Abstract
The spinal cord contains a diverse array of sensory and motor circuits that are essential for normal function. Spinal cord injury (SCI) permanently disrupts neural circuits through initial mechanical damage, as well as a cascade of secondary injury events that further expand the spinal cord lesion, resulting in permanent paralysis. Tissue clearing and 3D imaging have recently emerged as promising techniques to improve our understanding of the complex neural circuitry of the spinal cord and the changes that result from damage due to SCI. However, the application of this technology for studying the intact and injured spinal cord remains limited. Here, we optimized the passive CLARITY technique (PACT) to obtain gentle and efficient clearing of the murine spinal cord without the need for specialized equipment. We demonstrate that PACT clearing enables 3D imaging of multiple fluorescent labels in the spinal cord to assess molecularly defined neuronal populations, acute inflammation, long-term tissue damage, and cell transplantation. Collectively, these procedures provide a framework for expanding the utility of tissue clearing to enhance the study of spinal cord neural circuits, as well as cellular- and tissue-level changes that occur following SCI.
Highlights
The spinal cord mediates a complex repertoire of sensory and motor functions through a sophisticated network of neural circuits
This study describes a gentle and efficient method to clear the murine spinal cord through optimization of the passive CLARITY technique (PACT) tissue clearing procedure
While electrophoresis may further expedite clearing, we chose a passive approach to avoid the risk of tissue damage associated with electrophoretic tissue clearing, as well as the need for specialized equipment (Kim et al, 2015)
Summary
The spinal cord mediates a complex repertoire of sensory and motor functions through a sophisticated network of neural circuits. Passive Spinal Cord Tissue Clearing repair of damaged neural circuits (Kwon et al, 2011a,b; Tetzlaff et al, 2011; Griffin and Bradke, 2020), limited efficacy in the clinical setting has been achieved to date. Greater anatomical characterization of the intact and injured spinal cord may provide critical insight that can improve future studies to promote recovery after SCI. One of the major limitations to studying the spinal cord neural circuits, as well as the secondary pathological mechanisms that exacerbate tissue damage after SCI, has been the traditional use of labor-intensive and error-prone histological sectioning (Steward et al, 2003; Erturk et al, 2011)
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