Effects of hind limb unloading and reloading on nitric oxide synthase expression and apoptosis of osteocytes and chondrocytes.

In rat bone, the absence of mechanical load results in a reduction in bone formation, inhibition of longitudinal growth, and a decrease in the number of osteoblasts and osteoprogenitors in cancellous bone. Unloading has also been linked to an increase in apoptosis of osteocytes and chondrocytes through production of nitric oxide (NO) and increased expression of NO synthases (NOS). Reloading results in recovery of bone volume within 14 days, although osteoblast and osteoclast numbers remain below control values, suggesting decreased bone turnover. This study was designed to evaluate the effects of hind limb unloading and subsequent reloading on apoptosis, NOS expression, and histomorphometric parameters in trabecular and cortical bone, articular cartilage, and growth plate cartilage of the proximal tibia of the hind limbs. Compared to ambulatory controls, 2 weeks of unloading resulted in a 66% increase in the percentage of apoptotic osteocytes in the trabecular metaphysis, a 14% increase in osteoclast number and a 48% decrease in bone volume. The percentage of eNOS- or iNOS-positive osteocytes was unchanged. Upon reloading, the percentage of apoptotic osteocytes and bone volume returned to baseline whereas the percentage of iNOS-positive osteocytes increased by 50% and osteoclast number decreased by 30% compared to ambulatory controls. More striking changes were observed in articular and growth plate cartilage. Unloading resulted in a 230% increase in apoptotic chondrocytes, a 400% increase in iNOS-positive chondrocytes, and a 17% reduction in width in articular cartilage. Reloading for 2 weeks resulted in partial recovery. Chondrocytes in the proliferative and hypertrophic zones of the growth plate responded similarly to those in the articular cartilage. In summary, we observed that 14 days of unloading increased apoptosis of osteocytes and chondrocytes. This was associated with an increase in the proportion of iNOS-positive chondrocytes whereas the proportion of iNOS-positive osteocytes remained unchanged. Reloading for 14 days restored osteocyte apoptosis to control levels but the percentage of iNOS- and eNOS-positive osteocytes increased in reloaded bone compared to controls. This was associated with a decrease in osteoclast number. In cartilage, reloading for 2 weeks did not result in a return to baseline in any of the parameters measured, suggesting that the effects of unloading on articular cartilage and the growth plate last longer than those in bone and may have prolonged effects on joint biomechanics and longitudinal bone growth.

[1]  Theo H Smit,et al.  Strain-derived canalicular fluid flow regulates osteoclast activity in a remodelling osteon--a proposal. , 2003, Journal of biomechanics.

[2]  L. Vico,et al.  Rat Hindlimb Unloading by Tail Suspension Reduces Osteoblast Differentiation, Induces IL-6 Secretion, and Increases Bone Resorption in Ex Vivo Cultures , 2002, Calcified Tissue International.

[3]  N Loveridge,et al.  Identification of apoptotic changes in osteocytes in normal and pathological human bone. , 1997, Bone.

[4]  Wronski Tj,et al.  Recovery of the rat skeleton from the adverse effects of simulated weightlessness. , 1983 .

[5]  E Park,et al.  A simple hindlimb suspension apparatus. , 1993, Aviation, space, and environmental medicine.

[6]  P. Collin‐Osdoby,et al.  Bone cell function, regulation, and communication: A role for nitric oxide , 1995, Journal of cellular biochemistry.

[7]  I. Shapiro,et al.  Phosphate-induced chondrocyte apoptosis is linked to nitric oxide generation. , 2001, American journal of physiology. Cell physiology.

[8]  C. G. Bellows,et al.  The effect of reloading on bone volume, osteoblast number, and osteoprogenitor characteristics: studies in hind limb unloaded rats. , 2005, Bone.

[9]  S. Doty,et al.  Histomorphometric analysis of rat skeleton following spaceflight. , 1987, The American journal of physiology.

[10]  J M Polak,et al.  Mechanical Strain Stimulates Nitric Oxide Production by Rapid Activation of Endothelial Nitric Oxide Synthase in Osteocytes , 1999, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[11]  R. Globus,et al.  The role of 1,25-dihydroxyvitamin D in the inhibition of bone formation induced by skeletal unloading. , 1986, Endocrinology.

[12]  S. Ralston,et al.  Requirement of the inducible nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[13]  A. Zeiher,et al.  Nitric oxide and apoptosis: another paradigm for the double-edged role of nitric oxide. , 1997, Nitric oxide : biology and chemistry.

[14]  Toshitaka Nakamura,et al.  Role of Inducible Nitric Oxide Synthase in Skeletal Adaptation to Acute Increases in Mechanical Loading , 2002, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[15]  Emily R Morey-Holton,et al.  Hindlimb unloading rodent model: technical aspects. , 2002, Journal of applied physiology.

[16]  A. van der Plas,et al.  Sensitivity of osteocytes to biomechanical stress in vitro , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[17]  X. Holy,et al.  Systemic administration of transforming growth factor-beta 2 prevents the impaired bone formation and osteopenia induced by unloading in rats. , 1995, The Journal of clinical investigation.

[18]  A. Zeiher,et al.  Shear stress inhibits H2O2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase. , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[19]  D. Reid,et al.  Evidence for a Pathogenic Role of Nitric Oxide in Inflammation‐Induced Osteoporosis , 1999, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[20]  K. Brandt.  Response of joint structures to inactivity and to reloading after immobilization. , 2003, Arthritis and rheumatism.

[21]  P. Duke,et al.  Spaceflight and age affect tibial epiphyseal growth plate histomorphometry. , 1992, Journal of applied physiology.

[22]  J. Arokoski,et al.  Remobilization does not fully restore immobilization induced articular cartilage atrophy. , 1999, Clinical orthopaedics and related research.

[23]  Jenneke Klein-Nulend,et al.  Shear stress inhibits while disuse promotes osteocyte apoptosis. , 2004, Biochemical and biophysical research communications.

[24]  D. Reid,et al.  Activation of the inducible nitric oxide synthase pathway contributes to inflammation-induced osteoporosis by suppressing bone formation and causing osteoblast apoptosis. , 2001, Arthritis and rheumatism.

[25]  R. Ochs,et al.  Chondrocyte apoptosis induced by nitric oxide. , 1995, The American journal of pathology.

[26]  P. Cerri,et al.  Combined TUNEL and TRAP methods suggest that apoptotic bone cells are inside vacuoles of alveolar bone osteoclasts in young rats. , 2003, Journal of periodontal research.

[27]  T. Kasten,et al.  Potentiation of osteoclast bone-resorption activity by inhibition of nitric oxide synthase. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[28]  L. Lanyon,et al.  Mechanical loading: biphasic osteocyte survival and targeting of osteoclasts for bone destruction in rat cortical bone. , 2003, American journal of physiology. Cell physiology.

[29]  P. Nibbering,et al.  Inducible production of nitric oxide in osteoblast-like cells and in fetal mouse bone explants is associated with suppression of osteoclastic bone resorption. , 1994, The Journal of clinical investigation.

[30]  X. Holy,et al.  Insulin-like growth factor-I increases trabecular bone formation and osteoblastic cell proliferation in unloaded rats. , 1994, Endocrinology.

[31]  E. Burger,et al.  DNA fragmentation during bone formation in neonatal rodents assessed by transferase‐mediated end labeling , 1996, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[32]  E. Bonucci,et al.  In vivo incidence of apoptosis evaluated with the TdT FragEL DNA fragmentation detection kit in cartilage and bone cells of the rat tibia. , 1998, Tissue & cell.

[33]  P. Collin‐Osdoby,et al.  Decreased Nitric Oxide Levels Stimulate Osteoclastogenesis and Bone Resorption Both in Vitro and in Vivo on the Chick Chorioallantoic Membrane in Association with Neoangiogenesis , 2000, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[34]  T. Gross,et al.  Osteocyte hypoxia: a novel mechanotransduction pathway. , 1999, American journal of physiology. Cell physiology.

[35]  K. Brandt,et al.  Development and reversal of a proteoglycan aggregation defect in normal canine knee cartilage after immobilization. , 1979, Arthritis and rheumatism.

[36]  L. Vico,et al.  Bone histomorphometric comparison of rat tibial metaphysis after 7-day tail suspension vs. 7-day spaceflight. , 1991, Aviation, space, and environmental medicine.

[37]  J M Polak,et al.  Bidirectional regulation of osteoclast function by nitric oxide synthase isoforms. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[38]  J. Frangos,et al.  Venous ligation-mediated bone adaptation is NOS 3 dependent. , 2004, Bone.

[39]  O. Verborgt,et al.  Loss of Osteocyte Integrity in Association with Microdamage and Bone Remodeling After Fatigue In Vivo , 2000, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[40]  F. Blanco,et al.  IL-1-induced nitric oxide inhibits chondrocyte proliferation via PGE2. , 1995, Experimental cell research.

[41]  P. W. Johnston,et al.  Nitric oxide: A cytokine‐induced regulator of bone resorption , 1995, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[42]  E. Bonucci,et al.  Evaluation of apoptosis and the glucocorticoid receptor in the cartilage growth plate and metaphyseal bone cells of rats after high-dose treatment with corticosterone. , 2000, Bone.

[43]  D P Fyhrie,et al.  Intracortical remodeling in adult rat long bones after fatigue loading. , 1998, Bone.

[44]  X. Holy,et al.  TGF-beta2 prevents the impaired chondrocyte proliferation induced by unloading in growth plates of young rats. , 1997, Life sciences.

[45]  S. Ralston,et al.  Nitric oxide and bone , 2001, Immunology.

[46]  J. Polak,et al.  Endothelial nitric oxide synthase gene-deficient mice demonstrate marked retardation in postnatal bone formation, reduced bone volume, and defects in osteoblast maturation and activity. , 2001, The American journal of pathology.

[47]  A. Zeiher,et al.  Nitric Oxide Down-regulates MKP-3 mRNA Levels , 2000, The Journal of Biological Chemistry.

[48]  J. Wit,et al.  The Role of Estrogen in the Control of Rat Osteocyte Apoptosis , 1998, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[49]  S. Abramson,et al.  The role of nitric oxide in articular cartilage breakdown in osteoarthritis. , 1998, Current opinion in rheumatology.

[50]  D Montufar-Solis,et al.  In vivo and in vitro studies of cartilage differentiation in altered gravities. , 1996, Advances in space research : the official journal of the Committee on Space Research.

[51]  C. Nathan,et al.  Nitric oxide and macrophage function. , 1997, Annual review of immunology.

[52]  W. O'Brien,et al.  Perinatal Development of Endothelial Nitric Oxide Synthase-Deficient Mice1 , 2001, Biology of reproduction.

[53]  D. Marsh,et al.  Rapid new bone tissue remodeling during distraction osteogenesis is associated with apoptosis , 2003, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.