Cellular Characterization of OCT and Outer Retinal Bands Using Specific Immunohistochemistry Markers and Clinical Implications

Abstract

Purpose: OCT has been a technological breakthrough in the diagnosis, treatment, and follow-up of many ocular diseases, especially retinal and neuro-ophthalmologic pathologic conditions. Until now, several controversies have arisen over the specific cell types that the bands observed in the OCT represent, especially over the 4 outer retinal bands. Design: To correlate the 4 outer hyperreflective bands observed in the OCT with the histologic structures using human retinal sections and immunocytochemistry at the fovea level. Participants: Eyes from human donors. Methods: Vertical cryosections of human retinas were immunostained with antibodies specific for cones photoreceptors, bipolar cells, mitochondria, Müller cells, and retinal pigment epithelium (RPE) cells and were visualized using confocal microscopy. Main Outcome Measures: Morphological correlation between histology and OCT at the fovea level. Results: Triple immunolabeling allowed distinguishing between cells types and different cell compartments. Immunostaining with guanine nucleotide-binding protein β 3 (GNB3) and cellular retinaldehyde-binding protein (CRALBP) antibodies showed all retinal layers at the foveola, especially the separation between the outer nuclear layer and the Henle fiber layer. CRALBP and cytochrome C (Cyt C) immunolabeling revealed that hyperreflective bands 1 and 2, observed in the OCT, correspond to the outer limiting membrane and the cone ellipsoids, respectively, separated by the cone myoids. CRALBP, cytochrome C, and GNB3 showed that the RPE interdigitations extend along the entire external segment of the cones, we do not believe them to be the structure responsible for forming the third band. However, the identification of small fragments of cone outer segments within the RPE led us to characterize the third band as the cone phagosomes located in the top of the RPE. Finally, we propose that the fourth band corresponds to the accumulation of mitochondria at the basal portion of the RPE, as identified by cytochrome C immunoreactivity, and that the hyporeflective band between bands 3 and 4 corresponds to the RPE nuclei and melanosomes zone. Conclusions: This study proposes a new interpretation of the outer retinal bands that leads to a more accurate interpretation of OCT images, providing information about the health of cones and their relationship with the RPE, and could help to form a better understanding of retinal disease diagnosis and prognosis.