Electron Microscopic Analysis of Axonal Damage in the Optic Nerve of Mice with Brain Injury

Isolate the optic nerve from transgenic mice with brain injuries at defined intervals post-injury. The nerve contains neurons expressing a fluorescent protein.

Using fluorescence microscopy, visualize the injured nerve, which exhibits a progressive increase in axonal swellings and disconnections.

Fix the tissue to preserve cellular structures.

Rinse the tissue to remove excess fixative.

Treat with an osmium-ferrocyanide solution that binds to myelin sheath lipids and enhances contrast for imaging.

Rinse the tissue, then incubate in a heavy metal compound that binds to macromolecules and enhances contrast.

Dehydrate the tissue using increasing alcohol concentrations and embed in a resin.

Obtain transverse and longitudinal sections and treat them with heavy metal compounds, improving contrast.

Under an electron microscope, the longitudinal sections reveal a progressive development of axonal swellings with accumulated swollen mitochondria, vesicles, membrane blebbing, and disrupted cytoskeletal structures.

The transverse sections show a progressive disruption of myelin sheaths, indicating axonal degeneration.