned to one of three groups (n = 5/group): two bisphosphonate-treated groups [(alendronate sodium in doses of 10 and 20 µg/kg sc), Technodrugs and Intermediates (P), Gujarat, India] and a control group that received vehicle only (PBS sc).

Dose-response study. To determine whether pQCT scanning techniques could measure a dose-response effect on trabecular bone, we repeated the study described above by using graded doses of bisphosphonate. To assess this, we employed lower doses with smaller increments between doses to show sensitivity and repeatability of the study. Thirty rats were randomly assigned to one of six groups (n = 5/group): five groups received daily doses of bisphosphonate (alendronate sodium in 2.5, 5.0, 10.0, 20.0, and 25.0 µg/kg sc, respectively), and a control group received vehicle only (PBS sc).

Corticosteroid treatment to growing rats. Twenty-five rats were randomly assigned to one of five groups (n = 5/group): a low-dose group (methylprednisolone 3.5 mg · kg-1 · wk-1 sc), a standard-dose group (methylprednisolone 7 mg · kg-1 · wk-1 sc), two high-dose groups (methylprednisolone 10.5 and 14 mg · kg-1 · wk-1 sc), and a control group that received vehicle only (saline + methanol sc).

High-dose corticosteroid treatment. Eighteen rats were randomly assigned to one of two glucocorticoid-treated groups [one group received double the highest dose of our previous study (methylprednisolone 28 mg · kg-1 · wk-1 sc, n = 5), and another group received an even higher dose (methylprednisolone 42 mg · kg-1 · wk-1 sc, n = 5)] or to a control group that received vehicle only (saline + methanol sc, n = 8). For measurement of femoral length and weight, the left femur was removed and cleaned free of muscle and nonbone tissue. Because of the rat's quadruped stance, the highest point on the rat femur is not the head but the greater trochanter. The length of the femur was measured from the greater trochanter to the lateral condyle using a digital caliper (Pro-max, Japan Micrometer Manufacturing). The bones were then weighed (wet weight) on an Ohaus Voyager balance (Ohaus, Pine Brook, NJ).

pQCT measurements. All groups were treated in the same manner. On day 1, we took baseline BMD of the proximal tibia by pQCT (XCT Research, Stratec Medizintechnik, Pforzheim, Germany). The rats were given their supplements or vehicle daily on days 1-7. On the 8th day, the final BMD measurements were taken. The settings for pQCT scanning included research SA collimation at a voxel size of 0.1 mm3; therefore, the slice width was 0.1 mm. The voxel is equivalent to a pixel with three-dimensional volume. This small voxel size minimizes "partial volume effect" errors (i.e., including voxels that are not completely filled with bone). Before the pilot study, multiple slice scans were performed on the weanling rat to examine variations in slice densities at the proximal tibia. The slice placement that gave the most consistent density with least variability between adjacent slices at baseline was determined and is shown by the low variability of the baseline data in Fig. 1. Comparable placement of slices was ensured by measuring a slice in the metaphysis 2 mm from the reference line, which was placed at the proximal edge of the growth plate.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1.   Bone mineral density (BMD) in the growing rat---effect of bisphosphonate treatment. The 1st experiment shows effect of bisphosphonate treatment (alendronate sodium 10 and 20 µg · kg-1 · day-1; n = 5/group) on BMD in the growing rat. Trabecular BMD values at baseline were consistent between groups. After 7 days of bisphosphonate treatment, there was significant increase in trabecular BMD that was dose dependent. * Significant vs. baseline, P < 0.001; # significant difference from control final measurement, P < 0.001; dagger  significant difference between 2 doses of alendronate sodium, P = 0.03. Data are means ± SE.

pQCT analysis. All bone slices were analyzed with the same parameters by use of Stratec software (Stratec Medizintechnik). The analysis parameters of the Stratec software employed in the present study were an automatic ContourMode 1, which was used to define the outer edge of the cortical bone, and PeelMode 20 (an adaptation of PeelMode 2 that determines the threshold to be used by evaluating the BMD at a predefined percentage of total bone), which was used to define the inner edge of the cortical bone and the beginning of the trabecular bone. For determining trabecular BMD, the percent option was used, with trabecular area defined at 30% with a threshold of 280 mg/cm3. For cortical bone analysis, cortical bone was defined as any density >710 mg/cm3 within the defined region of interest. Thresholds were determined by using the "profile" function of the CT scanner to visualize the density at the edges of the cortical bone.

Statistical analysis of data was assessed using ANOVA, with Dunnett's test for treatments with a control, simple linear regression, and Pearson product-moment correlation coefficient (SigmaStat, SPSS Software, Chicago, IL). All data are reported as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Initial study. Baseline trabecular BMD data as measured by pQCT scans were highly consistent among groups (Fig. 1, baseline). After 7 days of treatment, the final pQCT measurements (BMD) were significantly different from their own baseline (P < 0.001), a finding characteristic of young, growing animals. A comparison of final trabecular BMD measurements showed that there was a significant difference between the treatment groups. Both of the treated groups had a significantly higher trabecular BMD than the untreated controls (P < 0.001). Also, alendronate produced a dose-related increase in trabecular BMD at 20 vs. 10 µg/kg (Fig. 1, final) that was statistically significant (P = 0.03). The final area measurements of cortical bone (volume) were insignificant among groups (0.18 ± 0.04 mm2 in control vs. 0.31 ± 0.07 mm2 and 0.27 ± 0.05 mm2 in the 10 and 20 µg/kg groups, respectively).

Alendronate dose-response study. Baseline BMD data as measured by pQCT were not significantly different among groups. After the 7 days of treatment, final trabecular BMD measurements were significantly different from those at baseline. Moreover, when we compared the groups on the basis of longitudinal trabecular bone density measurements (change from baseline), all doses showed a significant difference from control (P < 0.001, Fig. 2). The final measurements of cortical bone area (volume) demonstrated that, at the highest dose, the increase in cortical bone was significantly higher than control (P = 0.002, Fig. 3).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Trabecular BMD---dose-response effects with bisphosphonate. The 2nd experiment evaluated the dose-response effect of alendronate sodium in the growing rat model. Final trabecular BMD data at day 7 demonstrated a significant difference at all doses, P < 0.001. Dose-response data were linear (y = 247.304 + 6.247x; P < 0.001). Data are means ± SE.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3.   Cortical bone area---effect of bisphosphonate treatment. Although there was a trend toward a dose-response effect of bisphosphonate treatment on cortical bone area (volume) in the final day 7 measurements, significance was achieved only at the highest dose (25 µg · kg-1 · day-1) compared with control (* P = 0.002).

Corticosteroid treatment to growing rats. Baseline pQCT scans were not significantly different among groups. After 7 days of treatment, each rat's final trabecular BMD measurement was significantly greater than its respective baseline values. When we compared the final BMD (trabecular) measurements among all three groups, there were no significant differences. Because glucocorticoids (GC) have effects on cortical bone as well, we analyzed the bone slice for cortical bone content. The slices from the GC-treated animals had significantly higher areas of cortical bone than untreated controls (Fig. 4, A and B). The group receiving the highest dose showed a trend toward having a lower trabecular BMD than the midrange doses. When we repeated the study with high doses of GC, we observed a diminished growth rate in the GC-treated rats vs. control that was significant (P = 0.002). The final mean weight of the control rats was 92 ± 2 g vs. 76 ± 3 g and 61 ± 15 g in the 28 and 42 mg · rat-1 · wk-1 GC-treated rats, respectively (Fig. 5A). The left femur (long bone) was removed to determine whether longitudinal bone growth was also affected. There were no significant differences in the length of the femur among groups; however, there was a trend for the weight of the GC-treated femurs to be lower (Fig. 5B). This decrease in femur weight correlated positively with the diminished final mean weights of the rats (Pearson correlation coefficient 0.528, P = 0.024). Baseline trabecular BMD data as measured by pQCT scans were not significantly different among groups. After 7 days of treatment, each rat's final trabecular CT BMD measurement was significantly different from its own baseline. An ANOVA comparing the final trabecular BMD measurements among all three groups showed no significant difference, although there was a trend toward a lower BMD in trabecular bone in the groups treated with high doses of GC (Fig. 6A). A comparison of cortical area showed that, again, the slices from the GC-treated animals had a significantly higher volume of cortical bone than those from untreated controls (Fig. 6B).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4.   Trabecular BMD and cortical bone area---effect of glucocorticoid treatment. A: longitudinal changes (difference from baseline) in BMD of trabecular bone of the growing rat treated with glucocorticoids. There were no significant differences among groups. B: effects of glucocorticoids on the final 7-day measurements of cortical bone area per slice. At doses >7 mg · kg-1 · wk-1 there was a significant increase in area of cortical bone vs. control (7 mg · kg-1 · wk-1, P = 0.028; 10.5 mg · kg-1 · wk-1, P = 0.037; 14 mg · kg-1 · wk-1, P = 0.044); n = 5/group. Data are means ± SE.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 5.   Final weights and femur weights of rats. A: final weights after 7 days of glucocorticoid treatment. Treated rats had significantly attenuated weight gains, P = 0.002. B: graph of final femur weights, which show a trend toward attenuated growth in the treated rats, although there was no significance. Data are means ± SE. Femur weight correlated positively with final weights of rats (Pearson's correlation coefficient 0.528, P = 0.024).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 6.   Trabecular BMD and cortical bone area---effect of glucocorticoid treatment. A: a trend demonstrated toward a lower trabecular BMD in longitudinal measurements (difference from baseline); with high doses of glucocorticoids, however, significance was not achieved. B: effects of glucocorticoid treatment on final 7-day measurement of cortical bone area. Both doses showed significant increases in cortical area compared with control (28 mg · kg-1 · wk-1, P < 0.001; 42 mg · kg-1 · wk-1, P = 0.005). Data are means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrated that pQCT analysis can be utilized to quantitate changes in bone in the growing rat model. The data were consistent, repeatable, and statistically relevant. We observed a clear dose-response effect with small increments in the drug. Although the growing rat bone is not a disease model of osteoporotic bone, it can be used to detect resorption/antiresorption efficacy. The growing 21-day-old rat is an attractive model for the prescreening of bone density effects of antiresorptive drugs. The relatively small body weights of these animals mean that less compound is needed, and the ability to detect dramatically significant changes in a relatively short period of time is an invaluable tool in studies on bone. Employing pQCT scanning techniques allows for quicker evaluation, especially as a prescreening tool vs. histology or histomorphometry.

An important limitation of using the growing rat is the difficulty in finding the initial scanned area after a period of growth. We circumvented this problem by utilizing rats that were 44 ± 5 g in weight. The rate of growth over the 7-day period still permitted identification of the original site. The young Sprague-Dawley rat has growth spurts. We found that if we used rats that had an initial weight between 80 and 100 g, the rate of bone growth at this stage was prohibitive to obtaining consistent measurements (personal observation). The changes in bone are again quantifiable at weights between 120 and 145 g, but the dosages required at these weights negate the benefits of using a growing rat.

To our knowledge, this is the first report to use pQCT technology for assessing bone density in the growing rat. Our study investigates the use of the CT scanner to quickly assess bone changes in the growing rat for purposes of early drug discovery; however, this model has many potential applications in other areas of bone research. The advantages of using this model include observation of faster bone turnover due to the age of the animals, earlier quantification of changes to bone, and a noninvasive technique that allows for measurements in the same animals over multiple time points. For example, Barou et al. (3) used micro CT scanning techniques to quantify bone loss in a disuse rat model of bone loss (3). This group observed significant changes in 13 days. By using a weanling rat model, the study time could thus be halved. Furthermore, weanling rats are useful models for studying effects of chronic alcohol consumption (20) and binge drinking (19) (two major concerns of teenage alcohol consumption) on the growing bone. The use of pQCT scanning techniques would offer a noninvasive and quick way of assessing changes to bone in these studies. In our study, we killed the rats at 7 days, when significant changes were observed in trabecular bone density; however, this noninvasive technique can also be employed to scan animals multiple times until significant changes are observed. This feature would be helpful in studies such as those that evaluate effects of dietary changes on bone density (22). Our model also allows each animal to serve as its own control, rather than killing animals at different time points during a study.

Because the weanling rat is a model of growing bone and there is a great deal of concern regarding the affects of anti-inflammatory steroids on juvenile bones, we attempted to simulate the effects of GC on the growing bone by use of this in vivo assay. The effects of GC on human bone are multifactorial. Not only do steroids inhibit the secretion of gonadal hormones (7, 14), thereby eliminating the bone-sparing effects of estrogen and testosterone; they also affect the metabolism of calcium by decreasing intestinal calcium absorption and increasing renal calcium excretion (8, 13). The decrease in available circulating calcium stimulates secondary hyperparathyroidism. In the growing child, these side effects are magnified by the fact that peak bone mass has not yet been achieved, and the decreases in bone-promoting hormones result in smaller bones (5). The decrease in raw materials and bone turnover leads to a lower bone mass. Moreover, growth during toddler and prepuberty years is predominantly dependent on growth hormone (GH). Oral GC excess has direct and indirect effects on GH secretion. Endogenous GCs downregulate the hypothalamic relay mechanisms that are responsible for pituitary pulsatile GH secretion (1). GCs act directly to downregulate expression and binding of the GH receptor and interfere with the bioactivity of IGF-I, the primary second messenger of GH (1). Furthermore, GH therapy appears to counteract the adverse effects of GC therapy in children with juvenile chronic arthritis (4).

Whereas the major effect of steroids on the adult skeleton is in the axial skeleton and femoral neck (24, 25), in the growing preadolescent the effects on longitudinal bone growth and achieving peak bone mass are of concern. Using lower therapeutic doses of methylprednisolone, we observed no significant decreases in trabecular BMD in the growing rat model. We did, however, observe significant changes to cortical bone area in doses greater than 7 mg · kg-1 · wk-1, suggesting an effect on trabecular-to-cortical bone ratios. At the higher doses, we observed significant changes in bone size and body size/weights similar to the stunted growth observed in the preadolescent on severe steroidal regimens. Although there was a trend toward lower trabecular BMD in the animals subjected to higher doses, statistical significance was not achieved. The decrease in body weight may be explained by high-dose GC decreases in ad libitum food intake (18); however, even though animal size decreased in our study, the area of cortical bone (as defined by >710 mg/cm3) increased in a dose-dependent manner, suggesting that cortical bone growth/metabolism was enhanced. These data do not preclude the fact that there may be differential effects of GCs on other bones in the rat and bone loss occurrence in sections of the skeleton that were not presently assessed for BMD. We cannot ascertain on the basis of present data that extending the study beyond the 7-day period would yield different results on bone density; however, there are conflicting data regarding the effects of steroids on the BMD of the rat bone. Ferretti et al. (8) observed a dose-dependent decrease in femoral cortical BMD by pQCT scanning and a decrease in load-bearing capacity in older female Sprague-Dawley rats that were exposed to lower doses of steroids (dexamethasone) over 4 wk. By using an extremely sensitive (smaller) voxel size (0.0219 mm3; Stratec), they observed an increase in cortical porosity that compromised bone strength. In contrast, King et al. (11) observed an increase in bone, by histomorphometry, with GC treatment, and the biochemical markers of bone turnover (osteocalcin and deoxypyridinoline cross-links) were reduced. Differences in quantification techniques may account for the conflicting results. The increases in bone observed in the study by King et al. may reflect the larger cortical area that we observed. The increased porosity as measured by Ferretti et al. with the three-dimensional analysis by pQCT was not observed by King et al., who used histomorphometric techniques. Had porosities been present, they should have been visible by histomorphometry despite the fact that these techniques are not comparable, and histomorphometry cannot measure the true physical BMD of bone tissue. In the present study, we were unable to observe increased cortical porosity with our 0.1-mm3 voxel size compared with their voxel size of 0.0219 mm3. Alternatively, the differences may be due to the differences in ages of the animals. Our 21-day-old rats double their weight during the 7 days of the study and can be compared with humans at the GH-dependent stage of growth. Animals studied by Ferretti et al. were slightly older than those of the present study, and rats used in the study by King et al. were ~3 mo old.

Although we anticipated simulating adult human GC-induced osteoporosis at the trabecular level in this growing rat, this was not the case, possibly because GCs in rats do not inhibit absorption of calcium from the intestine as the human on steroid therapy does, and therefore they may not have the same bone loss effects on trabecular-rich areas that humans have (12, 26). Our findings did show, however, that normal weight gain (growth) and cortical bone growth (area) were significantly affected with GC treatment in the growing rat. We thus propose that the 7-day-rat model relates more to the effects of GCs on the juvenile bone and provides a useful tool for comparison of effects of prospective anti-inflammatory compounds on juvenile bone vs. standard steroid therapy.

In summary, although the relevance and appropriateness of this model to adult human disease may be limited because of the differences just mentioned that we have observed with steroid therapy, we propose the use of the weanling rat and pQCT techniques for early/first assessment of compounds on bone remodeling. Moreover, our data with alendronate demonstrate that the weanling rat may provide a useful model for mechanistic evaluation of nonglucocorticoid therapeutic modalities or efficacy assessment to help guide dose selection for chronic long-term osteoporosis models.


    FOOTNOTES

Address for reprint requests and other correspondence: N. A. McHugh, Schering-Plough Research Institute, Allergy, 2015 Galloping Hill Rd., K15-1-1600, Kenilworth, NJ 07033 (E-mail nansie.mchugh{at}spcorp.com).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

September 3, 2002;10.1152/ajpendo.00102.2002

Received 7 March 2002; accepted in final form 28 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allen, HD. Inhaled corticosteroid therapy for asthma in preschool children: growth issues. Pediatrics 109: 373-380, 2002[Abstract/Free Full Text].

2.   Allen, HD, Thong IG, Clifton-Bligh P, Holmes S, Nery L, and Wilson KB. Effects of high-dose inhaled corticosteroids on bone metabolism in prepubertal children with asthma. Pediatr Pulmonol 29: 188-193, 2000[ISI][Medline].

3.   Barou, O, Valentin D, Vico L, Barbier A, Alexandre C, and Lefage-Proust MH. High-resolution three-dimensional micro-computed tomography detects bone loss and changes in trabecular architecture early: comparison with DEXA and bone histomorphometry in a rat model of disuse osteoporosis. Invest Radiol 37: 40-46, 2002[ISI][Medline].

4.   Bechtold, S, Ripperger P, Muhlbayer D, Truckenbrodt H, Hafner R, Butenandt O, and Schwarz HP. GH therapy in juvenile chronic arthritis: results of a two-year controlled study on growth and bone. J Clin Endocrinol Metab 86: 5737-5744, 2001[Abstract/Free Full Text].

5.   Blodgett, FM, Burgin L, Lezzoni D, Gribetz D, and Talbot NB. Effects of prolonged cortisone therapy on the statural growth, skeletal maturation and metabolic status in children. N Engl J Med 254: 636-641, 1956[ISI].

6.   Boot, AM, de Jongste JC, Verberne AA, Pols HA, and de Muinck Keizer-Schrma SM. Bone mineral density and bone metabolism of prepubertal children with asthma after long-term treatment with inhaled corticosteroids. Pediatr Pulmonol 24: 379-384, 1997[ISI][Medline].

7.   Crilly, RG, Cawood M, Marshall DH, and Nordin BE. Hormonal status in normal, osteoporotic and corticosteroid-treated postmenopausal women. J R Soc Med 71: 733-736, 1978[ISI][Medline].

8.   Ferretti, JL, Gaffuri O, Capozza R, Cointry G, Bozzini C, Olivera M, Zanchetta JR, and Bozzini CE. Dexamethasone effects on mechanical, geometric and densitometric properties of rat femur diaphyses as described by peripheral quantitative computerized tomography and bending tests. Bone 16: 119-124, 1995[ISI][Medline].

9.   Harris, M, Hauser S, Nguyen TV, Kelly PJ, Rodda C, Morton J, Freezer N, Strauss BJ, Eisman JA, and Walker JL. Bone mineral density in prepubertal asthmatics receiving corticosteroid treatment. J Paediatr Child Health 37: 67-71, 2001[ISI][Medline].

10.   Hougardy, DM, Peterson GM, Bleasal MD, and Randall CT. Is enough attention being given to the adverse effects of corticosteroid therapy? J Clin Pharm Ther 25: 227-234, 2000[ISI][Medline].

11.   King, CS, Weir EC, Gundberg CW, Fox J, and Isogna KL. Effects of continuous glucocorticoid infusion on bone metabolism in the rat. Calcif Tissue Int 59: 184-191, 1996[ISI][Medline].

12.   Lane, NE. An update on glucocorticoid-induced osteoporosis. Rheum Dis Clin North Am 27: 235-253, 2001[ISI][Medline].

13.   Lukert, BP, Stanbury SW, and Mawer EB. Vitamin D and intestinal transport of calcium: effects of prednisolone. Endocrinology 93: 718-722, 1973[ISI][Medline].

14.   MacAdams, MR, White RH, and Chipps BE. Reduction of serum testosterone levels during chronic glucocorticoid therapy. Ann Intern Med 104: 648-651, 1986[ISI][Medline].

15.   Morris, HG. Growth and skeletal maturation in asthmatic children: effect of corticosteroid treatment. Pediat Res 9: 579-583, 1975[ISI][Medline].

16.   Muhlbauer, RC, Bauss F, Schenk R, Janner M, Bosies E, Strein K, and Fleisch H. BM21.0955, a potent new bisphosphonate to inhibit bone resorption. J Bone Miner Res 6: 1003-1011, 1991[ISI][Medline].

17.   Nishimura, J, and Ikuyama S. Glucocorticoid-induced osteoporosis: pathogenesis and management. J Bone Miner Metab 18: 350-352, 2000[ISI][Medline].

18.   Ohyama, T, Sato M, Wada Y, and Takahara J. Diverse effects of glucocorticoids on the hypothalamic pituitary axis in rat growth hormone secretion. Endocr J 43, Suppl: S115-S117, 1996[Medline].

19.   Sampson, HW, Gallager S, Lange J, Chondra W, and Hogan HA. Binge drinking and bone metabolism in a young actively growing rat model. Alcohol Clin Exp Res 23: 1228-1231, 1999[ISI][Medline].

20.   Sampson, HW, and Spears H. Osteopenia due to chronic alcohol consumption by young actively growing rats is not completely reversible. Alcohol Clin Exp Res 23: 324-327, 1999[ISI][Medline].

21.   Schenk, R, Eggli P, Fleisch H, and Rosini S. Quantitative morphometric evaluation of the inhibitory activity of new aminobisphosphonates on bone resorption in the rat. Calcif Tissue Int 38: 342-349, 1986[ISI][Medline].

22.   Shen, V, Birchman R, Xu R, Lindsay R, and Dempster DW. Short-term changes in histomorphometric and biochemical turnover markers and bone mineral density in estrogen- and/or dietary calcium-deficient rats. Bone 16: 149-156, 1995[ISI][Medline].

23.   Suzuki, Y, Ichikawa Y, Saito E, and Homma M. Importance of increased urinary calcium excretion in the development of secondary hyperparathyroidism of patients under glucocorticoid therapy. Metabolism 32: 151-156, 1983[ISI][Medline].

24.   Weinstein, RS, Jilka RL, Parfitt AM, and Manolagas SC. Inhibition of osteoblastogenesis and promotion of apoptosis of osteoblasts and osteocytes by glucocorticoids. J Clin Invest 102: 274-282, 1998[Abstract/Free Full Text].

25.   Wong, CA, Walsh LJ, Smith CJ, Wisniewski AF, Lewis SA, Hubbard R, Cawte S, Green DJ, Pringle M, and Tattersfield AE. Inhaled corticosteroid use and bone-mineral density in patients with asthma. Lancet 355: 1399-1403, 2000[ISI][Medline].

26.   Yasumura, S. Effect of adrenal steroids on bone resorption in rats. Am J Physiol 230: 90-93, 1976[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 284(1):E70-E75
0193-1849/03 $5.00 Copyright © 2003 the American Physiological Society