Automated 3D Axonal Morphometry of White Matter

Abstract

Axonal structure underlies white matter functionality and plays a major role in brain connectivity. The current literature on the axonal structure is based on the analysis of two-dimensional (2D) cross-sections, which, as we demonstrate, is precarious. To be able to quantify three-dimensional (3D) axonal morphology, we developed a novel pipeline for automated 3D segmentation and morphometric analysis (ACSON) of the white matter ultrastructure. The automated pipeline eliminates the need for time-consuming manual segmentation of 3D datasets. ACSON segments myelin, myelinated and unmyelinated axons, mitochondria, cells and vacuoles, and analyzes the morphology of axons. We applied the pipeline to serial block-face scanning electron microscopy images of the corpus callosum of sham-operated (n = 2) and brain injured (n = 3) rats 5 months after the injury. The 3D morphometry showed that axonal cross-sections were elliptic rather than spherical, and their diameter varied substantially along axons. It also showed a significant reduction in the axonal diameter of the ipsilateral corpus callosum of rats 5 months after brain injury, indicating ongoing axonal alterations even at this chronic time-point.