Imaging and Quantification of Axonal Regeneration in Axotomized Rat Retinal Ganglion Neurons

JoVE Encyclopedia of Experiments

Neurowetenschap

0 weergaven • 2:23 min • June 17th, 2025

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Take a co-culture of immortalized human olfactory ensheathing glia, or OEG cells, and axotomized rat retinal ganglion neurons, or RGNs, on a coverslip.

OEG cells exhibit neuroregenerative properties that facilitate axonal regeneration in RGNs.

RGNs are immunolabeled so that their somas, or cell bodies, fluoresce red while their axons fluoresce green.

Using mounting media, mount the coverslip containing the cells.

Use an epifluorescence microscope to capture multiple images of RGNs.

Open an image in imaging software.

Quantify the percentage of neurons with the axons relative to the total number of RGNs.

Next, trace an axon starting from the soma till the end and begin axon tracking to determine the lengths of all axons.

Quantify the axonal regeneration index by dividing the sum of all axonal lengths by the total number of RGNs.

Mount the coverslips with mounting medium and keep them at 4 degrees Celsius. Use a 40 times objective of an epifluorescence microscope to quantify axonal regeneration. Quantify the percentage of neurons with axons relative to the total population of retinal ganglion neurons.

Use the NeuronJ plugin in ImageJ to quantify the axonal regeneration index, or mean axonal length, which is the sum of the lengths of all identified axons divided by the total number of counted neurons, whether they presented an axon or not. After opening the image, click on Analyze and set scale according to microscope settings, working in pixels per micrometer. Select Add Tracings to begin axon tracking, then track the axons from the soma to the end. Click on Measure Tracings, display tracing measurements, and Run.

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Laatst bijgewerkt: 1 augustus 2026