Y.F. Ma

The deleterious effects of long-term cyclosporine a, cyclosporine g, and fk506 on bone mineral metabolism in vivo

Administration of cyclosporine A to male and female rats accelerates bone remodeling and causes bone loss, among other side-effects. The newer immunosuppressant drugs, FK506 and CsG, have been synthesized to counteract the toxic effects of CsA, yet maintain clinical efficacy. We investigated the in vivo effects of long-term administration of these drugs on bone mineral metabolism in the rat. Five groups of Sprague-Dawley rats, 15 per group, were allocated to receive by daily gavage for a period of 28 days: (1) Cs-vehicle; (2) CsA 15 mg/kg b.w.; (3) CsG 15 mg/kg b.w.; (4) FK506 vehicle; (5) FK506 5 mg/kg b.w. Blood was sampled on days 0, 14, and 28 for measurement of ionized calcium (Ca2+), parathyroid hormone (PTH), 1,25-(OH)2-vitamin D, and bone gla protein (BGP). Tibiae were removed on day 28 after double calcein labeling for histomorphometric analysis. Immunosuppressant groups were compared with the respective vehicle groups. Neither CsA or CsG affected the levels of Ca2+ or PTH, whereas by day 28 FK506 caused a decrease in Ca2+ and a corresponding rise in PTH (P < 0.05). The 1,25-(OH)2-vitamin D and BGP levels in both the CsA and CsG groups were increased on days 14 and 28 (P < 0.05), while FK506 had no effect on these serum levels. Tibial bone histomorphometry revealed that all 3 immunosuppressants increased measures of bone formation and bone resorption, accompanied by a significant reduction in percent trabecular area, most marked with FK506. This report demonstrates that all three immunosuppressants have adverse effects on bone--most deleterious with FK506.

Comparative effects of droloxifene, tamoxifen, and estrogen on bone, serum cholesterol, and uterine histology in the ovariectomized rat model

The purpose of this study was to compare the effects of droloxifene (DRO), tamoxifen (TAM), and 17 alpha-ethynyl estradiol (EE) on bone mineral density, bone histomorphometry, total serum cholesterol, and uterine histology in the ovariectomized (ovx) rat model. Sprague-Dawley female rats at five months of age were sham-operated and treated orally with vehicle (n = 8), or ovx (n = 56) and treated (p.o.) with either vehicle, DRO at 0.1 or 1.0 mg/kg daily, TAM at 0.1 or 1 mg/kg daily, or EE at 3 or 30 micrograms/kg daily for 4 weeks. The uterine wet weight and uterine histologic parameters (cross-sectional tissue area, stromal thickness, and luminal epithelial thickness) were determined. Femoral and lumbar vertebral bone mineral density was determined ex vivo using dual energy x-ray absorptiometry. Static and dynamic cancellous bone histomorphometry was performed on double-labeled, undecalcified longitudinal sections from proximal tibial metaphyses. Furthermore, the changes in total serum cholesterol and body weight gain were also determined. Compared to sham controls, ovx for four weeks significantly decreased uterine weight (-72%), uterine cross-sectional tissue area (-74%), stromal thickness (-52%), and luminal epithelial thickness (-53%). ovx rats treated with EE at 30 micrograms/kg/day maintained these parameters at the levels of sham controls. Uterine weight and uterine cross-sectional tissue area in 3 micrograms/kg/day of EE treated ovx rats were higher than that of vehicle-treated ovx rats. In ovx rats treated with TAM at both 0.1 and 1 mg/kg/day, these parameters were significantly less than sham controls but significantly higher than ovx controls. DRO at 0.1 mg/kg/day had no effects on all above parameters. Uterine weight and cross-sectional tissue area in 1 mg/kg/day of DRO treated ovx rats was slightly but significantly higher than that in ovx controls. However, DRO at 1 mg/kg/day had no effects on uterine stromal thickness and luminal epithelial thickness compared to ovx controls. The ovx-induced decrease in femoral and lumbar vertebral bone mineral density was prevented by treatment with EE at 30 micrograms/kg/day, TAM at both 0.1 and 1 mg/kg/day, or DRO at 1 mg/kg/day. Similarly, the decrease in bone mass and the increase in bone resorption and bone turnover in proximal tibial metaphyses were prevented by treatment with EE at 30 micrograms/kg/day or TAM at both 0.1 and 1 mg/kg/day, or DRO at 1 mg/kg/day. Total serum cholesterol decreased significantly in ovx rats treated with either EE, DRO, or TAM at all dose levels compared to vehicle treated ovx controls (-32% to -56%). The ovx-induced body weight gain was completely prevented by EE at 30 micrograms/kg/day, and partially prevented by DRO at 1 mg/kg/day. TAM at both 0.1 and 1 mg doses caused a significant decrease in body weight compared to both sham and ovx controls. Our results indicated that DRO prevented ovx-induced bone loss and lowered total serum cholesterol with an ED50 less than 1 mg/kg/day. The bone protective and cholesterol lowering effects of DRO were comparable to those observed with TAM and EE. However, DRO differed from TAM and EE in its lack of significant estrogenic effects on uterine tissue at doses which were bone protective. These data suggest that DRO may be a significant alternative to EE and TAM for prevention and treatment of postmenopausal osteoporosis.

Time responses of cancellous and cortical bones to sciatic neurectomy in growing female rats

Effects of unilateral sciatic neurectomy on the responses of both cancellous and cortical bones were studied in growing female rats at 0, 1, 4, 8, and 12 weeks after operation. Using double-fluorescent labeling techniques, histomorphometric analyses were performed on longitudinal sections of proximal tibial metaphyseal secondary spongiosa (PTM) and on cross sections of tibial shaft (TX). In PTM, sciatic neurectomy not only inhibited the age-related bone gain, but also reduced the trabecular bone mass by 46%, which was accompanied by decreases in trabecular number, thickness, and node to node density, and an increase in trabecular separation and free end to free end density. The bone loss occurred mainly between 1 and 4 weeks after operation. A sharp increase in bone formation indices was observed during the first week after nerve section. However, these endpoints quickly dropped to levels lower than those of sham-operated controls at 4 weeks, and were not different from the control levels at 8 weeks after operation. Eroded surface increased progressively after sciatic neurectomy during the 12 weeks experimental period. In TX, sciatic neurectomy inhibited the age-related increase in total tissue area that maintained it at the basal control level. However, the cortical bone area in neurectomized legs was lower than that in sham-operated controls. Sciatic neurectomy also stimulated the bone formation indices on both periosteal and endocortical surfaces during the first week after operation. These endpoints declined sharply between 1 and 4 weeks and then maintained at control levels between 8 and 12 weeks post surgery. Endocortical eroded surface increased 1 week after neurectomy, reached the peak at 8 weeks, and then decreased thereafter. These findings suggest that (1) sciatic neurectomy not only inhibited age-related bone gain but also induced marked bone loss in cancellous bone site and inhibited age-related bone gain in cortical bone site, which mainly resulted from the decrease in bone formation and the increase in bone resorption; (2) the changes in both cancellous and cortical bones responded to sciatic neurectomy occurred mostly within the first 4 weeks and stabilized between 8 and 12 weeks after surgical intervention. In conclusion, the unilateral sciatic neurectomized rat is a complex model in which to study osteopenia. Despite sciatic neurectomy being a simple operation, the interactions of skeletal responses to postsurgical regional acceleratory phenomenon (RAP) and disuse and adaptation changes cannot be clearly differentiated. Furthermore, the complications from growth and aging should be avoided.

Parathyroid Hormone and Mechanical Usage Have a Synergistic Effect in Rat Tibial Diaphyseal Cortical Bone

Previous reports showed that bone mass and architecture only partially recovered by remobilization (RM) after immobilization (IM)‐induced osteopenia, and that parathyroid hormone (PTH) had an anabolic effect on the skeleton. The aim of this study was to determine whether low doses of PTH could restore IM‐induced cortical bone loss and whether a combination of PTH plus loading (RM) treatment would be more effective than the PTH in unloaded (IM) limbs. One hundred and sixty 6‐month‐old rats were divided into aging and IM groups. The right hindlimb of the rat was immobilized by elastic bandage for 18 weeks, and then groups of rats were either kept IM or RM and treated with 30 μg or 80 μg of hPTH(1–38)/kg/day for 2, 10, and 20 weeks. Fluorescent‐labeled, undecalcified cross‐sections of right tibial shafts were studied. We found that RM for 20 weeks after 18 weeks of IM only partially recovered IM‐induced muscle weight loss and PTH had no effect on muscle weight in either IM or RM limbs; that RM for 20 weeks after 18 weeks of IM partially restored some minimal cortical width by stimulating periosteal and endocortical bone formation and decreasing endocortical resorption; that PTH treatment of IM limbs completely restored IM‐induced cortical bone loss and added extra bone by stimulating bone formation indices on all bone surfaces and depressing bone resorption on endocortical surface; that PTH treatment of RM limbs produced similar anabolic effects as in IM limbs with 30 μg/kg/day dose but the 80 μg/kg/day dose‐treated limbs had a higher periosteal bone formation rate, which created a larger cross‐sectional area, more cortical bone area, and a thicker cortex than the same dose treated IM limbs; and that PTH 80 μg/kg/day treatment produced more anabolic effect than the 30 μg/kg/day in both IM and RM limbs. We concluded that reloading the hindlimb by RM after long‐term IM could not recover the cortical bone mass. PTH at employed doses was able to completely restore IM‐induced cortical bone loss, and this effect was independent of mechanical stimulation. However, when PTH was combined with mechanical loading (RM), a synergistic anabolic effect on periosteal bone formation occurred which increased the cross sectional area that can increase bone strength.

The Role of Testosterone in Cyclosporine‐Induced Osteopenia

Our laboratory has demonstrated that the immunosuppressants Cyclosporin A (CsA) and tacrolimus (FK506), in vivo in the rat, produce a high‐turnover osteopenia. CsA is known to decrease serum testosterone (Test) levels both in the rat and in human transplant patients. Less is known of FK506s effect on androgens. CsA‐induced hypogonadism may contribute to the aforementioned bone loss because hypogonadism itself is a risk factor for osteoporosis and fracture. The aim of this study was to assess serum androgen levels following CsA and FK506 therapy and to see whether Test replacement therapy, in the form of 28‐day controlled release subcutaneous pellet implants, could prevent CsA‐induced osteopenia. Two experiments were conducted. In experiment I, four groups of 6‐month‐old male Sprague‐Dawley rats received the following: (A) CsA vehicle and placebo pellet, (B) Test 15 mg pellet and CsA vehicle, (C) CsA 10 mg/kg and placebo pellet, (D) Test 15 mg pellet and CsA 10 mg/kg. In experiment II, two groups of rats received (E) FK506 vehicle and (F) FK506 4 mg/kg. CsA, FK506, and vehicles were given for 28 days by daily oral gavage. The rats were weighed and bled on days 0, 14, and 28. All rats received double fluorescent labeling, and on day 28 the tibiae were removed for histomorphometry. Whole blood was assayed for CsA and FK506 levels. Serum was assayed for total and free Test as well as for osteocalcin (BGP), blood urea nitrogen (BUN), creatinine, and calcium. Whole blood monoclonal CsA levels measured by fluorescent immunoassay were in the therapeutic range, while a drug concentration profile showed good absorption of FK506. Those rats receiving Test and FK506 lost weight, while those receiving CsA remained constant. BUN was only marginally elevated in the CsA‐treated groups on day 28 (p < 0.05), while creatinine was unchanged. On day 28, total and free Test was significantly reduced in the CsA‐treated rats versus control (p < 0.05), while Test replacement therapy maintained total Test levels above vehicle (p < 0.01) and free Test levels similar to vehicle on day 28. FK506 did not lower total or free Test levels. BGP levels were significantly increased in the CsA (p < 0.01) and FK506 (p < 0.001) groups on day 28. BGP in the groups receiving Test alone and in combination with CsA remained similar to vehicle. Histomorphometry confirmed CsA‐ and FK506‐induced high‐turnover osteopenia. The Test alone group marginally increased bone formation. Test replacement failed to prevent the CsA‐induced bone loss. In conclusion, immunosuppressive doses of CsA, but not FK506, lowers serum total and free Test. Hypoandrogenemia does not seem to be a major factor in CsA‐induced osteopenia because bone loss occurs despite Test replacement.

Alendronate prevents cyclosporin A-induced osteopenia in the rat

Post-transplantation bone disease is an increasingly recognized clinical entity whose etiology is multifactorial. The immunosuppressant agent cyclosporine-A (CsA) has repeatedly been shown experimentally to induce a high-turnover osteopenic state. Alendronate (Alen.) is a new generation bisphosphonate having far greater antiresorptive potency than previous bisphosphonates. It inhibits osteoclast resorption in vitro and in vivo without adversely affecting bone mineralization. This study was designed to investigate whether alendronate could prevent CsA-induced osteopenia in the rat. Forty-eight 8-month-old male Sprague Dawley rats were randomized into four groups to receive the following for 28 days: (1) CsA vehicle (veh.) p.o. daily and alendronate vehicle subcutaneously (s.c.) twice/week, (2) CsA 15 mg/kg p.o. daily and Alen. veh. s.c. twice/week, (3) Alen. 70 micrograms/kg s.c. twice/ week and CsA veh. p.o. daily, and (4) CsA 15 mg/kg p.o. daily and Alen. 70 micrograms/kg s.c. twice/week. Rats were weighed and bled and serum was assayed serially for calcium, PTH, 1,25(OH)2vit.D, and osteocalcin. Tibiae were removed following sacrifice on day 28, after double demeclocycline and calcein labeling, for histomorphometric analysis. Treated groups were compared to the vehicle-treated control. We confirmed previous findings that CsA produces elevated 1,25(OH)2 vitamin D and serum osteocalcin levels. Alendronate treatment by itself decreased osteocalcin by day 28 and resulted in a marginal decrease in serum total calcium on day 14. The histomorphometry findings reconfirmed that the administration of CsA induces a state of high-turnover osteopenia. Alendronate prevented CsAs adverse effects, particularly in maintaining trabecular bone volume, presumably by decreasing bone remodeling. Alendronate would seem to hold therapeutic promise in post-transplantation bone disease.

Effect of the Interaction of Parathyroid Hormone and Cyclosporine A on Bone Mineral Metabolism in the Rat

Cyclosporine A (CsA) induces high turnover osteopenia in the rat and there is evidence for this in humans. Recent studies suggest that increases in parathyroid hormone (PTH) may be involved in posttransplantation bone loss. However, human studies are difficult to interpret since transplant patients usually receive a cocktail of immunosuppressants and have underlying disease. Our aim was to try to resolve the influence of the absence or presence of PTH on CsA-induced bone disease. Male Sprague Dawley rats aged 7-9 months, either sham operated or parathyroidectomized (PTX), were randomly divided into vehicle and CsA groups. All PTX rats were given oral calcium supplementation ad libitum. The rats were divided into groups: basal, sham/vehicle, sham/CsA, PTX/vehicle, and PTX/CsA. Serial biochemistry was performed 0, 14, and 28 days after the start of the experimental period; bone histomorphometry was performed 28 days after the start of the experimental period. Statistical analysis consisted of group comparisons and factorial analyses. The results showed that CsA alone produced a high turnover osteopenia consistent with previous studies. In the PTX animals there was an increase in bone mass. PTX also decreased osteoblast activity and recruitment, and serum 1,25OH2D levels. Serum levels of osteocalcin (BGP) were unaffected by PTX. The combination group (PTX/CsA) did not differ statistically from the controls in most of the histomorphometric parameters measured, with the exception of reduced mineral apposition and bone formation rates, reflecting the effects of PTX. Serum BGP and 1,25OH2D levels did not differ, but PTH was reduced from the control. Explanations for these results are (1) CsA and PTX exert their effects via separate mechanisms, negating each other; (2) in the absence of PTH, CsA managed to cause bone loss, and thus PTH may not be essential for CsA-induced bone loss; or (3) the profound accelerated bone loss produced by CsA in normal rats requires PTH. These findings may help explain the discrepancies found in clinical studies where bone loss occurs with either elevated or normal PTH levels.

Prostaglandin E2 Adds Bone to a Cancellous Bone Site with a Closed Growth Plate and Low Bone Turnover in Ovariectomized Rats

The objects of this study were to determine the responses of a cancellous bone site with a closed growth plate (the distal tibial metaphysis, DTM) to ovariectomy (OVX) and OVX plus a prostaglandin E2 (PGE2) treatment, and compare the sites response to previous findings reported for another site (the proximal tibial metaphysis, PTM). Thirty-five 3-month-old female Sprague-Dawley rats were divided into five groups: basal, sham-OVX, and OVX + 0, +1, or +6 mg PGE2/kg/d injected subcutaneously for 3 months and given double fluorescent labels before sacrifice. Cancellous bone histomorphometric analyses were performed on 20-microns-thick undecalcified DTM sections. Similar to the PTM, the DTM showed age-related decreases in bone formation and increases in bone resorption, but it differed in that at 3 months post-OVX, there was neither bone loss nor changes in formation endpoints. Giving 1 mg PGE2/kg/d to OVX rats prevented most age-related changes and maintained the bone formation histomorphometry near basal levels. Treating OVX rats with 6 mg PGE2/kg/d prevented age-related bone changes, added extra bone, and improved microanatomical structure by stimulating bone formation without altering bone resorption. Furthermore, after PGE2 administration, the DTM, a cancellous bone site with a closed growth plate, increased bone formation more than did the cancellous bone in the PTM.

Immunosuppressant use without bone loss--implications for bone loss after transplantation.

Cyclosporine A (CsA) is associated with posttransplantation bone disease. Immunosuppressant drugs such as sirolimus (SRL), which are more potent and less deleterious than CsA, are being developed. Previous experiments have shown that SRL although immunosuppressive, is relatively bone sparing. The use of low doses of CsA and SRL in combination has displayed in vivo synergism. This study was initiated to examine the effect of low‐dose CsA and SRL on bone metabolism, thereby hopefully providing a bone sparing immunosuppressive regimen for transplant recipients. One hundred and nineteen rats were divided into groups: basal, vehicle, CsA high dose, CsA low dose, SRL low dose, and combination low‐dose CsA and SRL. The basal group was killed on day 0 for histomorphometry. The experimental groups were weighed and bled on days 0, 28, 56, and 84 and were killed on day 84 for histomorphometry. Serial assays for blood urea nitrogen (BUN), creatinine, and osteocalcin were performed. Osteocalcin was raised on days 28 and 56 in the high dose CsA group. Histomorphometry showed osteopenia with high‐dose CsA. Low‐dose CsA was relatively bone sparing, while low‐dose SRL and combined low‐dose CsA did not cause bone loss. In conclusion, the synergistic combination of low‐dose CsA and SRL has the potential of providing both bone sparing and immunosuppressive benefits.

Effects of droloxifene on prevention of cancellous bone loss and bone turnover in the axial skeleton of aged, ovariectomized rats

The purpose of this study was to determine the efficacy of droloxifene (DRO), an estrogen antagonist/agonist, in preventing ovariectomy (OVX)-induced lumbar vertebral cancellous bone loss and bone turnover in aged female rats. Fifty-three Sprague-Dawley female rats were OVX or sham-operated at 19 months of age, and divided into 6 groups: (I) sham-operated controls; (II) OVX vehicle controls; (III) OVX rats treated with E2 at 30 micrograms/kg/day; (IV)-(VI) OVX rats treated with DRO at either 2.5, 5, or 10 mg/kg p.o. daily. The treatment period was 8 weeks. Static and dynamic cancellous bone histomorphometric parameters were determined on 4 and 10 microns thick, undecalcified, double-fluorescent labeled sections of the fourth lumbar vertebral body. Changes in body weight, uterine weight, and total serum cholesterol were also determined. OVX for 8 weeks in 19-month-old female rats resulted in reduced trabecular bone volume (-18%) and trabecular width (-10%) and increased labeling perimeter (+52%), bone formation rate/bone surface referent (+60%), bone formation rate/bone volume referent (+77%), osteoclast number (+41%), and osteoclast perimeter (+41%). E2 treatment at 30 micrograms/kg/day for 8 weeks prevented OVX-induced cancellous bone loss and decreased bone resorption, bone formation, and bone turnover to the values of sham controls. DRO at 2.5-10 mg/kg/day completely prevented bone loss and bone turnover associated with estrogen deficiency. Osteoclast number and perimeter were significantly decreased in DRO-treated-OVX rats compared to both sham and OVX controls. Trabecular bone volume, trabecular width, labeling perimeter, bone formation rate/bone surface referent, and bone formation rate/bone volume referent showed no differences in DRO-treated OVX rats compared to those of E2-treated OVX rats and sham controls. These histomorphometric results indicated that DRO is an estrogen agonist on cancellous bone of lumbar vertebral bodies of aged, OVX rats. Further, E2 treatment prevented the OVX-induced increase in body weight gain and nonsignificantly reduced total serum cholesterol compared to OVX controls. Body weight gain and total serum cholesterol did not differ between OVX rats treated with E2 and sham controls. In OVX rats treated with DRO, body weight decreased significantly in a dose-response manner, and total serum cholesterol was significantly reduced by 65% to 70% compared to both sham and OVX controls. In addition, treatment with E2 increased uterine weight to the value of sham controls in OVX rats. However, DRO had no effect on uterine weight at either 2.5 or 10 mg/kg/day, while it only slightly but significantly increased uterine weight over OVX controls at 5 mg/kg/day. We conclude that DRO was efficacious in the prevention of lumbar vertebral cancellous bone loss and in the decline of total serum cholesterol but had no effect on uterine weight in the aged, OVX female rats. Our data suggest that DRO is a potentially useful agent for the prevention of vertebral bone loss leading to spinal fractures in postmenopausal women.