Ultrastructural Evaluation of the Interaction of Glucocorticoids and Vitamin D on Bone Cells in Thyroparathyroidectomized Rats Steven E. Weisbrode, VMD, PhD, and Charles C. Capen, DVM, PhD
To determine the direct effects of cortisol on bone. rats were thyroparathyroidectomized (T.'PTx). fed a low-calcium diet, and given high (50 mg/kg) or low (8 mg/kg) pharmacologic levels of cortisol with or without excess vitamin D3 (15.000 IU). Rats given vitamin D had osteoblasts and osteocytes interpreted ultrastructurally to be actively engaged in matrix synthesis, mineralization of matrix, and in calcium mobilization. Osteoclasts were numerous on metaphyseal trabeculae and in vascular channels of cortical bone. In T.XPTX rats not given *itamin D. osteoblasts and osteocy-tes were interpreted to have reduced metabolic activity with minimal evidence of participation in bone formation or resorption. Cortisol at both dose levels failed to alter the electron microscopic appearance of osteoblasts, osteocytes, and osteoclasts with or without vitamin D. Bone turnover indicated by urinary hvdroxyproline excretion was unaffected by cortisol treatment. These findings suggest that glucocorticoids have little direct action on bone cells and that their effects on calcium metabolism are probably mediated by an interference in intestinal calcium transport and by secondary hyperparathyroidism. (Am J Pathol 84:457-468, 1976)
THE I\FLU-ENCE OF GLUCOCORTICOIDS on calcium metabolism is complex. Glucocorticoids are able to low-er serum calcium by inhibiting intestinal transport of calcium 1 and increasing urinary calcium excretion.2 In intact animals. this depression of serum calcium by lomv levels of glucocorticoids induces secondary hyvperparathy roidism with subsequent bone resorption.3 High lev-els of glucocorticoids inhibit bone resorption and formation bv decreasing precursor cell proliferation and modulation to osteoblasts and osteoclasts.3 Glucocorticoid inhibition of bone resorption is synergistic with endogenous calcitonin secretion.4 Although several in vitro studies hav-e sho,s-n a decrease in bone collagen synthesis due to glucocorticoids.5- the direct effect of glucocorticoids on bone cell function is poorly understood. To study the direct effects of cortisol on bone in the absence of endogenous calcitonin and parathy roid hormone secretion and independent of intestinal calcium absorption. the present investigation -was undertaken to evaluate the effects of cortisol alone and in combination s-ith v-itamin D on bone of thyroparathyroidectomized (TxPTx) rats fed a los- calcium diet. From the Departnmernt of V eterinar\ Patholopg. C (ollece oV\ eternar\ Medicine The Ohio State L ni' ersit\. Coluimbus. Ohi(). Suppixrted in part b\ General Research CGrant RR0O3446f3 from the \ational Inistittutes of Health. anid b\ the L,nisersit\ Small Grants Procram. The Graduate Sch(,xd. The Ohio State 1.nisersit\. Xccepted for publication xpril 9. 1976. Xddrecs reprinit requests to Dr. Ste\en W'seishrode. Departmernt (f Veterinar\ Patholo, C(ollece f \Veterinarv \Medicine. Ohio State Unisersitv. 1923 CoffeG Road. C.olumbus. OH 4:3210. 457
458
WEISBRODE AND CAPEN
American Journal of Pathology
Materials and Methos Annl ad
ernDein
Mtale Sprague-Dawley rats weighing 300 to 350 g were surgically thyroparathyroidectomized (TXPTX). Rats with serum calcium levels below 7.5 mg/ dl at 48 hours after operation were divided into six groups: Group 1-Cortisol placebo (11 rats). Group 2Low-dose cortisol: hydrocortisone acetate, 8 mg/'kg (14 rats). Group 3-High-dose cortisol: hvdrocortisone acetate, 50 mg/kg (11 rats). Group 4-Cortisol placebo and 15.000 ItU vitamin D), (11 rats). Group 5-Low-dose cortisol, 8 mg/kg, and 15,000 IU vitamin D (10 rats). Group 6-Cortisol, 50 mg/kg, and 15,000 IU vitamin D (11 rats). Cortisol was administered subcutaneously; vitamin D was administered in cotton seed oil, per os, daily for 7 days. All rats were control fed a low-calcium (0.05%), normal phosphorus (0.3%) diet (Teklad Test Diets, Madison, Wisc., TD 71077) and given daily subcutaneous injections of 10 ,gg sodium levothyroxine (Travenol Laboratories, Inc., Morton Grove, Ill.). Urine was collected during the 24 hours prior to euthanasia for determination of total 24-hour urinary hvdroxvproline excretion by the method of Kivirikko et al.- At the end of the 7-dav period, all rats were exsanguinated from the abdominal aorta under light ether anesthesia. Terminal serum calcium levels were measured by atomic absorption spectrophotometry (Perkin-Elmer Model 303) and serum phosphorus levels according to the method of Fiske and Subbarow.' Morphologic studies were performed on 7 rats from each group. For light microscopy. tibias were dissected free of soft tissue, divided longitudinally, fixed in buffered formalin. decalcified to effect in 1O0c ethylenediaminetetraacetic acid disodium salt (EDTA), dehy-drated in alcohol, embedded in paraffin, section at 6 A, and stained with hematoxylin and eosin.
Eecro M c Bone sections from the tibial diaphysis and metaphysis (approximately 1 cu mm) were quicklv dissected free of soft tissue and fixed in 3% glutaraldehyde with 0.1 Mt sodium cacodylate buffer at pH 7.4. The tissue was postfixed in 1% osmium tetroxide with scollidine buffer at pH 7.4, dehydrated through ascending concentrations of ethanol, transferred to propylene oxide, and embedded in "hard" Epon (Shell Chemical Company, New York, NY). Thin sections were cut with a diamond knife on an LKB ultramicrotome, and sections were floated on a water bath buffered at pH 7.4 to prevent demineralization. Sections were stained with uranyl acetate and lead citrate and examined with a Philips 300 electron microscope. Data were evaluated for statistical significance using the Student t test.
Results Low pharmacologic levels of cortisol did not produce significant light microscopic changes in tibial bone. In rats given high pharmacologic levels of cortisol, osteoclasts in the metaphysis were reduced in number, but tibial bone was otherwise unchanged. The length of growth plates in the proximal tibia was reduced in rats given low (151 i 15 A) and high (135 ± 9 i) doses of cortisol compared to controls (176 i 20 y), but these differences were not statistically significant. Cortisol did not alter the light microscopic appearance of tibial bone in rats given vitamin D. However, the growth plates were significantly narrowed in the rats treated with high
Vol. 84, No. 3 September 1976
GLUCOCORTICOIDS AND VITAMIN D IN BONE
459
(160 ± 20 A, P
To determine the direct effects of cortisol on bone. rats were thyroparathyroidectomized (T.'PTx). fed a low-calcium diet, and given high (50 mg/kg) or low (8 mg/kg) pharmacologic levels of cortisol with or without excess vitamin D3 (15.000 IU). Rats given vitamin D had osteoblasts and osteocytes interpreted ultrastructurally to be actively engaged in matrix synthesis, mineralization of matrix, and in calcium mobilization. Osteoclasts were numerous on metaphyseal trabeculae and in vascular channels of cortical bone. In T.XPTX rats not given *itamin D. osteoblasts and osteocy-tes were interpreted to have reduced metabolic activity with minimal evidence of participation in bone formation or resorption. Cortisol at both dose levels failed to alter the electron microscopic appearance of osteoblasts, osteocytes, and osteoclasts with or without vitamin D. Bone turnover indicated by urinary hvdroxyproline excretion was unaffected by cortisol treatment. These findings suggest that glucocorticoids have little direct action on bone cells and that their effects on calcium metabolism are probably mediated by an interference in intestinal calcium transport and by secondary hyperparathyroidism. (Am J Pathol 84:457-468, 1976)
THE I\FLU-ENCE OF GLUCOCORTICOIDS on calcium metabolism is complex. Glucocorticoids are able to low-er serum calcium by inhibiting intestinal transport of calcium 1 and increasing urinary calcium excretion.2 In intact animals. this depression of serum calcium by lomv levels of glucocorticoids induces secondary hyvperparathy roidism with subsequent bone resorption.3 High lev-els of glucocorticoids inhibit bone resorption and formation bv decreasing precursor cell proliferation and modulation to osteoblasts and osteoclasts.3 Glucocorticoid inhibition of bone resorption is synergistic with endogenous calcitonin secretion.4 Although several in vitro studies hav-e sho,s-n a decrease in bone collagen synthesis due to glucocorticoids.5- the direct effect of glucocorticoids on bone cell function is poorly understood. To study the direct effects of cortisol on bone in the absence of endogenous calcitonin and parathy roid hormone secretion and independent of intestinal calcium absorption. the present investigation -was undertaken to evaluate the effects of cortisol alone and in combination s-ith v-itamin D on bone of thyroparathyroidectomized (TxPTx) rats fed a los- calcium diet. From the Departnmernt of V eterinar\ Patholopg. C (ollece oV\ eternar\ Medicine The Ohio State L ni' ersit\. Coluimbus. Ohi(). Suppixrted in part b\ General Research CGrant RR0O3446f3 from the \ational Inistittutes of Health. anid b\ the L,nisersit\ Small Grants Procram. The Graduate Sch(,xd. The Ohio State 1.nisersit\. Xccepted for publication xpril 9. 1976. Xddrecs reprinit requests to Dr. Ste\en W'seishrode. Departmernt (f Veterinar\ Patholo, C(ollece f \Veterinarv \Medicine. Ohio State Unisersitv. 1923 CoffeG Road. C.olumbus. OH 4:3210. 457
458
WEISBRODE AND CAPEN
American Journal of Pathology
Materials and Methos Annl ad
ernDein
Mtale Sprague-Dawley rats weighing 300 to 350 g were surgically thyroparathyroidectomized (TXPTX). Rats with serum calcium levels below 7.5 mg/ dl at 48 hours after operation were divided into six groups: Group 1-Cortisol placebo (11 rats). Group 2Low-dose cortisol: hydrocortisone acetate, 8 mg/'kg (14 rats). Group 3-High-dose cortisol: hvdrocortisone acetate, 50 mg/kg (11 rats). Group 4-Cortisol placebo and 15.000 ItU vitamin D), (11 rats). Group 5-Low-dose cortisol, 8 mg/kg, and 15,000 IU vitamin D (10 rats). Group 6-Cortisol, 50 mg/kg, and 15,000 IU vitamin D (11 rats). Cortisol was administered subcutaneously; vitamin D was administered in cotton seed oil, per os, daily for 7 days. All rats were control fed a low-calcium (0.05%), normal phosphorus (0.3%) diet (Teklad Test Diets, Madison, Wisc., TD 71077) and given daily subcutaneous injections of 10 ,gg sodium levothyroxine (Travenol Laboratories, Inc., Morton Grove, Ill.). Urine was collected during the 24 hours prior to euthanasia for determination of total 24-hour urinary hvdroxvproline excretion by the method of Kivirikko et al.- At the end of the 7-dav period, all rats were exsanguinated from the abdominal aorta under light ether anesthesia. Terminal serum calcium levels were measured by atomic absorption spectrophotometry (Perkin-Elmer Model 303) and serum phosphorus levels according to the method of Fiske and Subbarow.' Morphologic studies were performed on 7 rats from each group. For light microscopy. tibias were dissected free of soft tissue, divided longitudinally, fixed in buffered formalin. decalcified to effect in 1O0c ethylenediaminetetraacetic acid disodium salt (EDTA), dehy-drated in alcohol, embedded in paraffin, section at 6 A, and stained with hematoxylin and eosin.
Eecro M c Bone sections from the tibial diaphysis and metaphysis (approximately 1 cu mm) were quicklv dissected free of soft tissue and fixed in 3% glutaraldehyde with 0.1 Mt sodium cacodylate buffer at pH 7.4. The tissue was postfixed in 1% osmium tetroxide with scollidine buffer at pH 7.4, dehydrated through ascending concentrations of ethanol, transferred to propylene oxide, and embedded in "hard" Epon (Shell Chemical Company, New York, NY). Thin sections were cut with a diamond knife on an LKB ultramicrotome, and sections were floated on a water bath buffered at pH 7.4 to prevent demineralization. Sections were stained with uranyl acetate and lead citrate and examined with a Philips 300 electron microscope. Data were evaluated for statistical significance using the Student t test.
Results Low pharmacologic levels of cortisol did not produce significant light microscopic changes in tibial bone. In rats given high pharmacologic levels of cortisol, osteoclasts in the metaphysis were reduced in number, but tibial bone was otherwise unchanged. The length of growth plates in the proximal tibia was reduced in rats given low (151 i 15 A) and high (135 ± 9 i) doses of cortisol compared to controls (176 i 20 y), but these differences were not statistically significant. Cortisol did not alter the light microscopic appearance of tibial bone in rats given vitamin D. However, the growth plates were significantly narrowed in the rats treated with high
Vol. 84, No. 3 September 1976
GLUCOCORTICOIDS AND VITAMIN D IN BONE
459
(160 ± 20 A, P
