Relationship between pre-radiographic cartilage damage following anterior cruciate ligament injury and biomarkers of cartilage turnover in clinical practice: a cross-sectional observational study.

OBJECTIVE: To determine whether differences in synovial fluid (SF) biomarkers of collagen and proteoglycan turnover are associated with pre-radiographic damage to articular cartilage and menisci following anterior cruciate ligament (ACL) injury and are of clinical value. METHOD: SF samples from ACL injured knees of 108 patients were obtained when damage to cartilages and menisci was evaluated arthroscopically. Concentrations of SF collagenase-generated cleavage neoepitope of type II collagen (C2C) were determined using ELISA and aggrecan-derived disaccharides of chondroitin-4-sulfate (Δdi-C4S), chondroitin-6-sulfate (Δdi-C6S), and keratan sulfate (KS), were measured in SF by High performance liquid chromatography (HPLC). RESULTS: Radiographic examination failed to detect any intra-articular degenerative changes. The number of high-grade cartilage lesions was positively associated with age, duration after injury and the level of C2C, and negatively with the level of KS. There was no association between the number of high-grade cartilage and meniscal lesions. Multivariable logistic regression revealed significant associations of increased C2C (adjusted Odds ratio (OR) of the upper quartile to remainder of 2.49, 95% Confidence interval (CI) = 0.85-7.27) and decreased KS (adjusted OR of the lower quartile to the remainder of 3.32, 95% CI = 1.19-9.24) with the presence of three or more high-grade cartilage lesions, independent of age and duration after injury. The combined impact of increased C2C and decreased KS was 22.8 (95% CI = 1.95-265.9), far exceeding the impact of each independent biomarker. CONCLUSION: Combinations of the C2C and KS as described here may offer greater ability to identify patients with early pre-radiographic high-grade cartilage damage compared to single clinical or biomarker parameters.

Atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knee: a prospective multicenter clinical trial in Japan.

BACKGROUND: New tissue-engineering technology was developed to create a cartilage-like tissue in a three-dimensional culture using atelocollagen gel. The minimum 2-year followup outcome of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in knees was reported from the single institution. The present multicenter study was conducted to determine clinical and arthroscopic outcomes in patients who underwent atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knees. METHODS: At six medical institutes in Japan, we prospectively evaluated the clinical and arthroscopic outcomes of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in 27 patients (27 knees) with cartilage lesions on a femoral condyle or on a patellar facet over 24 months. RESULTS: The Lysholm score significantly increased from 60.0 +/- 13.7 points to 89.8 +/- 9.5 points (P = 0.001). Concerning the ICRS grade for arthroscopic appearance, 6 knees (24%) were assessed as grade I (normal) and 17 knees (68%) as grade II (nearly normal). There were few adverse features, except for detachment of the graft in two cases. CONCLUSIONS: We concluded that transplanting chondrocytes in a newly formed matrix of atelocollagen gel can promote restoration of the articular cartilage of the knee.

TNF-alpha increases expression of IL-6 and ICAM-1 genes through activation of NF-kappaB in osteoblast-like ROS17/2.8 cells.

Tumor necrosis factor-alpha (TNF-alpha) plays a key role in inflammatory diseases such as rheumatoid arthritis and in postmenopausal osteoporosis. In various tissues, TNF-alpha action is mediated by a transcription factor, nuclear factor-kappa B (NF-kappaB). However, little is known about how TNF-alpha exerts its action in osteoblasts. We thus examined the effect of TNF-alpha on the activation of NF-kappaB in rat osteoblast-like osteosarcoma cells (ROS17/2.8). Electrophoretic mobility shift assay revealed that the activation of the p50-p65 heterodimer NF-kappaB was induced by TNF-alpha as early as 15 minutes followed by a persistent activation for 48 h. When the binding activity of NF-kappaB in cytosol was examined using detergents that dissociate NF-kappaB from an inhibitory protein IkappaB, it decreased during the initial 30 minutes and then increased to the unstimulated level. Northern blot analysis revealed a marked increase in the mRNA levels of p105, a precursor of p50, 6 h after TNF-alpha and a gradual increase in p65 mRNA levels during the initial 1 h. Significant increase in both mRNA levels continued until 24 h after TNF-alpha. These results suggest that the rapid activation of NF-kappaB by TNF-alpha is mainly due to the nuclear translocation of NF-kappaB pre-existing in cytosol, and that the subsequent increase in the expression of p50 and p65 may result in the persistent activation of NF-kappaB during TNF-alpha stimulation. TNF-alpha also increased the mRNA levels of interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1). An antioxidant, N-acetyl-L-cysteine, significantly attenuated the TNF-alpha-dependent increase in these mRNAs, and simultaneously reduced the activation of NF-kappaB by TNF-alpha, indicating that NF-kappaB mediates the TNF-alpha-dependent expression of IL-6 and ICAM-1 in ROS17/2.8 cells. These results suggest that the activation of NF-kappaB by TNF-alpha may play an important role in the production of cytokines and cell adhesion molecules from osteoblasts, leading to the promotion of bone resorption and inflammation.

Effects of glucocorticoids on tumor necrosis factor alpha-dependent activation of nuclear factor kappaB and expression of the intercellular adhesion molecule 1 gene in osteoblast-like ROS17/2.8 cells.

Recently, we showed that tumor necrosis factor alpha (TNF-alpha) stimulates expression of the intercellular adhesion molecule 1 (ICAM-1) and interleukin-6 (IL-6) genes through activation of p65-p50 heterodimer nuclear factor KB (NF-kappaB) in rat osteoblast-like ROS17/2.8 cells. In the present study, we investigated effects of a synthetic glucocorticoid, dexamethasone (Dex), on TNF-alpha-dependent activation of NF-kappaB and expression of the ICAM-1 gene. ROS17/2.8 cells were pretreated with Dex for 6 h and then exposed to TNF-alpha. Electrophoretic mobility shift assay (EMSA) revealed that TNF-alpha-dependent activation of NF-kappaB was almost completely suppressed by Dex treatment. Increase in ICAM-1 messenger RNA (mRNA) level by TNF-alpha also was markedly suppressed by Dex. Western blot and immunocytochemical analyses showed that Dex attenuated the TNF-alpha-induced nuclear translocation of p65. Treatment with protein synthesis inhibitor cycloheximide (CHX) reversed the Dex effect, indicating that Dex requires de novo protein synthesis for its action. Northern blot analysis revealed that Dex increased IkappaB-alpha mRNA level synergistically with TNF-alpha, whereas it decreased p65 mRNA level. The p105 and IkappaB-beta mRNA levels were not altered by Dex. Consistent with the mRNA level, Dex increased the amount of IkappaB-alpha protein in the cytoplasm in either the presence or the absence of TNF-alpha. Considering a role of IkappaB to sequester NF-kappaB in the cytoplasm, it was suggested that an increase in IkappaB-alpha protein and the concomitant decrease in p65 synthesis account for the Dex-induced suppression of NF-kappaB activation in osteoblastic cells.

Tumor necrosis factor alpha induces expression of genes for matrix degradation in human chondrocyte-like HCS-2/8 cells through activation of NF-kappaB: abrogation of the tumor necrosis factor alpha effect by proteasome inhibitors.

Tumor necrosis factor alpha (TNF-alpha) has been suggested to induce chondrocytic chondrolysis in both inflammatory and degenerative joint diseases. However, its intracellular signaling pathway leading to the chondrolysis has not been studied in detail. Thus, we investigated whether TNF-alpha activates a transcription factor nuclear factor kappaB (NF-kappaB) in human chondrocyte-like cells (HCS-2/8) and induces the expression of genes involved in the degradation of cartilage matrix. Treatment of the cells with TNF-alpha markedly increased the levels of matrix metalloproteinase 1 (MMP-1), MMP-3, intercellular adhesion molecule 1 (ICAM-1), and cyclo-oxygenase 2 (COX-2) messenger RNAs (mRNAs). The increase in the mRNAs was associated with the activation of p65/p50 heterodimer NF-kappaB. IkappaB-alpha and IkappaB-beta, cytoplasmic molecules preventing the nuclear translocation of NF-kappaB, were degraded rapidly by TNF-alpha followed by their synthesis to the basal level. Treatment with proteasome inhibitors inhibited the degradation of both IkappaB-alpha and IkappaB-beta and prevented the TNF-alpha-dependent nuclear translocation of p65. Furthermore, the inhibitors completely prevented the TNF-alpha-dependent induction of MMP-1, MMP-3, ICAM-1, and COX-2 mRNAs. Thus, it is shown that the activation of p65/p50 NF-kappaB by TNF-alpha plays a cardinal role in inducing the expression of MMP-1, MMP-3, ICAM-1, and COX-2 genes, which are involved in matrix degradation and inflammatory reaction in chondrocytes, leading to chondrocytic chondrolysis.

Immature platelet fraction measurement in patients with chronic liver disease: a convenient marker for evaluating cirrhotic change.

Platelet number is often used as an indicator of the severity of liver disease. Although inadequate thrombopoietin production and decreased platelet production have been proposed as major causes of cirrhotic thrombocytopenia, the underlying mechanism has not yet been fully clarified. We examined whether the measurement of the immature platelet fraction (IPF) in thrombocytopenic patients with liver dysfunction is useful as a rapid and noninvasive method for the differential diagnosis of chronic liver diseases. We examined 20 liver cirrhosis patients, 56 patients with chronic hepatitis, 9 patients with fatty liver, and 86 patients without liver disease. The percentage value of IPF (IPF%) was measured using an XE-2100 multiparameter automatic hematology analyzer. Using a receiver operating characteristic curve, we found diagnostic significance of the absolute platelet count and the absolute number of the IPF between cirrhotic patients and noncirrhotic patients, and developed a powerful multivariate discriminant analysis (MDA) function based on the platelet count and the IPF%. The diagnostic accuracy obtained by the MDA function was superior to that obtained by the absolute number of platelets and the IPF. We therefore propose our IPF% measurement for the diagnosis of liver cirrhosis.

Oscillating fluid flow inhibits TNF-alpha -induced NF-kappa B activation via an Ikappa B kinase pathway in osteoblast-like UMR106 cells.

Fluid flow plays an important role in load-induced bone remodeling. However, the molecular mechanism of flow-induced signal transduction in osteoblasts remains unclear. In endothelial cells, fluid flow alters activation of NF-kappaB resulting in changes in expression of cell adhesion molecules. To test the hypothesis that fluid flow alters NF-kappaB activation and expression of cell adhesion molecules in osteoblastic cells, we examined the effect of oscillating fluid flow (OFF) on tumor necrosis factor (TNF)-alpha-induced NF-kappaB activation in rat osteoblast-like UMR106 cells. We found that OFF inhibits NF-kappaB-DNA binding activities, especially TNF-alpha-induced p50-p65 heterodimer NF-kappaB activation and TNF-alpha-induced intercellular adhesion molecule-1 mRNA expression. The inhibitory effects of OFF on both TNF-alpha-induced NF-kappaB activation and intercellular adhesion molecule-1 mRNA expression were shear stress-dependent and also increased with OFF exposure duration, indicating that OFF has potent effects on mechanotransduction pathways. OFF also inhibited TNF-alpha-induced IkappaBalpha degradation and TNF-alpha-induced IkappaB kinase (IKK) activity in a shear stress-dependent manner. These results demonstrate that IKK is an initial target molecule for OFF effects on osteoblastic cells. Thus, OFF inhibits TNF-alpha-induced IKK activation, leading to a decrease in phosphorylation and degradation of inhibitory IkappaBalpha, which in turn results in the decrease of TNF-alpha-induced NF-kappaB activation and potentially the transcription of target genes.

Peripheral blood CD4-mediated enhancement and CD8-mediated suppression in the presence of recombinant hepatitis B virus core antigen.

The proliferative response of peripheral blood T cells to hepatitis B core antigen (HBcAg) was studied in hepatitis B patients. CD4+ T cells from patients with chronic active hepatitis type B (CAH-B) exhibited a significant proliferative response to HBcAg, especially in hepatitis B envelope antigen (HBeAg)-positive patients. In contrast, there was no apparent T cell reaction to HBcAg in patients with CAH non-A, non-B, HBeAg-positive healthy carriers and in healthy volunteers. The proliferative response to CD4+ cells to bacterial extracts of Escherichia coli was always insignificant in all patients and healthy volunteers. The CD8+ cells did not proliferate in response to HBcAg in any subject, even in the presence of autologous irradiated CD4+ responder cells. The CD8+ cells, preactivated with HBcAg and HBcAg-reactive irradiated autologous CD4+ cells, suppressed the proliferative response to autologous CD4+ cells to HBcAg but not the response to phytohemagglutinin in HBcAg-responder CAH-B patients. CD4-mediated HBcAg-specific enhancement and CD8-mediated HBcAg-specific suppression in the peripheral blood compartments of HBcAg-responsive CAH-B patients are possible.

mRNA expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in interface tissue around implants in loosening total hip arthroplasty.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) play an important role in tissue destruction and remodeling. Nine samples of cement interface tissues from nine patients who had failed cemented total hip arthroplasty (THA) were obtained for revision of THA and analyzed on mRNA expression of MMPs and TIMPs. The preoperative serial radiographic examinations revealed an apparent clear zone around all implants. We excluded septic loosening as one of the factors affecting THA. Three samples were obtained from three different sites of the acetabular interface tissue in each patient. After extraction of total RNA from 27 samples, we used the reverse-transcriptional polymerase chain reaction (RT-PCR). mRNA of MMP-1, -2, -3, -9, and TIMP-1 and -2 was detected in the interface tissue. MMP-10 mRNA was not detected, yet MMP-1 and MMP-3 mRNA were commonly observed. TIMP-2 mRNA was also strongly expressed compared to TIMP-1. It was thus demonstrated that MMPs and TIMPs were produced locally in the cemented tissue of THA loosening. These findings may suggest that MMPs and TIMPs expressed around the implants play a critical role in the progression of aseptic loosening of THA.

Protooncogene expression in osteogenesis induced by bone morphogenetic protein.

In this study, changes in the expression of protooncogenes c-fos and c-myc messenger ribonucleic acid were investigated in mice after implantation of bone morphogenetic protein. The expression of c-fos showed a biphasic pattern. The first increase was observed on Day 1 with the aggregation of round cells. The second increase was observed on Day 7 with the appearance of chondroblasts. The amount of c-myc messenger ribonucleic acid showed the sustained high levels from Days 2 to 7. During this period, the proliferation of mesenchymal cells was histologically evident. After Day 11, the expression of c-fos and c-myc decreased and remained at low levels despite the progress in chondroosteogenesis. The protooncogenes c-fos and c-myc appear to increase before calcification in the process of bone morphogenetic protein induced bone and cartilage development.

Changes in the markers of bone metabolism following skeletal unloading.

To elucidate the mechanism in disuse bone atrophy induced by skeletal unloading, we studied the indices of bone resorption and bone formation in the femur of tail-suspended rats. The duration of the suspension ranged from 1 to 14 days. Tartrate-resistant acid phosphatase mRNA, an index used to evaluate bone resorption, increased significantly more than the controls for the first 3 days of the tail-suspension experiments, compared those in controls. Osteocalcin and alkaline phosphatase, two common markers for bone formation, were also monitored. Osteocalcin mRNA started to decrease after 3 days of suspension. Five days later, alkaline phosphatase mRNA showed a decrease. Levels of both of these mRNAs remained low for the remaining suspension period. Sequential changes in the markers for bone metabolism indicate that the transient increase in bone resorption preceded the decrease in bone formation in the development of disuse bone atrophy induced by skeletal unloading.

Effects of bisphosphonate on bone metabolism in tail-suspended rats.

Our previous studies demonstrated that tail suspension causes early, transient increases in osteoclastic activity, followed by a decrease in osteoblastic activity in the hind limbs of rats. To assess whether this early increase in bone resorption is important in the development of disuse atrophy, the effect of YH529, a third generation bisphosphonate, was studied on hind limb atrophy in rats subjected to tail suspension. YH529 (YH group) or PBS (control group) were administered subcutaneously in 5-week-old male Wistar rats suspended for 7 days. In the control group, wet weight, calcium and phosphorus contents decreased significantly in the femur but they did not change in the humerus. In the YH group, however, these parameters did not change significantly in the femur, but both calcium and phosphorus increased significantly in the humerus. These results indicate that the inhibition of bone resorption by YH529 prevents the development of disuse atrophy induced by tail suspension. It is thus suggested that early increases in bone resorption are important for the development of disuse bone atrophy.

Changes in serum concentrations of calcium and its regulating hormones during tail suspension in rats.

To study the effects of mechanical unloading on systemic calcium homeostasis, we determined the changes in serum concentration of calcium, 1,25-dihydroxyvitamin D3 and parathyroid hormone (PTH) during tail-suspension experiments in rats. The serum concentration of ionized calcium significantly increased during the 14 days of the suspension, reflecting increased bone resorption in the hind limbs. This hypercalcemic condition should cause suppression in PTH secretion. Indeed, serum PTH levels decreased on Day 3 of the suspension. This decrease was associated with lower serum levels of 1,25-dihydroxyvitamin [correction of dihyroxyvitamin] D3 probably due to a decrease in the activity of 1 alpha-hydroxylase in the kidneys resulting from a decrease in PTH secretion. Since it is known that 1,25-dihydroxyvitamin D3 stimulates osteoblastic function, it is suggested that endocrine responses evoked by tail suspension aggravate disuse atrophy of the hind limbs.

Macrophage activation and migration in interface tissue around loosening total hip arthroplasty components.

The bone-cement interface tissue of failed total hip arthroplasty (THA) has inflammatory characteristics, such as the presence of prostaglandin E2 and interleukin 1 (IL-1). We considered that the bone-cement interface tissue could be the site of granulomatous inflammation caused by a foreign-body reaction. It has been demonstrated that inflammatory cytokines and chemokines have an important role in granulomatous inflammation. Bone-cement interface tissue was obtained at revision from nine patients with failed cemented THA, and the role of macrophages was assessed by immunohistochemistry, electron microscopy, and molecular biological techniques. We used the reverse-transcriptional polymerase chain reaction to examine the expression of mRNA for IL-1 alpha, IL-1 beta, tumor necrosis factor alpha (TNF alpha), macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, IL-8, and monocyte chemoattractant protein. Polyethylene debris surrounded by macrophages and phagocytosis of debris by macrophages was frequently observed in the interface tissue. Macrophage activation and the production of inflammatory cytokines such as IL-1 and TNF alpha might induce the development of interface tissue. Expression of chemokine mRNAs was also commonly seen, suggesting that this led to recruitment of macrophages into the bone-cement interface tissue. Debris released from implants appears to cause activation of macrophages and the production of inflammatory cytokines and chemokines that induce cellular recruitment into interface tissue. This mechanism might form a vicious cycle that aggravates THA loosening.

TNF-alpha-dependent activation of NF-kappa B in human osteoblastic HOS-TE85 cells is repressed in vector-averaged gravity using clinostat rotation.

Effects of vector-averaged gravity on tumor necrosis factor (TNF)-alpha-dependent activation of nuclear factor kappa B (NF-kappa B) in human osteoblastic HOS-TE85 cells were investigated by culturing the cells using clinostat rotation (clinorotation). Cell cultures were rotated for 72 h at 40 rpm in a clinostat. At the end of clinorotation, the cells were treated with TNF-alpha for 30 min under stationary conditions. Electrophoretic mobility shift assays revealed that TNF-alpha-dependent activation of NF-kappa B was markedly reduced in the clinorotated cells when compared with the cells in control stationary cultures or after horizontal rotation (motional controls). The NF-kappa B-dependent transactivation was also impaired in the clinorotated cells, as evidenced by a transient transfection assay with a reporter plasmid containing multimerized NF-kappa B sites. Consistent with these findings, the TNF-alpha-dependent induction of endogenous NF-kappa B-responsive genes p105, I kappa B-alpha, and IL-8, was significantly attenuate in clinorotated cells. These results demonstrate that vector-averaged gravity inhibits the responsiveness of osteoblasts to TNF-alpha by repressing NF-kappa B activation.

Effects of stress on Kv1.5 K+ channel gene expression in the left ventricle of rat hearts.

Voltage-dependent K+ channels contribute to the repolarization phase of the cardiac action potential. An increase in Kv1.5 K+ channel gene expression by a pharmacological dose of glucocorticoid was reported recently in the heart of an adrenalectomized rat. In this study, we examined whether physiological elevations of glucocorticoid induced by tail suspension affects the K+ channel gene expression. Five-week-old male Wistar rats were subjected to tail suspension for three days. Total RNA was extracted from the ventricle, and the level of Kv1.5 mRNA was determined by Northern blot analysis. On the first day, the Kv1.5 mRNA had increased over the non-suspended rat. By the third day, mRNA had increased significantly by more than 3-fold. knowing that urinary excretions of glucocorticoid increase in tail-suspended rats, these findings suggest that physiologically elevated glucocorticoid may upregulate Kv1.5 K+ gene expression.

Effects of vector-averaged gravity on NF-kappa B activation in human osteoblast-like cells.

We have recently demonstrated that a transcription factor, nuclear factor kappa B (NF-kappa B), plays a key role in the production of cytokine and cell adhesion molecules in osteoblastic cells. In the present study, we investigated the effects of vector-averaged gravity (clinostat rotation) on tumor necrosis factor (TNF)-alpha-induced activation of NF-kappa B in human osteoblastic HOS-TE85 cells. After a 72-hr clinostat culture, the cells were treated with TNF-alpha for 30 min. Electrophoretic mobility shift assay using the nuclear extracts revealed that the DNA-binding activity of NF-kappa B was substantially reduced in clinostat-cultured cells compared with the stationary control. Concomitantly, the transactivation of NF-kappa B was examined by a transfection study. The cells were transfected with a plasmid expressing a luciferase reporter gene driven by multimerized NF-kappa B sites. They were cultured in the clinostat for 48-hr, followed by a 4-hr treatment with TNF-alpha. Clinostat culture resulted in a significant decrease in the luciferase activities, being consistent with the decreased binding of NF-kappa B. These results indicate that exposure of HOS-TE85 cells to vector-averaged gravity impairs NF-kappa B activation by TNF-alpha.

Changes in urinary excretion of deoxypyridinoline in tail-suspended rats: effects of a bisphosphonate, YH529.

Urinary excretion of deoxypyridinoline (D-Pyr) has been shown to be a useful marker for bone resorption. In this study, we investigated whether D-Pyr could be used to monitor the changes in bone resorption of the hind limb induced by tail-suspension. Male Wistar rats 5-weeks old were tail-suspended in a metabolic cage to unload the hind limbs. The control rats were not suspended. YH529 (YH), an inhibitor of bone resorption, or a vehicle (phosphate buffered saline=PBS) was administered daily starting 3 days before the commencement of tail-suspension. In the non-suspended rats receiving PBS, urinary excretion of D-Pyr did not show any significant change during the one-week experimental period. In the non-suspended rats receiving YH, D-Pyr excretion significantly decreased on day 5 and 7 when compared with that observed on day 0, in accordance with the systemic inhibition of bone resorption by YH. In the tail-suspended rats receiving PBS, D-Pyr excretion showed a tendency to increase on day 1, which is in agreement with our previous report that tail-suspension causes an early (on day 1 of suspension) and transient increase in bone-resorption of the hind limbs. In the tail-suspended rats treated with YH, the increase in D-Pyr excretion on day 1 was not observed, and a significantly lower excretion was noted from day 3 to 7 during the tail-suspension. It was suggested that D-Pyr excretion might reflect the transient increase in hind limb bone resorption induced by tail-suspension. As observed in-YH treated rats, D-Pyr excretion could serve as a good marker for the inhibition of systemic bone resorption.

Effect of bisphosphonate administration on the excretion of stress hormones in tail-suspended rats.

We demonstrated that administration of a bisphosphonate, YH529, prevents the development of disuse atrophy of the hind limbs induced by tail-suspension in rats. Since tail suspension is accompanied by an increase in the secretion of stress hormones, we studied whether administration of bisphosphonate affects the secretion of stress hormones during that procedure. Tail suspension was carried out in a metabolic cage by connecting a wire inserted through tail bone to the ceiling of the cage. The control rat received the same treatment but was not suspended. YH529 or a vehicle (PBS=phosphate buffered saline) was administered daily starting 3 days before the commencement of tail suspension. Urine samples were collected before the wire was inserted (day 0), on the day of insertion (day 1) and 3, 5 and 7 days after. In the control rats receiving PBS, urinary excretion of corticosterone and epinephrine did not change throughout the 7-day experimental period. In the control rats receiving YH529, urinary excretion of corticosterone increased significantly on the day of tail-piercing and wiring but then returned to the prior level. This increase was not observed in the control group receiving PBS. In the tail suspended rats, excretion of corticosterone and epinephrine increased significantly in both PBS and YH529 groups, the highest level being observed on the first day of tail suspension. Although statistically not significant, corticosterone excretion on day 1 of tail suspension was higher in the YH529 groups than that in the PBS group. It is thus suggested that administration of YH529 causes an augmented response to stress load.

Administration of bisphosphonate prevents disuse bone atrophy induced by tail suspension.

We recently demonstrated that osteopenia induced by rat tail-suspension was associated with an initial increase in bone resorption. To study the significance of the increase in early bone resorption for osteopenia, we investigated whether administration of YH529, a third-generation bisphosphonate, prevents the development of osteopenia as evidenced by increased wet weight of the femur, together with its calcium and phosphorus contents, when compared with those of tail-suspended rats treated with the vehicle alone. These results suggested that the initial increase in bone resorption plays an important role in the development of osteopenia induced by tail suspension.