An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Journal of Visualized Experiments, 2014 · DOI: doi:10.3791/52173 · Published: November 25, 2014

Simple Explanation

This study introduces an ex vivo spinal cord model to observe how central myelinated axons respond to injury in real-time. The model uses transgenic mice expressing yellow fluorescent protein in axons and Nile Red to visualize myelin, combined with laser-induced spinal cord injury. The ex vivo model allows researchers to study dynamic processes like axonal retraction and myelin degeneration with excellent spatiotemporal resolution. It also permits controlled manipulation of the environment around the spinal cord, such as altering ion concentrations or introducing drugs. This model mimics aspects of clinical spinal cord injuries, including axonal swelling and transection. It serves as a surrogate model for assessing acute responses and protective interventions before testing them in live animals.

Study Duration

Not specified

Participants

Adult 6-10 week old thy1-YFP transgenic mice

Evidence Level

Not specified

Key Findings

1
Laser-induced spinal cord injury (LiSCI) causes immediate axonal retraction and formation of sigmoidal-like coils within swollen myelin. Myelin ensheathing axons remote to the ablation appear unchanged at this time point.
2
Following LiSCI, primary transected axons and those undergoing secondary degeneration form characteristic endbulbs as they retract from the lesion site. Peri-axonal swelling and vesicular degeneration of myelin is also evident.
3
Axons initially spared by the LiSCI undergo delayed secondary degeneration. The extent of axonal retraction differs between proximal axonal stumps and their distal segments.

Research Summary

The study presents an ex vivo laser-induced spinal cord injury (LiSCI) model for real-time assessment of axonal degeneration mechanisms. This model utilizes transgenic mice with fluorescently labeled axons and myelin, enabling high-resolution imaging of injury responses. The ex vivo model offers advantages over in vivo imaging, including reduced motion artifacts, controlled environmental manipulation, and the ability to differentiate between primary and secondary injury mechanisms. It also allows for the assessment of potential therapeutic interventions. Key findings include the observation of axonal retraction, endbulb formation, peri-axonal swelling, and myelin degeneration following LiSCI. The model facilitates the study of both primary and secondary axonal injury responses.

Practical Implications

Drug Screening

The model can be used for rapid and cost-effective screening of potential axomyelinic protective agents before in vivo testing.

Understanding Injury Mechanisms

The model allows detailed investigation of the pathophysiology of white matter injury and identification of molecular targets for therapeutic intervention.

Studying White Matter Injury

The protocol can be utilized to investigate other components of white matter injury including demyelination, neuroinflammation and anoxic/ischemic injury mechanisms.

Study Limitations

1
The model does not allow for the study of peripheral blood-derived cell effects following LiSCI.
2
Behavioral and regeneration outcomes following injury cannot be evaluated due to the terminal nature of the model.
3
Imaging depth is limited by the light scattering properties of spinal cord white matter.

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