Background: The spatial complexity of neuronal circuits in the central nervous system is a hurdle in understanding and treating brain and spinal cord injury (SCI). 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 for SCI researchers. 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 such as vacuum devices, can label specific circuits without genetic technology, and re-staining rounds can be easily implemented. 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.
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