RetroDISCO: Clearing technique to improve quantification of retrograde labeled motor neurons of intact mouse spinal cords

J Neurosci Methods, 2016 · DOI: 10.1016/j.jneumeth.2016.05.017 · Published: September 15, 2016

Simple Explanation

Current methods to count regenerated motor neurons involve retrograde labeling followed by cryosectioning. However, sectioning can lead to errors due to potential double counting of cells in adjacent sections. This paper introduces retroDISCO, a method for optically clearing whole mouse spinal cords without losing fluorescent signal. This allows imaging of retrograde labeled motor neurons using confocal microscopy, avoiding the need for sectioning. The retroDISCO technique is inexpensive, simple, and robust, utilizing commonly available microscopy techniques to determine the number of motor neurons extending axons across an injury site.

Study Duration

Not specified

Participants

C57BL/6J mice

Evidence Level

Not specified

Key Findings

1
Complete optical clearing of spinal cords using retroDISCO takes approximately four hours. Confocal microscopy can then obtain z-stacks of labeled motor neuron pools within 3–5 minutes.
2
The retroDISCO technique is able to detect anticipated differences in motor neuron number after cross-suture and conduit repair when compared to intact mice, demonstrating high repeatability.
3
The clearing procedure reduces the volume of mouse spinal cords by approximately 50%.

Research Summary

The study introduces retroDISCO, a novel optical clearing technique for intact mouse spinal cords, enabling efficient and accurate quantification of retrograde labeled motor neurons using confocal microscopy. RetroDISCO is shown to be a rapid, repeatable, and less labor-intensive method compared to traditional cryosectioning techniques, while also eliminating the risk of double-counting cells. The authors demonstrate the feasibility and application of retroDISCO in assessing nerve regeneration after injury and repair, as well as in evaluating age-related changes in motor neuron populations.

Practical Implications

Improved Motor Neuron Quantification

RetroDISCO provides a more accurate and efficient method for quantifying motor neurons in nerve regeneration studies, reducing the error associated with traditional sectioning techniques.

Enhanced Research Accessibility

The simplicity and use of commonly available equipment make RetroDISCO accessible to a wider range of researchers, facilitating advancements in peripheral nerve repair research.

Potential for Advanced Imaging Studies

The preservation of spatial relationships between motor neurons with RetroDISCO opens avenues for future mapping studies and the use of multiple labels to investigate complex nerve regeneration processes.

Study Limitations

1
The technique is tracer dependent, with FastBlue degrading rapidly during the clearing process, suggesting that only certain fluorescent tracers are compatible with retroDISCO.
2
Overlapping cell bodies in uninjured nerves can make distinguishing and counting individual cells more difficult, potentially leading to a modest underestimation of motor neuron numbers.
3
Some preliminary work was needed to optimize imaging and counting consistency.

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