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Inactivation mutation of the recently discovered klotho gene in mice causes a syndrome resembling aging. Manifestations include short life span, atherosclerosis, gonadal atropy, skin atropy, emphysema, ataxia and ectopic calcification. These mice also exhibit abnormally high bone density in the epiphyses of their tibiae based on X-ray and histological analyses. However, micro-structures of the trabecular bones in arbitrary two-dimensional planes or three-dimensional regions are difficult to analyze by these techniques. Therefore, we applied high resolution micro-computed tomography (microCT) to characterize the micro-structural abnormality in the trabecular bone in long bones as well as in vertebrae of four- to six-week-old klotho mutant mice. Two-dimensional microCT analyses in the mid-sagittal plane as well as three-dimensional microCT analyses indicated that the trabecular bone volume fraction measured in the proximal metaphyses of the tibiae was increased more than twofold in klotho mutant mice compared with the wild-type mice. Similarly, the trabecular bone area fraction in the mid-sagittal plane of the lumbar vertebral bodies was also increased by about 80% at the proximal and distal ends. No significant difference was observed with regard to the cortical thickness in the mid-shaft of femora between klotho mutant and wild-type mice. Three-dimensional microCT analyses also indicated that the trabecular number and thickness of the proximal metaphyses of the tibiae were increased by about 80% and 300% respectively in the klotho mutant mice, while trabecular separation was 60% less in klotho mutant mice compared with the wild-type mice. These quantitative microCT analyses indicate that the inactivation of klotho gene expression results in an increase in three-dimensional bone volume fraction, number and thickness of the trabecular bones in these mice.

High-resolution micro-computed tomography analyses of the abnormal trabecular bone structures in klotho gene mutant mice