3-D Microarchitecture of Organ (Cerebellum and Ovary) Studied by Synchrotron X-ray Microtomography

Abstract

Quantifying microstructures over a whole organ can provide a useful map of anatomical structure with the potential to give functional insight. However, an accurate quantitative description for a mammalian organ has not been generated so far, owing to both the limited imaging depth in light/ confocal/ electron microscopes and the restricted resolutions in CT/ MRI. By contrast, synchrotron X-ray microscopy based on phase contrast can examine thick specimens over a large field of view thanks to high penetration capability of the X-rays. Furthermore, strongly collimated synchrotron X-rays produce 3-dimensional (3-D) images with very high quality, giving access to quantitative information in 3-D geometry.Here we developed a 3-D organ imaging system based on synchrotron X-ray microtomography with optimized staining methods, and applied this to mammalian organs with complex microscopic geometries, specifically, cerebellum and ovary.First, we demonstrated 3-D microarchitectures of Purkinje cell (PC) dendrites in cerebellum based on synchrotron X-ray microtomography with Golgi staining: dendritic morphology has important functional implication in signal processing. In reeler, the model mice for neurological disorders, we visualized the shape alterations of planar PC dendrites (i.e., abnormal 3-D arborization) in 3-D geometry. Despite these alterations, the 3-D quantitative analysis of the branching patterns showed no significant changes of the 77 ± 8° branch angle, whereas the branch segment length strongly increased with large fluctuations, comparing to the control. The 3-D fractal dimension of the PCs decreased from 1.723 to 1.254, indicating a significant reduction of dendritic complexity. This study provides insights into etiologies and further potential treatment options for lissencephaly and various neurodevelopmental disorders.Second, we directly visualized ‘intact’ ovary in 3-D using the synchrotron X-ray microtomography, which could deliver accurate quantification of folliculogenesis. In VRK1-deficient mice with infertility, the numbers of pre-antral and antral follicles were significantly reduced by 38% and 46%, respectively, comparing to the control. The oocytes volumes in antral and Graffian follicles also decreased by 42% and 37% in the mutants, respectively, indicating defects in oocyte quality at preovulatory stage. Genetic analysis showed that gene expressions related to folliculogenesis were down-regulated in VRK1-deficient ovaries, implying defects in folliculogenesis. We suggest that VRK1 is required for both follicle development and oocyte growth in mammalian female reproduction system.