Sodium selenate treatment mitigates reduction of bone volume following traumatic brain injury in rats.

OBJECTIVES: Administration of sodium selenate to rats given traumatic brain injury (TBI) attenuates brain damage and improves long-term behavioural outcomes. We have previously provided evidence that TBI causes bone loss in rats, however the effect of sodium selenate treatment on bone quantity following TBI is unknown. METHODS: Rats were randomly assigned into sham injury or fluid percussion injury (FPI) groups and administered saline or sodium selenate for 12 weeks post-injury. Femora were analysed using histomorphometry, peripheral quantitative computed tomography (pQCT) and biomechanical testing. RESULTS: Distal metaphyseal trabecular bone volume fraction of FPI-selenate rats was higher than FPI-vehicle rats (41.8%; p<0.01), however, femora from selenate-treated groups were shorter in length (4.3%; p<0.01) and had increased growth plate width (22.1%; p<0.01), indicating that selenate impaired long bone growth. pQCT analysis demonstrated that distal metaphyseal cortical thickness was decreased in TBI rats compared to shams (11.7%; p<0.05), however selenate treatment to TBI animals offset this reduction (p<0.05). At the midshaft we observed no differences in biomechanical measures. CONCLUSION: These are the first findings to indicate that mitigating TBI-induced neuropathology may have the added benefit of preventing osteoporosis and associated fracture risk following TBI.

Sharpin is a key regulator of skeletal homeostasis in a TNF-dependent manner.

OBJECTIVES: SHARPIN is a subunit of LUBAC and regulates activation of NF-κB, a pivotal transcription factor in skeletal homeostasis. Mutated SHARPIN gene (cpdm) mice develop chronic proliferative dermatitis and systemic inflammation. Cpdm mice have an osteopaenic phenotype characterised by decreased cortical and trabecular bone volume, but whether this is a consequence of the hyper-inflammatory phenotype is unknown. The inflammatory phenotype of cpdm mice is prevented by Tnf deficiency so we examined cpdm.Tnf (-/-) mice to examine the role of SHARPIN in skeletal development. METHODS: This research determined the extent to which SHARPIN and TNF interact within the skeleton through analyses of gene expression, µCT and biomechanical properties of bones of control (CTRL), cpdm, Tnf (-/-) (TNF KO) and cpdm.Tnf (-/-) (cpdm/TNF KO) mice. RESULTS: Gene expression of IL-1ß, TNF and caspase-3 increased in cpdm mice but was comparable to control values in cpdm/TNF KO mice. Decreased cortical and trabecular bone in cpdm mice translated to a loss in bone strength (ultimate stress and peak force). Cpdm/TNF KO mice developed bones similar to, or stronger than, control bones. CONCLUSIONS: Our results suggest that SHARPIN plays a significant role in skeletal homeostasis and that this role is strongly regulated through TNF pathways.

Galnon, a galanin receptor agonist, improves intrinsic cortical bone tissue properties but exacerbates bone loss in an ovariectomised rat model.

OBJECTIVES: Previous studies have shown galanin (GAL) injections onto mouse calvaria increased bone thickness and osteoblast number. This study investigated the effects of the GAL receptor agonist galnon on bone loss using the ovariectomised (OVX) rat model. METHODS: OVX rats were treated with either vehicle or galnon for 6 weeks via mini-osmotic pumps. Plasma osteocalcin concentrations, osseous cell gene expression, morphological and biomechanical properties of the skeleton were compared between the two groups. RESULTS: Treatment with galnon increased RANKL:OPG gene ratio (p<0.001) plus expression of TNF-α (p<0.05) and cathepsin K (p<0.05). µCT analyses revealed galnon-treated OVX animals had reduced trabecular and cortical morphology compared to control animals. Biomechanically, galnon OVX animals required similar peak force to failure to that of control OVX animals although galnon treatment did enhance the mechanical properties of Young's modulus and ultimate tensile stress. CONCLUSIONS: Our research suggests that galnon, a GAL receptor agonist, may enhance osteoclastic bone resorption in OVX rats. Although galnon reduced bone volume, biomechanical testing revealed that bone of galnon-treated animals was mechanically superior per unit area. Taken together, galnon simultaneously improves the intrinsic quality of cortical bone whilst stimulating osteoclastic activity in the OVX rat model.

Galanin treatment offsets the inhibition of bone formation and downregulates the increase in mouse calvarial expression of TNFalpha and GalR2 mRNA induced by chronic daily injections of an injurious vehicle.

We have previously shown that after bone fracture, galanin (GAL) and GAL receptor expression is increased in osteoblast-like cells of callus; however, the role of elevated GAL/GAL receptors in this instance of bone injury is not known. We hypothesize that in injury, GAL may facilitate bone formation by suppressing the production of cytokines such as TNFalpha and IL-1alpha, thereby affecting bone collagen formation and collagenolysis by key matrix metalloproteinases (MMPs). In studies to explore this hypothesis, we used a mouse calvarial injection model to (1) investigate whether mild injury caused by a daily subcutaneous injection of a glycerol-containing vehicle onto calvaria affected osteoblast/bone formation-associated histomorphometric parameters and gene expression (mRNA encoding GAL, GAL receptors, TNFalpha, IL-1beta, collagen type I, MMP-2 and -13) compared to non-injected, control mice and (2) determine the effect of GAL+vehicle treatment on these entities. Five groups of 4-week-old mice were used: a non-injected control group; a vehicle (50/50 solution of 10 mM PBS+0.025% BSA/5.4 M glycerol)-treated group; and 3 GAL-treated groups (0.2, 2 and 20 ng doses). Solutions were injected subcutaneously onto calvaria in a 10 mul volume, every day for 2 weeks. Vehicle injection reduced calvarial periosteal osteoblast cell height (P<0.001), osteoblast number (P<0.001) and osteoid thickness (P<0.01), relative to values in non-injected animals at 2 weeks. Vehicle injection also inhibited BFR in this periosteal bone relative to values in non-injected animals at both 1 and 2 weeks (P<0.05 and P<0.001, respectively). Increasing concentrations of GAL reversed the above-listed inhibitory effects caused by vehicle. This reversal was demonstrated by a dose-dependent effect of GAL on osteoblast cell height (Pearson's r=0.330; P<0.05), osteoblast number (Pearson's r=0.715; P=0.000), osteoid thickness (Pearson's r=0.516; P=0.000) and BFR (Pearson's r=0.525; P<0.05) after 2 weeks of GAL+vehicle treatment; with the 20 ng/day GAL+vehicle injection schedule returning these measured parameters toward non-injected control values. All GAL+vehicle treatments had no effect on calvarial expression of GAL, GALR1, GALR3, collagen type 1 and MMP-2 mRNAs compared to levels in vehicle-injected controls. GAL treatment did, however, produce dose-dependent effects on calvarial expression of GALR2 (Pearson's r=0.763; P=0.000), MMP-13 (Pearson's r=0.806; P=0.000), IL-1beta (Pearson's r=0.807; P=0.000) and TNFalpha (Pearson's r=0.542; P=0.000) mRNAs with 20 ng/day of GAL+vehicle producing the strongest reversal of vehicle-associated changes. Thus, the 20 ng/day GAL+vehicle regimen offset the inhibition of osteoblastic activity, and therefore bone formation caused by daily glycerol-containing vehicle injection. This effect on bone formation may be due in part to the peptide suppressing the formation and associated activity of TNFalpha, IL-1beta and MMP-13, as TNFalpha and IL-1beta are known inhibitors of bone formation and MMP-13 is involved in collagenolysis. Furthermore, these effects may be due to the action of GAL via GALR2, as it was the only GAL receptor affected by this GAL treatment regimen. These results indicate that GAL can facilitate bone formation associated with injury and reveal potential efficacy for GAL in treating osseous conditions where bone formation may be inhibited due to excess TNFalpha and IL-1beta production.

The effects of hypothyroidism on nerve growth factor and norepinephrine concentrations in weight-bearing and non-weight-bearing bones of rats.

Thyroid hormones affect bone remodelling directly via receptors in osteoblasts. Previously, however, we have shown that the euthyroid and hyperthyroid states significantly influence the concentrations of both nerve growth factor (NGF) and norepinephrine (NE) in particular bones. Both NGF and NE directly affect bone metabolism and therefore it is possible that thyroid hormone action on bone may also be indirect via its actions on these two neural-related substances. In light of previous studies, the current experiments aimed to investigate whether hypothyroidism also influenced NGF and NE concentrations in weight-bearing and non-weight-bearing rat bones. Hypothyroidism was induced by oral ingestion of propylthiouricil (PTU; 3.8+/-0.2 mg/kg/day) for 21 days. Histological examination on distal femurs and microparticle enzyme immunoassayed plasma concentrations of T3 and T4 verified the hypothyroid status in treated rats. NGF concentrations were assayed via enzyme-linked immunosorbent assay (ELISA) and NE concentrations were measured via high performance liquid chromatography (HPLC) with electrochemical detection (ECD). NGF concentrations: Femoral NGF concentrations were 207% higher in hypothyroid rats (674.9+/-88.3 ng/g) than in euthyroid rats (326.7+/-63.6 ng/g; p < 0.05). Rib NGF concentrations in hypothyroid rats (3125.1+/-450.2 ng/g) were increased by 342% compared to euthyroid ribs (914.5+/-128.6 ng/g; p < 0.01). Rib NGF concentrations in hypothyroid rats were 463% higher than in femurs of hypothyroid rats (p < 0.001). NE concentrations: In hypothyroid rats, NE concentrations were reduced by approximately 50% in both ribs (38.9 ng/g) and calvaria (41.5 ng/g) compared to euthyroid rats (74.7 ng/g and 87.4 ng/g respectively; p < 0.05 for both). These findings on hypothyroid rats may be taken in conjunction with our companion work on hyperthyroid rats (Yao et al., 2002, JMNI 2:327-334) and put in context with other reports, to indicate that (i) there are several sources of NGF in bone, some of which are stimulated by hypothyroidism and others by hyperthyroidism and (ii) the concentrations of both NGF and NE in bone are sensitive to weight-bearing and thyroid hormone status.

Nerve growth factor and norepinephrine concentrations in weight-bearing and non-weight-bearing bones of euthyroid and hyperthyroid rats.

UNLABELLED: Thyroid hormone, nerve growth factor (NGF) and norepinephrine (NE) and weight-bearing affect bone metabolism, yet interactions between these factors and osseous tissue have not been investigated. Therefore, the aims of the study were to measure NGF and NE concentrations in weight-bearing and non-weight-bearing bones from euthyroid (control) and hyperthyroid (HT) rats. Hyperthyroidism was induced by oral intake of triiodothyronine (90 mg/kg/day) for 21 days. Histomorphometry on distal femurs verified significant trabecular bone loss in HT rats compared to euthyroid animals. NGF concentrations were assayed via ELISA, whilst NE concentrations were measured via HPLC and ECD. In euthyroid rats: (i) the concentration of NGF in ribs (914 ng/g) was almost 3-fold greater than in femurs (326 ng/g wet weight of tissue) (ii) the concentrations of NE in ribs (74.7 ng/g) and calvaria (87.4 ng/g) were 2.5-3.5-fold greater than either femurs (24.0 ng/g) or tibiae (30.5 ng/g) and (iii) NE concentrations were comparable between ribs (74.7 ng/g) and calvaria (87.4 ng/g) and similar between tibiae (30.5 ng/g) and femurs (24.0 ng/g). In HT rats: (i) the concentration of NGF in ribs (1802 ng/g) was 4-fold greater than in femurs (402 ng/g) (ii) NE concentrations in ribs (23.3 ng/g) and calvaria (13.6 ng/g) were 4.5-fold and 2.6-fold greater respectively than in tibiae (5.2 ng/g), while ribs had almost a 2-fold higher concentration of NE than calvaria. In HT rats compared to euthyroid animals: (i) NGF concentrations almost doubled in ribs but there was little change in the NGF concentration in femurs (ii) there was a reduction in NE concentrations in calvaria by 84%, in ribs by 69% in tibiae by 83% and 55% in femur (NS). CONCLUSIONS: (i) Non-weight-bearing is associated with higher concentrations of NGF and NE than weight-bearing in bones in euthyroid and HT rats; (ii) Hyperthyroidism exerts opposite effects on NGF and NE in bone and (iii) Hyperthyroidism interacts with weight-bearing to determine NGF and NE concentrations in bone. Therefore, the influence of thyroid hormone on NGF and NE in bone may need to be taken into account when considering the action of thyroid hormone on bone in either euthyroid or hyperthyroid states.

Effects of short-term use of ibuprofen or acetaminophen on bone resorption in healthy men: a double-blind, placebo-controlled pilot study.

Prostaglandins are known to be involved in the metabolism of bone, having a significant influence on bone resorption in cases of bone pathology. We therefore investigated the short-term effects of two commonly used nonsteroidal anti-inflammatory drugs (NSAIDs), ibuprofen and acetaminophen (paracetamol), on bone resorption in healthy men. In a randomized, double-blind pilot study, 28 healthy, age- and weight-matched male volunteers were treated with ibuprofen (n = 10), acetaminophen (n = 9), or a placebo (n = 9) for 3 days. As an indication of bone resorption rate, levels of the biochemical bone markers N-telopeptide (NTx) and free deoxypyridinoline (D-Pyr) were measured in urine. Differences in resorption marker levels pre- and post-NSAID use were then compared between groups. We found that NTx concentrations in the acetaminophen group were lower than placebo (p = 0.048), whereas NTx levels in the ibuprofen group were higher than in the acetaminophen group (p = 0.016). By contrast, D-Pyr concentrations in the ibuprofen group were significantly lower than in the placebo group (p = 0.009). A comparison of the percentage changes of D-Pyr:NTx ratios found that the ratio in the ibuprofen group was significantly lower than that of both the control (p = 0.0065) and acetaminophen (p = 0.01) groups. These results show the differential effects of ibuprofen and acetaminophen on urinary excretion of peptide-bound and free deoxypyridinoline cross-links of type I collagen. Short-term ibuprofen use may alter the renal handling of collagen cross-links and increase bone resorption to a greater extent than acetaminophen in normal men.

Can some physical therapy and manual techniques generate potentially osteogenic levels of strain within mammalian bone?

BACKGROUND AND PURPOSE: Although physical therapy techniques are used to alleviate pain and stiffness in joint injuries, whether these methods are capable of affecting bone is unknown. For example, can these techniques potentially influence bone formation or resorption? To begin exploring this possibility, this study investigated the ability of 4 manual techniques to generate levels of compressive strains that presumably can stimulate bone metabolism. SUBJECTS: Six 3,4 metacarpals from three 3-year-old Merino ewes were used. METHODS: A rosette strain gauge was implanted onto the dorsomedial cortex of each ovine 3,4 metacarpal. Four different manual procedures were applied on 2 occasions on each metacarpal in vivo and ex vivo. Mean peak principal compressive strains were calculated for each technique. RESULTS: Levered bending produced greater mean peak compressive strains than almost all other manual procedures tested in vivo or ex vivo. CONCLUSION AND DISCUSSION: Manual levered bending created levels of compressive strain similar in magnitude to those created by mechanical devices used in previous animal experiments to induce new bone formation (osteogenesis). This animal model appears to be suitable for investigating the effects of manually applied procedures on bone and may establish whether manual techniques can stimulate bone formation.

Immunohistochemical localization of nerve growth factor in fractured and unfractured rat bone.

We detected nerve growth factor (NGF) by immunohistochemical localization in both fractured and unfractured rat rib. In unfractured bone, periosteal mesenchymal osteoprogenitor cells appeared to be the only skeletal cells which stained for NGF. Adjacent skeletal muscle fibers exhibited NGF staining both in fractured and unfractured bone. Fracture callus periosteal osteoprogenitor cells, marrow stromal cells, osteoblasts, young osteocytes and endothelial cells of new capillaries had moderate to heavy staining for NGF at 1 and 3 weeks after fracture. Deeply positioned osteocytes and osteoclasts showed no NGF staining. Most chondrocytes of fracture calluses stained for NGF, however, some chondrocytes did not stain which may indicate that NGF is produced at particular stages of chondrocytic differentiation. In calluses, periosteal matrix stained heavily for NGF when juxtaposed to cartilage and less obviously when associated with new bone at both 1 and 3 weeks post-fracture. However, other fibrous, cartilaginous and osseous matrices did not stain for NGF at any time. At 6 weeks post-fracture, NGF staining was largely confined to periosteal osteoprogenitor cells. The detection of NGF in periosteal osteoprogenitor cells of unfractured rib points to these cells having a role in nerve maintenance in intact bone. Furthermore, the localization of NGF in osteoprogenitor cells, marrow stromal cells, osteoblasts, certain chondrocytes, endothelial cells, periosteal matrix of the fracture callus and skeletal muscle may mean that these entities participate in fracture innervation. The presence of NGF in the callus may also indicate a direct, as yet undefined action of this neurotrophin on skeletal cell metabolism.

Topical application of nerve growth factor improves fracture healing in rats.

The aim of the present study was to examine the effects of nerve growth factor on the healing of unsplinted fractured ribs. After fracture of a rib in male rats, nerve growth factor was delivered by a miniosmotic pump to the fracture site for 7 days at the rate of 1.4 micrograms/day. Callus catecholamine concentrations, bone callus size, histomorphometry, and biomechanical properties of the repairing rib were measured at 7, 21, and 42 days after fracture. After 21 days, concentrations of norepinephrine and epinephrine were significantly increased in the group treated with nerve growth factor compared with those in the control group (211% norepinephrine and 322% epinephrine). Also, the midline longitudinal area of non-osseous (fibrous tissue and cartilage) callus of the fracture was significantly smaller (54%) and had a higher proportion of cartilage in the treated group than in the controls. By 42 days, there was only bony callus between the fracture ends in both the control group and the treated group. The treated group, however, again showed significantly elevated concentrations of norepinephrine and epinephrine (286 and 382%, respectively) and significantly elevated breaking stress (50%) and Young's modulus (51%), together with a reduction in the transverse cross-sectional area of the repair site (57%). The resultant increases in effectiveness and rate of repair of bone with administration of nerve growth factor suggest that it may play an important role in the healing processes of fractured bone.

Identification of plasminogen activator in osteoclasts.

Plasminogen activator (PA) was located in newborn rat osteoclasts using a single-cell assay. Immunohistochemistry using biotin-streptavidin-peroxidase indicated the presence of both tissue-type plasminogen activator (tPA) and urokinase (uPA) within the cytoplasm of osteoclasts isolated from newborn rat long bones. Electron microscopic immunohistochemistry using the biotin-streptavidin-colloidal gold system on L.R. Gold thin resin sections of undecalcified, newborn rat tibial metaphyseal trabecular bone identified these proteases in the lysosomal network of osteoclasts. uPA was also localized in marrow macrophage lysosomes, but tPA was not detected in these cells. The localization of these enzymes within osteoclasts may imply their involvement in bone resorption.

Insulin release from a cloned precursor beta cell line.

This paper describes the establishment in long-term tissue culture of a functional, clonal beta (B) cell line UMR 407/3 derived from neonatal rat pancreas. Immunofluorescence demonstrated specific and uniform staining for insulin. Transmission electron microscopy showed the presence of microvilli and cytoplasmic granules. The doubling time in culture was approximately 60 h in 2% (v/v) fetal calf serum with inhibition of growth at confluence. Biochemical studies demonstrated the incorporation of [3H]leucine into proinsulin and insulin, with insulin comprising 43.6% of the total radioactivity incorporated into immune complexes. When incubated at 37 degrees C for 30 min with Krebs-Ringer bicarbonate buffer (pH 7.4), the amount of insulin released on stimulation by 16.7 mmol glucose/l, 20 mmol DL-glyceraldehyde/l or 20 mmol alpha-ketoisocaproate/l was significantly higher compared with 5.6 mmol glucose/l. The mean insulin content was equivalent to 99 +/- 0.4 fmol (S.E.M.)/5 X 10(5) cells. Regulated insulin release was maintained through at least 15 passages in culture. The cells showed morphological evidence of senescence after passage 26 and this was associated with significant reduction in stimulated insulin release as well as insulin content. The ability of the cells of this clonal line to grow in soft agar suggests that it is a precursor cell line. The clonal B cell lines isolated so far may thus represent variably committed rather than fully differentiated B cells in culture. These clonal non-neoplastic cell lines will be useful models with which to study the regulation of maturation/differentiation of B cells and insulin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)