The Laboratory Rat as an Animal Model for Osteoporosis Research

Osteoporosis is an important systemic disorder, affecting mainly Caucasian women, with a diverse and multifactorial etiology. A large variety of animal species, including rodents, rabbits, dogs, and primates, have been used as animal models in osteoporosis research. Among these, the laboratory rat is the preferred animal for most researchers. Its skeleton has been studied extensively, and although there are several limitations to its similarity to the human condition, these can be overcome through detailed knowledge of its specific traits or with certain techniques. The rat has been used in many experimental protocols leading to bone loss, including hormonal interventions (ovariectomy, orchidectomy, hypophysectomy, parathyroidectomy), immobilization, and dietary manipulations. The aim of the current review is not only to present the ovariectomized rat and its advantages as an appropriate model for the research of osteoporosis, but also to provide information about the most relevant age and bone site selection according to the goals of each experimental protocol. In addition, several methods of bone mass evaluation are assessed, such as biochemical markers, densitometry, histomorphometry, and bone mechanical testing, that are used for monitoring and evaluation of this animal model in preventive or therapeutic strategies for osteoporosis.Abbreviations: BMD, bone mineral density; DEXA, dual-energy X-ray absorptiometry; μCT, microcomputerized tomography; pQCT, peripheral quantitative computerized tomographyOsteoporosis is a multifactorial skeletal disease, characterized by reduction in bone mass and disruption of the microarchitectural structure of bone tissue, resulting in loss of mechanical strength and increased risk of fracture.² The disorder can be localized or involve the entire skeleton. Generalized osteoporosis can be primary (postmenopausal and senile) or secondary. In the European Union, osteoporosis is a leading cause of mortality and morbidity in the elderly and a key factor in the high cost of medical care.³⁴Although osteoporosis usually makes its appearance late in life, and age is a major risk factor, its roots can be tracked back into adolescence. Particularly during periods of rapid bone growth, dietary calcium levels are of high importance.³⁴ Other factors that contribute to the pathogenesis of osteoporosis are lifestyle and genetic and hormonal attributes.^13,71 Reduced physical activity increases the rate of bone loss, and muscle contraction is the prevailing source of skeletal loading. Regarding hormonal factors, women, especially in the decade after menopause, can show a severe reduction of bone mass, thus explaining the high incidence of osteoporotic fractures in women compared with men.³⁴The multiple factors implicated in osteoporosis, its obscure pathogenesis, the dramatic decline in quality of life, high incidence of the disorder (especially in postmenopausal women), financial cost, and high mortality, make the need for further experimentation in animal models imperative. Experimental research can improve our understanding of pathogenesis and of the activity of pharmaceutical agents in the prevention or treatment of the disease. Although many aspects of the disorder have been revealed, others remain unclear, including the mechanisms involved in calcium homeostasis in the extracellular space and its effect on bone physiology and disease⁶⁵ and the cell and molecular pathways triggered after mechanical loading to orchestrate bone renewal.⁵³ Current research is focused on new therapeutic possibilities targeting the osteolytic enzymes of the osteoclast and the mechanisms activating bone progenitor cells and those controlling apoptosis as new potential treatments.^63,64Many therapeutic advances in the management of osteoporosis were studied first in diverse animal models and then entered clinical practice.^31,67,69 All of these models should fulfill similar basic criteria: they must comply with national and local ethical and legislative considerations, be accessible to experimental centers, be easy and safe to handle, have a low cost of acquisition, require little maintenance, reliably reproduce the disease and the biological material to be examined should be readily available. Laboratory rats meet most of these criteria. In addition, the availability of detailed knowledge of the rat skeleton and protocols for rapid induction of osteopenia, have increased this model''s popularity. Here we review the advantages and limitations of the use of the laboratory rat in osteoporosis research.     

Decorin-induced growth inhibition is overcome through protracted expression and activation of epidermal growth factor receptors in osteosarcoma cells

Decorin is an established natural oncosuppressive factor whose action is being studied in detail. Recently, decorin gene therapy formulations using adenoviral vectors have been shown in several animal models with very promising results. The present study describes the first exception to the established oncosuppression model using human osteosarcoma cells. MG-63 osteosarcoma cells were found to constitutively produce decorin, and furthermore, to be resistant to decorin-induced growth arrest. On the contrary, decorin seemed to be beneficial to osteosarcoma cells because it was necessary for MG-63 cell migration and acted as a mediator, counteracting the transforming growth factor-beta2-induced cytostatic function. Efforts to determine how MG-63 cells could overcome the decorin-induced cytostatic effect established that decorin in MG-63 cells does not induce p21 expression nor does it cause protracted retraction and inactivation of the epidermal growth factor receptor. Conversely, epidermal growth factor receptor seemed to be overexpressed and continuously phosphorylated. In view of the proposed design of decorin-based anticancer therapeutic strategies, our study provides new data on pathways that cancer cells might employ to overcome the established decorin-induced growth suppression.     

Environmental Conditioning of Skeletal Anomalies Typology and Frequency in Gilthead Seabream (Sparus aurata L., 1758) Juveniles

In this paper, 981 reared juveniles of gilthead seabream (Sparus aurata) were analysed, 721 of which were from a commercial hatchery located in Northern Italy (Venice, Italy) and 260 from the Hellenic Center for Marine Research (Crete, Greece). These individuals were from 4 different egg batches, for a total of 10 different lots. Each egg batch was split into two lots after hatching, and reared with two different methodologies: intensive and semi-intensive. All fish were subjected to processing for skeletal anomaly and meristic count analysis. The aims involved: (1) quantitatively and qualitatively analyzing whether differences in skeletal elements arise between siblings and, if so, what they are; (2) investigating if any skeletal bone tissue/ossification is specifically affected by changing environmental rearing conditions; and (3) contributing to the identification of the best practices for gilthead seabream larval rearing in order to lower the deformity rates, without selections. The results obtained in this study highlighted that: i) in all the semi-intensive lots, the bones having intramembranous ossification showed a consistently lower incidence of anomalies; ii) the same clear pattern was not observed in the skeletal elements whose ossification process requires a cartilaginous precursor. It is thus possible to ameliorate the morphological quality (by reducing the incidence of severe skeletal anomalies and the variability in meristic counts of dermal bones) of reared seabream juveniles by lowering the stocking densities (maximum 16 larvae/L) and increasing the volume of the hatchery rearing tanks (minimum 40 m³). Feeding larvae with a wide variety of live (wild) preys seems further to improve juvenile skeletal quality. Additionally, analysis of the morphological quality of juveniles reared under two different semi-intensive conditions, Mesocosm and Large Volumes, highlighted a somewhat greater capacity of Large Volumes to significantly augment the gap with siblings reared in intensive (conventional) modality.     

Concentration-dependent diffusion of unlabeled protein within an in vitro hyaluronic acid matrix

Diffusion and movement of subcutaneously injected biologics and high-concentration immunoglobulin G (IgG) therapeutics away from the injection site and through the subcutaneous (SC) tissue may be concentration dependent. This possibility was confirmed by in situ measurement of diffusion coefficients of unlabeled bovine IgG in phosphate-buffered saline within an in vitro hyaluronic acid matrix that represents the SC electrostatic environment. Diffusion decreased from 2.67 to 0.05 × 10⁻⁷ cm²/s when IgG concentration increased from 25 to 73 mg/mL. The results demonstrated that in situ detection of unlabeled proteins within an in vitro SC environment provides another useful tool for the preclinical characterization of injectable biologics.     

Flow Measurements in a Blood-Perfused Collagen Vessel Using X-Ray Micro-Particle Image Velocimetry

Blood-perfused tissue models are joining the emerging field of tumor engineering because they provide new avenues for modulation of the tumor microenvironment and preclinical evaluation of the therapeutic potential of new treatments. The characterization of fluid flow parameters in such in-vitro perfused tissue models is a critical step towards better understanding and manipulating the tumor microenvironment. However, traditional optical flow measurement methods are inapplicable because of the opacity of blood and the thickness of the tissue sample. In order to overcome the limitations of optical method we demonstrate the feasibility of using phase-contrast x-ray imaging to perform microscale particle image velocimetry (PIV) measurements of flow in blood perfused hydrated tissue-representative microvessels. However, phase contrast x-ray images significantly depart from the traditional PIV image paradigm, as they have high intensity background, very low signal-to-noise ratio, and volume integration effects. Hence, in order to achieve accurate measurements special attention must be paid to the image processing and PIV cross-correlation methodologies. Therefore we develop and demonstrate a methodology that incorporates image preprocessing as well as advanced PIV cross-correlation methods to result in measured velocities within experimental uncertainty.     

Design and validation of a high-throughput assay to detect codon 146 polymorphisms in the caprine prion protein gene

In sheep, scrapie susceptibility is so strongly associated with single nucleotide polymorphisms (SNPs) in the gene encoding the prion protein (PrP) that this linkage constitutes the basis for selective breeding strategies directed toward controlling the disease. For goats, in contrast, the association between scrapie susceptibility/resistance and variations in the PrP gene is far weaker, with only a few identified SNPs showing an influence on scrapie susceptibility. A recent survey of PrP genotypes in Cypriot goats, however, revealed the existence of a robust association between polymorphisms at codon 146 of the caprine PrP gene and resistance/susceptibility to natural scrapie. Here we describe here a high-throughput assay, based on homogeneous MassExtend technology coupled with mass spectrometry, for genotyping codon 146 of the caprine PrP gene. Our results demonstrate that this assay exhibits high accuracy, reproducibility, and repeatability, thereby making it suitable for large-scale SNP genotyping, as required for scrapie surveillance programs.     

Caffeic acid phenethyl ester,an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of RANKL‐induced NF‐κB and NFAT activity

Receptor activator NF‐κB ligand (RANKL)‐activated signaling is essential for osteoclast differentiation, activation and survival. Caffeic acid phenethyl ester (CAPE), a natural NF‐κB inhibitor from honeybee propolis has been shown to have anti‐tumor and anti‐inflammatory properties. In this study, we investigated the effect of CAPE on the regulation of RANKL‐induced osteoclastogenesis, bone resorption and signaling pathways. Low concentrations of CAPE (<1 µM) dose dependently inhibited RANKL‐induced osteoclastogenesis in RAW264.7 cell and bone marrow macrophage (BMM) cultures, as well as decreasing the capacity of human osteoclasts to resorb bone. CAPE inhibited both constitutive and RANKL‐induced NF‐κB and NFAT activation, concomitant with delayed IκBα degradation and inhibition of p65 nuclear translocation. At higher concentrations, CAPE induced apoptosis and caspase 3 activities of RAW264.7 and disrupts the microtubule network in osteoclast like (OCL) cells. Taken together, our findings demonstrate that inhibition of NF‐κB and NFAT activation by CAPE results in the attenuation of osteoclastogenesis and bone resorption, implying that CAPE is a potential treatment for osteolytic bone diseases. J. Cell. Physiol. 221: 642–649, 2009. © 2009 Wiley‐Liss, Inc.     

Atypical membrane-embedded phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2)-binding site on p47(phox) Phox homology (PX) domain revealed by NMR

The Phox homology (PX) domain is a functional module that targets membranes through specific interactions with phosphoinositides. The p47(phox) PX domain preferably binds phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) and plays a pivotal role in the assembly of phagocyte NADPH oxidase. We describe the PI(3,4)P(2) binding mode of the p47(phox) PX domain as identified by a transferred cross-saturation experiment. The identified PI(3,4)P(2)-binding site, which includes the residues of helices α1 and α1' and the following loop up to the distorted left-handed PP(II) helix, is located at a unique position, as compared with the phosphoinositide-binding sites of all other PX domains characterized thus far. Mutational analyses corroborated the results of the transferred cross-saturation experiments. Moreover, experiments with intact cells demonstrated the importance of this unique binding site for the function of the NADPH oxidase. The low affinity and selectivity of the atypical phosphoinositide-binding site on the p47(phox) PX domain suggest that different types of phosphoinositides sequentially bind to the p47(phox) PX domain, allowing the regulation of the multiple events that characterize the assembly and activation of phagocyte NADPH oxidase.