Comparison of chorioretinal layers in rhesus macaques using spectral-domain optical coherence tomography and high-resolution histological sections

Abstract

Nonhuman primates are important preclinical models of retinal diseases because they uniquely possess a macula similar to humans. Ocular imaging technologies such as spectral-domain optical coherence tomography (SD-OCT) allow noninvasive, in vivo measurements of chorioretinal layers with near-histological resolution. However, the boundaries are based on differences in reflectivity, and detailed correlations with histological tissue layers have not been explored in rhesus macaques, which are widely used for biomedical research. Here, we compare the macular anatomy and thickness measurements of chorioretinal layers in rhesus macaque eyes using SD-OCT and high-resolution histological sections. Images were obtained from methylmethacrylate-embedded histological sections of 6 healthy adult rhesus macaques, and compared with SD-OCT images from 6 age-matched animals. Thicknesses of chorioretinal layers were measured across the central 3 mm macular region using custom semi-automated or manual software segmentation, and compared between the two modalities. We found that histological sections provide better distinction between the ganglion cell layer (GCL) and inner plexiform layer (IPL) than SD-OCT imaging. The first hyperreflective band between the external limiting membrane (ELM) and retinal pigment epithelium (RPE) appears wider on SD-OCT than the junction between photoreceptor inner and outer segments seen on histology. SD-OCT poorly distinguishes Henle nerve fibers from the outer nuclear layer (ONL), while histology correctly identifies these fibers as part of the outer plexiform layer (OPL). Overall, the GCL, inner nuclear layer (INL), and OPL are significantly thicker on histology, especially at the fovea; while the ONL, choriocapillaris (CC), and outer choroid (OC) are thicker on SD-OCT. Our results show that both SD-OCT and high-resolution histological sections allow reliable measurements of chorioretinal layers in rhesus macaques, with distinct advantages for different sublayers. These findings demonstrate the effects of tissue processing on chorioretinal anatomy, and provide normative values for chorioretinal thickness measurements on SD-OCT for future studies of disease models in these nonhuman primates.

Keywords: Histology; Optical coherence tomography; Primate; Retina; Rhesus macaque.

Figure 1

Comparison of high-resolution histological sections and spectral domain-optical coherence tomography (SD-OCT) images of a normal eye in an adult rhesus macaque. Histological sections stained with toluidine O (A) demonstrate individual chorioretinal layers corresponding to those seen on an SD-OCT image (B) along with the reflectivity profile measured from 10 adjacent A-scans at the left-hand side of panel B. Higher magnification (C) of the area outlined in panel A (yellow dashed box) shows greater details of the outer retinal and inner choroidal layers. The photoreceptor outer segments can be seen to overlap slightly with the RPE apical processes. The vacuolization of some cone pedicles in the outer plexiform layer and also some outer segments is artifact. Scale bars: 200 μm. Abbreviations: NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; ELM, external limiting membrane; IS, photoreceptor inner segments; OS, photoreceptor outer segments; RPE, retinal pigment epithelium; CC, choriocapillaris; OC, outer choroid.

Figure 2

Image segmentation and thickness of chorioretinal layers in rhesus macaques. A high-resolution histological section (A) and SD-OCT image (B) of normal maculae from adult rhesus macaque eyes before (top panels) and after (bottom panels) image segmentation. Retinal and choroidal thickness measurements across the central 3 mm segment around the fovea, as well as individual sublayers are compared between histology (C) and SD-OCT (D). Cumulative thicknesses are measured from Bruch’s membrane, which is designated at zero. Scale bars: 200 μm. The line graphs in (C) and (D) are not drawn to the same scale as the SD-OCT and histological images in (A) and (B). Abbreviations: NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; ELM, external limiting membrane; IS, photoreceptor inner segments; OS, photoreceptor outer segments; RPE, retinal pigment epithelium; CC, choriocapillaris; OC, outer choroid.

Figure 3

Comparison of chorioretinal layer thicknesses measured from high-resolution histological sections and SD-OCT across the central 3 mm segment around the fovea. Topographic plots of the nerve fiber layer (A), ganglion cell layer (B), inner plexiform layer (C), inner nuclear layer (D), outer plexiform layer (E); outer nuclear layer (F); photoreceptor inner segments (G); photoreceptor outer segments (H), retinal pigment epithelium (I), choriocapillaris (J), and outer choroid (K), along with total retinal thickness (L) show differences in measurements taken from histological sections (solid lines) and SD-OCT images (dashed lines).