High-resolution 3D imaging and analysis of axon regeneration in unsectioned spinal cord with or without tissue clearing.

Abstract

Here we present a protocol for analyses of axon regeneration and density in unsectioned adult mouse spinal cord. This includes methods for injury and tracing of dorsal column sensory and corticospinal axons; clearing and staining of unsectioned spinal cord; visualization of axon degeneration and regeneration in cleared and uncleared specimens using two-photon microscopy; and either manual or semi-automatic analysis of axon density and regeneration in 3D space using Imaris and ImageJ software. This protocol can be used to elucidate the molecular and cellular mechanisms underlying nervous system degeneration and regeneration and to establish the therapeutic efficacy of candidate neuroregenerative treatments. Because tissue sectioning is not required, this protocol enables unambiguous evaluation of regeneration and greatly accelerates the speed at which analyses can be conducted. Surgical procedures take <30 min per mouse, with a wait period of 2 weeks between axonal injury and tracing and 2-8 weeks between tracing and tissue processing. Clearing and immunolabeling take ~1-2 weeks, depending on the size of the sample. Imaging and analysis can be performed in 1 d. All these procedures can be accomplished by a competent graduate student or experienced technician.