Double labeling serial sections to enhance three-dimensional imaging of injured spinal cord

Abstract

A method of double labeling a set of serial histological sections was performed to produce multiple three-dimensional (3D) reconstructions from the same segment of injured spinal cord. Alternate groups of consecutive histological sections were stained with Luxol fast blue with cresyl violet and Mallory’s trichrome in order to reconstruct two different 3D images that reveal different pathological features of the same 1-month-old compression spinal cord injury. Three-dimensional visualization of the two reconstructions was accomplished using an isocontouring algorithm that automatically extracts surfaces of features of interest based on pixel intensity. The two 3D reconstructions demonstrated the sparing of myelinated nerve fibers and the composition of neuroglia through the chronic lesion of an adult guinea pig. The 3D images provided a comprehensive and explicit view of a chronically injured spinal cord that is not possible by the inspection of two-dimensional (2D) histological sections or from magnetic resonance imaging. Using every histological section, we believe this double labeling 3D reconstruction technique provides a more enhanced and accurate visualization of the entire spinal cord lesion than has been possible before. Furthermore, we contend that this double labeling technique can further elucidate the histopathological events of secondary injury at different time points post-injury by using different combinations of complementary histological makers.