Simultaneous labeling of projecting neurons and apoptotic state.

We describe a straightforward method that accomplishes both the labeling of projecting neurons and the identification of apoptosis in those neurons. A single dye, 4',6-diamidino-2-phenylindole (DAPI), is both retrogradely transported and binds DNA. When delivered to the sites of neuronal projections, DAPI travels via retrograde transport from neuronal projections to the soma and stain nuclei with little or no cytoplasmic labeling. The staining of the nuclei allows for visualization of their morphological characteristics; DAPI-stained living cells appear markedly different from DAPI-stained apoptotic cells, due to the nuclear changes that apoptotic cells undergo. This technique has been successfully employed with retinal ganglion cells in the retinocollicular pathway. The use of a single dye not only eliminates the need for secondary staining for apoptosis, but also allows for the use of a wider variety of non-overlapping fluorescent dyes for other studies.

Amplification of a reactive oxygen species signal in axotomized retinal ganglion cells.

Retinal ganglion cells (RGCs) undergo apoptosis after axonal injury. Elucidation of the sequence of intracellular events proximal to caspase activation may allow development of effective neuroprotective strategies. In this study, we explored the role that reactive oxygen species may have in signaling RGC apoptosis after axonal injury. Using the fluorescent probe dihydroethidium, we were able to measure intracellular superoxide anion production. We found that axotomized RGCs exposed to oxidative stress exhibited a secondary superoxide burst. The broad-spectrum caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethyl ketone did not block the burst, suggesting it is proximal to caspase activation, but it was inhibited by cycloheximide, consistent with a requirement for protein synthesis. These results are consistent with RGC axotomy inducing synthesis of one or more proteins that mediate oxidative amplification. This could be an early event in signaling of RGC apoptosis after axonal injury.