In vivo staining of oligodendroglia in the rabbit retina

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Abstract
We have discovered that a strongly fluorescent dye, sulforhodamine 101, when injected intravitreally in vivo, very effectively stains a class of star‐shaped cells in the innermost layers of the rabbit retina. The cells were strictly confined to the region containing medullated fibers and emitted dichotomously branching processes that ended up running some distance along the myelinated fibers. In favorable cases they could be seen to ensheath the fibers in a tube‐like fashion. No other retinal cells were stained. Shortly (hours) after the injection, the stain appeared in the cell cytoplasm, but it later became progressively more localized to intracellular granules. Most of the dye had disappeared after 2 days. Oligodendrocytes and astrocytes are the only cells known to be confined to the region of the medullated fibers in the rabbit retina, and hence the sulforhodamine 101‐stained cells should be one of these two types. Sulforhodamine 101‐stained cells were indistin‐guishable from oligodendrocytes identified by 2′,3′‐cyclic nucleotide phosphodiesterase (CNP) immunohistochemistry, and sequential staining showed them to be the same. Sulforhodamine 101‐stained cells were microinjected with lucifer yellow after lightly fixing rabbit retinas with formaldehyde and were found to be indistinguishable from oligodendrocytes. Glial fibrillary acidic protein staining for astrocytes showed fiber bundles that to some extent were similar to the bundles stained by sulforhodamine 101, but at the level of individual fibers, it was impossible to establish any concordance. Sulforhodamine 101 thus appears to stain oligodendrocytes rather than astrocytes in the rabbit retina. A related dye, rhodamine 123, also stained rabbit oligodendrocytes, but with poor contrast because many other cells and structures were also stained. A number of related rhodamine dyes did not stain the rabbit oligodendrocytes. The staining procedure is simple, has wide tolerance margins, gives robust results, and is well suited for cell microinjections and similar experiments.