biPACT: a method for three-dimensional visualization of mouse spinal cord circuits of long segments with high resolution

Background The spatial complexity of neuronal circuits in the central nervous system is a hurdle in understanding and treating brain and spinal cord injuries. Although several methods have recently been developed to render the spinal cord transparent and label specific neural circuits, three-dimensional visualization of long segments of spinal cord with high resolution remains challenging. New Method We present a method that combines tissue staining of neuronal tracts traced with biotinylated dextran amine (BDA) and a modified passive clarity clearing protocol to describe individual fibers in long segments of mouse spinal cord. Results Corticospinal tract was traced with BDA with a mouse model of thoracic spinal cord injury. The spinal cord was stained and cleared in two weeks with four solutions: staining solution, hydrogel solution, clearing solution, and observation solution. The samples were observed with a light-sheet microscope, and three-dimensional reconstruction was performed with ImageJ software. High resolution-images comparable with tissue sections were obtained continuously and circumferentially. By tiling, it was possible to obtain high-resolution images of long segments of the spinal cord. The tissue could be easily re-stained in case of fading, Comparison with Existing Methods The present method does not require special equipment, can label specific circuits without genetic technology, and re-staining rounds can be easily implemented. It enables to visualize individual neural fiber of specific neural circuit in long spinal cord segments. Conclusions By using simple neural staining, and clearing methods, it was possible to acquire a wide range of high-resolution three-dimensional images of the spinal cord. Highlights No special devices or genetic tracers are required for a new clearing method Neuronal fibers are individually depicted in long segments of mouse spinal cord. Re-staining of neuronal fiber is possible. Stereotaxic observation is achieved by 3-D reconstruction with open-source software.