Comparison between quantitative X-ray imaging, dual energy X-ray absorptiometry and microCT in the assessment of bone mineral density in disuse-induced bone loss.

OBJECTIVES: We recently introduced a new methodology called quantitative X-ray imaging (qXRI) to investigate bone mineral density in isolated rodent bones. The aims of the present study were to compare DXA and microCT with qXRI in a rat model of disuse osteoporosis. METHODS: Fourteen Copenhagen rats were injected with a single dose of botulinum toxin (BTX - 2 UI) in the right Mus quadriceps femoris. The left hindlimb serves as control. Areal BMD and vBMD were determined with a Hologic Discovery-W device and a Skyscan 1172 microcomputed tomograph (microCT). Absorbing material density (AMD) was determined on digitized X-ray images obtained with a Faxitron M020 device. RESULTS: All three methods highlighted significant lower values for aBMD, vBMD and AMD in trabecular and cortical bone in the BTX-injected side. In trabecular bone, aBMD, vBMD and AMD were significantly correlated with BV/TV. In cortical bone, only aBMD and vBMD were significantly correlated with cortical bone mass On the other hand, only AMD was significantly correlated with the mechanical parameters bending strength and bending modulus. CONCLUSIONS: qXRI is a rapid and cheap method to assess trabecular bone mass in isolated rodent bones and can be used as a surrogate for the densitometry of small animals.

Double incretin receptor knock-out (DIRKO) mice present with alterations of trabecular and cortical micromorphology and bone strength.

UNLABELLED: A role for gut hormone in bone physiology has been suspected. We evidenced alterations of microstructural morphology (trabecular and cortical) and bone strength (both at the whole-bone--and tissue-level) in double incretin receptor knock-out (DIRKO) mice as compared to wild-type littermates. These results support a role for gut hormones in bone physiology. INTRODUCTION: The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), have been shown to control bone remodeling and strength. However, lessons from single incretin receptor knock-out mice highlighted a compensatory mechanism induced by elevated sensitivity to the other gut hormone. As such, it is unclear whether the bone alterations observed in GIP or GLP-1 receptor deficient animals resulted from the lack of a functional gut hormone receptor, or by higher sensitivity for the other gut hormone. The aims of the present study were to investigate the bone microstructural morphology, as well as bone tissue properties, in double incretin receptor knock-out (DIRKO) mice. METHODS: Twenty-six-week-old DIRKO mice were age- and sex-matched with wild-type (WT) littermates. Bone microstructural morphology was assessed at the femur by microCT and quantitative X-ray imaging, while tissue properties were investigated by quantitative backscattered electron imaging and Fourier-transformed infrared microscopy. Bone mechanical response was assessed at the whole-bone- and tissue-level by 3-point bending and nanoindentation, respectively. RESULTS: As compared to WT animals, DIRKO mice presented significant augmentations in trabecular bone mass and trabecular number whereas bone outer diameter, cortical thickness, and cortical area were reduced. At the whole-bone-level, yield stress, ultimate stress, and post-yield work to fracture were significantly reduced in DIRKO animals. At the tissue-level, only collagen maturity was reduced by 9 % in DIRKO mice leading to reductions in maximum load, hardness, and dissipated energy. CONCLUSIONS: This study demonstrated the critical role of gut hormones in controlling bone microstructural morphology and tissue properties.

Thiazolidinediones induce osteocyte apoptosis and increase sclerostin expression.

AIMS: Thiazolidinediones (TZDs) are associated with a higher risk of bone fracture in women compared with men. The aim of the present study was to investigate whether TZDs could influence osteocyte behaviour and contribute to the skeletal phenotype observed in TZD-treated patients. METHODS: The murine MLO-Y4 cell line was used as a source of osteocytes. These cells were cultured for 24 h with 0, 10(-8) m, 10(-7) m, 10(-6) m, 10(-5) m or 10(-4) m of pioglitazone, rosiglitazone or troglitazone in the presence or absence of 17beta-oestradiol. The extent of osteocyte apoptosis was assessed, as was the expression of the bone formation inhibitor sclerostin and receptor activator for nuclear factor kappaB ligand (RANKL) also. RESULTS: In the absence of 17beta-oestradiol, pioglitazone, rosiglitazone and troglitazone induced osteocyte apoptosis dose-dependently even at the lowest concentration of 10(-8) m. Furthermore, the expression of sclerostin but not RANKL was significantly increased in TZD-treated cultures compared with untreated cultures. The presence of 17beta-oestradiol significantly reduced TZD-induced osteocyte apoptosis and also sclerostin up-regulation. CONCLUSIONS: These findings therefore raise the potential concern of using TZDs in post-menopausal women where the lack of oestrogen would not prevent osteocyte apoptosis and sclerostin up-regulation and may aggravate the reduction in bone mass in these patients.

Influence of dietary peas and organic acids and probiotic supplementation on performance and caecal microbial ecology of broiler chickens.

1. The effect of dietary pea and addition of organic acid blend (OA) or probiotic (Pro) on performance and caecal microbial ecology of broiler chickens was studied. 2. A growth trial was conducted with 160 Ross 308 female broilers from d 1 to 35 of age. There were 8 treatment groups based on either control (S) or white pea (P). Both S and P were supplemented with OA (Galliacid - fumaric acid, calcium formate, calcium propionate and potassium sorbate coated with plant triglycerides, Vetagro) and or with Pro (LABYuc-Probio - lactic acid bacteria, Saccharomyces cerevisiae and Yucca schidigeri extract, Mifarmex GmbH). 3. Inclusion of peas in the diet increased feed intake and decreased gain:feed ratio in comparison to the control diet. Neither probiotic nor OA supplementations affected broiler performance. 4. The caecal microbiota was characterised in 37-d-old birds by fluorescent in situ hybridisation (FISH) and terminal-restriction fragment length polymorphism (T-RFLP). Total bacterial counts in caecal contents were slightly higher for birds fed the pea diets, but were not affected by OA or Pro supplements. 5. Neither pea nor Pro affected the Lactobacillus/Enterococcus and Streptococcus/Lactococcus counts in caecal contents, whereas OA supplementation slightly increased the Lactobacillus/Enterococcus counts. The composition of the Lactobacillus/Enterococcus population was altered by inclusion of peas as revealed by the T-RFLP patterns. 6. The DNA fingerprint further suggested that the caecal microbiota was dominated by the lactic acid bacterium Streptococcus alactolyticus. 7. In ileal contents, the concentration of short-chain fatty acids (SCFA) was decreased only by Pro supplementation. In caecal contents, the SCFA concentration was higher for birds fed on the pea diets, and increased significantly with Pro supplementation 8. In conclusion, the results indicate that the use of pea and probiotics in broiler feed may stimulate the caecal commensal microbiota (growth and/or activity) to some extent and hence prevent establishment of pathogenic and zoonotic enterobacteria in these segments of the gut.

Effect of an organic acid blend and phytase added to a rapeseed cake-containing diet on performance, intestinal morphology, caecal microflora activity and thyroid status of broiler chickens.

The experiment was carried out on 96 female broilers, allocated to eight groups of 12 birds kept in individual cages. Two basal wheat- and soyabean meal-based diets containing 150 g/kg of rapeseed expeller cake were formulated, differing in the level of P: 7.1 g/kg in diet H or 5.9 g/kg in diet L. Rapeseed cake supplied 3.15 micromol alkenyl glucosinolates per gram of diet. The eight treatments were: basal diets only, basal diets + phytase (1000 U/kg), basal diets + organic acid blend (OA, 6 g/kg), or basal diets + both additives. Diets were fed from day 8 to 28 of life. The results showed that the lower dietary P content and OA supplementation did not significantly affect feed intake or BWG, while both increased (p < 0.001) after phytase supplementation. Tibia ash content as well as tibia ultimate strength were lower (p < 0.001) in birds fed diets L compared with diets H, and increased (p < 0.01) with phytase supplementation of diet L, while OA had no influence on either parameter. Dietary P levels and OA supplementation had no influence on the pH of gut digesta, but the pH of jejunal digesta increased following phytase supplementation (p < 0.01). Morphological measurements of the small intestinal mucosa of chicks indicated that OA added to diet L depressed villi height (p < 0.001) and crypt depth (p < 0.001); both parameters increased after phytase supplementation (p < 0.01). The lower total SCFA as well as acetic, propionic and butyric acid concentrations in caecal digesta indicated lower activity of caecal microflora in birds fed diets L compared with H. OA supplementation had no influence, while phytase supplementation increased the concentration of acetic acid in caecal digesta. Supplementation of diets with either phytase or OA increased thyroid weight by 16% (p < 0.01) and 11% (p < 0.05) respectively. The increase in thyroid weight because of phytase supplementation was greater at the lower dietary P level, and the greatest when both phytase and OA were added to the diet.

Response of animals to dietary gramine. I. Performance and selected hematological, biochemical and histological parameters in growing chicken, rats and pigs.

The effects of feeding varied levels of low- and high-gramine yellow lupin seeds (LG and HG, respectively), and of synthetic gramine added to the diets in amounts ranging from 0.15 to 1.2 g per kg were investigated in one experiment on growing chicken and in two experiments on growing rats. The comparison of LG and HG lupin and the effect of 0.5 g gramine per kg of LG diet were determined in a growth-balance experiment with pigs. Organ weights and histology, blood parameters and activity of liver enzymes were determined. The response to HG lupin and gramine concentration varied among the species, the rats being more affected than chicken; no adverse effects of HG lupin or gramine were found in growing pigs. The common reaction of rats and chicken to the high levels of gramine (native or synthetic) was the decrease of feed intake and body gain. The increase of the relative weight of liver or kidney, changes in hematological parameters and liver enzymes were found only in rats. The estimated NOAEL (no-observed-adverse-effect level) of gramine was about 0.3 g/kg diet for rats, 0.65 g for chicken and at least 0.5 g for growing pigs.