Stable in vivo imaging of densely populated glia, axons and blood vessels in the mouse spinal cord using two-photon microscopy

J Neurosci Methods, 2008 · DOI: 10.1016/j.jneumeth.2007.11.011 · Published: March 30, 2008

Spinal Cord Injury Neurology Medical Imaging

Simple Explanation

This paper describes a new method for imaging the living spinal cord in mice using a technique called two-photon microscopy. The method overcomes the problem of movement caused by breathing and heartbeat, which makes it difficult to get clear images of the spinal cord. The new method involves stabilizing the spinal column of the mouse using a special device and using a specific combination of anesthetic drugs to minimize breathing movements. This allows researchers to get stable, high-resolution images of cells and structures in the spinal cord without needing to intubate the animal or process the images afterwards. This technique can be used to study spinal cord injury, regeneration, and diseases, providing a better understanding of how these conditions affect the living spinal cord.

Study Duration

5 Days

Participants

Adult transgenic mice

Evidence Level

Not specified

Key Findings

1
A novel in vivo technique for two-photon microscopy was developed to allow direct, stable and repetitive imaging of densely populated cells and axons in the living spinal cord of transgenic mice.
2
The use of a custom-built spinal stabilization device, combined with a specific anesthetic mix (ketamine–xylazine–acepromazine), significantly improves the spinal column's stability under the two-photon microscope, minimizing respiratory movements.
3
The technique allows for detailed study of multiple axonal and glial processes, as well as their interactions with the vasculature, enabling the acquisition of functional information of cell–cell interactions in the living spinal cord.

Research Summary

The study introduces a novel in vivo imaging technique for the mouse spinal cord, addressing the challenge of movement artifacts caused by breathing and heartbeat. This technique uses a custom-built stabilization device and a specific anesthetic mix to minimize these movements. The developed method allows for stable, high-resolution, and repetitive imaging of densely populated cells, axons, and blood vessels in the living spinal cord without requiring animal intubation or extensive image post-processing. The technique's application in animal models of spinal cord injury and disease promises to enhance our understanding of axonal degeneration, regeneration, inflammatory processes, and neurodegenerative mechanisms in the spinal cord.

Practical Implications

Enhanced Spinal Cord Research

The technique facilitates the study of cellular and molecular functions in the spinal cord in vivo, accommodating sophisticated experimental designs such as local injections or electrophysiological recordings during imaging.

Improved Understanding of Spinal Cord Injury

Application of the technique in animal models of spinal cord injury can elucidate mechanisms of axonal degeneration and regeneration, allowing for the unequivocal identification of regenerating axons.

Advancements in Disease Modeling

The technique can be used to image inflammatory and neurodegenerative processes in animal models of diseases like multiple sclerosis and amyotrophic lateral sclerosis, where disease pathogenesis is prominent in the spinal cord.

Study Limitations

1
In vivo imaging inherently presents with slight movement between sequential timepoints in timelapse movies, mainly due to the animal heartbeat that is present even under deep anesthesia.
2
The results obtained using this in vivo imaging method may greatly depend on the use of proper anesthesia.
3
Repetitive imaging studies at later time points should take into consideration potential microglial responses to the laminectomy and/or the repetitive opening and closing of the wound.

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