Katie M. Litts

OUTER RETINAL TUBULATION IN ADVANCED AGE-RELATED MACULAR DEGENERATION: Optical Coherence Tomographic Findings Correspond to Histology.

Purpose: To compare optical coherence tomography (OCT) and histology of outer retinal tubulation (ORT) secondary to advanced age-related macular degeneration in patients and in postmortem specimens, with particular attention to the basis of the hyperreflective border of ORT. Method: A private referral practice (imaging) and an academic research laboratory (histology) collaborated on two retrospective case series. High-resolution OCT raster scans of 43 eyes (34 patients) manifesting ORT secondary to advanced age-related macular degeneration were compared to high-resolution histologic sections through the fovea and superior perifovea of donor eyes (13 atrophic age-related macular degeneration and 40 neovascular age-related macular degeneration) preserved ⩽4 hours after death. Results: Outer retinal tubulation seen on OCT correlated with histologic findings of tubular structures consisted largely of cones lacking outer segments and lacking inner segments. Four phases of cone degeneration were histologically distinguishable in ORT lumenal walls, nascent, mature, degenerate, and end stage (inner segments and outer segments, inner segments only, no inner segments, and no photoreceptors and only Müller cells forming external limiting membrane, respectively). Mitochondria, which are normally long and bundled within inner segment ellipsoids, were small and scattered within shrunken inner segments and cell bodies of surviving cones. A lumenal border was delimited by an external limiting membrane. Outer retinal tubulation observed in closed and open configurations was distinguishable from cysts and photoreceptor islands on both OCT and histology. Hyperreflective lumenal material seen on OCT represents trapped retinal pigment epithelium and nonretinal pigment epithelium cells. Conclusion: The defining OCT features of ORT are location in the outer nuclear layer, a hyperreflective band differentiating it from cysts, and retinal pigment epithelium that is either dysmorphic or absent. Histologic and OCT findings of outer retinal tubulation corresponded in regard to composition, location, shape, and stages of formation. The reflectivity of ORT lumenal walls on OCT apparently does not require an outer segment or an inner/outer segment junction, indicating an independent reflectivity source, possibly mitochondria, in the inner segments.

Inner Segment Remodeling and Mitochondrial Translocation in Cone Photoreceptors in Age-Related Macular Degeneration With Outer Retinal Tubulation

PURPOSE To quantify impressions of mitochondrial translocation in degenerating cones and to determine the nature of accumulated material in the subretinal space with apparent inner segment (IS)-like features by examining cone IS ultrastructure. METHODS Human donor eyes with advanced age-related macular degeneration (AMD) were screened for outer retinal tubulation (ORT) in macula-wide, high-resolution digital sections. Degenerating cones inside ORT (ORT cones) and outside ORT (non-ORT cones) from AMD eyes and unaffected cones in age-matched control eyes were imaged using transmission electron microscopy. The distances of mitochondria to the external limiting membrane (ELM), cone IS length, and cone IS width at the ELM were measured. RESULTS Outer retinal tubulation and non-ORT cones lose outer segments (OS), followed by shortening of IS and mitochondria. In non-ORT cones, IS broaden. Outer retinal tubulation and non-ORT cone IS myoids become undetectable due to mitochondria redistribution toward the nucleus. Some ORT cones were found lacking IS and containing mitochondria in the outer fiber (between soma and ELM). Unlike long, thin IS mitochondria in control cones, ORT and non-ORT IS mitochondria are ovoid or reniform. Shed IS, some containing mitochondria, were found in the subretinal space. CONCLUSIONS In AMD, macula cones exhibit loss of detectable myoid due to IS shortening in addition to OS loss, as described. Mitochondria shrink and translocate toward the nucleus. As reflectivity sources, translocating mitochondria may be detectable using in vivo imaging to monitor photoreceptor degeneration in retinal disorders. These results improve the knowledge basis for interpreting high-resolution clinical retinal imaging.

RefMoB, a Reflectivity Feature Model-Based Automated Method for Measuring Four Outer Retinal Hyperreflective Bands in Optical Coherence Tomography

PURPOSE To validate a model-driven method (RefMoB) of automatically describing the four outer retinal hyperreflective bands revealed by spectral-domain optical coherence tomography (SDOCT), for comparison with histology of normal macula; to report thickness and position of bands, particularly band 2 (ellipsoid zone [EZ], commonly called IS/OS). METHODS Foveal and superior perifoveal scans of seven SDOCT volumes of five individuals aged 28 to 69 years with healthy maculas were used (seven eyes for validation, five eyes for measurement). RefMoB determines band thickness and position by a multistage procedure that models reflectivities as a summation of Gaussians. Band thickness and positions were compared with those obtained by manual evaluators for the same scans, and compared with an independent published histological dataset. RESULTS Agreement among manual evaluators was moderate. Relative to manual evaluation, RefMoB reported reduced thickness and vertical shifts in band positions in a band-specific manner for both simulated and empirical data. In foveal and perifoveal scans, band 1 was thick relative to the anatomical external limiting membrane, band 2 aligned with the outer one-third of the anatomical IS ellipsoid, and band 3 (IZ, interdigitation of retinal pigment epithelium and photoreceptors) was cleanly delineated. CONCLUSIONS RefMoB is suitable for automatic description of the location and thickness of the four outer retinal hyperreflective bands. Initial results suggest that band 2 aligns with the outer ellipsoid, thus supporting its recent designation as EZ. Automated and objective delineation of band 3 will help investigations of structural biomarkers of dark-adaptation changes in aging.

Quantitative Analysis of Outer Retinal Tubulation in Age-Related Macular Degeneration From Spectral-Domain Optical Coherence Tomography and Histology.

Purpose To assess outer retinal tubulation (ORT) morphology from spectral-domain optical coherence tomography (SD-OCT) volumes and donor eye histology, analyze ORT reflectivity, and estimate the number of cones surviving in ORT. Methods In SD-OCT volumes from nine patients with advanced AMD, ORT was analyzed en face and in B-scans. The hyperreflective ORT border in cross-section was delineated and surface area calculated. Reflectivity was compared between ORT types (Closed, Open, Forming, and Branching). A flatmount retina from a donor with neovascular AMD was labeled to visualize the external limiting membrane that delimits ORT and allow measurements of cross-sectional cone area, center-to-center cone spacing, and cone density. The number of cones surviving in ORT was estimated. Results By en face SD-OCT, ORT varies in complexity and shape. Outer retinal tubulation networks almost always contain Closed cross-sections. Spectral-domain OCT volumes containing almost exclusively Closed ORTs showed no significant direction-dependent differences in hyperreflective ORT border intensity. The surface areas of partial ORT assessed by SD-OCT volumes ranged from 0.16 to 1.76 mm2. From the flatmount retina, the average cross-sectional area of cone inner segments was 49.1 ± 7.9 μm2. The average cone spacing was 7.5 ± 0.6 μm. Outer retinal tubulation cone density was 20,351 cones/mm2. The estimated number of cones in ORT in a macula ranged from 26,399 to 186,833 cones, which is 6% to 44% of the cones present in a healthy macula. Conclusions These first estimates for cone density and number of cones surviving in ORT suggest that ORT formation considerably distorts the photoreceptor mosaic. Results provide additional insight into the reflectivity characteristics and number of ORT cones observable in living patients by SD-OCT, as cones persist and disease progresses.

Exploring Photoreceptor Reflectivity Through Multimodal Imaging Of Outer Retinal Tubulation In Advanced Age-related Macular Degeneration.

Purpose: To investigate the microscopic structure of outer retinal tubulation (ORT) and optical properties of cone photoreceptors in vivo, we studied ORT appearance by multimodal imaging, including spectral domain optical coherence tomography (SD-OCT) and adaptive optics scanning laser ophthalmoscopy. Methods: Four eyes of four subjects with advanced age-related macular degeneration underwent color fundus photography, infrared reflectance imaging, SD-OCT, and adaptive optics scanning laser ophthalmoscopy with a high-resolution research instrument. Outer retinal tubulation was identified in closely spaced (11 &mgr;m) SD-OCT volume scans. Results: Outer retinal tubulation in cross-sectional and en face SD-OCT was a hyporeflective area representing a lumen surrounded by a hyperreflective border consisting of cone photoreceptor mitochondria and external limiting membrane, per previous histology. In contrast, ORT by adaptive optics scanning laser ophthalmoscopy was a hyporeflective structure of the same shape as in en face SD-OCT but lacking visualizable cone photoreceptors. Conclusion: Lack of ORT cone reflectivity by adaptive optics scanning laser ophthalmoscopy indicates that cones have lost their normal directionality and waveguiding property due to loss of outer segments and subsequent retinal remodeling. Reflective ORT cones by SD-OCT, in contrast, may depend partly on mitochondria as light scatterers within inner segments of these degenerating cells, a phenomenon enhanced by coherent imaging. Multimodal imaging of ORT provides insight into cone degeneration and reflectivity sources in optical coherence tomography.