Elsevier

Bone

Volume 39, Issue 4, October 2006, Pages 854-862
Bone

Monitoring individual morphological changes over time in ovariectomized rats by in vivo micro-computed tomography

https://doi.org/10.1016/j.bone.2006.04.017Get rights and content

Abstract

The ovariectomized (OVX) rat is a well established model for osteoporosis research. The recent development of in vivo micro-computed tomography (micro-CT) provides new possibilities to monitor individual bone changes over time. The purpose of this study was to establish the normal time course of bone loss in the OVX rat model, and to determine the ability to detect morphological changes in vivo compared to cross-sectional study designs where animals are sacrificed at each time point. Eight-month-old female Wistar rats were randomly assigned to one of two groups: OVX (N = 10) or sham-operated (N = 10). In vivo micro-CT scanning of the right proximal tibial metaphyses occurred at 1-month intervals for 6 months. Morphological analyses were performed at each time step for every animal, and a two-way ANOVA with repeated measures was used to analyze the data. A second statistical analysis was performed without repeated measures for analysis as a cross-sectional study design. The repeated measures analysis was more sensitive to early changes than the cross-sectional study analysis. Changes were detected by longitudinal analysis in the sham-operated and OVX animals over time (P < 0.001) with the exception of trabecular separation in the sham animals. The OVX animals had decreases of bone volume ratio of 33% after 1 month, and 72% after 3 months relative to baseline measurements. Significant changes in bone volume ratio, trabecular number and separation were detected early using a longitudinal analysis, thus in vivo assessment is well poised to enable the study of early treatment protocols on the effects of bone architecture. The in vivo analysis found significant changes in the sham animals which were not detected by the cross-sectional analysis, and the changes to the OVX animal morphology was detected sooner. A substantial variation of baseline morphometry within the homogenous group of rats and response to OVX was observed, thus emphasizing the advantage of performing in vivo analysis where each animal acts as its own control. These data provide new insight into individual bone changes following OVX, and can be used as baseline information upon which future in vivo studies can be designed.

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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