A reinvestigation of murine cranial suture biology: microcomputed tomography versus histologic technique.

BACKGROUND: Histology remains the standard form to analyze cranial suture in murine models, but this technique provides only limited "snapshots" of the entire suture and requires animal euthanasia with tissue destruction. Because of the bone complex microarchitecture, better methods are required to study the behavior of the cranial suture and its surrounding environment. The authors compared microcomputed tomography and histology as techniques to evaluate murine cranial sutures. METHODS: A total of 360 microcomputed tomography images and 160 to 170 histologic sections were processed from a mouse at postnatal days 22 and 45, respectively. After euthanasia, the posterior frontal and sagittal sutures were imaged with a microcomputed tomography system and subsequently processed for histologic analysis. Quantitative analysis of two-dimensional images was performed to determine the percentage of bone in a 1-mm sample. RESULTS: Quantitative analysis of the percentage of bone within the sutures showed identical patterns by microcomputed tomography and histology techniques. Both methods demonstrated the posterior frontal suture to have heavier fusion patterns in the anterior and endocranial portions, with variable skip areas of complete patency on the endocranial surface, ectocranial surface, or both at day 45. CONCLUSIONS: Cranial suture fusion in the murine model is not an "all-or-none" phenomenon. The posterior frontal suture, previously thought to be completely fused on day 45 by histological analysis, showed variable fusion along the length of the suture by both methods. Quantitative assessment of the percentage of bone within the posterior frontal and sagittal sutures and morphologic assessment of these sutures demonstrated similar findings by both methods. Whereas thorough histologic evaluation of an entire suture would be extremely labor intensive and impractical, these findings help to validate microcomputed tomography as a rapid and reliable method of examining the entire suture in murine models.

Microfocal CT: a method for evaluating murine cranial sutures in situ.

INTRODUCTION: The murine model is a well-established surrogate for studying human cranial suture biology. In mice, all sutures with the exception of the posterior frontal (PF) suture remain patent throughout life. Histology is regarded as the gold standard for analyzing sutures. On this basis, PF suture fusion begins on day of life 25 and is complete by day 45. Cranial suture histology, however, requires sacrifice of the animal to obtain tissue for analysis. As a result, knowledge of the kinetics of cranial suture fusion is based on a patchwork analysis of many sutures from many different animals. The behavior of a single suture through time is unknown. Our goal is to develop a noninvasive means to repeatedly image mouse cranial sutures in vivo. As a first step, the present study was performed to evaluate microfocal computer tomography (micro-CT) technology for the use of capturing images of a mouse cranium in situ. METHODS: The micro-CT system consists of a microfocal X-ray source and a large format CCD camera optically coupled to a high-resolution X-ray image intensifier, digitally linked to a computer. The PF and sagittal sutures lie in continuity along the midline of the skull. Holes were drilled in the calvaria on both sides of the PF and sagittal sutures of a 45-day-old euthanized mouse. A micro-CT scan of this animal was performed and hundreds of cross-sectional images were generated for the cranium. These images were used to reconstruct three-dimensional volumetric images of the entire cranium. Comparisons were made between (1). the gross specimen and the three dimensional reconstructions; (2). two-dimensional coronal images obtained by micro-CT and those obtained by histology. RESULTS: Analysis of PF and sagittal sutures demonstrated the following: (1). The drilled holes were accurately rendered by micro-CT, when compared to both the gross specimen and the histology. (2). The sagittal suture was found to be patent by both micro-CT and histology. (3). The PF suture is fused by histology, but unexpectedly, the PF suture appears incompletely fused by micro-CT. By micro-CT, however, the anterior and endocranial regions appear more extensively fused than the remainder of the PF suture, a finding consistent with published histologic analysis. CONCLUSIONS: We successfully imaged 45-day-old mouse cranial sutures in situ using micro-CT technology. Precise correlation between histologic sections and radiologic images is difficult, but convincing similarities exist between the gross specimen and images from micro-CT and histology. PF suture fusion in a 45-day-old animal appears different by micro-CT than by histology. One possible explanation for this apparent discrepancy is that suture fusion in histology is determined based on the appearance of bone morphology and not tissue density, as the specimens are necessarily decalcified to section the bone. Micro-CT, on the other hand, distinguishes tissues on the basis of density. Newly forming bone may require bone matrix formation prior to complete calcification; PF suture in 45-day-old mice may be morphologically complete but incompletely ossified. Studies correlating histologic and micro-CT assessment of suture development are underway. Micro-CT appears to be a promising method for noninvasive imaging of mouse cranial suture.