Monitoring individual morphological changes over time in ovariectomized rats by in vivo micro-computed tomography
Introduction
Osteoporosis is a disease characterized by low bone mass and an increased risk of fragility fractures [1]. The ovariectomized (OVX) rat is an important small animal model for studying the effects of estrogen loss on bone quality [2]. Although a consensus on the precise definition of bone quality is still emerging [3], three-dimensional (3D) micro-architecture is a significant component [4]. Measurement of 3D architecture provides unique insight into the underlying bone mineral density changes that are typically observed by planer densitometric methods such as dual X-ray absorptiometry (DXA). It has been shown in the rat model that there is a rapid decline of trabecular bone volume following ovariectomy [5], [6], and that 3D cancellous connectivity loss may be a marker of irreversible architectural damage [7], [8], [9].
The vast majority of studies investigating the time course of architectural changes in small animal models have utilized a cross-sectional study design where cohorts of animals are killed at strategic time points after an intervention such as ovariectomy or tail suspension [5], [6], [10], [11], [12]. Cross-sectional designs have been necessary, because, until recently, micro-computed tomography (micro-CT), which provides a non-destructive method to assess 3D bone micro-architecture, could only be performed in vitro. The advent of in vivo measurements was first introduced using synchrotron radiation microtomography to assess 3D trabecular bone architecture in laboratory animals [8], [13], [14], and very recently compact in vivo micro-CT systems have been developed which are more accessible to laboratories for performing longitudinal studies [15], [16]. The advantage of a longitudinal study design is that each animal acts as its own control. Thus, normal variations within a cohort are less prone to mask subtle morphological effects, and smaller numbers of animals are needed. The effects of interventions can be assessed on an individual basis, and this may provide valuable information for the increasing volume of research emphasizing the relation between genotype and phenotype [10], [17], [18], [19], [20], [21].
In this study, we applied the techniques of in vivo micro-CT to establish the time course of architectural changes in ovariectomized rats at the tibial metaphyses on an individual basis. These data establish a useful basis on which future in vivo study designs can be based. Three-dimensional morphological analysis tools were used to quantify the structural changes in a longitudinal (repeated measures) study design. To assess the merits of performing in vivo over in vitro measurement approaches for detecting morphological changes, these same data were reanalyzed by the statistical methods used for a cross-sectional study design.
Section snippets
Animals
Twenty-eight-month-old female Wistar rats (BRL, Füllinsdorf, Switzerland) weighing on average 260 g were included in the study. They were randomly assigned to one of two groups; ovariectomy was performed on one group (OVX; N = 10) by the dorsal approach, and sham operations were performed on the second group (SHAM; N = 10). Animals were housed at 25°C with a 12:12-hour light–dark cycle. Animals were housed in groups of five and provided with a standard laboratory diet containing 0.8% phosphorus
Results
All 20 rats completed the study and in vivo micro-CT measurements were obtained at each time point providing an assessment of the morphological changes within each individual rat (Fig. 1). The individual morphological change over the study duration as well as group averages and standard deviations are presented (Fig. 2), in addition to the fold-change from baseline of the morphological parameters for the OVX group (Fig. 3). A detailed description of the group averages, and change relative to
Discussion
The time course of bone architectural changes in a rat model of osteoporosis was determined in vivo by micro-CT measurements of the proximal tibia for 6 months. The rats receiving the OVX operation had a large decrease in bone volume ratio and associated morphological parameters as expected, and that decrease was greatest in the first 3 months. The sham-operated rats also changed, although the changes were much smaller than in the OVX group. The novelty of these data is that this is the first
Acknowledgments
The authors wish to acknowledge the support of Dr. Tak Fung for his assistance with the statistical analysis.
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