3D Imaging of Axons in Transparent Spinal Cords from Rodents and Nonhuman Primates

eNeuro, 2015 · DOI: http://dx.doi.org/10.1523/ENEURO.0001-15.2015 · Published: March 26, 2015

Spinal Cord Injury Neurology Medical Imaging

Simple Explanation

This research focuses on improving how we visualize nerve fibers (axons) in the spinal cord using advanced imaging techniques. Traditional methods involve examining thin slices of tissue, which can be time-consuming and may lead to inaccurate interpretations of the complex three-dimensional structure of the spinal cord. The study uses a technique called tissue clearing, which makes the spinal cord transparent, combined with fluorescent labeling of specific axons. This allows researchers to see the entire structure of the axons in three dimensions using microscopes like light sheet and confocal microscopes. By using viruses or chemical tracers to label axons, the method can be applied to different animal models, including those where genetic labeling isn't available. This approach helps scientists study axonal regeneration and connections in the spinal cord more effectively.

Study Duration

Not specified

Participants

Mice, rats, and a nonhuman primate

Evidence Level

Not specified

Key Findings

1
AAV8 serotype with UbC promoter expressing GFP provides the strongest fluorescence signal after tetrahydrofuran-based tissue clearing, referred to as 3D imaging of solvent-cleared organs (3DISCO).
2
The study demonstrates that supraspinal tracts traced with AAV2 or AAV8 expressing GFP or tdTomato under the UbC promoter can be visualized using LSFM or confocal microscopy using THF/BABB-based tissue clearing methods.
3
The method allows for the identification of mislabeled axons that could be mistaken for regenerating axons in traditional histological sections, enhancing the accuracy of axon regeneration studies.

Research Summary

The study introduces optimized methods for visualizing traced axons in cleared spinal cords from transgenic and nontransgenic animals, comparing different AAV serotypes, promoters, and fluorescent protein reporters to determine the best combination for strong fluorescence signal after tissue clearing. It demonstrates the application of these methods to examine specific supraspinal pathways like the CST and RST in injured spinal cords, showing the relationship between axons and scar tissue using transgenic mouse models of scar formation. The research extends the application of these techniques to rats and nonhuman primates, proving that virus-based tracing can be combined with tissue clearing to study axon trajectory in injured spinal cords of nontransgenic animals and providing detailed visualizations of CST axon projections in the nonhuman primate spinal cord.

Practical Implications

Improved Accuracy in Axon Regeneration Studies

The ability to distinguish regenerated axons from spared or mislabeled axons enhances the reliability of studies investigating spinal cord injury and recovery.

Enhanced Understanding of Axon-Scar Interactions

Visualizing the spatial relationship between axons and scar tissue provides insights into the mechanisms that hinder or promote axonal regeneration.

Broad Applicability Across Species

The optimized viral- and chemical-based tract-tracing strategies can be applied to various animal models, including those where transgenic labeling is unavailable, expanding the scope of research possibilities.

Study Limitations

1
THF-based tissue clearing can quench fluorescent signals, requiring optimization of labeling methods to achieve strong signals.
2
The study notes that certain experiments may require tract-tracing using other types of tracers because of limitations of fluorescent proteins.
3
The study acknowledges that for thinner axons such as CST axons, or for fine anatomical structures such as synaptic terminals, confocal microscopy was necessary to provide the required resolution.

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