Passive clearing and 3D lightsheet imaging of the intact and injured spinal cord in mice

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.

In Vivo and 3D Imaging Technique(s) for Spatiotemporal Mapping of Pathological Events in Experimental Model(s) of Spinal Cord Injury

“…Co-labeling of MRP8 with NeuN demonstrated loss of neurons at the lesion epicenter and loss of neutrophils and neurons near the epicenter and found the interaction between both types of cells. 37 3.3. In Vivo Imaging System.…”

EZ Clear for simple, rapid, and robust mouse whole organ clearing

“…In solvent-based clearing methods (e.g., 3DISCO and BABB), dehydrated and delipidated samples are equilibrated in high RI solvents, such as DBE (RI = 1.56) and BABB (RI = 1.55) to reduce light scattering. In aqueous-based clearing methods, solutions like RIMS (80% (v/v) Nycodenz, RI = 1.46) and sRIMS (80% D-sorbitol, RI = 1.43) are suitable for mounting and imaging of SDS-mediated delipidated tissue, although they are lower than the ideal RI of 1.52-1.56 14,33 . Given the aqueous nature of EZ Clear, we first tested whether RIMS or sRIMS were compatible with EZ Away.…”

Navigating the Light-Sheet Image Analysis Software Landscape: Concepts for Driving Cohesion From Data Acquisition to Analysis

“…Researchers have adapted the basic light-sheet body plan to different applications with different lens geometries ( Huisken and Stainier, 2007 ; Dunsby, 2009 ; Wu et al, 2011 , 2013 ; Tomer et al, 2012 ; Kumar et al, 2014 , 2018 ; Voleti et al, 2016 , 2019 ; Sapoznik et al, 2020 ), beam shaping strategies ( Keller et al, 2008 ; Planchon et al, 2011 ; Chen et al, 2014 ; Vettenburg et al, 2014 ; Liu et al, 2018 ; Chang et al, 2019 ), sample mounting and scanning techniques ( Bouchard et al, 2015 ; Royer et al, 2016 ; Wu et al, 2017 ; Fadero et al, 2018 ; Glaser et al, 2019 ), and contrast mechanisms ( Truong et al, 2011 ; Di Battista et al, 2019 ). The ability to image intact tissues, now made possible with advances in clearing protocols ( Richardson and Lichtman, 2015 ; Matryba et al, 2019 ; Ueda et al, 2020 ; McCreedy et al, 2021 ), and also the desire to image naturally dynamic 3D biological systems with live-cell imaging are the two main forces driving this unusual technological variety. This variety stands in comparison to the more purely performance driven development of, for example, confocal microscopy where samples are typically uniformly thin layers, sections, or cell cultures on a slide.…”